Patent Application
20200121725
Simple neural in vitro systems do not reflect the physiology, cellular interactions, or genetics of mammalian brain tissue. Accordingly, there is an unmet need to develop human models of brain disorders and/or diseases.
Aspects of the invention are directed to an in vitro brain microphysiological system (BMPS). In embodiments, the BMPS comprises a least one neural cell type aggregated into a spheroid mass and a population of microglia-like cells. The in vitro BMPS can be electrophysiologically active in a spontaneous manner.
In one embodiment, the micro-glia like cells comprise microglia, microglia precursor cells, or a combination thereof. The micro-glia like cells can comprise monocytes, human monocytes, pro-monocyte cell lines, hematopoietic stem cells, isolated microglia, immortalized microglia, or combinations thereof. In embodiments, the monocytes comprise adult cell-derived monocytes, embryonic cell-derived monocytes, or a combination thereof. The monocytes can comprise embryonic stem cell (ESC)-derived monocytes, induced pluripotent stem cell (iPSC)-derived monocytes, or a combination thereof. In certain embodiments, the isolated microglia comprise adult microglia, fetal microglia, or a combination thereof. The microglia-like cells can be derived from somatic cells, neuronal cells, myeloid progenitor cells, or a combination thereof. In embodiments, the microglia-like cells express one or more of the following biomarkers: HLA-DR, Iba1, CD14, CX3CR1, F4/80, CD80, CD86, CD36, iNOS, COX2, ARG1, PPARy, SOCS-3, TMEM119, Mertk, Ax1, CD11b, CD11c, P2RY12, CD45, CD68, CD40, B7, ICAM-1 or any combination thereof.
In some embodiments, the BMPS expresses receptors associated with microglia function. In embodiments, the receptors associated with microglia function comprise CCL2, CX3CL, RAGE, NLRP3, SR-AI, TREM2, FPRL1/FPR2, CD36, CD33, C5a, CR1, CR3/Mac-1, FcRs, FPRs, TLRs, or a combination thereof.
In embodiments, the BMPS is configured to elicit a pro-inflammatory response, an anti-inflammatory response, or a combination thereof. The BMPS can be configured to elicit a pro-inflammatory response to viral infection, LPS exposure, or a combination thereof. The BMPS being configured to elicit an anti-inflammatory response to IL-3, IL-4, IL-10, IL-13, IL-1β, IL-6, TNF-α, TGF-β, or a combination thereof.
In certain embodiments, the microglia-like cells comprise about 20% or less of the BMPS.
In embodiments, the at least one neuronal cell type comprises a mature neuron, a glial cell, or a combination thereof. The at least one neural cell type can further comprise astrocytes, polydendrocytes, oligodendrocytes, or combinations thereof.
In various exemplary embodiments, the in vitro BMPS has neural characteristics selected from the group consisting of synaptogenesis, neuron-neuron interactions, neuronal-glial interactions, axon myelination, cell migration, neurological development, disease phenotypes, or combinations thereof. Exemplary disease process phenotypes include autophagy, Integrated stress response, non-sense mediated decay, lesions, amyloid deposition, plaque formation, protein aggregation, or combinations thereof.
In embodiments, the at least one neural cell type express one or more biomarker selected from the group consisting of MBP, PLP, NG2, Olig1, Olig2, Olig 3, OSP, MOG, SOX10, neurofilament 200 (NF200), GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof. The at least one neuronal cell type can comprise one or more genetically modified cells. In embodiments, the one or more genetically modified cells comprise one or more reporter genes.
In certain embodiments, the spheroid mass comprises a diameter that is about 1000 μm or less. The spheroid mass can comprise a diameter that is about 500 μm or less.
In embodiments, the BMPS comprises one or more endothelial cells, pericytes, or a combination thereof capable of forming a blood-brain-barrier.
Other aspects are directed to a method of reproducibly producing an in vitro brain microphysiological system (BMPS). In embodiments, the method comprises any one or more of the following steps: inducing one or more pluripotent stem cell (PSC) types; differentiating the one or more PSC types to form one or more neural progenitor cell (NPC) types; exposing the one or more NPC types to gyratory shaking or stirring; differentiating the one or more NPC types into one or more neural cell types aggregated into a spheroid mass; and adding microglia-like cells. In embodiments, the micro-glia like cells comprise mature microglia, monocytes, human monocytes, pro-monocyte cell lines, PSC-derived monocytes, hematopoetic stem cells, isolated microglia, immortalized microglia, or combinations thereof. In embodiments, the one or more pluripotent stem cells are selected from the group consisting of human or animal embryonic stem cells, iPSC, adult stem cells, fibroblasts, embryonic fibrobflasts, peripheral blood mononuclear cells, neuronal precursor cells, mesenchymal stem cells, neuronal cells, glial cells, and combinations thereof. The microglia-like cells can be added during an early stage (before BMPS differentiation) or late stage (after BMPS differentiation). In alternate embodiments, the microglia are generated in parallel with the BMPS. In certain embodiments, gyratory shaking comprises constant or regular gyratory shaking or stirring for 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more weeks.
The present invention provides brain microphysiological systems (BMPS) that can be produced from induced pluripotent stem cells (iPSCs). Furthermore, the invention provides for reproducible BMPS that differentiate into mature neurons and glial cells (astrocytes and oligodendrocytes) in the central nervous system. This model is electrophysiologically active in a spontaneous manner and may be reproduced with patient cells. The derivation of 3D BMPS from iPSCs has applications in the study and treatment of neurological diseases.
In an aspect, the disclosure provides an in vitro brain microphysiological system (BMPS), comprising two or more neural cell types aggregated into a spheroid mass, wherein the spheroid mass has a diameter that is less than about 500 μm and the in vitro BMPS is electrophysiologically active in a spontaneous manner.
In an embodiment, the two or more neural cell types comprise at least a mature neuron and glial cell.
In an embodiment, the two or more neural cell types further comprise cells selected from the group consisting of astrocytes, polydendrocytes, oligodendrocytes, and combinations thereof.
In an embodiment, the in vitro BMPS has neural characteristics selected from the group consisting of synaptogenesis, neuron-neuron interactions, neuronal-glial interactions, axon myelination, and combinations thereof.
In an embodiment, two or more neural cell types of the in vitro BMPS express one or more biomarker selected from the group consisting of GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof.
In an aspect, the disclosure provides a synthetic neurological organ comprising two or more neural cell types aggregated into a spheroid mass, wherein the spheroid mass has a diameter that is less than 500 μm and the in vitro BMPS is electrophysiologically active in a spontaneous manner. In an embodiment, the two or more neural cell types comprise at least a mature neuron and glial cells.
In an embodiment, the mature neuron and glial cells further comprise cells selected from the group consisting of astrocytes, polydendrocytes, oligodendrocytes, and combinations thereof.
In an embodiment, the synthetic neurological organ further comprises neural characteristics selected from the group consisting of synaptogenesis, neuron-neuron interactions, neuronal-glial interactions, axon myelination, and combinations thereof.
In an embodiment, the synthetic neurological organ mimics the microenvironment of the central nervous system (CNS).
In an aspect, the disclosure provides a method of reproducibly producing an in vitro brain microphysiological system (BMPS), comprising: inducing one or more pluripotent stem cell (PSC) types; differentiating the one or more PSC types to form one or more neural progenitor cell (NPC) types; exposing the one or more NPC types to gyratory shaking or stirring; and differentiating the one or more NPC types into one or more neural cell types aggregated into a spheroid mass, wherein the spheroid mass has a diameter that is less than 500 μm.
In an embodiment, the one or more pluripotent stem cells are selected from the group consisting of human or animal embryonic stem cells, iPSC, adult stem cells, fibroblasts, embryonic fibroblasts, peripheral blood mononuclear cells, neuronal precursor cells, mesenchymal stem cells, and combinations thereof.
In an embodiment, inducing further comprises: adding micro-glia or micro-glia precursor cells.
In an embodiment, the micro-glia or micro-glia precursor cells are selected from the group consisting of monocytes, human monocytes, pro-monocyte cell lines, iPSC-derived monocytes, hematopoetic stem cells, isolated microglia, immortalized microglia, and combinations thereof.
In an embodiment, gyratory shaking comprises constant or regular gyratory shaking or stirring for 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more weeks.
In an embodiment, the one or more growth factors are selected from the group consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, and any combination thereof.
In an aspect, the disclosure provides a method of cryopreserving an in vitro brain microphysiological system (BMPS), comprising: differentiating BMPS aggregates into one or more mature neurons; incubating the aggregates in a cryopreserving medium; and exposing the aggregates to freezing temperatures of −60° C. or colder.
In an embodiment, differentiating further comprises: inducing differentiation of one or more pluripotent stem cell types by incubation with one or more growth factors.
In an embodiment, the one or more pluripotent stem cells are selected from a group consisting of human or animal embryonic stem cells, iPSC, adult stem cells, fibroblasts, embryonic fibroblasts, peripheral blood mononuclear cells, neuronal precursor cells, mesenchymal stem cells, and combinations thereof.
In an embodiment, inducing further comprises: adding micro-glia precursor cells.
In an embodiment, micro-glia precursor cells are selected from the group consisting of monocytes, human monocytes, iPSC-derived monocytes, hematopoetic stem cells, pro-monocyte cell lines, isolated microglia, immortalized microglia, and combinations thereof.
In an embodiment, the one or more growth factors are selected from the group consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, and any combination thereof.
In an embodiment, the cryopreserving medium is a medium selected from the group consisting of regular cryopreservation medium (95% FBS and 5% DMSO), STEMdiff Neural Progenitor Freezing Medium (Stem Cells Technologies), solutions with cryoprotectants, and combinations thereof.
In an embodiment, exposing the aggregates to freezing temperatures further comprises freezing aggregates over a temperature gradient of about 1° C. per hour to below −60° C. over up to 48 hours.
In an embodiment, cryopreserving further comprises additives selected from the group consisting of DMSO, HES, glycerol, serum, and any combination or derivative thereof.
