Patent Application
20200377885
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
The present disclosure generally relates to methods and compositions for generating neurons, conversion of fibroblasts, and treatment of motor neuron diseases.
Among the various aspects of the present disclosure is the provision of a method of generating a neuron. In some embodiments, the neuron is generated from an adult fibroblast cell comprising miR-9/9 and miR-124(miR-9/9*-124); and one or more transcription factors. In some embodiments, the transcription factors comprise ISL1 and LHX3.
An aspect of the present disclosure provides for a method of generating a neuron from an adult somatic cell. In some embodiments, the method comprises (i) providing an adult somatic cell, optionally, a fibroblast; (ii) providing at least one miRNA to the somatic cell; or (iii) providing one or more transcription factors to the somatic cell, resulting in conversion of the somatic cell into a converted neuron. In some embodiments, the adult somatic cell is an adult human fibroblast of mesodermal origin. In some embodiments, the miRNA is selected from miR-9/9* and miR-124 (miR-9/9*-124). In some embodiments, the one or more transcription factors comprise: a motor neuron transcription factor comprising ISL1 and LHX3; or a striatal-enriched factor comprising CTIP2, DLX1, DLX2, and MYT1L (CDM); or optionally further comprise a neurogenic transcription factors comprising NeuroD2, ASCL1 and Myt1L (DAM). In some embodiments, the one or more transcription factors initiate conversion of a somatic cell toward a clinically relevant cell type. In some embodiments, the miRNA, optionally miR-9/9*-124, is expressed in the somatic cell by transduction; the one or more transcription factors, optionally a motor neuron transcription factor or a striatal-enriched factor, are expressed in the somatic cell by transduction; or the converted neuron is a motor neuron or a medium spiny neuron (MSN). In some embodiments, the miRNA, optionally miR-9/9*-124, or the one or more transcription factors, optionally a motor neuron transcription factor or a striatal-enriched factor, is expressed in the somatic cell by viral vector transduction, optionally lentivirus transduction. In some embodiments, a viral vector expresses miRNA, optionally miR-9/9*-124 and an anti-apoptotic gene, optionally, BCL-XL, beneficial for neuronal conversion, under a doxycycline-inducible promoter. In some embodiments, the miRNA or transcription factors are cloned into a lentiviral plasmid; a lentivirus is produced and the somatic cells are infected; the lentivirus genome comprises miRNA or one or more transcription factors and is transfected into the fibroblast genome, resulting in a transduced fibroblast cell; or the miRNA or transcription factors are stably expressed by the transduced fibroblast cell. In some embodiments, the miRNA or the one or more transcription factors are administered exogenously to the somatic cells. In some embodiments, the miRNA coordinates epigenetic and transcriptional changes resulting in neuronal cell fate conversion; induces a generic neuronal state characterized by loss of fibroblast identity, presence of a pan-neuronal gene expression program, and absence of subtype specificity; initiates subunit switching within BAF chromatin remodeling complexes while separately repressing neuronal cell-fate inhibitors REST, CO-REST, and SCP1: or alters expression of genes involved in DNA methylation, histone modifications, chromatin remodeling, and chromatin compaction. In some embodiments, the converted neuron is selected from the group consisting of: a motor neuron, a spinal motor neuron, a cortical neuron, a cortical-like neuron, a striatal neuron, a medium spiny neuron (MSN), a striatal medium spiny neuron (MSN), a dopaminergic neuron, a GABAergic neuron, a cholinergic neuron, serotonergic neuron, and a glutamatergic neuron. In some embodiments, the converted neuron phenotypically resembles endogenous motor neurons when compared using immunostaining analysis or gene expression profiling; the converted neuron resembles endogenous motor neurons when compared using electrophysiological tests or co-culture tests; or the converted neuron retains donor age marks and positional information.
Another aspect of the present disclosure provides for a method of modeling a neurodegenerative disease. In some embodiments, the method comprises: (i) providing a fibroblast from a subject with a neurodegenerative disease; or (ii) providing miR-9/9* and miR-124 (miR-9/9*-124) or one or more transcription factors to the fibroblast. In some embodiments, the neurodegenerative disease, disorder, or condition is selected from one or more of the group consisting of: (i) a motor neuron disease; (ii) spinal cord injury (SCI); (iii) Amyotrophic Lateral Sclerosis (ALS) or Spinal Muscular Atrophy (SMA); or (iv) Huntington's Disease (HD) or Alzheimer's Disease (AD). In some embodiments, the transcription factors comprise striatal-enriched factors or motor neuron transcription factors. In some embodiments, the striatal-enriched factors comprise CTIP2, DLX1, DLX2, and MYT1L (CDM) or the motor neuron transcription factors comprise ISL1 and LHX3. An aspect of the present disclosure provides for a method of generating a Huntington's Disease (HD) cellular platform comprising: (i) providing adult fibroblasts from a subject with HD; and (ii) providing miR-9/9* and miR-124 (miR-9/9*-124) and CDM to the fibroblast; wherein providing miR-9/9*-124 and CDM to an adult fibroblast results in generation of HD-MSNs from adult fibroblasts (HD-FB). In some embodiments, the HD-MSNs exhibit an HD-associated phenotype selected from one or more of the group consisting of: formation of aberrant protein aggregates, mHTT-induced DNA damage, spontaneous degeneration over time in culture, decline in mitochondrial function, or CAG repeat lengths remain stable after neuronal conversion.
Another aspect of the present disclosure provides for a composition comprising a cell-conversion agent. In some embodiments, the composition comprises: (i) one or more micro RNA and one or more of a motor neuron transcription factor or a striatal-enriched factor; (ii) a viral vector comprising miR-9/9*-124; (iii) a viral vector comprising ISL1 and LHX3; or (iv) a viral vector comprising CDM. In some embodiments, the cell-conversion agent initiates lineage-specific neuronal reprogramming in an adult fibroblast and generating a human neuron subtype from an adult fibroblast.
Another aspect of the present disclosure provides for a method of screening a candidate drug for effectiveness in treating a neurodegenerative or motor neuron disease. In some embodiments, the method comprises: (i) providing a cellular platform, the cellular platform comprising a neuron generated from a fibroblast of a subject with a neurodegenerative or motor neuron disease: (ii) providing a candidate drug; (iii) contacting the candidate drug and the cellular platform; and (iv) assessing efficacy of the candidate drug. In some embodiments, the cellular platform comprises cells obtained from a subject with a motor neuron disease, Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Spinal Cord Injury (SCI), or Huntington's Disease (HD). In some embodiments, the efficacy is evaluated by monitoring the neurons for reversal of electrical impairment.
Another aspect of the present disclosure provides for a method of screening therapeutic efficacy. In some embodiments, the method comprises: (i) providing a Huntington's Disease (HD) cellular platform comprising HD-MSNs derived from an adult human fibroblast; (ii) contacting the HD-MSNs with a therapeutic agent; or (iii) evaluating HD-MSN response to the therapeutic agent. In some embodiments, the therapeutic agent comprises a pharmacological factor or a genetic factor; or evaluating HD-MSN response comprises detecting levels of spontaneous cell death, stress-induced cell death, or electrophysiological properties.
Another aspect of the present disclosure provides for a method of treating a neurodegenerative disease disorder, or condition in a subject. In some embodiments, the method comprises: (i) administering an SP9 regulating agent to the subject; (ii) expressing SP9 in MSNs by cloning the cDNA of SP9 downstream of a human EF1α promoter in a viral vector.
Another aspect of the present disclosure provides for a method of screening compositions for an SP9 modulating agent. In some embodiments, the method comprises: obtaining cells from a subject; contacting the cells with a suspected SP9 modulating agent; or measuring the expression of SP9 on the cells.
An aspect of the present disclosure provides for a method of treating a neurodegenerative disease disorder, or condition in a subject. In some embodiments, the method comprises: (i) providing an adult fibroblast; (ii) providing miR-9/9* and miR-124 (miR-9/9*-124) and one or more transcription factors to the adult fibroblast; (iii) administering a cell-conversion agent composition comprising miR-9/9* and miR-124 (miR-9/9*-124) and one or more transcription factors, optionally, a motor neuron transcription factor comprising ISL1 and LHX3 to a fibroblast or a subject; or (iv) administering a converted neuron, optionally a motor neuron, to a subject.
Other objects and features will be in part apparent and in part pointed out hereinafter.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
The present disclosure is based, at least in part, on the discovery that in addition to micro RNAs (e.g., miR-9/9*-124) transcription factors can be added to somatic cells (e.g., adult somatic cells, adult human fibroblasts, adult human fibroblast of mesodermal origin) to differentiate into clinically relevant neurons (e.g., motor neurons, MSNs). The present disclosure describes that motor neuron genes become accessible in response to miR-9/9*-124. More specifically, miR-9/9*-124 allows the subtype-specifying activities of ISL1 and LHX3. As described herein, chromatin profiling revealed a modular synergism between microRNAs and transcription factors allowing lineage-specific neuronal reprogramming, providing a platform for generating distinct subtypes of human neurons. More specifically, the present disclosure shows small non-coding RNAs, miR-9/9* and miR-124 (miR-9/9*-124), and motor neuron transcription factors ISL1 and LHX3, directly convert somatic cells, such as adult human fibroblasts into human motor neurons. The technology can be used for human motor neuron generation and a cellular platform for drug screening.
The present disclosure is also based at least in part on the discovery that Huntington's Disease (HD) patient fibroblasts can be converted to medium spiny neurons (MSNs) through microRNA-based neuronal conversion. One of the primary barriers in treating and studying devastating neurological diseases and traumas (e.g., ALS, SMA, spinal cord injury) is the inability to isolate and directly manipulate human motor neurons in the laboratory. The technology described herein enables the generation of motor neurons directly from patients, enabling disease modeling, and drug screening while simultaneously providing a source of patient specific cells for regenerative medicine.
As described herein, the disclosure provides for extensive characterization of the efficiency and specificity of fibroblast conversion. Briefly, through immunostaining analysis and gene expression profiling it has been demonstrated that converted motor neurons (Moto-miNs) phenotypically resemble endogenous motor neurons. It was also determined that Moto-miNs behave as motor neurons through functional testing through electrophysiological and co-culture tests. Furthermore, the expression of hallmark motor neuron genes in Moto-miNs derived from multiple donor ages was directly compared to the human spinal cord and verified similar expression levels.
As described herein, a method of using small non-coding RNAs, miR-9/9* and miR-124 (miR-9/9*-124), and motor neuron transcription factors ISL1 and LHX3 have been discovered and optimized to directly convert adult human fibroblasts into human motor neurons. This technology enables the direct study and of the cell type affected in diseases (e.g., Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA)) using patient-specific motor neurons. Unique to this method is its utility of combining microRNAs and transcription factors for optimal efficiency, specificity, speed, ease of use, and importantly, the retention of both donor age marks and positional information. These features are currently believed to be important components in designing a cellular platform for drug screening.
The present disclosure contributes to the fields of developmental biology, regenerative medicine, direct conversion, neuroscience, genetics, chromatin biology, and microRNA biology.
Recent studies on cell-fate reprogramming have demonstrated the capability of generating post-mitotic neurons by directly converting an unrelated cell type such as fibroblasts. While direct cell-fate conversion presents great potential in disease-modeling and regenerative medicine, its promise strictly depends on the feasibility of converting primary human somatic cells from adults —the ideal source of cells for patient-specific disease modeling and regenerative therapy. Most studies on neuronal reprogramming focus on the utility of transcription factors using mouse fibroblasts. Unfortunately, the transcription factor-based conversion approaches that work in mouse cells often fail to efficiently generate functionally mature neurons when tested in human adult fibroblasts. This difficulty is reflected in the current hypothesis that fully differentiated human somatic cells contain epigenetic barriers that need to be overcome to allow the transition of cell-fates.
It has been demonstrated that ectopically expressing small non-coding microRNAs (miRNAs), miR-9/9′, and miR-124 (miR-9/9*-124), with transcription factors in human adult fibroblasts is sufficient to generate functionally mature neuronal subtypes (Yoo et al., Nature, 2011; Victor et al., Neuron, 2014). Interestingly, the same transcription factors in the absence of miR-9/9*-124 do not display reprogramming activities, suggesting miRNAs are capable of opening the neurogenic potential of human fibroblasts. However, the molecular events underlying the miRNA-induced resolution of the cell-fate barrier remain poorly understood.
As described herein, a series of experiments were carried out employing genome-wide DNA methylation analysis, chromatin accessibility analysis by ATAC-seq, genome-wide transciptome analyses, and cellular/electrophysiological analyses of human adult fibroblasts expressing miR-9/9*-124 to reveal the surprising potency of these miRNAs alone in inducing epigenetic and cellular remodeling leading to the adoption of a neuronal ground state. It was further demonstrated how this miRNA-induced neuronal state can be specified into a highly pure population of human spinal cord motor neurons by expression of transcription factors, ISL1 and LHX3, with miR-9/9*-124. The results are summarized briefly in the points below.
As described herein, it was discovered that expression of miR-9/9*-124 alone without any transcription factors in human adult fibroblasts is sufficient to generate a neuronal fate characterized by mature functionality and activation of a pan-neuronal genetic program. MiR-9/9*-124 also evoked extensive changes in the expression of multiple genes encoding regulators of chromatin, such as DNA-methylation-modifying proteins, proteins involved in histone modifications, and components of chromatin remodeling complexes.
Surprisingly, miR-9/9* and miR-124 led to extensive epigenetic remodeling characterized by active reconfiguration of differentially methylated regions in the genome and changes (opening and closing) in chromatin accessibilities. The miRNA-induced epigenetic state that was detected is neuronally primed in that genes involved in neurogenesis and neuronal function activate, while genes associated with a fibroblast fate are repressed. Importantly, it was found that miR-9/9*-124 induced opening of neuronal gene loci embedded in the heterochromatic regions present in human fibroblasts. This is in contrast to “pioneer” transcription factors that are capable of binding closed loci, but are unable to open large regions of the genome, further demonstrating the potency of these miRNAs as neurogenic effectors.
During miRNA-induced neuronal conversion, genomic loci associated with pan-neuronal genes open up and are expressed, whereas loci for subtype-associated genes are induced to open but are not activated. These results presented molecular insight into how the miRNA-induced neuronal state is unspecified yet poised to receive inputs from subtype-lineage determinants (often referred to as terminal selectors).
