Patent Application
20180072988
This application claims the benefit of priority of Singapore application No. 10201502869T, filed 10 Apr. 2015, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention relates generally to the field of biotechnology. In particular, the present invention relates to methods for differentiating a pluripotent or multipotent stem cell into multiple cell lineages. The present invention further relates to culture mediums and kits for use in performing the methods as described herein.
At multiple developmental junctures, lineage-specifying transcription factors (TFs) direct multipotent progenitors towards a single lineage outcome and repress alternate fates, ensuring a unilateral lineage decision.
The ability to differentiate stem cells such as human pluripotent stem cells (hPSC) into committed cell-types holds great benefit for cell replacement therapy, drug screening and discovery, mechanistic studies of dysfunction and other downstream applications.
An example of a cell lineage that stem cells can differentiate to is the neuronal lineage. Various methods to differentiate stem cells into cells of the neuronal lineage are known. These methods mimic the developmental signaling that occurs during biogenesis of neurons to generate neural progenitors and subsequently differentiate these progenitors into functional neurons. However, these methods typically involve multiple intermediate stages that require varying combinations of recombinant growth factors and small molecules, and eventually yield mixtures of both non-neuronal and neuronal cells with variable functional properties. The protracted timeline required to attain neuronal maturity and synaptic competence is a further limitation because this process can take as long as 30 weeks.
Accordingly, there is a need for a method to obtain functional lineage specific cells from stem cells that overcomes, or at least ameliorates, one or more of the disadvantages stated above.
In one aspect, there is provided a method of directly converting a stem cell into a lineage specific cell comprising:
In another aspect, there is provided a method of generating a directly convertible stem cell, said method comprising the steps of:
In one aspect, there is provided a directly convertible stem cell comprising i) one or more reprogramming factors operably linked to an inducible promoter and ii) a selection marker operably linked to a constitutive promoter, wherein said directly convertible stem cell is capable of direct conversion into an induced lineage specific cell.
In one aspect, there is provided a method of screening one or more factors and/or one or more genetic mutations that modulate a pre-selected activity of the induced lineage specific cell according to any one of the preceding claims, comprising the steps of:
wherein the difference in the measurement of the pre-selected activity of the lineage specific cell in c) indicates that the one or more factors or genetic mutations modulates the pre-selected activity of the lineage specific cell.
In one aspect, there is provided a kit for generating an induced lineage specific cell, comprising,
In one aspect, there is provided a method of directly converting a stem cell into a lineage specific cell comprising:
In one aspect, there is provided a method of directly converting a stem cell into a lineage specific cell comprising:
In one aspect, there is provided a stem cell directly convertible into an inhibitory neuron comprising:
In one aspect, there is provided a stem cell directly convertible into an excitatory neuron comprising:
In another aspect, there is provided a method of screening an agent using a cell obtained by the method disclosed herein comprising:
As used herein, the term “stem cells” include but are not limited to undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. For example, “stem cells” may include (1) totipotent stem cells; (2) pluripotent stem cells; (3) multipotent stem cells; (4) oligopotent stem cells; and (5) unipotent stem cells.
As used herein, the term “totipotency” refers to a cell with a developmental potential to make all of the cells in the adult body as well as the extra-embryonic tissues, including the placenta. The fertilized egg (zygote) is totipotent, as are the cells (blastomeres) of the morula (up to the 16-cell stage following fertilization).
As used herein, the term “pluripotent stem cell” (PSC) refers to a cell with the developmental potential, under different conditions, to differentiate to cell types characteristic of all three germ cell layers, i.e., endodeini (e.g., gut tissue), mesoderm (including blood, muscle, and vessels), and ectoderm (such as skin and nerve). The developmental competency of a cell to differentiate to all three germ layers can be determined using, for example, a nude mouse teratoma formation assay. In some embodiments, pluripotency can also be evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency of a cell or population of cells generated using the compositions and methods described herein is the demonstration that a cell has the developmental potential to differentiate into cells of each of the three germ layers.
As used herein, the term “induced pluripotent stem cells” or, iPSCs, means that the stem cells are produced from differentiated adult cells that have been induced or changed, i.e., reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature.
As used herein, the term “embryonic stem cell” refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta, i.e., are not totipotent.
As used herein, the term “multipotent stem cell” refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers, but not all three. Thus, a multipotent cell can also be termed a “partially differentiated cell.” Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stern cells and neural stem cells. “Multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. Accordingly, the term “multipotency” refers to a state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.
As used herein, the term “differentiation” is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
As used herein, the term “undifferentiated cell” refers to a cell in an undifferentiated state that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types, without a specific implied meaning regarding developmental potential (i.e., totipotent, pluripotent, multipotent, etc.).
As used herein, the term “progenitor cell” refers to cells that have greater developmental potential, i.e., a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression) relative to a cell which it can give rise to by differentiation. Often, progenitor cells have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct cells having lower developmental potential, i.e., differentiated cell types, or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
As used herein, the term “reprogramming” in the context of a cell or cell lineage refers to the conversion of a specific cell type to another cell type. Accordingly, “reprogramming factor” refers to a molecule that is capable of reprogramming a specific cell type to another cell type.
As used herein, the term “efficiency” in the context of reprogramming means that conversion of a specific cell type to another cell type occurs at a frequency of at least about 50%. In other words, reprogramming efficiency of at least about 50% means that at least about 50% of cells of a specific cell type is converted to another cell type.
As used herein, the tenn “markers” refers to nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
As used herein, the term “inducible” in the context of a promoter refers to a promoter whose activity may be stimulated by an agent. Presence of the agent stimulates promoter activity which in turn drives expression of the gene that is under the control of the inducible promoter. In the absence of the agent, the promoter is inactive and the gene that is under the control of the inducible promoter is not expressed.
As used herein, the term “constitutive” in the context of a promoter refers to a promoter which is consistently active. A gene that is under the control of a constitutive promoter is continually expressed.
As used herein., the term “transcription factor” refers to proteins that bind to DNA and regulate transcription. Transcription factors may comprise DNA-binding domains which recognise and bind to specific sequences of DNA to regulate transcription. Transcription factors commonly recognise and bind to promoter and/or enhancer regions and may activate or repress gene expression.