In an aspect, the disclosure provides a method of transporting a brain microphysiological system (BMPS) or mini-brain, comprising: producing the BMPS or mini-brain of claim 1, incubating the BMPS or mini-brain at 37° C., and maintaining the temperature at 37° C. with constant application of heat while moving the BMPS or mini-brain.
In an embodiment, maintaining the temperature comprises use of heating pads, heaters, insulation, insulated boxes, heat packs, electric blankets, chemical pads, and combinations thereof.
In an aspect, the disclosure provides a method of studying a neurological disease or disorder comprising: producing an in vitro brain microphysiological system (BMPS); exposing the in vitro BMPS to conditions that replicate or induce the neurological disease or disorder; adding an agent to treat the neurological disease or disorder; and assessing the effect of the agent on the neurological disease or disorder.
In an embodiment, the neurological disease or disorder is selected from the group consisting of neurodegenerative disorder, muscular dystrophy, Parkinson's Disease, Huntington's Disease, Autism Spectrum Disorder and other neurodevelopmental disorders, Down's Syndrome, Multiple Sclerosis, Amyotrophic lateral sclerosis, brain cancer, encephalitis, infection, trauma, stroke, and paralysis.
In an aspect, the disclosure provides a method of treating a patient having a neurological disease or disorder, comprising: extracting a stem cell from the patient with a genetic background pre-disposed for the neurological disease or disorder; producing a brain microphysiological system (BMPS) or mini-brain with the genetic background; treating the BMPS or mini-brain with an agent targeting the neurological disease or disorder; and assessing the effect of the agent on the BMPS or mini-brain.
In an embodiment, the neurological disease or disorder is selected from the group consisting of neurodegenerative disorder, muscular dystrophy, Parkinson's Disease, Huntington's Disease, Autism Spectrum Disorder and other neurodevelopmental disorders, Down's Syndrome, Multiple Sclerosis, Amyotrophic lateral sclerosis, brain cancer, encephalitis, infection, trauma, stroke, and paralysis.
In an embodiment, the BMPS includes two or more neuronal cell types that include one or more genetically modified cells. The BMPS wherein the one or more genetically modified cells include one or more reporter genes. The BMPS further comprises one or more endothelial cells capable of forming a blood-brain-barrier.
In an embodiment, the synthetic neurological organ may include two or more neural cell types that include one or more genetically modified cells. The synthetic neurological organ including one or more genetically modified cells that include one or more reporter genes. The synthetic neurological organ further comprising one or more endothelial cells capable of forming a blood-brain-barrier.
In an aspect, the disclosure provides a method of reproducibly producing an in vitro brain microphysiological system (BMPS), comprising: exposing one or more NPC types to gyratory shaking or stirring; and differentiating the one or more NPC types into one or more neural cell types aggregated into a spheroid mass, wherein the spheroid mass has a diameter that is less than 500 μm.
In an embodiment, the spheroid mass has a diameter that is less than about 450 μm, 400 μm, 350 μm, or 300 μm, or a diameter that is between about 350 μm and about 300 μm, or a diameter that is between about 330 μm and about 300 μm, or a diameter that is about 310 μm.
In an embodiment, the two or more neural cell types of the in vitro BMPS express one or more biomarker selected from the group consisting of GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof.
In an embodiment, the two or more neural cell types of the in vitro BMPS express one or more biomarker selected from the group consisting of GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof.
In an embodiment, the two or more neural cell types of the in vitro BMPS express one or more biomarker selected from the group consisting of GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof.
In an embodiment, inducing comprises a single PSC.
In an embodiment, the an in vitro brain microphysiological system (BMPS) may be produced according to the above described method.
It is also contemplated within the scope of the invention that the addition of other cells inside (see e.g.,
By “agent” is meant any small compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like may have the meaning ascribed to them in U.S. Patent law and may mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “effective amount” is meant the amount of an agent needed to ameliorate the symptoms of a neurological disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a neurological disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids, or more.
By “gene” is meant a locus (or region) of DNA that encodes a functional RNA or protein product, and is the molecular unit of heredity.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By “modulate” is meant alter (increase or decrease). Such alterations are detected by standard art known methods such as those described herein.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition.
By “pluripotency” is meant stem cells with the potential to differentiate into any of the three germ layers: endoderm (e.g., interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g., muscle, bone, blood, urogenital), or ectoderm (e.g., epidermal tissues and nervous system). However, one of skill in the art will understand that cell pluripotency is a continuum, ranging from the completely pluripotent cell that can form every cell of the embryo proper, e.g., embryonic stem cells and iPSCs (see below), to the incompletely or partially pluripotent cell that can form cells of all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells. Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a “forced” expression of certain genes and transcription factors. These transcription factors play a key role in determining the state of these cells and also highlight the fact that these somatic cells do preserve the same genetic information as early embryonic cells. The ability to induce cells into a pluripotent state was initially pioneered using mouse fibroblasts and four transcription factors, Oct4, Sox2, Klf4 and c-Myc;—a process called reprogramming. The successful induction of human iPSCs derived from human dermal fibroblasts has been performed using methods similar to those used for the induction of mouse cells. These induced cells exhibit similar traits to those of embryonic stem cells (ESCs) but do not require the use of embryos. Some of the similarities between ESCs and iPSCs include pluripotency, morphology, self-renewal ability, a trait that implies that they can divide and replicate indefinitely, and gene expression.
By “stem cells” is meant undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cells—ectoderm, endoderm and mesoderm (see induced pluripotent stem cells)—but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. There are three known accessible sources of autologous adult stem cells in humans: 1. Bone marrow, which requires extraction by harvesting, that is, drilling into bone (typically the femur or iliac crest). 2. Adipose tissue (lipid cells), which requires extraction by liposuction. 3. Blood, which requires extraction through apheresis, wherein blood is drawn from the donor (similar to a blood donation), and passed through a machine that extracts the stem cells and returns other portions of the blood to the donor. Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a neurological disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an,” and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.
By “GRIN1 polypeptide” (or glutamate ionotropic receptor NMDA type subunit 1) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q05586.
By “GRIN1 nucleic acid molecule” (or glutamate ionotropic receptor NMDA type subunit 1) is meant a polynucleotide encoding an GRIN1 polypeptide. An exemplary GRIN1 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_007327.
By “GAD1 polypeptide” (or glutamate decarboxylase 1) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q99259.
By “GAD1 nucleic acid molecule” (or glutamate decarboxylase 1) is meant a polynucleotide encoding an GAD1 polypeptide. An exemplary GAD1 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC036552.
By “GABA polypeptide” (or gamma-Aminobutyric acid) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P30531.
By “GABA nucleic acid molecule” (or gamma-Aminobutyric acid) is meant a polynucleotide encoding an GABA polypeptide. An exemplary GABA nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. U76343.
By “TH polypeptide” (or Tyrosine Hydroxylase) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_002692.
By “TH nucleic acid molecule” (or Tyrosine Hydroxylase) is meant a polynucleotide encoding an TH polypeptide. An exemplary TH nucleic acid molecule is provided at NCBI Accession No. NG_008128.
By “LMX1A polypeptide” (or LIM homeobox transcription factor 1-alpha) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q8TE12.
By “LMX1A nucleic acid molecule” (or LIM homeobox transcription factor 1-alpha) is meant a polynucleotide encoding an LMX1A polypeptide. An exemplary LMX1A nucleic acid molecule is provided at NCBI Accession No. AH011517.
By “FOXO1 polypeptide” (or Forkhead box protein 01) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q12778.
By “FOXO1 nucleic acid molecule” (or Forkhead box protein 01) is meant a polynucleotide (e.g., mRNA) encoding an FOXO1 polypeptide. An exemplary FOXO1 nucleic acid molecule is provided at NCBI Accession No. NM_002015.
By “FOXA2 polypeptide” (or Forkhead box protein A2) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q9Y261.
By “FOXA2 nucleic acid molecule” (or Forkhead box protein A2) is meant a polynucleotide (e.g., mRNA) encoding an FOXA2 polypeptide. An exemplary FOXA2 nucleic acid molecule is provided at NCBI Accession No. NM_021784.
By “FOXO4 polypeptide” (or Forkhead box protein 04) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P98177.
By “FOXO4 nucleic acid molecule” (or Forkhead box protein 04) is meant a polynucleotide (e.g., mRNA) encoding an FOXO4 polypeptide. An exemplary FOXO4 nucleic acid molecule is provided at NCBI Accession No. NM 005938.
By “CNP polypeptide” (or 2′,3′-cyclic-nucleotide 3′-phosphodiesterase) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P09543.
By “CNP nucleic acid molecule” (or 2′,3′-cyclic-nucleotide 3′-phosphodiesterase) is meant a polynucleotide (e.g., mRNA) encoding an CNP polypeptide. An exemplary CNP nucleic acid molecule is provided at NCBI Accession No. BC011046.
By “MBP polypeptide” (or myelin basic protein) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P02686.
By “MBP nucleic acid molecule” (or myelin basic protein) is meant a polynucleotide (e.g., mRNA) encoding an MBP polypeptide. An exemplary MBP nucleic acid molecule is provided at NCBI Accession No. M13577.
By “TUBIII polypeptide” (or TUBB3, tubulin beta chain 3) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001184110.
By “TUBIII nucleic acid molecule” (or TUBB3, tubulin beta chain 3) is meant a polynucleotide (e.g., mRNA) encoding an TUBIII polypeptide. An exemplary TUBIII nucleic acid molecule is provided at NCBI Accession No. BC000748.
By “NEUN polypeptide” (or Feminizing Locus on X-3, Fox-3, RNA-binding protein fox-1 homolog 3, or Hexaribonucleotide Binding Protein-3) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001076044.
By “NEUN nucleic acid molecule” (or Feminizing Locus on X-3, Fox-3, RNA-binding protein fox-1 homolog 3, or Hexaribonucleotide Binding Protein-3) is meant a polynucleotide (e.g., mRNA) encoding an NEUN polypeptide. An exemplary NEUN nucleic acid molecule is provided at NCBI Accession No. NM_001082575.
By “SLC1A6 polypeptide” (or Excitatory amino acid transporter 4; Sodium-dependent glutamate/aspartate transporter; Solute carrier family 1 member 6) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P48664.