Further building on these findings, transcription factors expressed in motor neurons were screened to identify transcription factors that would synergize with miR-9/9*-124 to specifically generate human motor neurons, the cell type affected in devastating neurological diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal cord injury. By transcriptome analyses, motor neuron-specific programs activated by ISL1 and LHX3 in the background of miR-9/9*-124 were also identified.
Electrophysiology analyses revealed that 45/45 motor neurons patched, from young and old donors, had the ability to fire action potentials demonstrating the impressive functional maturity of converted motor neurons. To further validate the authenticity of motor neuron conversion, translating ribosome affinity purification (TRAP)-seq on in vivo mouse motor neurons were performed and directly compared their gene expression to converted motor neurons. Collectively, the results showed an unprecedented level of subtype specificity and functional maturity of human spinal cord motor neurons generated by the direct conversion of human adult fibroblasts.
Altogether, here is presented important epigenetic insights into how the miRNA-based reprogramming modality may serve as a platform for generating multiple clinically relevant neuronal subtypes. The present findings also provide further insights into previously demonstrated subtype-specific conversion (Yoo et al., Nature, 2011; Victor et al., Neuron 2014). Understanding such control for subtype-specificity is important particularly for modeling late onset neurological diseases that selectively affect distinct subtypes of neurons, as it was recently found that miRNA-based neuronal conversion retains the cellular age stored in the original human fibroblasts (Huh et al., eLife, 2016). This is in contrast to the resetting of age to an embryonic stage seen in induced pluripotent stem cells.
Mechanistic insights into the biological phenomena of transdifferentiation have broad implications in diverse fields of biology. First, the observation that miRNAs alone are sufficient to elicit cell-fate conversion phenotypically demonstrates that small non-coding RNAs can function as potent cell-fate regulators, much more than fine-tuning gene expressions. Indeed, the present study will provide novel insights into understanding how small RNA molecules regulate chromatin states during cellular differentiation or reprogramming. Second, due to the conversion efficiency, specificity, speed, and functional maturity seen in converted motor neurons, many researchers will employ the neuronal conversion technique presented in this study to derive spinal cord motor neurons from patients with inherited motor neuron diseases towards the goal of modeling the disease using patient neurons. Lastly, the epigenome and transcriptome datasets, provided herein, describing the epigenetic changes in the miRNA-induced neuronal state, provides a platform to derive additional neuronal subtypes directly from adult human fibroblasts. Indeed, the motivation to devise a reprogramming approach to generate human motor neurons started with the discovery that motor neuron genes become accessible in response to miR-9/9*-124, which has been successfully achieved as shown in the present disclosure.
Advances in the understanding of genetic pathways that specify neuronal cell fates during development have has enabled the directed differentiation of pluripotent stem cells into specific neuronal subtypes. This knowledge has been further leveraged to directly convert (reprogram) non-neuronal somatic cells into neurons. These direct conversion modalities can be valuable in the study of late-onset neurodegenerative diseases, as the original age of human fibroblasts is maintained in converted neurons in contrast to the cellular rejuvenation observed in induced pluripotent stem cells (Horvath, 2013; Miller et al., 2013). However, little is known about the epigenetic and molecular events that accompany direct cell-fate conversion limiting the utility of these features.
The present disclosure provides for cell-conversion agents. Cell conversion agents can convert cells (e.g., somatic cells) and convert them into neurons (e.g., MSNs). Cell conversion agents can comprise small non-coding RNA (micro RNA) and transcription factors.
Small Non-Coding RNA (Micro RNA)
As described herein, neuronal microRNAs, such as miR-9/9* and miR-124 (miR-9/9*-124) to direct cell-fate conversion of adult human fibroblasts to post-mitotic neurons and, with additional transcription factors, enable the generation of discrete neuronal subtypes. Previously, the molecular events underlying the neurogenic switch mediated by microRNAs during neuronal reprogramming were unknown. Here, the neurogenic state induced by miR-9/9*-124 expression alone was systematically dissected alone and reveal the surprising capability of miR-9/9*-124 in coordinately stimulating the reconfiguration of chromatin accessibility, DNA methylation and mRNA levels, leading to the generation of functionally excitable miRNA-induced neurons, yet uncommitted towards a particular subtype-lineage.
miRNA (e.g., miR-9/9*)
As described herein, it was discovered that micro RNAs (miRNAs) (e.g., miR-9/9*-124) concertedly and separately target components of genetic pathways that antagonize neurogenesis and promote neuronal differentiation during neural development.
The miRNAs capable of converting neurons are capable of converting somatic cells into neurons can coordinate epigenetic and transcriptional changes resulting in neuronal cell fate conversion; induce a generic neuronal state characterized by the loss of fibroblast identity, the presence of a pan-neuronal gene expression program, and absence of subtype specificity; initiate subunit switching within BAF chromatin remodeling complexes while separately repressing the neuronal cell-fate inhibitors REST, Co-REST, and SCP1; or alter the expression of genes involved in DNA methylation, histone modifications, chromatin remodeling, and chromatin compaction.
The miRNAs as described herein can concertedly and separately target components of genetic pathways that antagonize neurogenesis and promote neuronal differentiation during neural development; open the neurogenic potential of adult human fibroblasts and thus provides a platform for subtype-specific neuronal conversion of human cells; orchestrate widespread neuronal chromatin reconfiguration; or promote the opening of neuronal subtype-specific loci, but are not expressed.
A microRNA is a small non-coding RNA molecule (e.g., containing about 22 nucleotides) that can be found in plants, animals, and some viruses, that can function in RNA silencing and post-transcriptional regulation of gene expression. While the majority of miRNAs are located within the cell, some miRNAs, commonly known as circulating miRNAs or extracellular miRNAs, have also been found in an extracellular environment, including various biological fluids and cell culture media.
miRNAs can be encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA. miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced, by one or more of the following processes: cleavage of the mRNA strand into two pieces, destabilization of the mRNA through shortening of its poly(A) tail, or less efficient translation of the mRNA into proteins by ribosomes.
miRNAs can resemble the small interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway, but miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs can derive from longer regions of double-stranded RNA. The human genome can encode over 1000 miRNAs, which are abundant in many mammalian cell types and appear to target about 60% of the genes of humans and other mammals.
MicroRNAs (miRNAs) can regulate genetic pathways by binding to their target transcripts and repressing their expression. Target specificity can be governed largely through short sequence complementarity within the 5′ end of a miRNA enabling a single miRNA to target hundreds of mRNA transcripts. Moreover, a single mRNA can be targeted by multiple miRNAs, markedly enlarging the effect on single gene repression (Wu et al., 2010). These attributes position miRNAs to affect broad changes in gene expression and genetic programs despite their limited size. The convergence of genetic controls by miRNAs towards a specific biological process is exemplified by miR-9/9*- and miRNA-124 miRNAs activated at the onset of neurogenesis. For example, miR-9* and miR-124 can synergistically act as a molecular switch to initiate subunit switching within BAF chromatin remodeling complexes while separately repressing the neuronal cell-fate inhibitors REST, Co-REST, and SCP1. These examples suggest that miR-9/9* and miR-124 target components of genetic pathways that antagonize neurogenesis to promote a neuronal identity during development.
It has been shown that co-expressing the neuronal miRNAs, miR-9/9*-124, with TFs enriched in the cortex and striatum is sufficient to directly convert primary adult human fibroblasts to cortical and striatal medium spiny neurons, respectively. Furthermore, the expression of region-specific TFs alone or substituting miR-9/9*-124 with ASCL1 is insufficient to convert human fibroblasts. Therefore, the use of subtype-specifying TF activity to confer terminal identity during miRNA-mediated neuronal conversion may be reminiscent to in vivo terminal selector TFs which, upon determination of a neuronal fate, initiate and advance mature subtype-identities. However, the existence and utility of such a neuronal state during microRNA-induced neuronal reprogramming has yet to be determined.
Transcription Factors
In addition to showing the surprising capability of miRNAs, such as miR-9/9′-124, in coordinately stimulating the reconfiguration of chromatin accessibilities, it has been further shown that the microRNA-induced neuronal state enables additional transcription factors, such as ISL and LHX3, to selectively commit conversion to a highly homogenous population of human spinal cord motor neurons (Moto-miNs). Furthermore, it has been shown striatal-enriched factors (e.g., CTIP2, DLX1, DLX2 and MYT1L (CDM)) with miR-9/9-124 generate MSNs from adult human fibroblasts. Taken together, the disclosure reveals a modular synergism between microRNAs and transcription factors that allows lineage-specific neuronal reprogramming, providing a platform for generating distinct subtypes of human neurons.
The transcription factors as described herein can be administered in any method known in the art. For example transcription factors can be provided exogenously or expressed ectopically.
Striatal-Enriched Transcription Factors
As described herein, striatal-enriched factors (e.g., CTIP2, DLX1, DLX2 and MYT1L (CDM)) with miR-9/9*-124 have been shown to generate MSNs from adult human fibroblasts, yielding a neuronal population comprised of about 70-80% of MSNs.
Motor Neuron Transcription Factors
As described herein, motor neuron factors, ISL1 and LHX3, can function as terminal selectors to specify neuronal conversion to a highly enriched population of human spinal cord motor neurons. Plasticity of the miRNA-induced state was further demonstrated by directly converting adult human fibroblasts into a highly pure population of motor neurons through the addition of motor neuron enriched TFs, ISL, and LHX3, thereby presenting a modular method to directly convert human fibroblasts into desired neuronal subtypes.
Neurogenic Transcription Factors
As described herein, neurogenic transcription factors, CTIP2, DLX1, DLX2, and MYT1L (CDM) can reprogram fibroblasts into cortical-like neurons (CN).
Converted Neurons and Disease Models Derived from Patient Fibroblast Cells
The present disclosure provides for converted neurons and uses thereof for models of disease using a patients fibroblast cells, ectopic expression of microRNAs, and transcription factors (see e.g., Example 2).
As described herein, the identification of miRNA-induced neurogenic state has provided molecular insights into how multiple neuronal subtypes can be generated from patient fibroblasts for modeling neurological diseases. For example, the methods described herein can be used to convert a somatic cell such as a fibroblast cell (e.g., human fibroblast cells) into a converted neuron. The somatic cell or fibroblast cell can be any somatic cell or fibroblast capable of being converted using any of the methods as described herein. The converted neuron can be a microRNA-induced neuron (miN). For example, the converted neuron can be a motor neuron, a spinal motor neuron, a cortical neuron, a cortical-like neuron, a striatal medium spiny neuron (MSN), a dopaminergic neuron, a GABAergic neuron, a cholinergic neuron, serotonergic neuron, or a glutamatergic neuron.
Ectopic expression of brain-enriched microRNAs (miRNAs), such as miR-9/9′ and miR-124 (miR-9/9*-124), in human adult fibroblasts have been shown to directly convert fibroblasts to neurons. The miR-9/9-124-mediated conversion, partially afforded by their activity in controlling chromatin remodeling complexes, can be guided to specific and mature neuronal subtypes with the co-expression of transcription factors. As such, striatal factors (or striatal-enriched factors) CTIP2, DLX1, DLX2, and MYT1L (CDM) with miR-9/9-124 have been shown to generate MSNs from adult human fibroblasts, yielding a neuronal population comprised of 70-80% of MSNs. Given that iPSC-based protocols have reported MSN conversion efficiencies of only 5%-10% and more recently 20-40%, the MSN-specific neuronal conversion that generates a neuronal population highly enriched with MSNs from HD patients will offer a useful tool to model HD. Moreover, in contrast to neurons differentiated from iPSCs in which the age stored in original fibroblasts is erased during the induction of pluripotency20,21, directly converted neurons has been shown to retain age-associated marks of starting adult human fibroblasts, including the epigenetic age (also known as the epigenetic clock), oxidative stress, DNA damage, miRNAome, telomere lengths and transcriptome22,23. This unique feature offers potential advantages in modeling adult-onset disorders using directly converted neurons, yet the value of MSNs converted from HD patients' fibroblasts in disease modeling has not been determined.
The generation of HD patient-derived MSNs (HD-MSNs) through miR-9/9′-124-CDM-based conversion of fibroblasts is reported herein. The present disclosure focused on HD samples with CAG repeat ranges in the 40s, which represent the majority of HD cases, in contrast to previously reported studies on modeling HD with CAG repeat numbers longer than 60 CAG repeats. It was found that HD-MSNs captured many HD-associated phenotypes, including formation of aberrant protein aggregates, mHTT-induced DNA damage, spontaneous degeneration over time in culture, and decline in mitochondrial function. Furthermore, by inducing HD fibroblasts into iPSCs and redifferentiating them back into fibroblasts for miRNA-based neuronal conversion, it was discovered that differences in mHTT aggregation propensity observed in these two distinct cellular reprogramming methods are the result of drastically different levels of proteasome activity. Intriguingly, modifying the terminal neuronal cell fate to cortical neurons in directly reprogrammed HD cells alleviated mHTT-induced toxicity through reduced DNA damage and reduced cell death. Furthermore, MSNs reprogrammed from six pre-symptomatic HD patients, sampled at least 13 years before the clinical onset of the disease, were less vulnerable to mHTT-induced toxicity despite the marked presence of mHTT aggregates. These data highlight the advantages of direct neuronal conversion offers for modeling age-related phenotypes of late-onset diseases with enriched populations of specific neuronal subtypes. While the applicability of iPSCs for the development of stem cell-based therapies and modeling of developmental processes remains unequivocal, the present findings address many of the challenges for modeling adult-onset HD.
In Huntington's disease (HD), expansion of CAG codons within the Huntington gene (HTT) leads to the aberrant formation of protein aggregates and the differential degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-specific MSNs has been challenging, as neurons differentiated from induced pluripotent stem cells are free of aggregates and lack an overt cell death phenotype. Here MSNs from HD patient fibroblasts were generated through microRNA-based neuronal conversion, which has previously been shown to bypass the induction of pluripotency and retain age signatures of original fibroblasts. It was found that patient MSNs consistently exhibited mutant HTT (mHTT) aggregates, and spontaneous degeneration overtime in culture that was preceded by mHTT-dependent DNA damage. Further evidence is provided that erasure of age stored in starting fibroblasts or diverting conversion into cortical neurons resulted in differential manifestation of cellular phenotypes associated with HD, highlighting the importance of age and neuronal subtype specificity in modeling late-onset neurological disorders.