As used herein, “microRNAs” or a “microRNA” molecule refers to a short, non-coding RNA which can negatively regulate expression of one or more genes at post-transcriptional level.
As used herein the phrase “culture medium” refers to a liquid substance used to support the growth of stem cells and any of the cell lineages. The culture medium used by the invention according to some embodiments can be a liquid-based medium, for example water, which may comprise a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones.
As used herein, the term “feeder cell” refers to feeder cells (e.g., fibroblasts) that maintain stem cells in a proliferative state when the stem cells are co-cultured on the feeder cells or when the pluripotent stem cells are cultured on a matrix (e.g., an extracellular matrix, a synthetic matrix) in the presence of a conditioned medium generated by the feeder cells. The support of the feeder cells depends on the structure of the feeder cells while in culture (e.g., the three dimensional matrix formed by culturing the feeder cells in a tissue culture plate), function of the feeder cells (e.g., the secretion of growth factors, nutrients and hormones by the feeder cells, the growth rate of the feeder cells, the expansion ability of the feeder cells before senescence) and/or the attachment of the stem cells to the feeder cell layer(s).
As used herein, the term “cortical network” refers to a group of neurons that are interconnected via one or more synapses. A cortical network may comprise of a group of neurons in vitro or ex vivo. An in vitro or ex vivo cortical network mimics the human cortex in the type of neurons present.
Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Before the present inventions are described, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure.
In one embodiment, there is provided a method of directly converting a stem cell into a lineage specific cell. In particular, the method comprises: transfecting said stem cell with at least one expression vector comprising i) one or more cell lineage reprogramming factors operably linked to an inducible promoter and ii) a selection marker; and inducing said transfected stem cells from step a) with an inducing agent to directly convert said stem cell into a lineage specific cell.
In one embodiment, the at least one expression vector may comprise a selection marker operably linked to a constitutive promoter.
The method as described herein may further comprise the step of selecting the transfected stem cell for expression of the selection marker, prior to inducing the cells.
In some embodiments, the selection marker may be an antibiotic resistance gene selected from the group consisting of puromycin, blasticidin, hygromycin, zeocin and neomycin.
In a preferred embodiment, the antibiotic resistance gene is blasticidin and/or hygromycin.
The constitutive promoter may be selected from the group consisting of phosphoglycerate kinase (PGK), elongation factor 1-α(EF1α), ubiquitin C (UBC), (β-actin and cytomegalovirus (CMV) enhancer/chicken β-actin promoter (CAG).
In a preferred embodiment, the constitutive promoter is PGK or EF1α.
In some embodiments, the method may further comprise the step of transfecting the stem cell with an expression vector comprising a transactivator capable of inducing the inducible promoter in the presence of an inducer. The inducing agent may be selected from the group consisting of doxycycline and cumate. It will be understood that when the inducing agent is doxycycline, the transactivator is a reverse tetracycline-controlled transactivator (rtTA). It will also be understood that when the inducing agent is cumate, the transactivator is a reverse cumate activator (rcTA).
The expression vector may be an integrating or non-integrating vector. In some embodiments, the integrating vector may be a retroviral or lentiviral expression vector. In some embodiments, the non-integrating vector is a sendai virus, adeno-associated virus (AAV) or episomal DNA. In a preferred embodiment, the vector is a lentiviral expression vector.
The method as described herein may further comprise the step of enriching the selected cells using one or more selection steps. In some embodiments, the selection step may be selected from the group consisting of antibiotic selection, fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), or single clone isolation and expansion.
The stem cell may be an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC). In some embodiments, the stem cell may be a primate or non-primate stem cell. Where a primate stem cell is selected, the stem cell may be a human stem cell. In a preferred embodiment, the embryonic stem cell is a human embryonic stem cell.
In some embodiments, the stem cell may be a stem cell line. The stem cell line may be cultured as a two-dimensional cell culture or a three-dimensional cell culture.
It is an advantage of the method as described herein that the lineage specific cell is generated at an efficiency of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% and about 100%. In a preferred embodiment, the efficiency is at least about 70%. In yet another preferred embodiment, the efficiency is at least about 99%.
The lineage specific cell may be a population of cells cultured as a two-dimensional cell culture or a three-dimensional cell culture. In some embodiments, the lineage specific cell may be a cell of the ectoderm, mesoderm or endoderm lineage. In a preferred embodiment, the cell of the ectoderm lineage may be a neural cell. The neural cell may be selected from the group consisting of excitatory neurons, inhibitory neurons, dopamine neurons, serotonin neurons, medium spiny neurons, basal forebrain cholinergic neuron, oligodendrocytes, astrocytes and motor neurons. In a further preferred embodiment, the neural cell may be an excitatory neuron or an inhibitory neuron. In another embodiment, the neural cell is at least one cell of a cortical network. A minimal cortical network comprises an excitatory and an inhibitory neuron. It will be understood that the ratio of types of neurons within a cortical network may vary. For example, a cortical network may comprise at least about 70% excitatory neurons and at least about 30% inhibitory neurons, at least about 75% excitatory neurons and at least about 25% inhibitory neurons, at least about 80% excitatory neurons and at least about 20% inhibitory neurons, at least about 85% excitatory neurons and at least about 15% inhibitory neurons and at least about 90% excitatory neurons and at least about 10% inhibitory neurons. In a preferred embodiment, a cortical network may comprise at least about 75% excitatory and at least about 25% inhibitory neurons. In yet another preferred embodiment, a cortical network may comprise at least about 80% excitatory and at least about 20% inhibitory neurons.
In one embodiment, the inhibitory neuron may be selected from the group consisting of parvalbumin (PV) type, somatostatin (SOM) type, calbindin (CB) type, calretinin (Cr) type, vasoactive intestinal polypeptide (VIP) type, Reelin type, neuropeptide Y (NPY) type, neuronal nitric oxide synthase (nNOS) type and 5HT3aR expressing neurons. In a preferred embodiment, the inhibitory neuron is a SOM type, CR type, CB type or NPY type neuron.