By “SLC1A6 nucleic acid molecule” (or Excitatory amino acid transporter 4; Sodium-dependent glutamate/aspartate transporter; Solute carrier family 1 member 6) is meant a polynucleotide (e.g., mRNA) encoding an SLC1A6 polypeptide. An exemplary SLC1A6 nucleic acid molecule is provided at NCBI Accession No. BC040604.
By “NOGOA polypeptide” (or neurite outgrowth inhibitor A; neurite outgrowth inhibitor isoform A; human reticulon-4; human reticulon-4 isoform A) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_065393.
By “NOGOA nucleic acid molecule” (or neurite outgrowth inhibitor A; neurite outgrowth inhibitor isoform A; human reticulon-4; human reticulon-4 isoform A) is meant a polynucleotide encoding an NOGOA polypeptide. An exemplary NOGOA nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_020532.
By “oligodendrocyte 01 polypeptide” (or oligodendrocyte marker 01; oligodendrocyte transcription factor 1; olig1) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q8TAK6.
By “oligodendrocyte 01 nucleic acid molecule” (or oligodendrocyte marker 01; oligodendrocyte transcription factor 1; olig1) is meant a polynucleotide encoding an oligodendrocyte 01 polypeptide. An exemplary oligodendrocyte 01 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_138983.
By “oligodendrocyte 02 polypeptide” (or oligodendrocyte marker 02; oligodendrocyte transcription factor 2; olig2) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q13516.
By “oligodendrocyte 02 nucleic acid molecule” (or oligodendrocyte marker 02; oligodendrocyte transcription factor 2; olig2) is meant a polynucleotide encoding an oligodendrocyte 02 polypeptide. An exemplary oligodendrocyte 02 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_005806.
By “oligodendrocyte 04 polypeptide” (or oligodendrocyte marker 04; oligodendrocyte transcription factor 4; olig4) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. Q05586.
By “oligodendrocyte 04 nucleic acid molecule” (or oligodendrocyte marker 04; oligodendrocyte transcription factor 4; olig4) is meant a polynucleotide encoding an oligodendrocyte 04 polypeptide. An exemplary oligodendrocyte 04 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_007327.
By “GFAP” (or Glial fibrillary acidic protein) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P14136.
By “GFAP nucleic acid molecule” (or Glial fibrillary acidic protein) is meant a polynucleotide encoding an GFAP polypeptide. An exemplary GFAP nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_002055.
By “s100b” (or S-100 protein beta chain; S-100 protein subunit beta; S100 calcium-binding protein B) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P04271.
By “s100b nucleic acid molecule” (or S-100 protein beta chain; S-100 protein subunit beta; S100 calcium-binding protein B) is meant a polynucleotide encoding an s100b polypeptide. An exemplary s100b nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006272.
By “SOX10 polypeptide” (or SRY-related HMG-box transcription factor) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_008872.1.
By “SOX10 nucleic acid molecule” (or SRY-related HMG-box transcription factor) is meant a polynucleotide encoding an SOX10 polypeptide. An exemplary SOX10 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006941.3.
By “SYN1 protein” (or Synaptin I protein) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank: AH006533.2.
By “SYN1 nucleic acid molecule” (or synapsin I gene) is meant a polynucleotide encoding an SYN1 polypeptide. An exemplary SYN1 nucleic acid molecule (e.g., mRNA) is provided at GenBank: AH006533.2.
By “SYP protein” (or synaptophysin protein) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Reference Sequence: NM_003179.2.
By “SYP nucleic acid molecule” (or synaptophysin gene) is meant a polynucleotide encoding an SYN1 polypeptide. An exemplary SYP nucleic acid molecule (e.g., mRNA) is provided at NCBI Reference Sequence: NM_003179.2.
By “NOGOA polypeptide” (or neurite outgrowth inhibitor A; neurite outgrowth inhibitor isoform A; human reticulon-4; human reticulon-4 isoform A) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_065393.
By “NOGOA nucleic acid molecule” (or neurite outgrowth inhibitor A; neurite outgrowth inhibitor isoform A; human reticulon-4; human reticulon-4 isoform A) is meant a polynucleotide encoding an NOGOA polypeptide. An exemplary NOGOA nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_020532.
By “GFAP” (or Glial fibrillary acidic protein) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P14136.
By “GFAP nucleic acid molecule” (or Glial fibrillary acidic protein) is meant a polynucleotide encoding an GFAP polypeptide. An exemplary GFAP nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_002055.
By “s100b” (or S-100 protein beta chain; S-100 protein subunit beta; S100 calcium-binding protein B) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. P04271.
By “s100b nucleic acid molecule” (or S-100 protein beta chain; S-100 protein subunit beta; S100 calcium-binding protein B) is meant a polynucleotide encoding an s100b polypeptide. An exemplary s100b nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006272.
By “PAX6 polypeptide” (or paired box protein PAX6) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAK95849.1.
By “PAX6 polynucleotide” (or paired box protein PAX6) is meant a polynucleotide encoding an PAX6 polypeptide. An exemplary PAX6 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. AY047583.
By “Nestin polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_006608.1.
By “Nestin polynucleotide” is meant a polynucleotide encoding an Nestin polypeptide. An exemplary Nestin nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006617.
By “LHX6 polypeptide” (or LIM homeobox 6) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI03937.1.
By “LHX6 polynucleotide” (or LIM homeobox 6) is meant a polynucleotide encoding an LHX6 polypeptide. An exemplary LHX6 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC103936.
By “LHX8 polypeptide” (or LIM homeobox 8) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAH40321.1.
By “LHX8 polynucleotide” (or LIM homeobox 8) is meant a polynucleotide encoding an LHX8 polypeptide. An exemplary LHX8 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC040321.
By “TBR1 polypeptide” (or T-box, brain 1 (TBR1)) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_006584.1.
By “TBR1 polynucleotide” (or T-box, brain 1 (TBR1)) is meant a polynucleotide encoding an TBR1 polypeptide. An exemplary TBR1 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006593.
By “SLC1A3 polypeptide” (or solute carrier family 1; glial high affinity glutamate transporter member 3 (SLC1A3)) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. BAG35230.1.
By “SLC1A3 polynucleotide” (or solute carrier family 1; glial high affinity glutamate transporter member 3 (SLC1A3)) is meant a polynucleotide encoding an SLC1A3 polypeptide. An exemplary SLC1A3 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. AK312304.
By “TH polypeptide” (or tyrosine hydroxylase) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI43612.1.
By “TH polynucleotide” (or tyrosine hydroxylase) is meant a polynucleotide encoding an TH polypeptide. An exemplary TH nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC143611.
By “Neurofilament 200 polypeptide” (or neurofilament heavy (NEFH)) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_066554.2.
By “Neurofilament 200 polynucleotide” (or neurofilament heavy (NEFH)) is meant a polynucleotide encoding an Neurofilament 200 polypeptide. An exemplary Neurofilament 200 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_021076.
By “Map2” (or microtubule-associated protein 2) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAH38857.1.
By “Map2 polynucleotide” (or microtubule-associated protein 2) is meant a polynucleotide encoding an Map2 polypeptide. An exemplary Map2 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC038857.
By “DCX” (or doublecortin) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_835366.1.
By “DCX polynucleotide” (or doublecortin) is meant a polynucleotide encoding an DCX polypeptide. An exemplary DCX nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. NM 178153.
By “GABRA1” (or gamma-aminobutyric acid (GABA) A receptor) is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAH30696.1.
By “GABRA1 polynucleotide” (or gamma-aminobutyric acid (GABA) A receptor) is meant a polynucleotide encoding an GABRA1 polypeptide. An exemplary GABRA1 nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No. BC030696.
Other features and advantages of the invention will be apparent to those skilled in the art from the following detailed description and claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The viral load (RNA copies/mL) are shown with standard deviation.
The present invention is based, at least in part, upon the discovery that brain microphysiological systems (BMPS) can be produced from induced pluripotent stem cells (iPSCs). Furthermore, the invention provides for reproducible BMPS that differentiate into mature neurons and glial cells (astrocytes and oligodendrocytes) in the central nervous system. This model is spontaneously electrophysiological active and may be reproduced with patient or genetically modified cells. The derivation of 3D BMPS from iPSCs has applications in the study and treatment of neurological and neurodevelopmental diseases. In some embodiments, the present disclosure provides for compositions and methods to study and/or treat neurodevelopmental and neurodegenerative disorders. In some cases, the neurodevelopmental and neurodegenerative disorders treated and/or studied by the present disclosure include, but are not limited to, autism, encephalitis, trauma, brain cancer, stroke, Amyotrophic lateral sclerosis, Huntington's Disease, muscular dystrophy, neurodegenerative disorder, neurodevelopmental disorder, Multiple Sclerosis, infection, Parkinson's Disease and Alzheimer's Disease.
As described herein, the present disclosure provides for the derivation of a multitude of identical brain microphysiological systems (BMPS) from stem cells, preferably of human origin, but including stem cells from animal origin. The preferred starting material are human induced pluripotent stem cells or embryonic stem cells, although other pluripotent stem cells such as, for example, neuronal precursor cells and mesenchymal stem cells may also be employed. Human in-vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. The techniques herein provide a reproducible iPSC-derived human 3D BMPS that includes differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over about eight weeks and show the critical elements of neuronal function including, but not limited to, synaptogenesis and neuron-to-neuron (e.g. spontaneous electric field potentials) and neuronal-glial interactions (e.g. myelination). Advantageously, the BMPS described herein include mature neurons (e.g., glutamatergic, dopaminergic and GABAergic neurons) and glial cells (e.g., astrocytes and oligodendrocytes). Quantification of the different cell types exhibited high reproducibility between experiments. Moreover, the BMPS disclosed herein present neuron and glial functions such as spontaneous electrical activity and axon myelination. The BMPS described herein are able to mimic the microenvironment of the central nervous system, which is a significant advance in the field of neurobiology as this ability has not been achieved at this level of functionality, reproducibility, and consistency in prior art in vitro systems.