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by the abnormal expansion of CAG codons within the first exon of the Huntington (HTT) gene1,2. HD symptoms typically manifest in midlife, and include motor deficits, psychiatric symptoms and cognitive decline3. While healthy individuals have an average HTT CAG tract size of 17-20 repeats, HD patients have an expansion of 36 or more CAGs4. Moreover, CAG repeat length is directly correlated to severty of the disease and inversely related to age of onset, with abnormally large CAG expansions (>60 repeats) leading to juvenile onset5,6. Expanded CAG trinucleotides encode a polyglutamine stretch (PolyQ) that can accumulate into proteinaceous cytoplasmic and intranuclear aggregates that are generally thought to be neurotoxic7, although the formation of inclusion bodies has also been suggested as a neuroprotective mechanism8.
A striking characterstic of HD pathology is the selective degeneration of stratal medium spiny neurons (MSNs), while other neuronal subpopulations are relatively spared. Due to the clinical importance of MSNs and the lack of treatment capable of halting HD onset and progression, many protocols have been developed to generate MSNs from induced pluripotent stem cells (iPSCs) to establish patient-specific platforms for disease modeling10-12.
Studies modeling HD with patient-specific iPSC-derived MSNs have, however, only uncovered mild phenotypes, often requiring additional cellular insults10,11,13-15. For example, striatal neurons generated through HD-iPSCs demonstrated elevated levels of caspase activity only upon trophic factor withdrawal, treatment with hydrogen peroxide or high levels of glutamate, but otherwise displayed no overt cell death phenotype11,13,16. In addition, neurons differentiated from iPSCs of HD patients did not display mutant HTT (mHTT) aggregates even after the addition of cellular stressors10, and in other studies required culturing for at least 6-8 months and treatment with proteasome inhibitors before aggregates were detected14,15. Therefore, a different reprogramming approach that generates a homogenous population of patient-derived MSNs that display HD phenotypes more robustly will offer an alternative cellular platform for disease modeling and drug screening.
Neurodegeneratve Diseases, Disorders, and Conditions
The present disclosure provides for methods and compositions for treating or modeling neurodegenerative (e.g., neurological, motor neuron) diseases, disorders, or conditions or screening for therapeutics for neurological diseases, disorders, or conditions. For example, a neurodegenerative disease, disorder, or condition can be Abulia; Agraphia; Alcoholism; Alexia; Alien hand syndrome; Allan-Hemdon-Dudley syndrome; Alternating hemiplegia of childhood; Alzheimer's Disease (AD); Amaurosis fugax; Amnesia; Amyotrophic lateral sclerosis (ALS); Aneurysm; Angelman syndrome; Anosognosia; Aphasia; Apraxia; Arachnoiditis; Amold-Chiari malformation; Asomatognosia; Asperger syndrome; Ataxia; Attention deficit hyperactivity disorder; ATR-16 syndrome; Auditory processing disorder; Autism spectrum; Behcets disease; Bipolar disorder Bell's palsy; Brachial plexus injury; Brain damage; Brain injury; Brain tumor, Brody myopathy: Canavan disease; Capgras delusion; Carpal tunnel syndrome; Causalgia; Central pain syndrome; Central pontine myelinolysis; Centronuclear myopathy; Cephalic disorder; Cerebral aneurysm; Cerebral arteriosclerosis; Cerebral atrophy; Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); Cerebral dysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK syndrome); Cerebral gigantism; Cerebral palsy; Cerebral vasculitis; Cervical spinal stenosis; Charcot-Marie-Tooth disease; Chiari malformation: Chorea; Chronic fatigue syndrome; Chronic inflammatory demyelinating polyneuropathy (CIDP); Chronic pain; Cockayne syndrome; Coffin-Lowry syndrome; Coma; Complex regional pain syndrome; Compression neuropathy; Congenital facial diplegia: Corticobasal degeneration; Cranial arteritis; Craniosynostosis; Creutzfeldt-Jakob disease; Cumulative trauma disorders; Cushing's syndrome; Cyclothymic disorder Cyclic Vomiting Syndrome (CVS); Cytomegalic inclusion body disease (CIBD); Cytomegalovirus Infection; Dandy-Walker syndrome; Dawson disease; De Morsier's syndrome; Dejerine-Klumpke palsy; Dejerine-Sottas disease; Delayed sleep phase syndrome; Dementia; Dermatomyositis; Developmental coordination disorder; Diabetic neuropathy; Diffuse sclerosis; Diplopia; Disorders of consciousness; Down syndrome; Dravet syndrome; Duchenne muscular dystrophy; Dysarthria; Dysautonomia; Dyscalculia; Dysgraphia; Dyskinesia; Dyslexia; Dystonia; Empty sella syndrome; Encephalitis; Encephalocele; Encephalotrigeminal angiomatosis: Encopresis; Enuresis; Epilepsy; Epilepsy-intellectual disability in females; Erb's palsy; Erythromelalgia; Essential tremor; Exploding head syndrome; Fabry's disease; Fahr's syndrome; Fainting: Familial spastic paralysis; Febrile seizures: Fisher syndrome; Friedreich's ataxia; Fibromyalgia; Foville's syndrome; Fetal alcohol syndrome; Fragile X syndrome; Fragile X-associated tremor/ataxia syndrome (FXTAS); Gaucher's disease; Generalized epilepsy with febrile seizures plus; Gerstmann's syndrome; Giant cell arteritis: Giant cell inclusion disease; Globoid Cell Leukodystrophy; Gray matter heterotopia; Guillain-Barre syndrome; Generalized anxiety disorder; HTLV-1 associated myelopathy; Hallervorden-Spatz syndrome; Head injury; Headache; Hemifacial Spasm; Hereditary Spastic Paraplegia; Heredopathia atactica polyneuritiformis; Herpes zoster oticus; Herpes zoster; Hirayama syndrome; Hirschsprung's disease; Holmes-Adie syndrome; Holoprosencephaly; Huntington's disease; Hydranencephaly; Hydrocephalus; Hypercortisolism; Hypoxia; Immune-Mediated encephalomyelitis; Inclusion body myositis; Incontinentia pigmenti; Infantile Refsum disease; Infantile spasms; Inflammatory myopathy; Intracranial cyst; Intracranial hypertension; Isodicentric 15; Joubert syndrome: Karak syndrome: Kearns-Sayre syndrome; Kinsboume syndrome; Kleine-Levin syndrome; Klippel Feil syndrome; Krabbe disease; Kufor-Rakeb syndrome; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; Lateral medullary (Wallenberg) syndrome; Learning disabilities; Leigh's disease; Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; Leukodystrophy; Leukoencephalopathy with vanishing white matter; Lewy body dementia; Lissencephaly; Locked-in syndrome; Lou Gehrig's disease (See amyotrophic lateral sclerosis); Lumbar disc disease; Lumbar spinal stenosis; Lyme disease-Neurological Sequelae; Machado-Joseph disease (Spinocerebellar ataxia type 3); Macrencephaly; Macropsia; Mal de debarquement; Megalencephalic leukoencephalopathy with subcortical cysts; Megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; Meningitis; Menkes disease; Metachromatic leukodystrophy; Microcephaly; Micropsia; Migraine; Miller Fisher syndrome; Mini-stroke (transient ischemic attack); Misophonia; Mitochondrial myopathy; Mobius syndrome; Monomelic amyotrophy: Morvan syndrome; Motor Neurone Disease—see amyotrophic lateral sclerosis; Motor skills disorder; Moyamoya disease; Mucopolysaccharidoses; Multi-infarct dementia; Multifocal motor neuropathy; Multiple sclerosis; Multiple system atrophy; Muscular dystrophy; Myalgic encephalomyelitis; Myasthenia gravis: Myelinoclastic diffuse sclerosis; Myodonic Encephalopathy of infants; Myoclonus; Myopathy; Myotubular myopathy; Myotonia congenita; Narcolepsy; Neuro-Behget's disease; Neurofibromatosis; Neuroleptic malignant syndrome: Neurological manifestations of AIDS; Neurological sequelae of lupus; Neuromyotonia; Neuronal ceroid lipofuscinosis; Neuronal migration disorders; Neuropathy; Neurosis; Niemann-Pick disease; Non-24-hour sleep-wake disorder; Nonverbal leaming disorder; O'Sullivan-McLeod syndrome; Occipital Neuralgia; Occult Spinal Dysraphism Sequence; Ohtahara syndrome; Olivopontocerebellar atrophy; Opsoclonus myoclonus syndrome; Optic neuritis; Orthostatic Hypotension; Otosclerosis: Overuse syndrome; Palinopsia; Paresthesia; Parkinson's disease; Paramyotonia congenita; Paraneoplastic diseases; Paroxysmal attacks; Parry-Romberg syndrome; PANDAS; Pelizaeus-Merzbacher disease; Periodic paralyses; Peripheral neuropathy; Pervasive developmental disorders; Phantom limb/Phantom pain; Photic sneeze reflex; Phytanic acid storage disease; Pick's disease; Pinched nerve; Pituitary tumors; PMG; Polyneuropathy; Polio; Polymicrogyria; Polymyositis; Porencephaly; Post-polio syndrome; Postherpetic neuralgia (PHN); Postural hypotension; Prader-Willi syndrome; Primary lateral sclerosis; Prion diseases; Progressive hemifacial atrophy; Progressive multifocal leukoencephalopathy; Progressive supranuclear palsy; Prosopagnosia; Pseudotumor cerebri; Quadrantanopia; Quadriplegia; Rabies; Radiculopathy: Ramsay Hunt syndrome type I; Ramsay Hunt syndrome type II; Ramsay Hunt syndrome type III—see Ramsay-Hunt syndrome; Rasmussen encephalitis; Reflex neurovascular dystrophy; Refsum disease; REM sleep behavior disorder; Repetitive stress injury; Restless legs syndrome; Retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Rhythmic Movement Disorder Romberg syndrome; Saint Vitus dance; Sandhoff disease; Schilder's disease (two distinct conditions); Schizencephaly; Sensory processing disorder; Septo-optic dysplasia; Shaken baby syndrome; Shingles; Shy-Drager syndrome; Sjögren's syndrome; Sleep apnea; Sleeping sickness; Snatiation; Sotos syndrome; Spasticity; Spina bifida; Spinal cord injury (SCI); Spinal cord tumors; Spinal muscular atrophy; Spinal and bulbar muscular atrophy; Spinocerebellar ataxia; Split-brain; Steele-Richardson-Olszewski syndrome; Stiff-person syndrome; Stroke; Sturge-Weber syndrome; Stuttering; Subacute sclerosing panencephalitis; Subcortical arteriosclerotic encephalopathy; Superficial siderosis; Sydenham's chorea; Syncope; Synesthesia; Syringomyelia; Tarsal tunnel syndrome; Tardive dyskinesia; Tardive dysphrenia; Tarlov cyst; Tay-Sachs disease; Temporal arteritis; Temporal lobe epilepsy; Tetanus; Tethered spinal cord syndrome; Thomsen disease; Thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; Toxic encephalopathy; Transient ischemic attack; Transmissible spongiform encephalopathies; Transverse myelitis; Traumatic brain injury; Tremor; Trichotillomania; Trigeminal neuralgia; Tropical spastic paraparesis; Trypanosomiasis; Tuberous sclerosis; 22q13 deletion syndrome; Unverricht-Lundborg disease; Vestibular schwannoma (Acoustic neuroma); Von Hippel-Lindau disease (VHL); Viliuisk Encephalomyelitis (VE); Wallenberg's syndrome; West syndrome; Whiplash; Williams syndrome; Wilson's disease; Y-Linked Hearing Impairment; or Zellweger syndrome.
Motor Neuron Disease
As described herein, the present disclosure provides for the treatment of a neurodegenerative disease (e.g., a motor neuron disease) using a converted neuron, by expression of miR-9/9* and miR-124 (miR-9/9-124) and transcription factors (i.e., ISL1 and LHX3) in a human adult fibroblast.
In some embodiments, the neurodegenerative disease, disorder or condition can be a motor neuron disease (MND). Motor neuron diseases (MNDs) are a group of progressive neurological disorders that can destroy motor neurons, the cells that control essential voluntary muscle activity such as speaking, walking, breathing, and swallowing. A motor neuron disease can be an inherited disease with symptoms including difficulty or inability to grip, walk, speak, swallow, or breathe; a weakened grip, which can cause difficulty picking up or holding objects; weakness at the shoulder that makes lifting the arm difficult: a “foot drop” caused by weak ankle muscles; dragging of the leg; or slurred speech (dysarthra).
As another example, a motor neuron disease can be Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), or Spinal Cord Injury (SCI). Other motor neuron diseases can include frontotemporal dementia, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis (PLS), progressive muscular atrophy, Spinal muscular atrophy (SMA) (e.g., SMA type 1, also called Werdnig-Hoffmann disease; SMA type II; congenital SMA with arthrogryposis; Kennedy's disease, also known as progressive spinobulbar muscular atrophy), or post-polio syndrome (PPS).
Molecular Engineering
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The term “transduction”, as used herein, is a process by which foreign DNA is introduced into a cell (e.g., by a virus, viral vector, bacteriophage, naked DNA). Transduction methods are well known; see e.g., Transduction, Genetic at the US National Library of Medicine Medical Subject Headings (MeSH). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes. For example, as described herein, miRNAs and transcription factors can be cloned into a viral vector (e.g., a lentivirus plasmid, Sendai virus). For example, after cloning into the viral vector, a virus (e.g., lentivirus) is produced, and the fibroblasts are infected. The virus then integrates its genome (containing the miRNAs and TFs) into the fibroblast genome. As such, these ectopic genes are stably expressed by the transduced cells. A viral vector can be any viral vector known in the art. For example, the viral vector can be a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus.
The terms “heterologous DNA sequence”, “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cel nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
A “promoter is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position+1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
“Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
“Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
“Wild-type” refers to a virus or organism found in nature without any known mutation.
Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95/6-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by lle, Leu by lle, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
“Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm=81.5° C.+16.6(log10[Na+])+0.41(fraction G/C content)−0.63(% formamide)−(600/). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Methods of downregulation or silencing genes are known in the art. For example, expressed protein activity can be downregulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyrbonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoom and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, Tex.; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
Formulation
The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
The term “formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
The term “pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md., 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.