In some embodiments, the cell of the mesoderm lineage may be a cardiac cell. The cardiac cell may be selected from the group consisting of cardiomyocytes, endothelial cells, vascular smooth muscle cells (VSMCs) and cardiac fibroblasts.
In some embodiments, the cell of the endoderm lineage may be a hepatic cell. The hepatic cell may be selected from the group consisting of hepatocytes, Kupffer cells, stellate cells and sinusoidal endothelial cells.
In one embodiment, the lineage specific cell is present in a homogenous population of cells. In another embodiment, the lineage specific cell is present in a substantially homogenous population of cells. For example, the substantially homogenous population of lineage specific cells may be at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% homogenous.
In some embodiments, the homogenous population of lineage specific cells generated using the method of the present invention may be mixed. The mixture of homogenous populations may give rise to cellular networks, for example, a cortical network.
In some embodiments, the one or more reprogramming factors may be selected from the group consisting of a transcription factor, a chromatin remodeler, an epigenetic modifier and/or a non-coding RNA. The non-coding RNA may be microRNA. The transcription factor may be a neural transcription factor. In some embodiments, the neural transcription factor may be one or more transcription factors selected from the group consisting of Ngn1, Ngn2, Ngn3, Neuro D1, Neuro D2, Brn1m Brn2m Brn3A, Brn3B, Brn3C, Brn4, Dlx1, Dlx2, Asc11, phospho-dead mutant of the transcription factor Asc11 (SA/SV-Asc11), CTIP2, MYT1L, Olig1, Zic1 Nkx2.1, nkx2.2, Lhx2, Lhx3, Lhx6, Lhx8, SATB1, SATB2, Dlx5, Dlx6, Fezf2, Fev, Lmx1b, Lmx1a, Pitx3, Nurr1, FoxA2, Sox11, Atoh7, Olig2, Ptf1a, MEF2c, p55DD (dominant negative), Nkx6.1, Nkx6.2, Sox10, ST18, Myrf, Myt1, Zfp536, hes1, hes5, hes6, SOX2, SOX9, PAX6, NFIA, NFIB, NFIX, NICD, Islet1, Islet2, Irx3, Dbx2 and TAL1.
In a preferred embodiment, the one or more transcription factors are Asc11 (SA/SV-Ascl1) and Dxl12. In a further preferred embodiment, Ascii (SA/SV-Ascl1) and Dxl12 are linked by the T2A peptide. In another preferred embodiment, the transcription factor is NeuroD2.
The transcription factor may be a cardiac transcription factor. In some embodiments, the cardiac transcription factor is one or more transcription factors selected from the group consisting of Isl1, Mef2, Gata4, Tbx5, Nppa, Cx40, MESP1, MYOCD and ZFPM2, Baf60c, Hand2, Hopx, Hrt2, Pitx2c and nkx2.5.
In some embodiments, the transcription factor may be a hepatic transcription factor. The hepatic transcription factor may be one or more transcription factors selected from the group consisting of Hnf-1a, Hnf-1β, Hnf-3β, Hnf-3γ, Dbp, Lrh-1, Fxra, C/Ebpβ, Pxr, FOXA1, FOXA2, PROX1, HNF6, GATA6, PPARA, ZHX2, ONECUT2, ATF5, USF2, USF1, ZGPAT and NFIA.
In some embodiments, the microRNA is microRNA-9/9* and/or microRNA-124, miRNA-219, miRNA-338, miRNA-1, miRNA-133 and miRNA-187. In one embodiment, microRNA may be microRNA-9 and microRNA-124. In some embodiments, the one or more microRNAs may be linked to a reporter gene. In one embodiment, microRNA-9 and microRNA-124 are linked to a red fluorescent protein (RPF) gene.
The method as described herein may further comprise the step of contacting the population of non-lineage specific cells with an expression vector comprising a fluorescent indicator. The fluorescent indicator may be a calcium indicator, for example GCaMP6.
In another embodiment, there is provided a method of generating a directly convertible stem cell. In particular the method comprises the steps of: transfecting a stem cell with an expression vector comprising i) one or more cell lineage reprogramming factors operably linked to an inducible promoter and ii) a selection marker operably linked to a constitutive promoter; and screening the transfected stem cell for expression of the selection marker to generate said directly convertible stem cell, wherein said directly convertible stem cell is capable of direct conversion into an induced lineage specific cell.
In another embodiment, there is provided a directly convertible stem cell comprising i) one or more reprogramming factors operably linked to an inducible promoter and ii) a selection marker operably linked to a constitutive promoter, wherein said directly convertible stem cell is capable of direct conversion into an induced lineage specific cell.
In some embodiments, the directly convertible stem cell may be a cell line.
In another embodiment, there is provided a method of screening one or more factors and/or one or more genetic mutations that modulate a pre-selected activity of the induced lineage specific cell according to any one of the preceding claims, comprising the steps of: culturing said induced lineage specific cell in the presence of one or more factors and/or one or more genetic mutations; measuring the pre-selected activity of the lineage specific cell of step a); and comparing the measurement of b) relative to the measurement of the pre-selected activity in the lineage specific cell that has not been cultured in the presence of the said one or more factors or genetic mutations, wherein the difference in the measurement of the pre-selected activity of the lineage specific cell in c) indicates that the one or more factors or genetic mutations modulates the pre-selected activity of the lineage specific cell.
In some embodiments, the one or more factors may be selected from the group consisting of a drug, a growth factor, a small molecule, a biologic, a toxin, a stressor or a cell.
The one or more genetic mutations may be an engineered mutation or a naturally occurring mutation. In some embodiments, the engineered mutation may be selected from the group consisting of site-directed mutation, deletion, duplication, inversion, copy-number variation, imprinting and random mutation. The naturally occurring mutation may be a polymorphism selected from the group consisting of single nucleotide polymorphism (SNP), microsatellite variation, small-scale insertion/deletion and polymorphic repetitive element.
In some embodiments, the pre-selected activity of the lineage specific cell may be a genetic activity or susceptibility to a disorder. The susceptibility to a disorder may be determined by one or more intracellular or extracellular assays or combinations thereof, selected from the group consisting of Ca2+ imaging, cell survival, intrinsic firing properties, measurement of Na+channels, measurement of Ca2+ channels, measurement of K+ channels, synaptic activity, dendritic arborisation, axonal growth and targeting, neurotransmitter release and uptake, and intracellular Ca2+ activity.