In particular, the high amount of myelination of axons (up to 40%) in the disclosed BMPS represents a significant improvement over the prior art. Myelin pathology is a rather frequent condition in demyelinating and inflammatory disorders such as multiple sclerosis and post-infection diseases as well as other neurological diseases such as acute and post-traumatic brain injury, stroke and neurodegenerative disorders (see e.g., Fumagalli et al., 2016; Tse and Herrup, 2016). Moreover, the myelination process can be perturbed by exposure to chemicals and drugs (see e.g., Garcia et al., 2005; Brubaker et al., 2009; Creeley et al., 2013) during brain development and adulthood. For example, the BMPS disclosed herein show 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglia function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. Thus, the BMPS provides a suitable and reliable model to investigate neuron-neuroglia function in neurotoxicology or other pathogenic mechanisms that has heretofore not been available in the prior art.
The method disclosed combines gyratory shaking or regular stirring and the addition of growth factors to obtain the basic model. Suitable conditions as to how to achieve reproducible brain composition are disclosed herein. In contrast to earlier models, identical units of BMPS are produced, which allow comparative testing for the purpose of product development or safety assessments.
According to the techniques herein, a number of additional measures complement the basic BMPS to increase their completeness in modeling the human brain and improve its usefulness for such testing, for example:
1. The addition of microglia: All stem-cell-derived brain models described so far lack micro-glia. The techniques herein provide that the addition of micro-glia precursor cells and suitable growth factors may allow microglia to be added to the model. Suitable cells may be monocytes (e.g., human monocytes), hematopoetic stem cells, respective (pro-)monocyte cell lines, and isolated microglia.
2. The addition of a blood-brain-barrier: The human brain is protected by a tight blood-brain-barrier that excludes many substances from the brain. For the first time, the techniques herein provide a method to form a blood-brain-barrier to the BMPS via cells such as, for example, human endothelial cells.
3. Addition of reporter and reporter cells: During the generation of the BMPS, cells carrying reporter for testing purposes may be used or added. These include, but are not limited to, fluorescent or luminescent markers to indicate a certain cell lineage or cell response. Genetic transient or permanent transfections are the primary, but not only, method of choice.
4. The BMPS may also be produced, entirely or in its components, from cells from a specific genetic background, e.g. from patients with a specific disease or after selective genetic manipulation of the cells.
5. The versatility of the BMPS may be improved by combining it with electrodes including, but not limited to, micro-electrode arrays (MEA).
6. The versatility of the BMPS may be improved by combining it with other MPS (organ models) platforms such as, for example, microfluidic human-on-chip systems, perfusion chambers and others.
7. Transportability of BMPS: Methods to cryopreserve BMPS were developed, which allow transport to other laboratories and testing or integration into multi-MPS platforms.
Simplified neural in vitro systems do not reflect physiology, interactions between different cell types, or human genetics. Induced pluripotent stem cells (iPSC)-derived human-relevant microphysiological systems (MPS) better mimic the organ level, but are too complex for chemical and drug screening. As described herein, a reproducible 3D brain MPS (BMPS) that differentiates into mature neurons and glial cells (astrocytes and oligodendrocytes), which reproduces the topology of neuronal-glial interactions and connectivity in the central nervous system was developed. BMPS from healthy donors or patients evolve from a period of differentiation to maturity over about 8 weeks, including synaptogenesis, neuron-neuron interactions (e.g. spontaneous electric field potentials) and neuronal-glial interactions (e.g. myelination of axons), which mimic the microenvironment of the central nervous system. Effects of substances on neurodevelopment may be studied during this phase of BMPS development. In an exemplary embodiment, the techniques herein were used to study Parkinson's disease (PD) by evaluating neurotoxicants with a link to PD pathogenesis. Exposure to 5 μM rotenone or 100 μM 1-methyl-4-phenylpyridinium (MPP+) (or 1 mM 1-methyl-4-phenylpyridinium (MPP+) for gene expression studies) disrupted dopaminergic neurons, as observed by immunohistochemistry and altered expression of PD-related genes (TH, TBR1, SNCA, KEAP1, NDU1-131, ATP5C1, ATP50 and CASP1), thus recapitulating hallmarks of PD pathogenesis linked to toxicant compounds in the respective animal models. The BMPS, as described herein, provide a suitable and reliable model to investigate neuron-neuroglia function in neurotoxicity or other pathogenic mechanisms.
There is growing concern about the continuing increase in neurodevelopmental and -degenerative disorders such as autism [1, 2], Parkinson's [3] and Alzheimer disease [4]. Although genetic factors play an important role, environmental factors such as pesticides, air pollution, cigarette smoke, and dietary toxicants appear to contribute [5, 6, 7]. Due to a lack of mechanistic understanding, it is difficult to study their contributions and interactions with respect to neurotoxicity and neurological disorders. The complexity of the CNS makes it challenging to find appropriate in vitro human-relevant models, ideally from different genetic backgrounds, that are able to recapitulate the relevant pathophysiology. The poor predictive ability of animal-based models for human health, which may fail to mimic human pathology as outlined in the costly and time-consuming current developmental neurotoxicity (DNT) guidelines, contributes to the lack of reliable information on DNT mechanisms [8]. At the same time, more than 90% of all drugs fail clinical trials after extensive animal testing [9] due, in part, to the fact that animal studies often do not reflect human physiology and inter-individual differences. Simple in vitro systems do not represent physiology and organ function [10], which creates a critical demand for better models in drug development, study of disease mechanisms and progression, bioengineering and toxicological testing.
Attempts to generate more complex organotypic cultures or microphysiological systems (MPS) [11, 12, 13, 14] have resulted in more physiological multicellular 3D co-culture models able to simulate a functional part of the brain [15, 16]. 3D MPS have shown increased cell survival, differentiation, cell-cell interactions and can reproduce the complexity of the organ more closely [18]. Recent US research programs by NIH, FDA, DARPA, and DTRA have initiated the systematic development of MPS, including the model presented here, and their combinations to human-on-a-chip technologies to assess the safety and efficacy of countermeasures to biological and chemical terrorism and warfare [19].
The discovery of induced pluripotent stem cells (iPSC) and new protocols to differentiate them into various cell types have boosted the development of human in vitro models [20, 21]. iPSC from healthy or patient donors with a specific disease [22, 23, 24, 12] used in MPS promise more human-representative models, e.g. the brain organoids by Lancaster et al. and Kadoshima et al., have been able to recapitulate features of human cortical development [15, 16]. These complex systems present novel tools to study biological mechanisms in the CNS, however, they have certain limitations: 1) an elaborate and complex protocol, 2) size differences between organoids, 3) necrosis in the center of the organoid, 4) low reproducibility in cell differentiation. The human BMPS described herein overcomes these limitations. The reproducible in vitro iPSC-derived human 3D brain microphysiological system (BMPS) is comprised of differentiated and mature neurons and glial cells (astrocytes and oligodendrocytes).
The techniques herein provide a reproducible BMPS that contains several different cell types of the human brain, such as glutamatergic, dopaminergic and GABAergic neurons, astrocytes and oligodendrocytes. Moreover, the system has shown neural functionality as observed by spontaneous electrical activity and myelination of axons. Furthermore, the BMPS is reproducible from batch to batch and displays differences between healthy and patient donors. In addition, the obtained results demonstrate the application of such BMPS to the study of neurological disorders such as, for example, Parkinson's Disease (PD).
The brain MPS described herein is a versatile tool for more complex testing platforms and strategies as well as research into neurotoxicity (e.g., developmental), CNS physiology and pathology. Some stem cell-derived brain microphysiological systems have been developed in the latest years showing the capability to recapitulate some of the in vivo biological process [36, 37, 38]. These models have an enormous advantage over the classical in vitro models to study various differentiation mechanisms, developmental processes and diseases [15]. However, they are mostly based on human embryonic stem cells raising ethical concerns and not allowing the use of patient cells. Moreover, they require complicated protocols that may reduce the reproducibility of the system and make it difficult to use in other fields such as chemical and drug screening. Some of these complex organoids have a large diameter, which can lead to extensive cell death, visible in the core of these tissues [15]. This may be due to insufficient diffusion of nutrients and oxygen in these non-vascularized systems, which may generate artifacts in toxicological and disease measurements and make it difficult to study different endpoints in a medium- to high-throughput manner. In addition, it will be challenging to adapt endpoints, established for relative simple 2D cultures, to such complex models. In the study described herein, the ability to generate a high number of viable (about 800 per batch), BMPS that are homogeneous in size (e.g., about 300 μm) and shape using iPSC by applying a constant or regular gyratory shaking or stirring technique as described earlier for rat re-aggregating brain cell cultures [40] is shown. Control of the size using specific shaker speed allowed the aggregates to be maintained below 350 μM in diameter (
The 3D differentiation protocol described herein covered stages from neuronal precursors to different cell types of the mature CNS. After 2 weeks, BMPS consisted of an immature population of cells, showing minimal neuronal networks, low percentage of mature astrocytes and oligodendrocytes, with no myelin basic protein expression (
Most of the brain MPS published so far are entirely focused on neurons and not glia populations [45, 46]; the brain MPS described herein is the first 3D model with fully characterized mature human oligodendrocytes, astrocytes and neurons, derived from iPSC. Astrocytes and oligodendrocytes play an important role during neuronal development, plasticity and neuronal injury. Astrocytes have a role in protecting neurons, increasing neuronal viability and mitochondrial biogenesis from both exogenous (e.g. chemicals) or endogenous (such as glutamate-induced excitotoxicity or the Alzheimer related A131-42) toxicity [47, 48, 49, 50]. Astrocytes have an especially important role in neuroprotection from oxidative stress. Oxidative stress is known to be involved in a number of neuropathological conditions (such as neurodegenerative diseases) [51, 52, 53]. Thus, the presence of astrocytes in a biological system to study disease is crucial due to their role in detoxification and neuronal protection. Immunochemistry results from the iPSC-derived BMPS showed low numbers of astrocytes (GFAP-positive cells) at 2 weeks of differentiation, which increased continuously throughout differentiation (
The presence of astroglia and oligodendroglia in the model described herein brings the system closer to the in vivo brain physiology, which is a crucial component to study neurodegeneration and neurotoxicity. In addition, the system has shown functionality as seen by imaging of cell-cell junctions, myelination, a rich astroglial network and electrical activity (
An assessment of the myelination process by quantification of MBP immunostaining along axons showed an increase over time reaching 42% of myelinated axons at 8 weeks (
In one embodiment, the model described herein is useful for studying Parkinson's disease (PD). Traditionally, PD has been described as a pre-synaptic degenerative process that affects dopaminergic neurons and induces a fundamental motor disorder [66], however, non-motor symptoms can also be present [67]. Research in Parkinson's disease is experiencing an upswing at the moment, owing to a lack of curative drugs for the large number of patients. Drug testing is nearly exclusively performed in vivo in the so-called MPTP (the parent compound to the metabolite MPP+ used here), rotenone, methamphetamine and 6-hydroxydopamine models requiring tens of thousands of animals [68, 69, 70]. These model toxins are mainly used in mice and primates (and less in cell cultures) to model a disease state resembling PD. Human neurons, which would be most relevant, are not usually available and existing cell lines are only very poor substitutes. The model described herein shows that treatment with MPP+ or rotenone induced specific degeneration of dopaminergic neurons in agreement with Parkinson patients and current animal models of the disease (
This disclosure provides for a description of a brain microphysiological system aiming to study various aspects of brain development, pathophysiology and disturbance by genetic and environmental factors. The possibilities to study developmental and neurodegenerative disorders, infections, toxicity and trauma are emerging with such a system. Furthermore, the potential to use iPSC from different donors adds a personalized component to these studies. The high reproducibility and relatively easy protocol, enables future higher throughput testing of chemicals, and drugs and their potential to induce or treat diseases.