The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
A “stable” formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0° C. and about 60° C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
Therapeutic Methods
Also provided is a process of treating a neurodegenerative disease, disorder, or condition in a subject in need administration of a therapeutically effective amount of converted neurons, so as to substantially inhibit a neurodegenerative disease, disorder, or condition, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition.
Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neurodegenerative disease, disorder, or condition. A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans. For example, the subject can be a human subject.
Generally, a safe and effective amount of converted neurons is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of converted neurons described herein can substantially inhibit a neurodegenerative disease, disorder, or condition, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition.
According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
When used in the treatments described herein, a therapeutically effective amount of converted neurons can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to substantially inhibit a neurodegenerative disease, disorder, or condition, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition.
The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
Administration of converted neurons can occur as a single event or over a time course of treatment. For example, converted neurons can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a neurodegenerative disease, disorder, or condition.
Converted neurons can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, converted neurons can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of converted neurons, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of converted neurons, an antibiotic, an anti-inflammatory, or another agent. Converted neurons can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, converted neurons can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.
Administration
Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.
Screening
Also provided are methods for screening for SP9 modulating agent or a candidate drug or therapeutic agent (e.g., a pharmacological factor, a genetic factor). An SP9 modulating agent can be any agent that can modulate SP9 expression.
For example, a method of screening compositions for an SP9 modulating agent can comprise obtaining cells from a subject; contacting the cells with a suspected SP9 modulating agent; and measuring the expression of SP9 on the cells.
The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45,177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc.).
Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about −2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.
When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical successful if it is drug-like.
Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.
The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 Å to about 15 Å.
Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363: Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
The following example describes microRNA-induced epigenetic remodeling during direct cell-fate conversion of adult human fibroblasts. The following example shows (1) microRNAs open the neurogenic potential in human adult fibroblasts; (2) miR-9/9* and miR-124 orchestrate widespread neuronal chromatin reconfiguration; (3) neuronal subtype-specific loci open in response to microRNAs but are not expressed; and (4) terminal selector genes guide this modular neuronal state to human motor neurons.
As evidenced in this example, miR-9/9*-124 concertedly and separately targets components of genetic pathways that antagonize neurogenesis and promote neuronal differentiation during neural development.
Advances in the understanding of genetic pathways that specify neuronal cell fates during development have enabled the directed differentiation of embryonic and induced pluripotent stem cells (iPSCs) into specific neuronal subtypes. This knowledge has been further leveraged to directly convert (or reprogram) non-neuronal somatic cells into neurons via ectopic expression of pro-neural transcription factors (TFs) or neurogenic miRNAs with TFs, bypassing the induction of pluripotency. Specifically, ectopically expressing small non-coding microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), with transcription factors in human adult fibroblasts is sufficient to generate functionally mature neuronal subtypes. These direct conversion modalities may prove invaluable in the study of late-onset neurodegenerative diseases, as the original age of human fibroblasts is maintained in converted neurons in contrast to the cellular rejuvenation observed in iPSCs. Interestingly, the miRNA-mediated reprogramming approach boasts high conversion efficiency in adult human fibroblasts, which may provide unique opportunities in modeling neurological disorders using patient derived neurons (Victor et al., 2014). However, despite the advantages of direct reprogramming, little is known about the epigenetic and molecular events that accompany direct cell-fate conversion. Here, mechanistic insights into the cell-fate pioneering activity of miR-9/9*-124 are provided. The present results demonstrate that miRNAs induce remodeling of chromatin accessibilities, DNA methylation and the transcriptome leading to the generation of functionally excitable neurons. Surprisingly, during neuronal reprogramming, miR-9/9*-124 opens neuronal gene loci embedded in heterochromatic regions while simultaneously repressing fibroblast loci, revealing how miRNAs may overcome the cell-fate barrier that exists in human fibroblasts. These findings led to the discovery of a miRNA-induced permissive neurogenic ground state capable of generating multiple, clinically relevant neuronal subtypes. As such, it has been shown that the addition of motor neuron factors, ISL1 and LHX3, can function as terminal selectors to specify neuronal conversion to a highly enriched population of human spinal cord motor neurons. Altogether, these studies identify miRNA-mediated epigenetic remodeling events underlying direct neuronal conversion of human fibroblasts.
Co-expressing miR-9/9* and miR-124 (miR-9/9*-124), with TFs enriched in the cortex and striatum directly converts primary adult human fibroblasts to cortical and striatal medium spiny neurons, respectively (Victor et al., 2014; Yoo et al., 2011). However, the same TFs without miR-9/9*-124 fail to trigger neuronal conversion (Victor et al., 2014: Yoo et al., 2011), suggesting that the miRNA-induced neuronal state is permissive to terminal selector TFs which, upon determination of a neuronal fate, initiate and advance mature subtype-identities.
As described herein, the miRNA-induced neuronal state in adult human cells was identified and systematically investigated.
Longitudinal analyses of the transcriptome, genome-wide DNA-methylation and chromatin accessibilities revealed that miR-9/9*-124 induced extensive remodeling of the epigenome, including simultaneous activation of a pan-neuronal program and the reconfiguration of chromatin accessibilities. These changes precede the emergence of differentially methylated genomic regions. Because miR-9/9*-124 also led to the opening of genomic loci for multiple subtype-specific genes including established motor neuron markers, it was postulated that motor neuron-enriched transcription factors would cooperate with miR-9/9*-124 to specify a motor neuron lineage. As such, it was demonstrated that co-expressing Ts ISL1 and LHX3 along with miR-9/9*-124 generate a highly pure population of human spinal cord motor neurons. Taken together, these results demonstrate that miR-9/9*-124 opens the neurogenic potential of adult human fibroblasts and provides a platform for subtype-specific neuronal conversion of human cells.
(A) Neuronal Conversion of Human Adult Fibroblasts with miR-9/9*-124 Alone
To dissect how miRNAs alone contribute to neuronal conversion, the ability of miR-9/9*-124 was first tested to convert primary human fibroblasts collected from multiple adult individuals from ages 22 to 68 into microRNAs-induced neurons (miNs). The multiple fibroblast samples were transduced with lentivirus containing a doxycycline-(Dox-) inducible promoter driving miR-9/9*-124 and BCL-XL (Victor et al., 2014) (see e.g.,
(B) Functional Properties and Stability of MiRNA-Induced Neurons
To determine if miNs displayed membrane excitability, whole-cell recording on miNs in a mono-culture condition was performed without co-culturing with glial or primary neurons. 100% of the cells recorded (23 out of 23 cells) exhibited fast TTX-sensitive inward currents upon depolarization (see e.g.,
The minimum duration of miRNA expression required for neuronal conversion was determined by inactivating the doxycycline-inducible promoter at 3-day intervals by Dox removal beginning at day 9 until reprogramming day 30 (
(C) Transcriptional Profiling of miNs
To further explore the miR-9/9*-124-mediated neuronal output, the transcriptome of starting human adult fibroblasts and miNs were profiled after 30 days of neuronal conversion by RNA-Seq. 2,692 differentially expressed genes (DEGs) were identified in miNs representing 1,251 up-regulated and 1,441 downregulated genes in comparison to fibroblasts (log fold change 22; adj.P-value <0.01) (see e.g.,
Analysis of top 10 gene ontology (GO) terms revealed that upregulated genes in miNs are primarily enriched with terms related to neuronal development and functionality (see e.g.,
(D) transcriptional Changes in Epigenetic Machinery
Epigenetic modifications can markedly affect gene expression and developmental programs (Cantone and Fisher, 2013). The presently disclosed gene expression studies showed that when compared to fibroblasts, miNs had markedly altered expression of genes involved in DNA methylation, histone modifications, chromatin remodeling, and chromatin compaction (see e.g.,
(E) Dynamic Regulatory Events During Neuronal Reprogramming
Because transcriptome profiling at day 30 only provided a snapshot of the functional output of neuronal reprogramming, transcriptome dynamics were explored by profiling intermediary timepoints (days 3, 6, 10, and 20) by RNA-seq. The Dynamic Regulatory Events Miner (DREM) (Schulz et al., 2012) reports 13 paths of co-regulated, differentially expressed genes during the first 20 days of neuronal conversion (
Finally, there were no significant changes in ASCL1 or SOX2, TFs that have been used to reprogram somatic cells into neurons (Niu et al., 2013; Pang et al., 2011), suggesting miR-9/9*-124-induced neuronal conversion activates a neuronal program through mechanisms distinct from those previously reported.
(F) DNA Methylation Profiling of miNs
After observing numerous changes in DNA methylation machinery, genome-wide DNA methylation was assessed at an early (day 10), intermediate (day 20), and late stage (day 30) of neuronal reprogramming fibroblasts by combining methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation sensitive restriction enzyme sequencing (MRE-seq;
(G) Chromatin Remodeling in miNs
Extensive expression changes in diverse chromatin remodeling genes during neuronal reprogramming (see e.g.,
(H) Erasure of Fibroblast Epigenetic Identity and Gain of Neuronal Chromatin Architecture
To gain insight into biological relevance of changes in chromatin accessibility after miR-9/9*-124 expression, gene enrichment analysis was performed on genes with differential ATAC signals around the transcription start site ±2 Kb (TSS). 4,915 genes were identified with gradual increases and 1,763 genes were identified with gradual decreases in ATAC signals during conversion. Top GO terms associated with genes with increased ATAC signals are enriched in neuronal terms (including nervous system development, generation of neurons and neurogenesis) (see e.g.,
Next, whether miR-9/9*-124-induced chromatin accessibility is correlated with changes in mRNA levels was examined. DEGs (logFC>2 or <−2 adjusted P-value <0.01) were compared to genes with altered chromatin accessibility around the TSS and identified 501 upregulated and 184 downregulated genes that coincide with open and closed regions, respectively (see e.g.,
(I) MicroRNA-induced Chromatin Remodelinq at Heterochromatin Regions in Fibroblasts
To gain a more complete understanding of the epigenetic architecture within opened and closed chromatin sites the relationship of these regions to pre-existing histone marks present in fibroblasts was examined. It was hypothesized that regions that close during reprogramming would overlap with the active enhancer/euchromatin marks, H3K27ac and H3K4me1 in fibroblasts. Conversely, regions that open during neuronal reprogramming may overlap with heterochromatic H3K9me3 and H3K27me3 signatures pre-existing in fibroblasts.
70,661 regions commonly marked by H3K27ac and H3K4me1 and 5,843 regions commonly marked by H3K9me3 and H3K27me3 in human fibroblasts were selected based on the Roadmap Epigenome database (Roadmap Epigenomics et al., 2015). The regions with altered chromatin accessibility overlapped with these histone marks. Strikingly, it was found that 1,128 ATAC signal peaks present in day 20 miNs overlapped with regions of fibroblasts marked by H3K9me3/H3K27me3, which were heterochromatic regions in fibroblasts. Whereas H3K27ac/H3K4me1 marked euchromatic regions in fibroblasts overlapped with 16,207 peaks that were closed in miNs (see e.g.,
(J) Chromatin Remodeling is Required for Direct Conversion
To determine if chromatin changes were necessary for cell fate conversion the expression BRG1 was knocked down. BRG1 is a core component of BAF chromatin remodeling complex whose reduced function has been shown to collapse the overall chromatin architecture (Kadoch et al., 2017). After 20 days of neuronal conversion, loss of BRG1 markedly decreased the amount of MAP2 positive cells when compared to a control shRNA (
(K) Instructing miRNA-Induced Neurogenic State to Motor Neuron Fate
The deposition and removal of nucleosomes along regulatory elements within DNA inhibits or enables the binding of TFs, simultaneously facilitating and reinforcing cell-type specific gene expression programs (Jiang and Pugh, 2009). It was noted chromatin regions in miNs with enhanced accessibility were proximal to MNX1 and choline acetyl transferase CHAT, two of the hallmark genes expressed by motor neurons (Fonnum, 1973; Tanabe et al., 1998) (see e.g.,
Interestingly, ISL1 and LHX3 alone were not sufficient to induce neuronal conversion when co-expressed with a non-specific miRNA (miR-NS) (see e.g.,
(L) Electrophvsioloaical properties of Moto-miNs
Motor neurons produced from fibroblasts by co-expression of miR-9/9*-124, ISL1, and LHX3 (Moto-miNs) demonstrated robust inward and outward currents in response to depolarizing steps (see e.g.,
The ability of motor neurons to control voluntary muscle movement stems from their ability to form neuromuscular junctions (NMJs), unique synapses formed between motor neurons and muscle cells. The formation of neuro-muscular junctions (NMJs) was visualized through the co-localization of EGFP-labeled Moto-miNs, Alexa-fluor-594, Bungarotoxin (BTX, a toxin that binds to the nicotinic acetylcholine receptor (AChR) of NMJs), and myosin heavy chain. BTX puncta were not observed in the absence of Moto-miNs (see e.g.,
(M) Transcriptional Profiling of Moto-miNs
To fully characterize the acquisition of a motor neuron fate and assess the contribution of ISL1 and LHX3, the transcriptome of 22-yr-old starting fibroblasts, miNs and Moto-miNs were profiled by microarray. The loss of fibroblast gene expression (for example, S100A4, VIM and COL13A1) was again observed, and the gain of a pan-neuronal identity (for example, MAP2, NEFL, SNAP25 and SCN1A) 35 days after the expression of miR-9/9*-124 (see e.g.,
Next, the cell type-specific enrichment analysis tool (CSEA) was used (Xu et al., 2014) to test whether the gene expression profile within each population of miNs and Moto-miNs would be associated with distinct subtypes of in vivo neurons. When queried with a gene list, CSEA identifies neuronal subtypes that show significant enrichment in genes from the input list through curated transcriptomic data. The CSEA analysis of the 100 most enriched genes within Moto-miNs identified two subtypes, brainstem motor neurons and spinal motor neurons, to be significantly associated. No subtype specificity found in the miN transcriptome (see e.g.,
Lastly, HOX gene expression patterns were compared by qRT-PCR between starting fibroblasts and Moto-miNs derived from the conversion. Interestingly, a high correlation (R2=0.88) was observed between the expression levels of HOX genes before and after conversion within each of the defined spinal cord regions tested, indicating Moto-miNs retain the positional identity that existed in original fibroblasts (see e.g.,
(N) Transcriptional Activation of ISL1 and LHX3 Genomic Targets
An alternative approach for generating motor neurons is forced expression of NGN2, ISL1, and LHX3 in human embryonic stem cells (ESCs) (Mazzoni et al., 2013). The genomic targets of ISL and LHX3 identified by Mazzoni et al. were compared through ChIP-seq to genes whose expression increases in Moto-miNs compared to miNs. Surprisingly, a large cohort of overlapping genes (323) were identified that included numerous hallmark motor neuron markers (see e.g.,
(O) Comparison of Moto-miNs to Endogenous Spinal Cord Motor Neurons
The obvious difficulty in obtaining a pure population of motor neurons within human individuals prevents direct transcriptional comparisons between Moto-miNs and their human in vivo counterpart. Therefore, Moto-miNs were directly compared to fully differentiated in vivo mouse motor neurons. To interrogate the gene expression of motor neurons within the large heterogeneity of cell-types present in the spinal cord, Translating Ribosomal Affinity Purification (TRAP) followed by RNA-seq was performed (see e.g.,
This includes expression of canonical motor neuron markers such as, SLIT2 and SLIT3, host genes for the motor neuron specific miRNA, miR-218 (Amin et al., 2015) (
The process by which a fully differentiated fibroblast of mesodermal origin is directly converted into a functional neuron—a highly specialized cell normally arising from neuroectoderm—has remained largely enigmatic. Exploring the mechanism of cell fate conversion, and determining the manner in which reprogramming factors synergize, presents a unique opportunity in the study of lineage commitment, and provides a molecular foundation for choosing factors that guide conversion towards clinically relevant cell types.