In some embodiments, the genetic activity may be selected from the group consisting of gain-of-gene-function, loss-of-gene-function, gene knockdown, gene knockout and gene activation. The genetic activity may be achieved by small hairpin RNA (shRNA), small interfering RNA (siRNA) or CRISPR-associated (Cas) endonuclease.
In some embodiments, the disorder may be a neural disorder. The neural disorder may be selected from the group consisting of schizophrenia, autism, Alzheimer's disease, Parkinson's, Depression, ADHD, dementia, epilepsy, Huntington's, Angelman syndrome, motor neuron disease (MND) and Dravet syndrome. It will be generally understood that motor neuron disease encompass a group of diseases that includes but is not limited to amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis (PLS), progressive muscular atrophy, spinal muscular atrophy and post-polio syndrome (PPS).
In another embodiment, there is provided a kit for generating an induced lineage specific cell, comprising, a directly convertible stem cell as described herein; an inducer; and optionally instructions for use.
In another embodiment, there is provided a method of directly converting a stem cell into a lineage specific cell comprising: transfecting said stem cell with at least one expression vector comprising i) one or more cell lineage reprogramming factors operably linked to a constitutive promoter.
In some embodiments, the at least one expression vector may comprise a selection marker operably linked to a constitutive promoter. Alternatively, the selection marker may be operably linked to a constitutive promoter.
In another embodiment, there is provided a method of directly converting a stem cell into a lineage specific cell comprising: transfecting said stem cell with at least one expression vector comprising i) one or more cell lineage reprogramming factors operably linked to an inducible promoter.
In another embodiment, there is provided a stern cell directly convertible into a GABAergic neuron comprising: i) SA-ASCL1, DLX2, LHX6 and miR-9/9*-124 linked to a doxycycline inducible promoter; and ii) a selection marker operably linked to a constitutive promoter.
In another embodiment, there is provided a stem cell directly convertible into an excitatory neuron comprising: i) NeuroD2 linked to a doxycycline inducible promoter; and ii) a selection marker operably linked to a constitutive promoter.
In another embodiment, there is provided a method of screening an agent using a cell obtained by the method as disclosed herein comprising: i) contacting said cell with the agent; ii) measuring a pre-selected activity of the agent on the cell and comparing this to a cell that has not been contacted with the agent; and iii) detecting the activity of the agent on said cell.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
One-way ANOVA followed by Tukey's test shows significant differences, ***P<0.001. (D) Efficiencies of pan-neuronal conversion (black bars) and GABAergic neuronal conversion (white bars) of hESCs (line H1) upon indicated TF combinations at 10 dpt. Data are means±SEMs (n=3 independent experiments). The optimal combination, ASADL, is shown in red. (E) Efficiency of neuronal conversion of ASADL-converted hESCs transduced with or without miR-9/9*-124 (shown as miR for simplicity throughout the manuscript) at 10 dpt. Data are means±SEMs (n>3 independent experiments). Two-tailed, unpaired t-test shows significant difference, ***P<0.001. (F) Efficiencies of pan-neuronal conversion (black bars) and GABAergic neuronal conversion (white bars) of two hESC lines (H1 and H9) and three hiPSC lines (iPSC1-3) by ASADL+miR at 10 dpt. Data are means±SEMs (n=3 independent experiments). (G) Representative images of H1 derived induced GABA-positive neuronal cells (iGNs) immunolabeled with MAP2 and markers of neuronal subtypes (GABA, gamma-aminobutyric acid; TH, tyrosine hydroxylase; CHAT, cholineacetyltransferase; 5-HT, 5-hydroxytryptamine (also known as serotonin); VGLUT1/2, vesicular glutamate transporter 1 and 2). Data were collected at 42-50 dpt. Scale bar=20 μm. The percentage of neuronal subtype marker-positive cells that also express neuronal marker MAP2 is shown in (H). Data are means±SEMs (n=3 independent experiments). One-way ANOVA followed by Tukey's test shows significant differences, ***P<0.001.
Gephyrin illustrating morphological inhibitory synapses. (E) Immunostaining of iGNs with antibodies against interneuronal subtype markers, including somatostain (SST), calretinin (CR), calbindin (CB), and neuropeptide-Y (NPY), and parvalbumin (PV). Data were collected between 42-56 dpt, except for PV, which was collected at 70-90 dpt. Scale bar=20 μm. (F) Immunostaining of iGNs with antibodies against interneuronal subtype markers, including Reelin (RELN), neuronal nitric oxide synthetase (nNOS), and vasoactive intestinal peptide (VIP). Arrowheads point to neuronal cells that also expressed subtype makers as indicated. Scale bar=20 μm. (G) Quantification of percentage of interneuronal subtype markers shown in (E) and (F) as a bar graph. Data are means±SEMs (n=3 independent experiments).
(J-K) Cumulative plots and histograms of sIPSCs frequency (J) and amplitude (K) recorded in iGNs with control or MDGA1 overexpression (n=13 and 12 for control and MDGA1 overexpression, respectively). All histogram data are shown as the mean±SEMs. Statistical significance was assessed by two-tailed, unpaired t-test except for (F), two-tailed, paired t-test, (*p<0.05).
(H). Data are means±SEMs (n=3 independent experiments). Two-tailed, unpaired t-test shows significant difference, ***P<0.001. (C) Quantification of total dendritic length (left) and primary branch number (right) based on analysis of MAP2 staining in ASADL or ASADL+miR transduced H1 hESCs at 10 dpt. Between 60-80 cells were analyzed in each case. Two-tailed unpaired t-test shows significant difference, *P<0.05, ***P<0.00. (D) Quantitative RT-PCR analysis showed that iGNs (transduced by ASADL+miR-9/9*-124 express genes required for GABA transport and synthesis (VGAT, GAD1 and GAD2) at 14 dpt, and the expression of all three genes robustly further increased at 35 dpt. Results are shown as normalized to HN (fetal human neurons, purchased from Sciencell), which contained about 10-15% of GABAergic neurons and served as positive controls. (E) Immunostaining images showed that some iGNs expressed interneuron subtype markers SST and CR respectively, while some cells expressed both markers. Scale bar=20 μm.