Autism
Autism is a highly variable neurodevelopmental disorder that first appears during infancy or childhood, and generally follows a steady course without remission. Patients with autism may be severely impaired in some respects but normal, or even superior, in others. Overt symptoms gradually begin after the age of six months, become established by age two or three years, and tend to continue through adulthood, although often in more muted form.
It is distinguished not by a single symptom, but by a characteristic triad of symptoms: impairments in social interaction; impairments in communication; and restricted interests and repetitive behavior. Other aspects, such as atypical eating, are also common but are not essential for diagnosis. Autism's individual symptoms occur in the general population and appear not to associate highly, without a sharp line separating pathologically severe from common traits.
While autism is highly heritable, researchers suspect both environmental and genetic factors as causes. In rare cases, autism is strongly associated with agents that cause birth defects. Controversies surround other proposed environmental causes; for example, the vaccine hypotheses have been disproven. Autism affects information processing in the brain by altering how nerve cells and their synapses connect and organize; how this occurs is not well understood. It is one of three recognized disorders in the autism spectrum (ASDs), the other two being Asperger syndrome, which lacks delays in cognitive development and language, and pervasive developmental disorder, not otherwise specified (commonly abbreviated as PDD-NOS), which is diagnosed when the full set of criteria for autism or Asperger syndrome are not met.
Globally, autism is estimated to affect 21.7 million people as of 2013. As of 2010, the number of people affected is estimated at about 1-2 per 1,000 worldwide. It occurs four to five times more often in boys than girls. About 1.5% of children in the United States (one in 68) are diagnosed with ASD as of 2014, a 30% increase from one in 88 in 2012. The rate of autism among adults aged 18 years and over in the United Kingdom is 1.1%. The number of people diagnosed has been increasing dramatically since the 1980s, partly due to changes in diagnostic practice and government-subsidized financial incentives for named diagnoses; the question of whether actual rates have increased is unresolved.
Autism has a strong genetic basis, although the genetics of autism are complex and it is unclear whether ASD is explained more by rare mutations with major effects, or by rare multigene interactions of common genetic variants. Complexity arises due to interactions among multiple genes, the environment, and epigenetic factors which do not change DNA but are heritable and influence gene expression. Studies of twins suggest that heritability is 0.7 for autism and as high as 0.9 for ASD, and siblings of those with autism are about 25 times more likely to be autistic than the general population. However, most of the mutations that increase autism risk have not been identified. Typically, autism cannot be traced to a Mendelian (single-gene) mutation or to a single chromosome abnormality, and none of the genetic syndromes associated with ASDs have been shown to selectively cause ASD. Numerous candidate genes have been located, with only small effects attributable to any particular gene. The large number of autistic individuals with unaffected family members may result from copy number variations—spontaneous deletions or duplications in genetic material during meiosis. Hence, a substantial fraction of autism cases may be traceable to genetic causes that are highly heritable but not inherited: that is, the mutation that causes the autism is not present in the parental genome.
Several lines of evidence point to synaptic dysfunction as a cause of autism. Some rare mutations may lead to autism by disrupting some synaptic pathways, such as those involved with cell adhesion. Gene replacement studies in mice suggest that autistic symptoms are closely related to later developmental steps that depend on activity in synapses and on activity-dependent changes. All known teratogens (agents that cause birth defects) related to the risk of autism appear to act during the first eight weeks from conception, and though this does not exclude the possibility that autism can be initiated or affected later, there is strong evidence that autism arises very early in development.
Exposure to air pollution during pregnancy, especially heavy metals and particulates, may increase the risk of autism. Environmental factors that have been claimed to contribute to or exacerbate autism, or may be important in future research, include certain foods, infectious diseases, solvents, diesel exhaust, PCBs, phthalates and phenols used in plastic products, pesticides, brominated flame retardants, alcohol, smoking, illicit drugs, vaccines, and prenatal stress, although no links have been found, and some have been completely disproven.
Autism does not have a clear unifying mechanism at either the molecular, cellular, or systems level; it is not known whether autism is a few disorders caused by mutations converging on a few common molecular pathways, or is (like intellectual disability) a large set of disorders with diverse mechanisms. Autism appears to result from developmental factors that affect many or all functional brain systems, and to disturb the timing of brain development more than the final product. Neuroanatomical studies and the associations with teratogens strongly suggest that autism's mechanism includes alteration of brain development soon after conception. This anomaly appears to start a cascade of pathological events in the brain that are significantly influenced by environmental factors. Just after birth, the brains of children with autism tend to grow faster than usual, followed by normal or relatively slower growth in childhood. It is not known whether early overgrowth occurs in all children with autism. It seems to be most prominent in brain areas underlying the development of higher cognitive specialization. Hypotheses for the cellular and molecular bases of pathological early overgrowth include the following: an excess of neurons that causes local over connectivity in key brain regions, disturbed neuronal migration during early gestation, unbalanced excitatory—inhibitory networks, and abnormal formation of synapses and dendritic spines, for example, by modulation of the neurexing neuroligin cell-adhesion system, or by poorly regulated synthesis of synaptic proteins.
The immune system is thought to play an important role in autism. Children with autism have been found by researchers to have inflammation of both the peripheral and central immune systems as indicated by increased levels of pro-inflammatory cytokines and significant activation of microglia. Biomarkers of abnormal immune function have also been associated with increased impairments in behaviors that are characteristic of the core features of autism such as deficits in social interactions and communication. Interactions between the immune system and the nervous system begin early during the embryonic stage of life, and successful neurodevelopment depends on a balanced immune response. It is thought that activation of a pregnant mother's immune system such as from environmental toxicants or infection can contribute to causing autism through causing a disruption of brain development. This is supported by recent studies that have found that infection during pregnancy is associated with an increased risk of autism.
The relationship of neurochemicals to autism is not well understood; several have been investigated, with the most evidence for the role of serotonin and of genetic differences in its transport. The role of group I metabotropic glutamate receptors (mGluR) in the pathogenesis of fragile X syndrome, the most common identified genetic cause of autism, has led to interest in the possible implications for future autism research into this pathway. Some data suggests neuronal overgrowth potentially related to an increase in several growth hormones or to impaired regulation of growth factor receptors. Also, some inborn errors of metabolism are associated with autism, but probably account for less than 5% of cases.
The mirror neuron system (MNS) theory of autism hypothesizes that distortion in the development of the MNS interferes with imitation and leads to autism's core features of social impairment and communication difficulties. The MNS operates when an animal performs an action or observes another animal perform the same action. The MNS may contribute to an individual's understanding of other people by enabling the modeling of their behavior via embodied simulation of their actions, intentions, and emotions. Several studies have tested this hypothesis by demonstrating structural abnormalities in MNS regions of individuals with ASD, delay in the activation in the core circuit for imitation in individuals with Asperger syndrome, and a correlation between reduced MNS activity and severity of the syndrome in children with ASD. However, individuals with autism also have abnormal brain activation in many circuits outside the MNS and the MNS theory does not explain the normal performance of children with autism on imitation tasks that involve a goal or object.
The under connectivity theory of autism hypothesizes that autism is marked by under functioning high-level neural connections and synchronization, along with an excess of low-level processes. Evidence for this theory has been found in functional neuroimaging studies on autistic individuals and by a brainwave study that suggested that adults with ASD have local over connectivity in the cortex and weak functional connections between the frontal lobe and the rest of the cortex. Other evidence suggests the under connectivity is mainly within each hemisphere of the cortex and that autism is a disorder of the association cortex.
From studies based on event-related potentials, transient changes to the brain's electrical activity in response to stimuli, there is considerable evidence for differences in autistic individuals with respect to attention, orientation to auditory and visual stimuli, novelty detection, language and face processing, and information storage; several studies have found a preference for nonsocial stimuli. For example, magnetoencephalography studies have found evidence in children with autism of delayed responses in the brain's processing of auditory signals.
Relations have been found between autism and schizophrenia based on duplications and deletions of chromosomes; research showed that schizophrenia and autism are significantly more common in combination with 1q21.1 deletion syndrome. Research on autism/schizophrenia relations for chromosome 15 (15q13.3), chromosome 16 (16p13.1) and chromosome 17 (17p12) are inconclusive.