In this study, the extensive neurogenic potential of miR-9/9* and miR-124 has been dissected, two brain-enriched miRNAs that when ectopically expressed in adult human fibroblasts directly evoke a neuronal state characterized by morphological changes, chromatin remodeling and DNA methylation, neuronal protein expression, and importantly, the adoption of intrinsic functional properties. The identification of miRNA-induced neurogenic state has provided molecular insights into how multiple neuronal subtypes can be generated from patient fibroblasts for modeling neurological diseases. The observation that miRNAs alone can stimulate direct conversion—leading to epigenetic, transcriptome and functional remodeling—simultaneously demonstrated the substantial neurogenic information embedded in small non-coding RNAs.
MiRNA-mediated neuronal conversion appears to be distinct from current models of cell fate reprogramming. Two models of lineage reprogramming have been proposed: one based on transcription factor cooperativity and positive feedback loops (Jaenisch and Young, 2008; Soufi et al., 2012; Vierbuchen and Wemig, 2012), and the other proposes that the “on-target” pioneer activity of a TF initiates and enables additional TFs to assist in cellular conversion (Wapinski et al., 2013). In stark contrast, canonical gene regulation by miRNAs requires the removal of information through translational repression and transcript degradation. This mode of repression in conjunction with the multitude of anti-neurogenic genes targeted by miR-9/9*-124 suggests miRNA-mediated reprogramming acts through an alternative mechanism. It is currently believed that miR-9/9*-124 expression in non-neuronal somatic cells initiates gradual, yet active changes in the activities of multiple chromatin modifiers while simultaneously repressing anti-neuronal genes and activating neuronal genes culminating in a binary cell-fate switch. This model is supported by the rapid cell cycle exit observed upon ectopic miR-9/9*-124 expression, the subsequent neuronal switching within chromatin modifiers, steady increase in epigenetic and transcriptional changes, and the time scale in which conversion takes place.
Chromatin Remodeling Accompanies Cell Fate Conversion.
In this study, the surprising potency of miR-9/9*-124 for remodeling chromatin and altering DNA methylation was revealed. Surprisingly, preexisting neuronal loci within the heterochromatic regions in human fibroblasts opened up in response to miR-9/9*-124. These data suggest the robustness of miRNA-mediated reprogramming observed in human cells could stem from their ability to induce epigenetic changes. Cellular processes and identity are governed by the cumulative action of multiple levels of genome regulation and it is unlikely a single genetic component downstream of miR-9/9*-124 mediates these changes and ultimately cell fate conversion. For example, almost every level of epigenetic remodeling participates in the induction of pluripotency (Takahashi and Yamanaka, 2016). Instructions operating through multiple levels of genetic and epigenetic regulation are likely required for true cell-fate conversion. The thorough characterization of miR-9/9*-124 induced transdifferentiation of human fibroblasts into functional neurons highlights molecular processes that are critical to cell fate conversion.
A Modular Neuronal State.
Importantly, the plastic neuronal platform presented here affords modularity to direct conversion. The synergism between miR-9/9*-124 and TFs was shown by generating a neuronal population highly enriched with spinal cord motor neurons from human adult fibroblasts through the coexpression of miR-9/9*-124, ISL1, and LHX3. Because MNs are a clinically relevant subtype affected in Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy, the robustness and specificity of neuronal conversion employing miRNAs and motor neuron TFs may pave the way towards generating patient-specific MNs for disease modeling. Unfortunately, the potent reprogramming capabilities of miR-9/9*-124 are likely restricted to neuronal identities. The brain-restricted expression of both miR-9/9* and miR-124 coupled with the data presented here, suggests this reprogramming paradigm is restricted to generating cells within the neuronal compartment, and it is unlikely that TFs important in other specialized non-neuronal lineages would enable miR-9/9*-124-mediated reprogramming towards the corresponding cell types.
The plastic neuronal platform presented here affords modularity to direct cell fate conversion. Numerous studies in developmental neuroscience have identified subtype-specific TFs or terminal selector genes that could be incorporated in neuronal reprogramming technology. Yet, identifying molecules capable of overcoming the cell-fate barrier present in human somatic cells and eliciting a permissive environment in which terminal selector genes can act has proven to be challenging. Here, this property was demonstrated by generating a neuronal population highly enriched in spinal cord motor neurons from human adult fibroblasts through the coexpression of miR-9/9*-124, ISL1, and LHX3. Because motor neurons are the major neuronal subtype affected in Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA), the robustness and specificity of neuronal conversion employing miRNAs and motor neuron TFs may pave the way towards generating patient-specific MNs for disease modeling.
(i) Plasmid Construction and Virus Production.
Complementary cDNA was generated from adult human spinal cord (Clontech) from which individual motor neuron transcription factors were subcloned into the N174 (Addgene 60859) and N106 (Addgene, 66808) lentiviral vectors using standard techniques. Lentivirus was produced in 2931e cells plated in 10 cm dishes (6.5×106 cells per dish) via polyethylineimine (48 μL of 2 mg/mL, Polysciences) assisted transfection of 3rd generation packaging vectors (1.5 μg pMD2.G, Addgene, 12259 and 4.5 μg psPAX2 Addgene, 12260), and 6 μg of lentiviral backbone plasmid (e.g. pT-BCL-9/9*-124; Addgene, 60859) 16 hours after initial plating. Media was changed the next day. After 2 days, media was harvested, filtered through a 0.45 μm polyethersulfone (PES) syringe filter and then concentrated by centrifugation at 70,000×G for 2 hours at 4C. Virus collected from a single 10 cm dish was resuspended in 1 mL of sterile PBS then aliquoted and stored at −80° C. Before each transduction, virus aliquots were spun at 5,000×G for 5 minutes at 4° C. to remove debris. Control vector expressing non-specific (NS) miRNA and BCL-XL was generated previously (Victor et al., 2014).
(ii) Cell Culture
Adult and Neonatal Human Fibroblasts were obtained from commercial sources and maintained in fibroblast media comprised of Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 15% fetal bovine serum (Life Technologies) 0.01% β-mercaptoethanol (Life Technologies), 1% non-essential amino acids 1% sodium pyruvate, 1% GlutaMAX, 1% 1M HEPES buffer solution and 1% penicillin/streptomycin solution (all from Invitrogen) and never passaged more than 15 times. Fibroblasts utilized in this study: 1 yr (PCS-201-010, ATCC), 22 yr (GM02171, NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research) 42 yr (F09-238, Washington University in St. Louis School of Medicine iPSC core facility), 56 yr (AG04148, NIA Aging Cell Repository at the Coriell Institute for Medical Research), 68 yr (ND34769, NINDS Cell Line Repository at the Coriell Institute for Medical Research)
(iii) Direct Conversion
To initiate direct conversion, 1.8×106 cells were seeded onto Costar 6 well cell culture vessels (Corning; 300,000 cells/well). The following day, each plate was transduced with the following reprogramming cocktail: 750 μL of concentrated lentivirus containing the reverse tetracycline-controlled transactivator (rtTA; Addgene, 66810) and 500 μL of virus containing pT-BCL-9/9′-124 or pT-BCL-9/9-124 and 500 μL of each individual TF driven by the EF1α promoter, and polybrene (8 μg/mL; Sigma-Aldrich) all diluted up to 18 mL (3 mL per well) then spinfected at 37° C. for 30 minutes at 1,000×G using a swinging bucket rotor. The following day media was changed to fresh fibroblast media (2 mL per well) supplemented with doxycycline (Dox; Sigma Aldrich, 1 μg/mL). After 2 days, fresh fibroblast media was changed and supplemented with Dox and antibiotics for respective vectors (Puromycin, 3 μg/ml; Blasticidin 5 μg/ml; Geneticin, 400 μg/mL; all from Invitrogen). Five days post-transduction cells were replated on to poly-omithine/laminin/fibrobnectin (PLF) coated glass coverslips. Before PLF coating, glass coverslips were acid treated according to (Richner et al., 2015). For each well of a 6 well plate, cells were first washed 2× with 1 mL sterile PBS, then 320 μL of 0.25% Trypsin (Gibco) was added to each well then placed in an incubator. Cells were monitored every 2 minutes, as soon as cells began to detach (no more than 6 minutes) 1 ml of MEF media supplemented with 1 μg/mL Dox was added to each well. One by one, each well was gently triturated three times to remove remaining attached cells then transferred to a sterile 1.5 mL eppendorf tube. Cells were then spun at 200×G for 5 minutes at 37° C. The supernatant was aspirated and cells were gently resuspended in 300 μL MEF media supplemented with Dox. Cells were then drop-plated onto either 18 mm (150 μL per/c.s.; placed in 12 well plate) or 12 mm (60 μL per c.s.; placed in 24 well plate) coverslips. Cells were left to settle for 15 minutes in an incubator then each well was flooded with MEF media supplemented with 1 μg/mL Dox. The following day media was then changed to Neuronal Media (Sciencell) supplemented with Dox, valproic acid (1 mM; EMD Millipore) dibutyryl cAMP (200 μM; Sigma-Aldrich), BDNF, NT-3, CNTF, GDNF (all 10 ng/ml, Peprotech), and Retinoic Acid (1 μM: Sigma-Aldrich) and antibiotics for each vector. Dox was replenished every two days and half the media was changed every 4 days. Drug selection was halted 14 days into conversion. A diagram of the reprogramming protocol is available in
For DNA methylation profiling, 1.8×106 human neonatal fibroblasts were seeded onto 10 cm plates (Corning). The following day, each plate was transduced with 10 ml of un-concentrated lentivirus containing a doxycycine inducible miR-9/9*-124 vector (Victor et al., 2014) and polybrene (8 μg/mL; Sigma-Aldrich). The following day media was changed to fresh fibroblast media (2 mL per well) supplemented with Dox. After 2 days, fresh fibroblast media was changed and supplemented with Dox and antibiotics for respective vectors (see TABLE 1). Seven days post-transduction, cells were first washed 2× with 3 mL sterile PBS, then 1 ml of 0.25% Trypsin (Gibco) was added to each plate then placed in an incubator. Cells were monitored every 2 minutes, as soon as cells began to detach (no more than 6 minutes), 4 ml of MEF media supplemented with 1 μg/mL Dox was added to each plate. Cells were transferred to Primaria modified 10 cm plates (Corning) and 5 ml fresh fibroblast media supplemented with 1 μg/mL Dox was added to a final volume of 10 ml. The following day media was changed to Neuronal Media (Sciencell) supplemented with Dox, valproic acid (1 mM; EMD Millipore) dibutyryl cAMP (200 μM; Sigma-Aldrich), BDNF, and NT-3 (all 10 ng/ml, Peprotech), Retinoic Acid (1 μM; Sigma-Aldrich), and 4% FBS. Dox was replenished every two days and half the media was changed every 4 days.
(iv) Myotube Differentiation
Human myotubes were generated by differentiating human myoblasts using defined culture conditions (Steinbeck et al., 2016). Briefly, human skeletal myoblasts were cultured according to manufacturer's recommendations (HSMM; CC-2580, Lonza) then were plated on matrigel (0.1 mg/mL) coated 12 mm glass coverslips at a density of 80,000 cells/well. The following day HSMM's were differentiated by switching media to skeletal muscle differentiation media comprised of a 1:1 mixture of DMEM F12 (Gibco) and Complete Neuronal Media+2% Horse Serum (Gibco). Every 2 days % of the media was replaced with fresh differentiation media. After 10-14 days of differentiation, day 14 Moto-miNs labeled with synapsin-eGFP via lentiviral transduction were replated onto myotubes at a 1:1 ratio (i.e. one 12 mm Moto-miN coverslip was replated on top of a 12 mm myotube coverslip). The following day media was changed to complete neuronal media and cells were cultures for 2 weeks. Dox was replenished every two days and half the media was changed every 4 days. After two weeks cells were fixed with 4% paraformaldehyde and processed for immunocytochemistry.
(v) Immunofluorescence and Cell Counting
Cells were fixed using 4% formaldehyde for 18 minutes at room temperature (RT) then blocked and permeabilized for one hour at RT in PBS containing 0.3% Triton-X100, 5% bovine serum albumin (Sigma-Aldrich), and 2% of either goat or donkey serum (Sigma-Aldrich). Primary antibodies were incubated overnight at 4° C. in blocking buffer. Cells were then washed 3× then incubated with secondary antibodies conjugated to either Alexa-488, -594 or -647, for one hour at RT. The following antibodies were used for immunostaining: MAP2 (Sigma-Aldrich, 1:500), TUBB3B (Covance, 1:7000), NeuN (AVES, 1:300), SCN1A (Sigma-Aldrich, 1:300) ANKG (NeuroMAB, 1:1000) SV2 (DSHB, 1:250), HB9 (DSHB, 1:200) CHAT (Millipore, 1:100) SMI-32 (Biolegend, 1:2000) Ki-67, (Abcam, 1:200), Myosin (DSHB, 1:50), a-Bungarotoxin (ThermoFisher, 1:200), NCAM (eric1) (Santa-Cruz, 1:100). Images were obtained on a Leica SP-2 Confocal Microscope. Quantifications were performed on at least 10 random fields of view in duplicate experiments.