Non-limiting examples of the invention, including the best mode, and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
Materials and Methods
Generation of Plasmid Constructs
A bi-cistronic lentiviral backbone as described (addgene #31780) was used to clone cDNAs encoding hASCL1 (NM_004316.3), hASCL1-phosphmutant, hNKX2.1 (NM_003317.3), hDLX2 (NM_004405.3), hLHX6 (NM_014368.4), and hNeuroD2 (addgene #31780) under the EF1 a promoter, respectively. Doxycycline (Dox)-inducible lentiviral miR-9/9*-124 construct was as described (addgene #31874). Lentiviral expression vector of rtTA was modified from addgene #20342 (FUW-rtTA-pGK-hygromycin).
Upon identification of optimal genetic elements (ASADL+miR-9/9*-124), four reprogramming factors were packed into two dox-inducible lentiviral constructs (TetO-ASA-T2A-DLX2-pGK-blastsicidin, modified from addgene #27151, and pTight-hLHX6-miR-9/9*-124-IRES-puro, modified from addgene #31874).
For optogenetic experiments, cDNA encoding ChETA-EYFP was obtained from Addgene #26967 and subcloned into a lentiviral construct with a human synapsin promoter (Synapsin-ChETA-EYFP). For MDGA1 overexpression, cDNA encoding human MDGA1 was subcloned to lentiviral backbone of addgene #20342 (FUW-MDGA1).
To generate lentiviral particles, lentiviral expression vectors together with psPAX2, and pMD2.G were co-transfected into Lenti-X 293T cells (Clontech) using Fugene HD (Roche). Supernatants were collected from culture media and lentiviral particles were concentrated using a PEG-it kit (# LV810A-1, System Biosciences), following manufacturer's protocol.
hPSCs Cell Culture
Human ESC lines H1 and H9 were originally obtained from WiCell Research Institute (Madison, Wis.), and have been maintained in the laboratory. hiPSC line #1 was obtained from Kerafast (AG1-0). hiPSC line #2 and #3 were GM23338 and GM23279, respectively, both purchased from Coriell Institute. All lines (hESCs, hiPSCs, and iGN inducible lines) were cultured in mTeSR1 media under feeder-free conditions in matrigel-coated, cell culture plates and are routinely passaged (1:6 to 1:10) using Dispase or ReLeSR (all from Stemcell Technologies). All lines used displayed normal karyotypes.
Generation of induced neuronal cells from hPSCs
hESCs and hiPSCs were dissociated with TrypLE (Life Technologies) to single cells and plated onto matrigel coated cell culture plates in mTeSR1 media supplemented with thiazovivin (1 μM, TOCRIS). On the following day, cells were transduced with lentiviruses expressing various transcription factors and microRNAs as indicated in the study, and this was designated as day 0. Next day (1 dpt), culture media was completed changed Neuronal Media (Sciencell), which was used until the end of the experiments. For experiments using Dox-induced iGN lines, doxycycline was added into the media at 1 μg/ml, and maintained for 3 weeks. Cells were selected with appropriate antibiotics from 3-7 dpt to enrich transduced cells. For molecular and functional characterization of induced neurons (dpt 14 or later), cells were dissociated at 7-10 dpt and replated onto poly-L-lysine/laminin-coated glass coverslips or onto cell culture plates. Primary rat glial cells (derived from P1 neonatal rat cortices, cultured more than 2 passages in vitro) and neurotrophic factors (BDNF, GDNF, NT3, and IGF1, each at 10 ng/ml, and all from Peprotech) were added to human induced neuronal cells at 14-20 dpt to enhance survival and synaptic maturity of the induced neurons.
For the generation of inducible iEN/iGN hESC line, H1 hESC stably expressing rtTA was first established (lentiviral transduction with hUbc-rtTA-pGK-hygro and subsequently selected with hygromycin). This line was further transduced with TetO-hNeuroD2-pGK-puro and selected with puromycin to generate the inducible iEN line. Similarly, we generated dox-inducible iGN line by transducing the rtTA-expressing H1 line mentioned above with TetO-ASA-T2A-DLX2-pGK-blasticidin and TetO-miR-9/9*-124-pGK-puromycin, and stably selected with blasticidin and puromycin.
Fluorescent Immunocytochemistry (Cultured Cells)
Immunostaining experiments were performed. Cells were fixed in 4% PFA for 20 minutes, and permeabilized with 0.2% triton X-100 for 15 minutes before blocking for 60 minutes. Blocking buffer was made up of 5% BSA and 2% FBS in PBS. Primary antibodies were diluted in blocking buffer and incubated with cells overnight at 4 degrees. The secondary antibodies were donkey or goat anti-rabbit, mouse, or chicken IgG conjugated with Alexa-488, -594, or -647 (Invitrogen). The following primary antibodies were used: chicken anti MAP2 (Abeam AB5392), mouse anti MAP2 (Abcam AB112670), mouse anti beta III tubulin (Covance, MMS-435P), mouse anti NeuN (Millipore MAB377), mouse anti Ankyrin G (NeuroMab 75-146), mouse anti SMI-312 (Covance SMI-312R), rabbit anti FOXG1 (Abeam AB18259), mouse anti Reelin (Millipore MAB5364), goat anti ChaT (Millipore AB144P), guinea pig anti VGLUTI (Millipore AB5905), guinea pig anti VGLUT2 (Millipore AB5907), rabbit anti DARPP-32 (Santa Cruz sc-11364), mouse anti Gephyrin (SYSY 147021), rabbit anti Synapsin (Millipore MAB355), rabbit anti nNOS (Immunostar, 24287), mouse anti GAD1 (Millipore MAB5406), rabbit anti VGAT (SYSY 131003), rabbit anti NPY (Abeam 10980), rabbit anti VIP (Immunostar 20077), mouse anti PV (SWANT, PV235), goat anti Calretinin (Millipore MAB1550), mouse anti Calbindin 1(SWANT 300), rabbit anti SST (Peninsula/Bachem T4103), rabbit anti GABA (Sigma A2052), mouse anti TH (Immunostar 22941), rabbit anti 5-HT (Immunostar 20080). Images were acquired using Observer Z.1 or LSM 710 (Zeiss). VGAT and gephyrin boutons were analyzed with MetaMorph (Universal Imaging) and Image J (National Institutes of Health). Areas with similar density of neurons were randomly chosen for analyses of VGAT density. VGAT fluorescence signals that were less than 0.22 μm2 (in area) were excluded from analyses. Same intensity threshold was used for both control and MDGA1 overexpression neurons. Total density length was quantified using MAP2 signal in each chosen area to confiini the VGAT density calculation is reliable by respecting to image area.