Diagnosis is based on behavior, not cause or mechanism. Under the DSM-5, autism is characterized by persistent deficits in social communication and interaction across multiple contexts, as well as restricted, repetitive patterns of behavior, interests, or activities. These deficits are present in early childhood, typically before age three, and lead to clinically significant functional impairment. Sample symptoms include lack of social or emotional reciprocity, stereotyped and repetitive use of language or idiosyncratic language, and persistent preoccupation with unusual objects. The disturbance must not be better accounted for by Rett syndrome, intellectual disability or global developmental delay. ICD-10 uses essentially the same definition. A pediatrician commonly performs a preliminary investigation by taking developmental history and physically examining the child. If warranted, diagnosis and evaluations are conducted with help from ASD specialists, observing and assessing cognitive, communication, family, and other factors using standardized tools, and taking into account any associated medical conditions. A pediatric neuropsychologist is often asked to assess behavior and cognitive skills, both to aid diagnosis and to help recommend educational interventions.
Clinical genetics evaluations are often done once ASD is diagnosed, particularly when other symptoms already suggest a genetic cause. Although genetic technology allows clinical geneticists to link an estimated 40% of cases to genetic causes, consensus guidelines in the US and UK are limited to high-resolution chromosome and fragile X testing. Metabolic and neuroimaging tests are sometimes helpful, but are not routine.
Many medications are used to treat ASD symptoms that interfere with integrating a child into home or school when behavioral treatment fails. More than half of US children diagnosed with ASD are prescribed psychoactive drugs or anticonvulsants, with the most common drug classes being antidepressants, stimulants, and antipsychotics. Antipsychotics, such as risperidone and aripiprazole, have been found to be useful for treating some conditions associated with autism, including irritability, repetitive behavior, and sleeplessness. A person with ASD may respond atypically to medications, the medications can have adverse effects, and no known medication relieves autism's core symptoms of social and communication impairments. Experiments in mice have reversed or reduced some symptoms related to autism by replacing or modulating gene function, suggesting the possibility of targeting therapies to specific rare mutations known to cause autism. Although many alternative therapies and interventions are available, few are supported by scientific studies. Some alternative treatments may place the child at risk. A 2008 study found that compared to their peers, autistic boys have significantly thinner bones if on casein-free diets; in 2005, botched chelation therapy killed a five-year-old child with autism. There has been early research looking at hyperbaric treatments in children with autism.
Parkinson's Disease
Parkinson's disease (PD, also known as idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans) is a degenerative disorder of the central nervous system mainly affecting the motor system. The motor symptoms of Parkinson's disease result from the death of dopamine-generating cells in the substantia nigra, a region of the midbrain. The causes of this cell death are poorly understood. Early in the course of the disease, the most obvious symptoms are movement-related; these include shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, thinking and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease, and depression is the most common psychiatric symptom. Other symptoms include sensory, sleep and emotional problems. Parkinson's disease is more common in older people, with most cases occurring after the age of 50; when it is seen in young adults, it is called young onset PD (YOPD).
The main motor symptoms are collectively called “parkinsonism,” or a “parkinsonian syndrome.” The disease can be either primary or secondary. Primary Parkinson's disease is referred to as idiopathic (having no known cause), although some atypical cases have a genetic origin, while secondary parkinsonism is due to known causes like toxins. The pathology of the disease is characterized by the accumulation of a protein into Lewy bodies in neurons, and insufficient formation and activity of dopamine in certain parts of the midbrain. Where the Lewy bodies are located is often related to the expression and degree of the symptoms of an individual. Diagnosis of typical cases is mainly based on symptoms, with tests such as neuroimaging being used for confirmation.
Diagnosis of Parkinson's disease involves a physician taking a medical history and performing a neurological examination. There is no lab test that will clearly identify the disease, but brain scans are sometimes used to rule out disorders that could give rise to similar symptoms. People may be given levodopa and resulting relief of motor impairment tends to confirm diagnosis. The finding of Lewy bodies in the midbrain on autopsy is usually considered proof that the person had Parkinson's disease. The progress of the illness over time may reveal it is not Parkinson's disease, and some authorities recommend that the diagnosis be periodically reviewed. Other causes that can secondarily produce a parkinsonian syndrome are Alzheimer's disease, multiple cerebral infarction and drug-induced parkinsonism. Parkinson plus syndromes such as progressive supranuclear palsy and multiple system atrophy must be ruled out. Anti-Parkinson's medications are typically less effective at controlling symptoms in Parkinson plus syndromes. Faster progression rates, early cognitive dysfunction or postural instability, minimal tremor or symmetry at onset may indicate a Parkinson plus disease rather than PD itself. Genetic forms are usually classified as PD, although the terms familial Parkinson's disease and familial parkinsonism are used for disease entities with an autosomal dominant or recessive pattern of inheritance.
The PD Society Brain Bank criteria require slowness of movement (bradykinesia) plus either rigidity, resting tremor, or postural instability. Other possible causes for these symptoms need to be ruled out prior to diagnosis with PD. Finally, three or more of the following features are required during onset or evolution: unilateral onset, tremor at rest, progression in time, asymmetry of motor symptoms, response to levodopa for at least five years, clinical course of at least ten years and appearance of dyskinesias induced by the intake of excessive levodopa. Accuracy of diagnostic criteria evaluated at autopsy is 75-90%, with specialists such as neurologists having the highest rates. Computed tomography (CT) and conventional magnetic resonance imaging (MRI) brain scans of people with PD usually appear normal. These techniques are nevertheless useful to rule out other diseases that can be secondary causes of parkinsonism, such as basal ganglia tumors, vascular pathology and hydrocephalus. A specific technique of MRI, diffusion MRI, has been reported to be useful at discriminating between typical and atypical parkinsonism, although its exact diagnostic value is still under investigation. Dopaminergic function in the basal ganglia can be measured with different PET and SPECT radiotracers. Examples are ioflupane (123I) (trade name DaTSCAN) and iometopane (Dopascan) for SPECT or fluorodeoxyglucose (18F) and DTBZ for PET. A pattern of reduced dopaminergic activity in the basal ganglia can aid in diagnosing PD.
Treatments, typically the medications L-DOPA and dopamine agonists, improve the early symptoms of the disease. As the disease progresses and dopaminergic neurons continue to be lost, these drugs eventually become ineffective at treating the symptoms and at the same time produce a complication marked by involuntary writhing movements. Surgery and deep brain stimulation have been used to reduce motor symptoms as a last resort in severe cases where drugs are ineffective. Although dopamine replacement alleviates the symptomatic motor dysfunction, its effectiveness is reduced as the disease progresses, leading to unacceptable side effects such as severe motor fluctuations and dyskinesias. Furthermore, there is no therapy that will halt the progress of the disease. Moreover, this palliative therapeutic approach does not address the underlying mechanisms of the disease.
The term parkinsonism is used for a motor syndrome whose main symptoms are tremor at rest, stiffness, slowing of movement and postural instability. Parkinsonian syndromes can be divided into four subtypes according to their origin: primary or idiopathic, secondary or acquired, hereditary parkinsonism, and Parkinson plus syndromes or multiple system degeneration. Usually classified as a movement disorder, PD also gives rise to several non-motor types of symptoms such as sensory deficits, cognitive difficulties or sleep problems. Parkinson plus diseases are primary parkinsonisms which present additional features. They include multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration and dementia with Lewy bodies.
In terms of pathophysiology, PD is considered a synucleiopathy due to an abnormal accumulation of alpha-synuclein protein in the brain in the form of Lewy bodies, as opposed to other diseases such as Alzheimer's disease where the brain accumulates tau protein in the form of neurofibrillary tangles. Nevertheless, there is clinical and pathological overlap between tauopathies and synucleinopathies. The most typical symptom of Alzheimer's disease, dementia, occurs in advanced stages of PD, while it is common to find neurofibrillary tangles in brains affected by PD. Dementia with Lewy bodies (DLB) is another synucleinopathy that has similarities with PD, and especially with the subset of PD cases with dementia. However, the relationship between PD and DLB is complex and still has to be clarified. They may represent parts of a continuum or they may be separate diseases.
Mutations in specific genes have been conclusively shown to cause PD. These genes encode alpha-synuclein (SNCA), parkin (PRKN), leucine-rich repeat kinase 2 (LRRK2 or dardarin), PTEN-induced putative kinase 1 (PINK1), DJ-1 and ATP13A2. In most cases, people with these mutations will develop PD. With the exception of LRRK2, however, they account for only a small minority of cases of PD. The most extensively studied PD-related genes are SNCA and LRRK2. Mutations in genes including SNCA, LRRK2 and glucocerebrosidase (GBA) have been found to be risk factors for sporadic PD. Mutations in GBA are known to cause Gaucher's disease. Genome-wide association studies, which search for mutated alleles with low penetrance in sporadic cases, have now yielded many positive results.
The role of the SNCA gene is important in PD because the alpha-synuclein protein is the main component of Lewy bodies. The histopathology (microscopic anatomy) of the substantia nigra and several other brain regions shows neuronal loss and Lewy bodies in many of the remaining nerve cells. Neuronal loss is accompanied by death of astrocytes (star-shaped glial cells) and activation of the microglia (another type of glial cell). Lewy bodies are a key pathological feature of PD.
Alzheimer's Disease
Alzheimer's disease (AD) accounts for 60% to 70% of cases of dementia. It is a chronic neurodegenerative disease that often starts slowly, but progressively worsens over time. The most common early symptom is short-term memory loss. As the disease advances, symptoms include problems with language, mood swings, loss of motivation, disorientation, behavioral issues, and poorly managed self-care. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the average life expectancy following diagnosis is three to nine years. The cause of Alzheimer's disease is poorly understood. About 70% of the risk is believed to be genetic with many genes involved. Other risk factors include a history of head injuries, hypertension, or depression. The disease process is associated with plaques and tangles in the brain.
Alzheimer's disease is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyrus Alzheimer's disease has been hypothesized to be a protein misfolding disease (proteopathy), caused by accumulation of abnormally folded A-beta and tau proteins in the brain. Plaques are made up of small peptides, 39-43 amino acids in length, called beta-amyloid (also written as A-beta or Aβ). Beta-amyloid is a fragment from a larger protein called amyloid precursor protein (APP), a transmembrane protein that penetrates through the neuron's membrane. APP is critical to neuron growth, survival and post-injury repair. In Alzheimer's disease, an unknown process causes APP to be divided into smaller fragments by enzymes through proteolysis. One of these fragments gives rise to fibrils of beta-amyloid, which form clumps that deposit outside neurons in dense formations known as senile plaques.