(vi) Electrophysiology Whole-cell patch-damp recordings were performed 35-40 days post-transduction. Data was acquired using pCLAMP 10 software with multiclamp 700B amplifier and Digidata 1550 digitizer (Molecular Devices). Electrode pipettes were pulled from borosilicate glass (World Precision Instruments) and typically ranged between 5-8 MO resistance. Intrinsic neuronal properties were studied using the following solutions (in mM): Extracellular: 140 NaCl, 3 KCl, 10 Glucose, 10 HEPES, 2 CaCl2 and 1 MgCl2 (pH adjusted to 7.25 with NaOH). Intracellular: 130 K-Gluconate, 4 NaCl, 2 MgCl2, 1 EGTA, 10 HEPES, 2 Na-ATP, 0.3 Na-GTP, 5 Creatine phosphate (pH adjusted to 7.5 with KOH). Membrane potentials were typically kept at −65 mV. In voltage-clamp mode, currents were recorded with voltage steps ranging from −20 mV to +90 mV. In current-clamp mode, action potentials were elicited by injection of step currents that modulated membrane potential from −10 mV to +35 mV. Data was collected in Clampex and initially analyzed in Clampfit (Molecular Devices). Further analysis was done in GraphPad Prism 7 (GraphPad Software). Liquid junction potential was calculated to be 15.0 mV and corrected in calculating resting membrane potential according to previously published methods (Barry, 1994).
(vii) RNA-Seq Library Preparation and Sequencing
Day 30 miNs and starting human adult fibroblasts (22 yr old) were extracted by RNeasy plus micro kit (Qiagen). The RNA samples with >9.5 of RIN based on a 2100 Bioanalyzer were used for RNA-Seq library preparation. Library preparation and sequencing were performed by Genome Technology Access Center in Washington University School in St. Louis. Briefly mRNA was isolated by using SMARTer Ultra Low RNA Kit for Illumina sequencing (Clontech). All cDNA libraries, based on two biological replicates for each condition, were sequenced on Illumina Hi-Seq 2500 with single-end 50 bp read length.
(viii) RNA-Seq Data Analysis
More than 35 million reads of each RNA-seq data were aligned to human genome assembly GRCh 37. For differential expression analysis, edgeR and limma were used. Genes with low read counts, regarded as genes not expressed at a biologically meaningful level were filtered out before read normalization. The cut-off for low read count was counts per million (CPM)<1 in at least any two samples across the experiment Reads for each sample were normalized by the edgeR method of trimmed mean of M-values (TMM). The quantitative difference of read counts between miNs and starting fibroblast samples were evaluated by carrying out limma and graphically represented by Glimma. Gene enrichment analysis for differentially expressed genes was performed using Metascape Gene Annotation and Analysis Resource tool.
(ix) MicroArray Analyses
Total RNA was extracted from miNs and Moto-miNs derived from 22 yr old donor fibroblasts alongside corresponding starting fibroblast controls using TRIzol (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer's instruction and extracted using chloroform and ethanol precipitation. RNA quality was determined by the ratio of absorbance at 260 nm and 280 nm to be approximately 2.0. Samples for RNA microarray were then standardly prepped and labeled with Illumina TotalPrep kits (Thermo Fisher Scientific, Waltham, Mass.) for Agilent Human 4×44Kv1. Standard hybridization and imagine scanning procedure were performed according to the manufacturer's protocol at Genome Technology Access Center at Washington University School of Medicine, St. Louis. The intensity of the probes was imported into Partek and quantile normalized. Differentially expressed genes were identified using Partek with a cut-off of adjusted p-value <0.05 and over 2.5 log2 fold expression change.
(x) Methylated DNA Immunoprecipitation Sequencing
MeDIP-seq was performed as in Maunakea et al. (Maunakea et al., 2010). Five micrograms of genomic DNA was sonicated to a fragment size of ˜100-400 bp using the Bioruptor sonicator (Diagenode). End-repair, addition of 3′-A bases and PE adapter ligation with 2 μg of sonicated DNA was performed according to the Illumina Genomic DNA Sample Prep Kit protocol. Adapter-ligated DNA fragments were size selected to 166-366 bp and purified by gel electrophoresis. DNA was heat denatures and then immunoprecipitated with 5-methylcytidine antibody (Eurogentec; 1 μg of antibody per 1 μg of DNA) in 500 μl of immunoprecipitation buffer (10 μM sodium phosphate, pH 7.0, 140 mM sodium chloride and 0.05% Triton X-100) overnight at 4° C. Antibody/DNA complexes were isolated by addition of 1 μl of rabbit anti-mouse IgG secondary antibody (2.4 mg ml−1, Jackson Immunoresearch) and 100 μl protein A/G agarose beads (Pierce Biotechnology) for 2 h at 4° C. Beads were washed nine times with immunoprecipitation buffer and then DNA was eluted in TE buffer with 0.25% SDS and 0.25 mg ml−1 of proteinase K for 2 h at 50° C. DNA was then purified with the Qiagen Qiaquick kit and eluted in 30 μl EB buffer. Ten microliters of DNA was used for a PCR-enrichment reaction with PCR PE Primers 1.0 and 2.0. PCR products were size selected (220-420 bp) and purified by gel electrophoresis. Methylated DNA enrichment was confirmed by PCR on known methylated (SNRPN and MAGEA1 promoters) and unmethylated (a CpG-less sequence on chromosome 15 and glyceraldehyde 3-phosphate dehydrogenase promoter) sequences. DNA libraries were checked for quality by Nanodrop (Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent). Reads were aligned to hg19 using BWA and pre-processed using methylQA (an unpublished C program; available at http://methylqa.sourceforge.net/). Detailed library construction protocols for MRE-seq and MeDIP-seq are publically available at the NIH Roadmap Epigenomics project website (http://www.roadmapepigenomics.org/protocols/type/expermental/.
(xi) Methylation-Sensitive Restriction Enzyme Sequencing
MRE-seq was performed as in Maunakea et al. (Maunakea et al., 2010), with modifications as detailed below. Five parallel restriction enzyme digestions (Hpall, Bsh12361, Ssl(Acil) and Hin6l (Fermentas), and HpyCH41V (NEB)) were performed, each using 1 μg of DNA per digest for each of the samples. Five units of enzyme were initially incubated with DNA for 3 h and then an additional five units of enzyme were added to the digestion for a total of 6 h of digestion time. DNA was purified by phenol/chloroformisoamyl alcohol extraction, followed by chloroform extraction using phase lock gels. Digested DNA from the different reactions was combined and precipitated with one-tenth volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. The purified DNA was size selected and purified (50-300 bp) by gel electrophoresis and Qiagen MinElute extraction. Library construction was performed as per the Illumina Genomic DNA Sample Prep Kit protocol with the following modifications. During the end-repair reaction, T4 DNA polymerase and T4 PNK were excluded and 1 μl of 1:5 diluted Klenow DNA polymerase was used. For the adapter ligation reaction, 1 μl of 1:10 diluted PE adapter oligo mix was used. Ten microliters from the 30 μl of purified adapter ligated DNA was used for the PCR enrichment reaction with PCR PE Primers 1.0 and 2.0. PCR products were size selected and purified (170-420 bp) by gel electrophoresis and Qiagen Qiaquick extraction. DNA libraries were checked for quality by Nanodrop (Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent). Reads were aligned to hg19 using BWA and pre-processed using methylQA. MRE reads were normalized to account for differing enzyme efficiencies and methylation values were determined by counting reads with CpGs at fragment ends (Maunakea et al., 2010).
(xii) Differential DNA-Methylated Region Analysis
The M&M statistical model (Zhang et al., 2013), which integrates MeDIP-seq and MRE-seq data to identify differentially methylated regions between two samples was implemented with a window size of 500 bp and a q-value (false discovery rate (FDR)-corrected P-value) cutoff of 5e-2. This cutoff was determined from
(xiii) GO Enrichment Analyses
DNA methylation GO analyses of MGI (Mouse Genome Informatics) expression (Smith et al., 2014) presented in
(xiv) Genomic Features
DMRs from day 30 (miN day 30 vs. Ctrl day 30) were segregated into exons, introns, intergenic regions, 3′ UTRs, 5′ UTRs, non-coding regions, promoter-TSSs, and TTSs by using the annotatePeaks program provided by HOMER (Heinz et al., 2010).
(xv) ATAC-Sequencing Library Preparation and Data Processing
ATAC-seq was performed as previously described (Buenrostro et al., 2013). Briefly, 20,000 cells were collected for ATAC-seq library preparation at ctrl D10, miNs D10 and miNs D20. Transposition reaction was carried out with Nextera Tn5 Transposase for 30 min at 37° C. Library fragments were amplified for optimal amplification condition. Final libraries were purified using Ampure XP beads (Ampure) and sequenced with 50 bp paired-end reads on Illumina HiSeq 2500.
More than 50 million ATAC-seq reads were trimmed for Nextera adapter sequences using TrimGalore and aligned to hg19 human genome assembly using bowtie2 with parameters—very-sensitive—maxins 2000—no-discordant—no-mixed. Duplicate reads were discarded with Picard and uniquely mapped reads were used for downstream analysis. Peaks were called using Homer with parameters findPeaks-region-size 150-minDist 300. Peaks called from all the samples were combined together and raw reads mapped on the combined peaks were counted using HTSeq count. Differential peaks between any two different samples were identified using edgeR with a cut-off: a fold-change threshold of 1.5 and FDR<0.01. Differential peaks were regarded as peaks that are gained or lost at each time point.
Gained peaks at miNs D10 and D20 were combined together and defined as open chromatin regions. Conversely, all lost peaks at miNs D10 and D20 were defined as close chromatin regions. The genomic features in the differential open and close chromatin regions were distributed by the CEAS software (Shin et al., 2009). Ref-seq genes that are most nearest located from differential peaks with Homer annotatePeaks command were annotated. Based on those genomic distribution and peak annotation, the promoter regions (−/+2Kb of TSS) and distal regions (all peak positions except the promoter regions) were defined. GO enrichment analysis was performed by Metascape or the Gene Ontology. All heatmaps were generated based on normalized signal intensity values (i.e. log2CPM) of each sample on relevant specific regions.
All histone mark ChIP-seq data were obtained from Roadmap Epigenome database of human fibroblasts (Roadmap Epigenomics et al., 2015). To identify histone mark-occupied chromatin accessibility during reprogramming, each histone ChIP-seq data was compared with open and close chromatin regions based on ATAC-seq. It was confirmed that most open and closed chromatin peaks overlapped with histone mark-ChIP peaks were found in regions outside of promoter regions (+/−2Kb of TSS). Open and closed chromatin regions excluding the promoter regions were then used to compare with histone ChIP-seq data and perform further GO enrichment analysis.
(xvi) Translating Ribosome Affinity Purification
Translating ribosome affinity purification (Heiman et al., 2014) was performed on spinal cord dissections pooled from 3-4 mice 21 days post birth that were positive for the eGFP-L10A fusion ribosomal marker protein under the expression of either the Chat promoter (Tg(Chat-EGFP/Rpl10a)DW167 Htz) or the Snap25 promoter (Tg(Snap25-EGFP/Rpl10a)JD362Jdd). TRAP samples underwent immunopurification for four hours at 4° C. Both TRAP and pre-immunopurification control RNA samples were extracted through TRIzol purification, DNase treatment, and Qiagen RNeasy Mini columns (74104). Quality and quantity of RNA was assessed using a Bioanalyzer 2100 RNA Pico Chip. Sequencing libraries were amplified using Nugen Amplification Kit Ovation@ RNA-Seq System V2 (7102). Genome Technology Access Center at Washington University in St. Louis performed adapter ligation and sequencing of the libraries on the Illumina Hiseq2500. Three replicates of this procedure were analyzed.
(xvii) Analysis of TRAP RNA-Seq Data
RNA-Seq reads were mapped to Ensembl release 76 using STAR (analysis performed by Genome Technology Access Center at Washington University in St. Louis). For downstream analyses, only those genes with >1 CPM in at least 3 samples, with an Ensembl gene biotype of “protein_coding,” were retained. For gene symbols mapping to multiple Ensembl gene IDs, only the ID with the highest number of mapped reads was retained, resulting in a total of 14,009 genes used for downstream analyses. Using edgeR, read counts were fit to a negative binomial generalized log-linear model, and a likelihood ratio test was done to determine differential expression.
(xviii) Comparative Analysis of RNA-Seq and Microarray Data
For comparative analysis, only probes with a detected call in at least 1 of 6 samples was retained, resulting in 23,775 probes mapping to a gene symbol. Expression level was then averaged over all probes for each gene, resulting in a total of 15,333 genes that were used for comparative analysis, 10,736 of which were also present in the gene set retained from the RNA-seq dataset (described above) after CPM filtering. Within the genes retained in both datasets, the top differentially expressed genes between motor neurons and controls-CHAT IP vs. SNAP25 IP (logFC>1 and p<0.05) in the RNA-seq dataset, and Moto-miN vs. miN (logFC>2.5 and p<0.05) in the microarray dataset-were assessed for significant contingency using a one-tailed Fisher's exact test.