Gene Expression Analyses
For quantitative RT-PCR analyses of pooled cultured cells, RNA was extracted using DirectZol (Zymo), treated with DNAse, and converted to cDNA using High Capacity cDNA Reverse Transcription kit (Life Technologies). Real-time PCR assay was performed using the Applied Biosystems 7900HT Fast real-time PCR system. Multiplex Single cell qPCR was performed. Cytoplasm of single induced neuronal cells (7 weeks after transduction) growing on coverslips was aspirated into patch pipette and ejected into 2× cells-direct buffer (Life Technologies), flash-frozen, and kept at −80° C. until processing. Thawed cytoplasm was subjected to reverse transcription (Superscript III, Life Technologies) and 18 cycles of PCR pre-amplification with pooled primers specific to the target genes (STA). Unused primers were then digested away using Exonuclease I (New England BioLabs, PN M0293). The cleaned-up cDNA of individual cells was processed for real-time PCR analysis on Biomark 96:96 Dynamic Array (Fluidigm) on a Biomark HD System (Fluidigm) using the indicated primer pairs (
Electrophysiology
Whole cell patch clamp recordings were performed on iGNs and iENs with voltage or current clamp mode. Recording pipettes with resistances of 4-6 MΩ were filled with an internal solution containing (in mM): 120 K-gluconate, 9 KCl, 10 KOH, 3.48 MgCl2, 4 NaCl, 10 HEPES, 4 Na2ATP, 0.4 Na3GTP, 17.5 Sucrose, 0.5 EGTA with an osmolarity of 290 mOsm and pH 7.3. Neurons were bathed in the extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.3 NaH2PO4, 10 dextrose, 2 MgCl2, 2 CaCl2, 10 HEPES at pH 7.4. Bicuculline (20 μM, Tocris) and CNQX (50 μM, Tocris) were used to block inhibitory or excitatory synaptic responses, respectively. Neurons were held at −70 mV except otherwise indicated or at required currents using an Axon MultiClamp 700B amplifier (Axon Instruments). Signal was sampled at 40 kHz and filtered at 2 kHz (Digidata 1440A, Molecular Devices). Recordings with serial resistance higher than 20 MO or leaking current more than 200 pA were not analyzed. Data were analyzed offline using the ClampFit (Molecular Devices) or MiniAnalysis (Synaptosoft). Voltage clamp was used for recording I/V curve of Na/K current using a protocol of increasing holding potential 10 mV for each step from −80 mV. Membrane potential of patched neurons was held at −70 mV for spontaneous synaptic current. The reversal potential for ions was −49 mV in our recording system, calculated using the simplified Nernst equation: E(Cl−)=−59*log(extracellular[Cl−]/intracellular[Cl−]),(T=25° C.). Spontaneous action potentials were recorded at I=0 mode. Action potentials also induced by current injection with steps at 10 pA lasting 800 ms after manually adjusting the membrane potential around −70 mV by current injection under current clamp mode. For cell-attached recordings, pipettes with resistances of 2-4 MΩ were filled with extracellular solution described above. Attachment between pipettes and neuron membrane was formed with 50-200 MΩ seal resistance. Voltage clamp mode was used to record current response by spontaneous spikes firing with holding pipettes at 0 mV.
Optogenetic or Chemical GABA Evoked IPSC Recording
iGNs that priorly transduced with Synapsin-ChETA-EYFP were co-cultured with iENs expressing turboRFP (FUW-tRFP). For optogenetical evoked IPSC recording, two nearly neurons with EYFP and tRFP, respectively, were visually identified with fluorescent microscope (Olympus) with DIC. Patch clamp recordings were performed on tRFP-positive neurons for evoked IPSC or on EYFP-positive neurons for identification of optical stimulation mediated by ChETA. Optical stimuli (5 ms duration, 30s interval) were provided with blue (470 nm) LED (Thorlabs, M470F1) controlled by digital input from Digidata 1440A (Molecular Devices).
For chemical GABA evoked IPSC recording, tip of glass pipettes filled with freshly made GABA (1 mM, Sigma) was put approximately 100 μm away from soma along dendrites of recorded neurons. Air puff to trigger GABA release was provided with PICOSPRITZER III (Parker) controlled by Digidata 1440A.
Transplantation
Immunodeficient NOD scid gamma (NSG) mice used for transplantation studies and breeding were a kind gift from Dr. David Virshup at Duke-NUS Graduate Medical School. Mice were housed in a specific pathogen free environment, maintained under 22° C., 55% humidity, with food and water provided ad libitum, on a 12-hr light/dark cycle (lights on at 0700 h). All procedures followed national guidelines for the care and use of laboratory animals for scientific purposes with approved protocols from the Institutional Animal Care and Use Committee of Duke-NUS Graduate Medical School.
P1 NSG pups were anesthetized on ice for 1-2 minutes before being secured with tape onto a prechilled ice block. Human iGNs were priorly labeled with RFP as described, and trypsinized to single cells at 8 dpt. Concentrated cell suspensions (˜2.5-5×104 cells/μl) were front loaded into a microliter syringe (26s gauge, Hamilton Company) and injected (200 nl, 250 nl/min) bilaterally to a depth of 0.2 mm near the centre of the anterior/posterior axis and 1 mm away from the midline.