A probable diagnosis is based on the history of the illness and cognitive testing with medical imaging and blood tests to rule out other possible causes. Initial symptoms are often mistaken for normal ageing. Examination of brain tissue is needed for a definite diagnosis. Alzheimer's disease is diagnosed through a complete medical assessment. There is no one clinical test that can determine whether a person has Alzheimer's. Usually several tests are performed to rule out any other cause of dementia. The only definitive method of diagnosis is examination of brain tissue obtained from a biopsy or autopsy. Tests (such as blood tests and brain imaging) are used to rule out other causes of dementia-like symptoms. Laboratory tests and screening include: complete blood cell count; electrolyte panel; screening metabolic panel; thyroid gland function tests; vitamin B-12 folate levels; tests for syphilis and, depending on history, for human immunodeficiency antibodies; urinalysis; electrocardiogram (ECG); chest X-ray; computerized tomography (CT) head scan; and an electroencephalogram (EEG). A lumbar puncture may also be informative in the overall diagnosis.
There are no known medications or supplements that decrease risk of Alzheimer's. Additionally, no known treatments stop or reverse Alzheimer's progression, although some may temporarily improve symptoms.
This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference.
According to the techniques herein, the BMPS model established herein follows a stepwise differentiation protocol (
In order to characterize different stages of the differentiation and maturation process, BMPS were collected every week up to 8 weeks of differentiation (FIGS. 1C1-C5). Analysis of different neuronal and glial cell-specific genes by real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to characterize the presence of neurons, astrocytes, oligodendrocytes and neural precursor cells (NPC). NPC are self-renewing and proliferating multi-potent cells able to generate different cell types of the central nervous system. The differentiation of NPC in 3D was initiated by changing the medium to differentiation medium. Gene expression of the cell proliferation marker Ki67 decreased 95% after 2 weeks of differentiation (FIG. 1C1, proliferation and stem cell markers). The remaining Ki67 expression appears to be due to the presence of a small population of NPC and other proliferating cell types such as oligodendrocytes and astrocytes (FIG. 1C2, astroglia and oligodendroglia). Astrocyte-specific genes (S100B and GFAP) showed a constant increase after two weeks, while, differentiation of oligodendrocytes was induced later, after six weeks of differentiation as shown by OLIG2 gene expression (FIG. 1C2).
Gene expression of specific neurotransmitters or their receptors was used to characterize the identity of different neuronal populations and the differentiation patterns of the human iPSC derived BMPS (FIG. 1C4, neuronal markers; right y-axis relative quantification of GRIN1 and GABRA1; MBP, FOXA2, and SLC1A3). GRIN1 encodes the essential Glutamate [NMDA] receptor subunit zeta-1 [25] was increased at very early stages of differentiation (one week after induction of differentiation) and continued to increase up to 5 weeks when it reached a plateau (FIG. 1C4). Similarly, GAD1, a GABAergic neuronal gene marker which encodes the Glutamate decarboxylase 1, and catalyzes decarboxylation of glutamate to GABA, showed an increase in expression during the first 4 weeks of differentiation, reaching a plateau thereafter (FIG. 1C4). The expression of tyrosine hydroxylase (TH) a gene, which identifies dopaminergic neurons, was observed first after three weeks, showing delayed differentiation compared to glutamatergic neurons. The expression of TH increased constantly thereafter reaching an 86-fold increase at seven weeks compared to NPC (week 0; FIG. 1C4). GABRA1, which encodes the gamma-aminobutyric acid (GABA) receptor, showed a steady increase of expression after 2 weeks and reached its maximum increase of a 150-fold change at 8 weeks compared to week 0 (FIG. 1C4). Moreover other markers for specific part of the brain, such as ventral midbrain neuron marker LMX1A, FOXO1 and FOXA2 (Hedlund et al., 2016; Stott et al., 2013), cerebral cortex marker FOXO4, or markers for myelination CNP and MBP (Li and Richardson, 2008; Agrawal et al., 1994) and L-glutamate transport SLC1A6 (Sery et al., 2015) has been studied (
To prove that BMPS can be generated from different IPCs, another healthy line (IPS IMR90) and Down syndrome line (DYP0730) were used (FIG. 1C5). Both lines were able to generate BMPS and differentiated to neurons (MAP2 marker), astrocytes (GFAP marker) and oligodendrocytes (OLIG1 marker).
In order to quantify cell populations in the iPSC-derived BMPS and verify the reproducibility between experiments and batches of the cell line (C1, CRL-2097), flow cytometry was performed using CNS-specific antibodies for identification of neural markers (Table 1). Flow cytometry allowed quantifying 60% of cells with proliferation marker (Ki67) at the NPCs stage (week 0), which was reduced during differentiation down to 9% at 2 weeks, 7% at 4 weeks and 1% at 8 weeks (
Quantification of the cell population in at least three independent experiments showed low variability between cultures, demonstrating the reproducibility of the system. The variation (standard deviation, SD) between experiments decreased with the cell differentiation process and was very small at the latest maturation stage (eight weeks); DCX SD 0.9%, Ki67 SD 0.2%, SOX1 SD 0.7%, SOX2 SD 1.2%, NES SD 0.7% and Tuj1 SD 9.8% (
MicroRNAs (miRNA), known as posttranscriptional regulators of developmental timing, have recently been established as markers to study the differentiation process [26]. Expression of neural-specific miRNAs showed strong induction of miRNAs involved in neurogenesis (FIG. 1C3, miRNA). mir-124, the most abundant brain miRNA, was strongly induced in the earlier stages of differentiation, then slightly down-regulated at eight weeks of differentiation. This finding correlates with previous studies, where mir-124 was shown to promote neuronal lineage commitment at earlier stages of neural stem cells specification by targeting anti-neuronal factors [26]. mir-128, a modulator of late neural differentiation, was strongly up-regulated after 5 weeks of differentiation. mir-137, the most induced miRNA over time in the system described herein, is known as a regulator of neural differentiation of embryonic stem cells (ESCs) [27]. mir-132 and mir-133b which are involved in regulation of dopaminergic neuron maturation and function, were induced in week three of differentiation, a finding which correlates with the expression pattern of TH. Moreover, mir-132 is involved in dendritic spine formation [28]. These results support the view of a coordinated mechanism of neuronal differentiation as reflected by the patterns of neuronal gene and miRNA expression and neuronal and neurotransmitter identity.
In order to assess the cellular composition and the process of maturation of the cells within the human BMPS, the expression of markers for different CNS cell populations including neurons and glial cells at 2, 4 and 8 weeks of differentiation were evaluated using immunohistochemistry and electron microscopy techniques. A reproducible pattern of expression consistent with maturation of the BMPS towards mature neural phenotypes was found. After 4 weeks of differentiation, the BMPS showed positive staining for mature neuronal markers such as microtubule-associated protein 2 (MAP2), neurofilament-heavy chain (NF, SMI32) and synaptophysin (
A subset of neuronal cells exhibited immunoreactivity for markers such as NOGOA, 01, 02, and CNPase (
GFAP-positive cells formed numerous cell processes organized in a network typical for human astrocyte glial processes in vivo, which established contacts with other glial cells and neurons (
The morphology of cell nuclei observed by immunocytochemistry and electron microscopy showed some variation in nuclear morphology attributed to (i) cell proliferation as seen by positive staining for Ki67 and Nestin markers, and (ii) nuclear fragmentation likely associated with apoptosis as indicated by caspase 3 staining (
Further analysis of neuronal cell populations and morphology presented a pattern of evolution that suggests BMPS maturation as seen by Nestin-positive cells decreasing over time of differentiation while MBP expressing cells increased (
To test the neurophysiological properties of the cells within the BMPS model, spontaneous electrical activity in BMPS was analyzed by micro-electrode array (MEA)(see
Due to the presence of TH-positive dopaminergic neurons in the iPSC-derived BMPS (
Peripheral blood mononuclear cells (PBMCs) are isolated from fresh or commercially available cryo-preserved whole blood of pooled healthy donors by Ficoll or Percoll gradient centrifugation. Monocyte populations are obtained by negative magnet-antibody selection after Ficoll or Percoll gradient and then re-suspend in RPMI 1640. Monocytes are cultured in macrophage serum-free medium, stimulated with a cocktail of cytokines, GM-CSF and IL-34. Monocytes may also be obtained by differentiation of iPSCs, hematopoetic or other stem cells. The microglia-like cells are combined with neuronal precursor cells in shaker cultures to preferably arrive at a final concentration of 5-8% microglia.
Primary monocytes or iPSC-derived monocytes may be incorporated into the system, both at early and later stages of BMPS differentiation. For the early stages, a number of 2×106 NPCs mixed with 2×104 monocytes are plated per 1 well (6 well-plate). Gyratory shaking is used at 88 rpms to generate spheres. After 2 days media are replaced with ½ CNS differentiation medial (Neurobasal® electro Medium (Gibco) supplemented with 5% B-27® Electrophysiology (Gibco), 1% glutamax (Gibco), 10 μg human recombinant GDNF (Gemini), 10 μg human recombinant BDNF (Gemini)) and ½ macrophage differentiation media (Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FCS, 0.055 mM β-mercaptoethanol, M-CSF (50 ng/ml), and IL-3 (25 ng/ml) (R&D Systems). The medium is replaced every 3 days.
Monocytes can also be incorporated after BMPS differentiation. For that, BMPS are differentiated up to 8 weeks. BMPS spheres are separated in 500 μl Eppendorf tubes. 2×104 monocytes are added to the Eppendorf with the BMPS. Tubes are shaking manually every hour, up to 8 hours. After that, BMPS-monocytes are collected and plated in 6 well plates. Cells are kept on constant shaking until use.