(xix) Quantitative PCR
Total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's instruction. Reverse-transcribed complementary DNA (cDNA) was synthesized from 500 ng of RNA with SuperScript III First-Strand Synthesis SuperMix (Invitrogen, USA) or from 10 ng of RNA for microRNAs expression analyses using specific stem-loop primer probes from TaqMan MicroRNA Assays (Invitrogen, USA). Subsequently, the cDNA was analyzed on a StepOnePlus Real-Time PCR System (AB Applied Biosystems, Germany). Expression data were normalized to housekeeping genes HPRT1 and RNU44 for coding genes and microRNAs, respectively, and analyzed using the 2-ΔΔCT relative quantification method. The following primers were utilized:
To identify the genes regulated by LHX3 and ISL in motor neurons, ISL- and LHX3-ChIP sequencing data (Mazzoni et al., 2013) were used. The regions co-occupied by ISL and LHX3 were selected during ES to motor neuron differentiation, accounting for 84.2% of peak regions called in each ChIP-seq data. Based on the peaks co-occupied by ISL and LHX3, 3,486 closest genes with peaks located within 5Kb upstream of TSS and intragenic regions were annotated. Comparing those annotated genes with genes selectively enriched in Moto-miNs vs miNs (log2 fold change=>2.5, p<0.01), identified 323 genes co-occupied by ISL1 and LHX3 that are also upregulated when miR-9/9*-124 is co-expressed with ISL1/LHX3.
The following example describes the generation of medium spiny neurons (MSNs) from Huntington's Disease (HD) patient fibroblasts through microRNA-based neuronal conversion.
Generation of MSNs from HD Patient Fibroblasts
The efficacy of miR-9/9*-124+CDM-based neuronal conversion in HD patient samples was first tested. Fibroblasts were obtained from ten symptomatic HD patients including both males and females, ranging from 6 to 71 years of age with various CAG-repeat expansions (40 to 180) in HTT (see e.g., TABLE 2). It was found that HD fibroblasts could be directly reprogrammed to MSNs regardless of age or number of CAG repeats (see e.g.,
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Electrophysiological studies in mouse models of HD have collectively demonstrated alterations in synaptic properties of striatal MSNs at different stages of disease progression. In order to determine potential functional differences, the properties of HD.47-MSNs and Ctrl. 16-MSNs were assessed by whole-cell recording. All recorded cells displayed neuronal properties, including multiple action potentials and robust inward and outward currents upon stimulation (see e.g.,
To further analyze the proper acquisition of striatal fate, RNA sequencing (RNA-seq) analysis was first performed at PID 32 and compared the gene expression profile between fibroblasts and converted neurons in HD and control samples. Analysis of 15 representative fibroblast-associated genes and of 53 genes highly enriched in the striatum revealed the successful acquisition of MSN fate in neurons converted from HD and control samples (see e.g.,
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Mutant HTT Aggregates in MSNs Directly Converted from HD Fibroblasts
Although a two-fold upregulation of HTT mRNA levels was observed in MSNs in comparison to starting fibroblasts, there are no significant changes in HTT mRNA levels in the analysis of HD-MSNs versus Ctrl-MSNs. This finding is not surprising given that HTT expression levels are comparable in the brains of HD and healthy patients. However, at the protein level, polyglutamine expansion within HTT leads to the formation of insoluble structures of aggregated mHTT, or inclusion bodies (IBs). The formation of mHTT inclusions in directly reprogrammed MSNs was then assessed by immunocytochemistry, ultrastructural, and biochemical analysis.
Noticeably, HD-MSNs exhibited mHTT aggregates in contrast to their corresponding fibroblasts or Ctrl-MSNs (see e.g.,
Induction of Pluripotency Alters mHTT Aggregation Propensity
Because the findings contrasted with previous studies that report the lack of mHTT aggregates in iPSC-derived neurons from HD patients, it was decided to directly test if altering the cellular state of adult HD fibroblasts to an embryonic-like stage, a process that effectively erases aging markers, would affect the aggregation propensity of mHTT in HD-MSNs. HD-iPSCs were derived from adult HD fibroblasts and these stem cells were differentiated back into fibroblasts using a method for generating human embryonic fibroblasts (HEFs) (see e.g.,
Although iPSCs have been previously shown to possess higher proteasome activity than their originating fibroblasts, differentiation of iPSCs into neurons also was shown to reduce proteasome activity. Therefore, to determine if changes in proteasome activity could account for the detection of mHTT aggregation in MSNs but not in heMSNs, the functional activity of the proteasome was assessed with a fluorogenic peptide LLVY-AMC assay in converted neurons. Surprisingly, it was discovered that proteostasis was collapsed in HD-MSNs in comparison to heMSNs, which retained proteasome activity more comparable to iPSCs levels (see e.g.,
mHTT-Mediated DNA Damage and Spontaneous Degeneration
Because aging contributes to the onset of HD, it was decided to test if direct conversion would also offer advantages in modeling spontaneous degeneration of HD patient's MSNs, a phenotype that has not been previously described in iPSC-derived neurons from adult-onset HD patients with low repeat numbers. DNA damage was first measured in HD-MSNs converted from three independent HD patients in comparison to starting fibroblasts and Ctrl-MSNs. At PID 30, HD-MSNs exhibited increased oxidative DNA damage determined by levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) (see e.g.,
Mitochondrial Dysfunction, Oxidative Stress and Metabolic Deficits in HD-MSNs
TEM analysis of HD-MSNs and Ctrl-MSNs (HD.40 and Ctrl. 19) identified high levels of mitophagy, the selective degradation of dysfunctional mitochondria, and many swollen mitochondria typical of apoptotic cells (see e.g.,
Differential Vulnerability of Neuronal Subtypes to mHTT Toxicity
Although HTT is ubiquitously expressed throughout the brain, mHTT leads to selective mass degeneration of MSNs and to a lesser extent, cortical neurons as the disease progresses. Human postmortem studies have shown that at a stage when neuronal loss was low in the cortex but high in the striatum, mHTT aggregates were more common in the cortex than in the striatum37. Additionally, the formation mHTT inclusion bodies was also reported to correlate positively with neuronal survival and hence may be a protective cellular response. It was hypothesized that by generating cortical neurons (CNs) from HD fibroblasts (HD-CNs), the selective vulnerability of HD-MSNs can be modeled with directly reprogrammed human neurons and the relationship could be examined between aggregate formation and toxicity. Control and HD-patient fibroblasts were transduced either with miR-9/9*-124+CDM or with miR-9/9*-124 in conjunction with NeuroD2, ASCL1 and MYT1L (DAM) (miR-9/9*-124+DAM), a combination that has been shown to convert human fibroblasts into neurons that express markers associated with cortical neurons (see e.g.,
Manifestation of HD Cellular Phenotypes is Dependent on Patient Age
Unlike iPSC-derived neurons, directly converted neurons do not undergo rejuvenation during cell fate conversion. The maintenance of aging signatures upon neuronal conversion has long been postulated to be an important advantage of using directly converted patient neurons to model late-onset diseases. However, no functional studies have provided empirical evidence that age information stored within donor's somatic cells actually contributes to the differential manifestation of HD-related cellular phenotypes. Even though the findings of deriving HD-heMSNs were insightful, the induction of pluripotency is likely altering many cellular properties and not just erasing aging signatures. Therefore, the ability to attribute the importance of aging to phenotypic manifestation of HD in our cellular model is limited. To further evaluate the importance of cellular age to our phenotypic analyses, the properties of MSNs reprogrammed from HD patients but sampled before the disease onset were investigated. Six fibroblasts lines were acquired from pre-symptomatic HD patients (Pre-HD), sampled 13 to 17 years prior to the onset of clinical symptoms, with CAG tract sizes of 42-49 repeats (see e.g., TABLE 2). All six Pre-HD fibroblasts were reprogrammed using miR-9/9*-124+CDM to generate MSNs (Pre-HD-MSNs), alongside fibroblasts from three controls and three symptomatic HD patients (see e.g.,
The ability to model neurological disorders and neuronal function in human neurons in vitro has proved to be a valuable approach for dissecting disease pathogenesis. However, since many neurological disorders primarily affect distinct neuronal subpopulations, studies using generic protocols to induce unrestricted neuronal cell fates are likely only capturing a global snapshot of factors that contribute to disease onset and progression. This is especially true for the study of HD, in which MSNs are differentially susceptible to cell death. The microRNA-based neuronal conversion for generating MSNs offers an experimental means to generate a highly enriched population of human MSNs from HD patients.
In HD, the accumulation of protein aggregates and neurodegeneration is observed in an age-dependent manner. Moreover, forced expression of mHTT leads to more severe pathological changes in the striatum of old rats than in young rats, including increased aggregate load and striatal cell loss58. Several other lines of evidence in HD patients and animal models suggest that deficits caused by HD pathogenesis are age-related, such as mitochondrial dysfunction, oxidative stress, and DNA damage59. To test the contribution of cellular age to the manifestation of disease-relevant phenotypes in MSNs derived from patient fibroblasts, two distinct cellular reprogramming approaches were applied that diverge in the maintenance of age signatures from donor cells. The induction of pluripotency has been well established to erase aging marks and reset the phenotypic age of donor cells to an embryonic state, while direct conversion has been shown to maintain age-related transcriptional, cellular and epigenetic signatures. In this study, it was established that the retention of age status through direct neuronal conversion is a critical component in effectively modeling HD, demonstrated by the detection of mHTT aggregates and determination that the propensity of mHTT to aggregate is directly related to the age- and cell fate-related functionality of proteostasis.
It was also found that mHTT induced DNA damage contributed to the cell death of HD-MSNs, as treating the cells with an inhibitor of the DNA damage response protein ATM rescued the cell death phenotype and protected the cells against oxidative stress, similar to iPSC-derived neurons undergoing degeneration upon BDNF withdrawal. Moreover, evidence was provided that generating MSNs with high specificity is critical for the manifestation of disease phenotypes, as altering the terminal neuronal cell fate of HD fibroblasts to HD-CNs drastically reduced levels of DNA damage and cell death, despite the presence of mHTT aggregates. Although cortical cells are not spared in HD, it has been observed that cortical neurons degenerate at a much slower rate with disease progression relative to MSNs and that mHTT aggregates are more common in the cortex than in the striatum. Accordingly, postmortem studies in HD patients have also shown significantly lower levels of DNA damage in the cortex than in the striatum. The cellular properties that render MSNs differentially vulnerable to mHTT-induced toxicity are poorly understood. The reprogramming approach described herein offers a platform to examine neuroprotective attributes conferred by acquisition of cortical fate, an important aim of further studies. Finally, the mechanistic roles of DNA damage response pathways in the modification of HD pathogenesis remain largely unknown. Importantly, recent human genetic studies reveal crucial DNA damage repair pathway gene loci (e.g. FAN1 and MLH1) are significantly associated with altered onset of motor symptoms in HD. The robust MSN-specific DNA repair pathway deficits in this HD-MSN model in conjunction with phenotype-free MSNs derived from pre-symptomatic HD patients may offer a new patient-derived neuronal paradigm to study human genetic modifier genes for HD.
Plasmids and Lentiviral Preparation
The construction of all plasmids used in this study has been previously described19,63 and are publicly available at Addgene: pTight-9-124-BclxL (#60857), rtTA-N144 (#66810), pmCTIP2-N106 (#66808), phMYT1L-N174 (#66809), phDLX1-N174 (#66859), and phDLX2-N174 (#66860). With the exception of hSP9-N174 which was cloned in house and not in prior publications. Polyglutamine fusion proteins, pEGFP-23Q and pEGFP-74Q were generated and acquired by from Addgene (#40261 and #40262), and transfected into human fibroblasts. Lentiviral production was carried out separately for each plasmid but transduced together as a single cocktail as previously described63. Briefly, supernatant was collected 60-70 hours after transfection of Lenti-X 293LE cells (Clontech) with each plasmid, in addition to psPAX2 and pMD2.G (Addgene), using polyethyleneimine (Polysciences). Collected lentiviruses were filtered through 0.45 μm PES membranes and concentrated at 70,000×g for 2 hours at 4° C. Viral pellets were re-suspended in 1× Dulbecco's phosphate-buffered saline (DPBS, Gibco) and stored at −80° C. until transduction.
Cell Lines and Culture
Adult dermal fibroblasts of symptomatic HD patients (Coriell NINDS and NIGMS Repositories: ND33947, ND30013, GM02173, GM09197, GM04687, GM04230, GM04194, GM04196, GM04198, GM02147. GM04687) and healthy controls (Coriell NINDS, NIA, and NIGMS Repositories: ND34769, AG04148, GM02171, GM05879, AG16409, AG11357, AG11483, GM05879, AG16409, AG05265, AG04062, AG04060) were acquired from the Coriell Institute for Medical Research. One additional healthy control adult dermal fibroblast line was acquired from the Washington University School of Medicine iPSC Core Facility (#F09-238). The International Cell Line Authentication Committee (ICLAC) lists none of these primary cells as commonly misidentified cell lines. In regards to de-identified skin fibroblasts samples and induced pluripotent stem cells (iPSCs) acquired from the Coriell Institute for Medical Research, the master list to re-identify subjects was not accessible. This activity is not considered to meet federal definitions under the jurisdiction of an Institutional Review Board, and thus exempt from the definition of human subject. All fibroblasts were cultured in fibroblast media (FM): Dulbecco's Modified Eagle Medium (DMEM) with high glucose containing 15% fetal bovine serum (FBS; Gibco), 0.01% β-mercaptoethanol (BME), 1% non-essential amino acids (NEAA), 1% sodium pyruvate, 1% GlutaMAX, 1% 1M HEPES buffer solution and 1% penicillin/streptomycin solution (all from Invitrogen). Cell cultures are routinely checked and confirmed to be free of mycoplasma contamination. The step-by-step MSN conversion protocol has been previously presented63. Briefly, the lentiviral cocktail of rtTA, pTight-9-124-BclxL, CTIP2, MYT1L, DLX1, and DLX2 was added to fibroblasts for 16 hours, then cells were washed and fed with FM with 1 μg/mL doxycycline (DOX). Cells were fed at post-induction day (PID) 3 with FM+puromycin (3 μg/mL)+blasticidin (3 μg/mL)+DOX and re-plated PID 5 onto poly-omithine/fibronectin/laminin-coated glass coverslips in FM+DOX. Media was switched PID 6 to Reprogramming Neuronal Medium (RNM): Neuronal Medium (NM; ScienCell Research Laboratories) with 200 μM dibutyl cyclic AMP, 1 mM valproic acid, 10 ng/mL BDNF, 10 ng/mL NT-3, and 1 μM retinoic acid, supplemented with DOX. Half volume media changes with RNM were performed every 4 days with addition of DOX every 2 days thereafter until PID 30-35. Addition of puromycin and blasticidin was terminated after PID 14.