Slice Recording
After isofluorane anesthesia, brains were quickly cut out and 300-μm coronal slices were sectioned using vibrating microtome (VF-200 Microtome, Precisionary Instruments). Slices were incubated at 30° C. in artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 26 NaHCO3, and 10 glucose, saturated with 95% 02 and 5% CO2 at 30-32° C. for recovery for at least 1 hour. During recording, slices were kept in a recording chamber with continuous perfusion of ACSF. CNQX (50 μM, Tocris) was used to block sEPSC. Patch recordings were done by using IR-DIC visualization techniques with an Olympus BX51WI upright microscope with a ×60 water-immersion lens. Patch clamp recording method was same with that of recording in cultured cell.
Fluorescent Immunocytochemistry (Brain Slices)
2 months post transplantation, mice were transcardially perfused with ice cold PBS followed by 4% PFA (Sigma) in 0.1M PBS. Brains were dissected, post-fixed in 4% PFA overnight and cryoprotected with 30% sucrose in 0.1M PB until they sunk. Sections 30 μm thick were cut on a sliding microtome (Leica), washed with PBS, permeabilized with 0.2% Triton-X in PBS for 10 minutes, blocked in PBS with 2% BSA (Sigma), 5% donkey serum (Invitrogen) and 0.2% Triton X-100 at room temperature for 1 hour, and subsequently incubated with primary antibodies overnight. Following incubation, sections were washed three times with 0.2% Triton-X, and incubated for 2 hours at room temperature with goat anti-rabbit, mouse or chicken IgG conjugated with Alexa-488 or 647 (Invitrogen). Sections were washed once with 0.2% Triton-X, incubated with DAPI (Life-Technologies) for 10 minutes followed by two additional washes. Sections were mounted on glass slides with Fluor Save (Millipore).
Electron Microscopy
Mice were anesthetized and intracardially perfused with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.15M cacodylate buffer. The brain was extracted from the skull and then postfixed overnight in the same fixative. Cortical tissues transplanted with RFP-expressing iGNs were sliced using a vibratome (˜100 μm). After targeting the location of fluorescent signals under a epifluorescent microscope, cortical slices were immunostained with a rabbit polyclonal anti-RFP antibody (MBL, 1:500), a biotinylated goat anti-rabbit secondary antibody, and the ABC-peroxidase kit (Vector Labs) and developed with DAB and hydrogen peroxidase. The slices were further prepared for serial block face-SEM (SBF-SEM) observation. Briefly, small pieces of immunostained slices were postfixed in 2% osmium tetroxide containing 1.5% potassium ferrocyanide and 2 mM calcium chloride in 0.15M cacodylate buffer, and then incubated in thiocarbohydrazide solution. Following the second exposure to 2% osmium tetroxide, tissue samples were en bloc stained in 1% uranyl acetate (Ted Pella), incubated in the lead aspartate solution, and then dehydrated in an ascending series of ethanol solutions. Samples were transferred to acetone and flat-embedded in Epon-812 (EMS). Epon-embedded specimens containing DAB-labeled neurons were glued on an aluminum stub (Gatan), painted with colloidal silver paste (Ted Pella), and then sputter-coated with gold/palladium to reduce charge artifacts. 11 stacks of serial images (tens to hundreds of 30-nm-thick sections/stack) were obtained using a scanning electron microscope (Merlin VP, Carl Zeiss NTS GmbH, Oberkochen, Germany) combined with the Gatan 3View2 diamond knife cutting system at the accelerating voltage of 1.5 kV. To cover the large area, low magnification images of the sample block were acquired with the both back-scattered and secondary electron detectors of the column.
Calcium Imaging
Neurons were incubated in extracellular solution containing (in mM): 124 NaCl, 5 KCl, 1.3 NaH2PO4, 10 dextrose, 2 MgCl2, 4 CaCl2, 10 HEPES at pH 7.4 with Fluo-4 AM (2 μM, ThermoFisher Scientific, F-14201) in incubator (37oC, 5% CO2) for 30 mins before acquiring images. Imaging solution was identical to extracellular solution. Live images were acquired with an LSM 710 (Zeiss) confocal microscopy using 20x objective at 1 Hz at 37° C. Calcium spikes sorting and analysis was done referred to previous works 6,7. Calcium spikes were identified with more than 20% change of fluorescence baseline on individual neurons. Spikes from two neurons with interval no more than ±2 frames were considered as occurring at the same time. A burst was counted when there were 50% neurons having calcium spikes at the same time in the network. For estimating network synchronization, pair-wise synchronization index of each two-neuron-pair was computed to get synchronization matrix based on either pairwise correlation coefficients or spikes instantaneous phase, which gave consistent results. Network synchronization index was acquired from the eigenvalues of the synchronization matrix. Higher synchronization index signified more synchronized activity in the neural network.
Identification of Genetic Elements that Directly Convert Human Pluripotent Stem Cells (hPSCs) to GABAergic Neurons.
To identify genetic elements that can directly convert hESCs to GABAergic neurons, focus was placed on four transcription factors (TFs), ASCL1, DLX2, NKX2.1, and LHX6. These TFs are expressed in the medial ganglionic eminence (MGE), a major site of GABAergic neurogenesis, and are important for the differentiation and functional maturation of cortical interneurons (
Lentiviruses expressing each of the four TFs, infected hESCs (line H1) were generated, and their conversion to neuronal cells was assessed by staining the cells for the pan-neuronal marker MAP2 at 10 days post-transduction (dpt) (
A phospho-mutant form of ASCL1 (in which 5 serine resides are substituted with alanine, denoted ASA) is more potent than the wild-type ASCLI in ectopic neural induction in Xenopus embryos and in the trans-differentiation of human fibroblasts to neurons. Accordingly, ASA was overexpressed in hESCs and it was found that it resulted in the production of approximately 2-fold more MAP2-positive neurons than A (
Next, the possibility that the addition of other factors could enhance neuronal conversion and, more specifically, GABAergic neuronal conversion was investigated. To that end, hESCs were transduced with different pools of lentiviruses that contained ASA as an obligatory factor together with various combinations of the other 3 TFs (D, N, and L) and quantified MAP2- and GABA- positive cells at 10 dpt (
To further improve the overall conversion efficiency, miR-9/9*-124, which we increase neuronal conversion from non-neuronal cells were co-expressed, together with the ASA, D, and L TFs. Strikingly, the addition of miR-9/9*-124 significantly increased the percentage of MAP2-positive cells from 50.3±4.7% to 81.3±3.1% (
To promote survival and functional maturation, cells that were transduced with ASADL+miR-9/9*-124 were co-cultured with rat glia Immunostaining at 42 dpt revealed that the majority of the induced neuronal cells were GABAergic (84.5±3.5%) (
Molecular Characterization of Human Induced GABAergic Neurons (iGNs)
To gain insight into the kinetics of how iGNs acquire an inhibitory neuronal fate, mRNA levels of key genes responsible for the GABAergic phenotype (VGAT, GAD1, and GAD2) were measured in iGNs using quantitative RT-PCR at 14 and 35 dpt. At 14 dpt, the iGNs expressed all three markers at levels similar to those observed in fetal human brains, which contained approximately 10-15% GABAergic interneurons (
To further characterize the iGNs, multiplexed gene expression analysis of single iGNs was performed at 48-52 dpt (
To confirm and corroborate the mRNA expression data, immunostaining analyses of the iGNs were performed. The iGNs strongly expressed NeuN, a mature neuronal marker, the axonal marker SMI-312 and Ankyrin G, an axon initial segment marker (
Mature cortical interneurons can be divided into different subgroups based on their expression of neuropeptides and calcium-binding proteins, including SST, PV, calretinin (CR), calbindin (CB), neuropeptide Y (NPY), reelin (RELN), neuronal nitric oxide synthetase (nNOS), and vasoactive intestinal peptide (VIP). Immunostaining revealed that a subset of iGNs expressed SST (24.3%), CR (11.6%), CB (6.5%), and NPY (5.4%) (
Functional Characterization of Induced GABAergic Neurons
To explore whether the iGNs exhibited functional membrane properties similar to those of neurons, patch-clamp recordings of iGNs at 42 and 56 dpt were performed. In voltage-clamp mode, the iGNs showed fast, inactivating inward and outward currents, which likely correspond to the opening of voltage-dependent potassium (K+)- and sodium (Na+)-channels, respectively (
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Next, both spontaneous and elicited action potentials (APs) were recorded from iGNs in cell-attached mode (
Next, to further confirm that the iGNs expressed the functional presynaptic machinery needed to release GABA and induce inhibitory postsynaptic responses, iGNs were co-cultured with iENs that were generated via a protocol similar to that used in a previous study (
Whether the iGNs exhibited functional postsynaptic mechanisms, which would enable synaptic transmission in situ was further investigated. First, exogenous application of GABA (1 mM, 100 ms) triggered inhibitory postsynaptic currents (IPSCs) in iGNs (
Functional Maturation and Synaptic Integration of Human iGNs In Vivo
To test whether the iGNs were able to undergo synaptic maturation and functional integration in vivo, RFP-expressing iGNs were stereotaxically transplanted at 8 dpt into cortices of P1 neonatal immunodeficient NOD SCID mice. Two months later, NeuN expressing iGNs were dispersed in mouse cortex, mostly in layer 5/6 (
To determine whether transplanted iGNs develop into functional neurons and integrate into host neural circuitry, whole-cell patch-clamp recordings were used in acute cortical slices obtained from transplanted mice. Grafted iGNs, identified by RFP expression, displayed repetitive AP firings (
Potential Use of iGNs in Large-Population Calcium Imaging and Interneuron-Specific Mechanistic Studies
To explore the potential use of the iGNs to assess cell type-specific drug effects or to model human disease states, iGNs were tested to see if they could form functional synaptic connections with other excitatory glutamatergic neurons. iGNs (20%) were co-cultured with iENs (80%), which mimics the proportions found in mammalian cortical networks, and measured spontaneous PSCs from the iGNs (
Next, spontaneous activity-dependent Ca2+ transients was examined either in homogenous populations of iENs or in mixtures of 80% iENs and 20% iGNs. In the homogenous population of iENs, the addition of bicuculline did not change the network activity, as measured by the synchronization of individual Ca2t transients (
Moreover, whether the iGNs could be used for interneuron-specific mechanistic studies was tested. Overexpression of MDGA1 (MAM-domain-containing glycosylphosphatidylinositol anchors 1) has been linked to autism and schizophrenia, in cortical neurons reduced inhibitory synapse numbers. However, whether the overexpression of MDGA1 in excitatory neurons or inhibitory neurons resulted in the reduced the inhibitory synaptic input remained unclear. To this end, MDGA1 was expressed in a homogenous population of iGNs via lentiviral transduction and measured inhibitory synapse density based on the number of VGAT-positive clusters. It was observed that MDGA1 overexpression significantly reduced inhibitory synapse density (
In the present study, a single-step, efficient, and reproducible method of generating a nearly pure population of human forebrain GABAergic neuronal cells (iGNs) is described based on the overexpression of selected TFs and microRNAs. Starting from hPSCs, we provided evidence that ASCL1, a proneural bHLH factor that is broadly expressed in the ventral brain, can induce a small fraction of MAP2-expressing neuronal cells within 7-10 days, consistent with previous reports (
Compared to previous attempts to derive GABAergic neurons from non-neuronal human cells, the present method offers several advantages. First, the genetic gain-of-function approach bypasses the neural progenitor stage, thereby eliminating the need for various patterning factors and recombinant proteins (saving on costs and reducing experimental variability). Second, the protocol generates functional iGNs within a significantly shorter period of time (6-8 weeks, compared to 10-30 weeks), which enables more rapid turnaround of experiments. Third, the method primarily generates GABAergic neurons; few cells of other lineages are produced. These salient features enable a unique opportunity for in vitro assembly of microcircuits with neurons of defined identities and densities.
Indeed, distinct patterns of spontaneous neuronal network activity in dishes that contained discrete percentages of human excitatory (iENs) or inhibitory neurons (iGNs) were observed, and drug-induced alterations of the activity of the networks en bloc using Ca2+ imaging (
More importantly, the single-step nature of the present method permitted the generation of generate dox-inducible iGN (as well as iEN) hESC lines that can be synchronously differentiated into GABAergic neurons upon the addition of dox (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201502869T | Apr 2015 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2016/050176 | 4/11/2016 | WO | 00 |