DNA Extraction and CAG Sizing
Fibroblasts were expanded in culture, collected by cell scraper, pelleted, and lysed for DNA extraction and ethanol precipitated following typical lab procedures with Proteinase K (Roche). DNA samples were CAG sized by Laragen, Inc (Culver City, Calif.).
Immunocytochemistry
Cells were fixed using 4% paraformaldehyde (PFA) for 20 minutes and permeabilized using 0.2% Triton-X solution for 10 minutes following three phosphate-buffered saline (PBS) washes. Cells were blocked for 1 hour at room temperature using 1% Normal Goat Serum (NGS) and 5% bovine serum albumin (BSA) in 1×PBS solution. Primary antibodies were added in the presence of blocking buffer overnight at 4° C. Secondary antibodies were added following three PBS washes at 1:1000 in blocking buffer at room temperature for 1 hour. The following primary antibodies were used for the immunofluorescence studies: mouse anti-MAP2 (Sigma-Aldrich #M9942 Clone HM2, 1:750), rabbit anti-β-III tubulin (BioLegend, #MMS-435P, 1:2,000), chicken anti-NeuN (Aves, #NUN, 1:500), rabbit anti-GABA (Sigma #A2052, 1:2,000), mouse anti-GABA (Sigma #A0310 Clone GB-69, 1:500), rabbit anti-DARPP32 (Santa Cruz Biotechnology #so-11365, 1:400), rabbit anti-S100A4 (FSP1) (Abcam #124805, 1:200), mouse anti-HTT (mEM48, Millipore #MAB5374, 1:50) (MW8, Developmental Studies Hybridoma Bank, 1:100), rabbit anti-ubiquitin (Abcam #ab7780, 1:50), mouse anti-vimentin (Sigma-Aldrich #V6630, 1:500), rabbit anti-fibronectin (Sigma-Aldrich #F3648, 1:500), mouse anti-phospho-histone H2A.X (Millipore #05-636-I, 1:200), rabbit anti-lap2 alpha (Abcam #ab5162, 1:500), rabbit anti-53BP1 (Abcam #ab21083, 1:200), mouse anti-80H-dG (Santa Cruz Biotechnology #so-139586, 1:1,000), rabbit anti-LC3B (Sigma-Aldrich #L7543, 1:1,000). The secondary antibodies were goat anti-rabbit or mouse IgG conjugated with Alexa-488, -594, or -647 (Invitrogen). Images were captured using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS) Advanced Fluorescence 2.7.3.9723. All staining quantification was performed by counting number of positive-stained cells over DAPI signal. Antibodies were validated by staining fibroblasts as negative controls, and exhibited low background.
Immunoblot Analysis
At post-infection day 28, cells were lysed in SDS-Lysis buffer (1 M Tris-HCl pH 6.8, 2% SDS, 30% Glycerol) supplemented with protease inhibitors (Roche, #04693132001). The concentrations of whole cell lysates were measured using the Pierce BCA protein assay kit (Thermo Scientific, #23227). Equal amounts of whole cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, #10600006) using a transfer apparatus according to the manufacturer's protocols (Bio-rad). After incubation with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 30 min, the membrane was incubated with primary antibodies at 4° C. overnight; MW8 (Developmental Studies Hybridoma Bank, 1:500) and MW1 (Developmental Studies Hybridoma Bank, 1:500). Following incubation, membranes were incubated with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody for 1 hr. Blots were developed with the ECL system (Thermo Scientific, #34080) according to the manufacturer's protocols.
Mitochondrial Assays
The cell permeant mitochondrial indicator, MitoTracker Red CMXRos (ThermoFisher Scientific #M7512) was added directly to live cells at final concentration of 50 nm in serum-free media. After 20 minutes of incubation in 37° C., cells were imaged with an epifluorescent microscope and then fixed and processed for immunostaining as described above. Analysis of colocalization of MitoTracker Red and LC3-II (Anti-LC3B antibody, Sigma-Aldrich #L7543) was performed using Metamorph bioimaging software after image acquisition using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS). Mitochondrial membrane potential was assayed with TMRE-Mitochondrial Membrane Potential Assay Kit (abcam #ab113852) following the manufacturer's protocol. Briefly, TMRE was added to live cells at a final concentration of 20 nm in serum-free media. After 15 minutes of incubation in 37° C., coverslips were removed from media and Vaseline was applied to edges of coverslips to create a rim for live mounting and microscopy (Fischer et al., CSH Protocols, 2008) and imaged using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS). Lipid droplets were stained with BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (ThermoFisher Scientific #D3922) at a final concentration of 0.1 μm in serum-free media. After 30 minutes of incubation in 37° C., cells were imaged with an epifluorescent microscope and quantified with Leica Application Suite (LAS) quantification tools.
Electrophysiology
Whole-cell patch-clamp recordings were performed 28-35 days post-induction (PID) with miR-9/9*-124-CDM. At PID 14, cells undergoing reprogramming were transduced with pSYNAPSIN tRFP or GFP, and the next day trypsinized and plated together on top of rat primary neurons and glia isolated from perinatal pups with the exception of recordings shown in
RNA Extraction and Gene Expression Profiling
Total RNA was extracted and isolated with TRIzol reagent (Thermo Fisher Scientific) according to manufacturer's instructions. cDNA was generated from isolated RNA with Superscript III Reverse Transcriptase (Thermo Fisher Scientific) primed with random hexamers. qPCR was performed with the following primer sets listed in TABLE 5. For RNA-seq, reads were aligned to the human genome (assembly hg38) with STAR version 2.4.2a [23104886]. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount [23558742], version 1.4.6, with GENCODE gene annotation (V23) [22955987]. All gene-level transcript counts were then imported into the RBioconductor package EdgeR [19910308] and TMM normalized to adjust for differences in library size. Genes not expressed in any sample were excluded from further analysis. The fit of the trended and tagwise dispersion estimates were then plotted to confirm proper fit of the observed mean to variance relationship where the tagwise dispersions are equivalent to the biological coefficients of variation of each gene. Differentially expressed genes were then filtered for those having fold-changes (FC)>1.5 together with false-discovery rate (FDR) adjusted p-values less than or equal to 0.05. Gene expression heat maps were generated using Z-scores for expression values of each gene among different samples (GENE-E Matrix Visualization and Analysis Platform, Broad Institute). MSN-specific genes were selected from previous studies that have profiled transcriptome profiles of various neuronal subtypes64,65. RNA-seq data is publicly available at GEO (Accession number GSE84013).
Dead-Cell Staining
SYTOX Green nucleic acid staining (Thermo Fisher Scientific) was performed following manufacturer's suggestions, and adapted as follows: A final concentration of 0.1 μM SYTOX green was added directly to the media of live cells. In addition, Hoechst 33342 solution (Thermo Fisher Scientific) was added as a counterstain to label all nuclei at a final concentration of 1 μg/ml in culture media. Samples were incubated for at least 10 minutes in 37° C. Images were captured using a Leica DMI 400B inverted microscope with Leica Application Suite (LAS) Advanced Fluorescence. Three images were taken from random areas of each coverslip for at least three biological replicates per experiment. Quantification performed by counting number of SYTOX-positive cells over total Hoechst signal.
Comet Assay
DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, Md. USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1×DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Approximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1×TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1×TE buffer pH 8.0 (Ambion) with 1:10 of 10,000×SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
Generation of iPSCs and Derivation of HEFs
iPSC lines used in this study were either directly acquired from the Coriell Institute for Medical Research NINDS Biorepository (#ND42235) or derived from adult dermal fibroblast acquired from the Coriell NINDS Biorepository (#ND33947) with the assistance of the Washington University School of Medicine Genome Engineering and iPSC Center (GEiC). For the generation of ND33947 iPSCs, fibroblasts were transduced with integration-free Sendai reprogramming vectors for Oct3/4, Sox2, Klf4, and c-Myc and characterized by the expression of the pluripotency markers Oct4, SSEA4, SOX2 and TRA-1-60 (PSC 4-Marker Immunocytochemistry Kit, Molecular Probes). Cytogenic analysis was performed on twenty G-banded metaphase cells from iPSC line at passage 5 and all twenty cells demonstrated an apparently normal karyotype (Cell Line Genetics, Madison Wis.). In addition, embryoid body formation assay confirmed the potential for acquisition of all three germ layers. iPSCs were expanded on ES grade Matrigel (Corning) coated plates cultured in mTeSR medium (STEMCELL Technologies) or DMEM/F-12 with 20% KnockOut Serum Replacement, 1% GlutaMAX, 0.1 mM NEAA, 10 ng/mL fibroblast growth factor-basic (bFGF) and 55 μM BME. To differentiate iPSCs into human embryonic fibroblasts (HEFs), culture media was replaced with DMEM+20% FBS without bFGF for at least three passages. HEFs were transduced and reprogrammed to MSNs following the established previously reported protocol (Richner, 2015).
Drug Treatment
The ATM-Kinase inhibitor KU-60019 was obtained from Abcam (ab144817), solubilized in DMSO and directly added to the cell culture media for a final concentration of 0.5 μM at 30 days post miR-9/9*-124 induction, then cell death was assessed by SYTOX at PID 35. Controls were treated with the same volume of DMSO but no drug. At day 35, cells treated with DMSO or KU-60019 also were treated with 1 mM of H2O2 for three hours. SYTOX green/Hoechst stain was added as already described and imaged for scoring.
20s Proteasome Activity Assay
Adherent cells were dissociated with 0.25% trypsin, pelleted by centrifugation and washed in cold 1×PBS twice. Cell pellets were then resuspended in chilled cell lysis buffer (50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 2 mM ATP) and incubated on ice for 30 minutes, and vortexed every 10 minutes. Cell lysate were then centrifuged at 15,000 RPM for 15 minutes at 4° C. Lysate was then transferred to a microcentrifuge tube, and 10 μL of each sample was used to determine protein concentration with a BCA protein assay kit (Thermo Scientific, Prod. #23227) following manufacturer's recommendations. Proteasome activity was assayed with 10 μg of each lysate with a 20 s Proteasome activity assay kit (Millipore, APT280). Fluorescent intensity was measured every 5 minutes for 1 hour with a microplate reader. Data was analyzed following previously reported methods49.
Electron Microscopy
Cells cultured in gridded glass bottom μ-dishes (Ibidi, Madison, Wis.) were fixed with EM grade 4% PFA+0.05% glutaraldehyde (GA) (Electron Microscopy Sciences) in 1×PBS with 2 mM CaCl2 at 37° C. for 5 minutes (min) then transferred to ice for 1 hour. Samples were then incubated for 5 min in 50 mM glycine in 1×PBS and permeabilized with 0.05% saponin with 1% BSA in PBS for 30 min. Cells were blocked with 1% BSA in PBS for 15 min and incubated with primary antibodies (mouse anti-HTT (MW8), 1:100 and rabbit anti-R-III tubulin BioLegend, 1:2,000) at room temperature for 2 hours with gentile agitation. After washing in PBS-BSA three times for 10 min each, cells were incubated for an additional 2 hours with Alexa Fluor 594 fluoronanogold secondary antibody (Nanoprobes, Yaphank, N.Y.) at a 1:250 dilution in PBS and 1% BSA at room temperature with gentle agitation while wrapped in foil. After washing in PBS three times for 10 min each, cells were fixed with 1% GA for 5 min and labeled with DAPI (1:10,000) for 5 min. Post fluorescent imaging, the samples were rinsed twice in ultrapure water for 1 min each and then rinsed in 0.02 M citrate buffer (pH 4.8) three times for 5 min each. The fluoronanogold label was silver enhanced using HQ Silver (Nanoprobes, Yaphank, N.Y.) for 9-11 min and immediately rinsed with ultrapure water twice for 5 min each. The culture dishes were then rinsed in PBS buffer three times for 10 minutes each, and subjected to a secondary fixation step for one hour in 1% osmium tetroxide/0.3% potassium ferrocyanide in PBS on ice. The samples were then washed in ultrapure water three times for 10 minutes each and then en bloc stained for 1 hour with 2% aqueous uranyl acetate. After staining was complete, samples were briefly washed in ultrapure water, dehydrated in a graded ethanol series (50%, 70%, 90%, 100%×2) for 10 minutes in each step, and infiltrated with microwave assistance (Pelco BioWave Pro, Redding, Calif.) into LX112 resin. Samples were cured in an oven at 60° C. for 48 hours. Once the resin was cured, the gridded glass coverslips were etched away with concentrated hydrofluoric acid and the exposed cells were excised with a jewelers saw and mounted onto blank resin blocks with epoxy, oriented in the coverslip growing plane. 70 nm thick sections were then taken and imaged on a TEM (JEOL JEM 1400 Plus, Tokyo, Japan) at 80 KeV.
Statistics
For all quantified data, multiple cells were counted from at least three biological replicates from multiple independent experiments or multiple lines. Statistical analyses were performed in GraphPad Prism using a two-tailed Students t-test or a one-way ANOVA followed by a post hoc Tukey's test with *P<0.05 considered significant Multiple comparisons were corrected with Bonferroni or Holm-Sidak method as described in the figure legends. Studies were performed blindly and automated whenever possible with the aid of ImageJ cell counting tools, and multiple investigators confirmed quantification results. Normality was tested with D'Agostino-Pearson test. In addition, Brown-Forsythe test did not identify significantly different standard deviations (P<0.05) for groups tested. Data in graphs are expressed as mean and error bars represent s.e.m. unless noted otherwise. Outliers were detected and excluded with Grubbs' test for alpha levels of 0.05. In total for this study, only 2 data points were excluded from
Step-by-step protocols used herein can be found in Richner, M., Victor, M. B., Liu, Y., Abernathy, D. & Yoo, A. S. MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons. Nature protocols 10, 1543-1555, doi:10.1038/nprot.2015.102 (2015).
This application claims priority from U.S. Provisional Application Ser. No. 62/541,858 filed on 7 Aug. 2017, U.S. Provisional Application Ser. No. 62/562,222 filed on 22 Sep. 2017, and U.S. Provisional Application Ser. No. 62/541,858 filed on 14 Dec. 2017, each of which is incorporated herein by reference in its entirety.
This invention was made with government support under grant number NS083372 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/044317 | 7/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62541858 | Aug 2017 | US | |
| 62562222 | Sep 2017 | US | |
| 62598368 | Dec 2017 | US |