The characterization of the immune-competent human organoids can be carried out by immunocytochemically assessing the presence of markers such as HLA-DR, and the ionized calcium-binding adapter molecule 1 (Iba1), specific microglial markers. Measures of cytokines and chemokines release and expression of receptors associated with microglia function (e.g., CCL2 and CX3CL) demonstrates successful engrafting of the microglia cells. This modified model is more suitable to investigate the neuroimmunological component associated with many substance exposures and diseases.
The blood brain barrier (BBB) has a crucial role in neurotoxicity, being the last barrier for substances before reaching the brain. Moreover, the BBB is the bottleneck in brain drug development and is the single most important factor limiting the future growth of neurotherapeutics [81]. Most of the in vitro models do not incorporate BBB.
Human brain microvascular endothelial cells (hBMECs) from human iPSCs are incorporated into the BMPS by two techniques. In the first approach, mature BBB endothelial cells and neuronal precursors cells (NPCs) are combined in a single cells suspension in a ratio of 1:5, gyratory shaking or stirring are used to generated spheroids and aggregates are cultured up to 8 weeks. In the second technique, mature BMPS (8 weeks of differentiation) are covered by BBB endothelial cells using gravity systems (aggrewell, gravity well or hanging drops). Cells may be covered as well with other cell types, such as fluorescent LUHMES cells (
The BMPS gives the opportunity to develop cell-based assays allowing for high-content imaging (HCI) that can be adapted to high-throughput platforms, to evaluate the effects of toxicants on key cellular processes of neural development and physiology in the culture system.
Example of establishing fluorescent iPSC cell line: Creation of reporter cells lines greatly assists imaging efforts by allowing us to avoid complications associated with staining 3D cultures, to image subsets of cells, and to perform functional assays. Differentiated 3D aggregates from iPSC cultures spiked with 1-2% of iPSCs ubiquitously expressing fluorescent protein allow visualizing individual cells within the aggregates aiding quantification of phenotypic parameters, including neurite outgrowth and migration. Lines expressing markers allow measurement of synapse formation (PSD95, Synapsin 1), proliferation (Ki67), glial maturation (GFAP), and calcium signaling (GCaMP). Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR) were used to create the various lines. Similar in function to the well-established zinc-finger (ZFNs) and TALEN nucleases, the Cas9-CRISPR system is a new entrant into the rapidly emerging field of genome engineering and has been quickly adopted and validated across a wide array of human stem cells. Gene-editing in hiPSCs has traditionally been a technically difficult task but with these advances it is now possible to generate reporter and mutant cell lines with genetically matched controls [83, 84, 85, 86]; essential tools not only for this project but also for the future success of using human iPSC-derived cells in quantitative live-cell phenotypic assays of toxicant testing.
Using the CRISPR-Cas9 system, fluorescent protein (FxP) reporter cell lines were generated by generating gRNAs targeting the gene of interested. In this system as described herein, an RNA guided Cas9 endonuclease is used in conjunction with customizable small guide RNAs (gRNAs) to target and cleave any DNA template with a GN21GG sequence; the first G is for the U6 polymerase promoter while the N21GG is for the protospacer adjacent motif (PAM) sequence requirement of Cas9 [86, 87, 89].
For reporter cell generation, homology-directed repair (HDR) guides the insertion of the appropriate DNA donor fragment into a target site at regions of homology between the donor fragment and the genomic DNA target. An ES line that ubiquitously expresses GFP was created by introducing CAG promoter-driven GFP into the AAVS1 safe harbor locus, and can use these constructs to transfect iPSC cells. For other reporters, constructs may be created that will direct the integration of a self-cleaving P2A peptide sequence [90] targeted fluorescent protein cassette in frame at the stop codon of the gene of interest. The P2A sequence engineered between the C-terminus of the endogenous protein and the fluorescent protein may minimize possible fusion protein functional defects. Plasmids encoding the Cas9 nuclease, the targeting gRNA, and appropriate donor DNA will be introduced by electroporation, recombinant hiPSC clones will be manually selected and screened for the desired insertion by PCR, and the genotype may be verified by sequencing. Reporter hiPSCs will be subjected to a differentiation protocol and expression of the reporter validated by examining expression patterns and through immunohistochemistry experiments where it may be determined whether the FxP expressing cells co-label with known markers.
The use of iPSCs, as described herein, has created new opportunities to study human diseases and gene/environment interaction [20, 21]. Fibroblasts or other somatic cells from healthy and diseased individuals can be reprogrammed into iPSCs, and subsequently be differentiated into all neural cell types. Similarly, iPSC can be genetically modified before creating the BMPS. As a proof-of-principle, iPSCs were obtained from patients with Down's syndrome (FIGS. 1C5 and 5A-D), Rett Syndrome and from individuals with mutations in disrupted in schizophrenia 1 (DISCI). DISCI may have some functional overlap with TSC-iPSCs as both are involved in the mTOR cell signaling pathway.
The Down's syndrome model is further characterized (see
In some embodiments, BMPS may be combined with other organs and/or organ model systems. Several groups have been developing organ-on-a-chip platforms for different organs by using microfluidic techniques. Those platforms are designed to mimic in-vivo fluidic flows in the organs by separating cell culture chambers and perfusion channels, and successfully demonstrate recapitulation of iPSC-based organ functions. Together with other organ models on these platforms, the BMPS can be integrated, which allow us to untwine the complex toxicology from organ interactions. Such platforms allow (1) in-situ and high-throughput production of mini-brains on chip, (2) in-vivo like fluidic flow around mini-brains with enough supply of nutrient and small molecule through diffusion, (3) a large number of parallel test of toxic materials, and (4) a real-time monitoring of electrophysiological activities from BMPS with integrated electrodes. Companies such as TissUse GmbH have designed microfluidics platform that allow culture of floating spheres like the BMPS as described herein.
In order to e.g. incorporate the BMPS into platforms or enable any use in other laboratories, transportability of the system was optimized. Preliminary studies have shown possible recovery of the neuronal 3D aggregates after cryopreservation (
Human iPSC derived mini-brains are kept in culture at 37° C. In order to transport the live mini-brains, temperature must be controlled. Different methods can be used to control temperature during transport. Heating pads combined with an insulated box have been used to transport live biological material. Disposable chemical pads employ a one-time exothermic chemical reaction such as catalyzed rusting of iron, or dissolving calcium chloride. The most common reusable heat pads are based on a chemical reaction that transforms a liquid into a solid thus releasing energy. Some new heating pads (such as Deltaphase Isothermal Pad 3SET, from Braintree Scientific, Inc.) have been able to maintain 37° C. for more than 6 hours. 3D mini-brains cultured up to 8 weeks are sent in an insulated material box with heating pads. After transport, viability may be measured.
The techniques herein provide a human BMPS model that is a versatile tool for more complex testing platforms, as well as for research into CNS physiology, mechanisms associated with (developmental) neurotoxicity, and pathogenesis of neurological disorders. Prior art stem cell-derived brain model systems developed in the past few years have shown the capability to recapitulate some of the in vivo biological processes (Juraver-Geslin and Durand, 2015; Nakano et al., 2012; Krug et al., 2014) and have an advantage over other classical in vitro models as they facilitate the study of various differentiation mechanisms, developmental processes and diseases (Lancaster et al., 2013). Unfortunately, these prior art systems require complicated protocols that reduce the reproducibility of the system and make it difficult to use in other fields such as chemical toxicity and drug screening. Additionally, these prior art models are also limited by large diameters, which lead to extensive cell death in the interior regions due to insufficient diffusion of oxygen and nutrients (Lancaster et al., 2013) and other artifacts.
The techniques herein overcome the limitations of the prior art by developing a human in vitro model by the gyratory shaking technique that enables reliably generation of a high number (about 500 per six-well plate) of viable BMPS that are homogeneous in size and shape. Control of size makes it possible to keep cell aggregates below 350 μM in diameter (
As described herein, the 3D differentiation protocol for the BMPS covers stages from neuronal precursors to different cell types of the mature CNS. As discussed in detail above, at two weeks, BMPS consisted of an immature population of cells, showing minimal neuronal networks, a low percentage of mature astrocytes and oligodendrocytes, and minimal but early stages of myelin basic protein (MBP) expression. iPSC differentiation into mature BMPS was indicated by decreasing NES expression over time and a progressive expression of mature neuronal and glial markers such as MAP2, GFAP, 01 and MBP. Gene expression studies, flow cytometry, image analysis, immunostaining and miRNA studies have shown increase of cell maturation markers, which follow the BMPS differentiation. The presence of GABAergic neurons, dopaminergic neurons and glutamatergic neurons was documented by immunohistochemistry and real-time PCR data. Moreover, the BMPS showed spontaneous electrical activity, indicating neuronal functionality of the system.
Since astrocytes and oligodendrocytes play important roles during neuronal development, plasticity and injury, the presence of glial cell populations in the presently disclosed BMPS model provides an excellent opportunity for the evaluation of neuronal-glial interactions and the role of glia in pathogenesis and toxicity processes. Astrocytes have an important role in protecting neurons, increasing neuronal viability and mitochondrial biogenesis from both exogenous (e.g. chemicals) and endogenous toxicity (Shinozaki et al., 2014; Aguirre-Rueda et al., 2015), especially against oxidative stress (Shao et al., 1997; Schwab and McGeer, 2008). Thus, their presence in a biological system to study disease and neurotoxicity is crucial Immunohistochemistry and RT-PCR results showed increasing numbers of astrocytes (GFAP-positive cells) in the BMPS model reaching 19% astrocytes of the total cell population at eight weeks, which is earlier than in previously described cortical spheroids, where similar proportions of GFAP-positive cells were observed first at day 181, at day 86 the number of GFAP+ cells was below 10% (Pasca et al., 2015).
The most novel element of this BMPS is the presence of mature human oligodendrocytes with myelination properties, which has not been achieved in the prior art. Immunocytochemical and ultrastructural studies confirmed the morphological identity of these cells (
In conclusion, the techniques herein provide a BMPS that replicates crucial aspects of brain physiology and functionality. The potential for studying developmental and neurodegenerative disorders, brain infections, toxicity and trauma with such a system is growing. Furthermore, the potential to use iPSCs from different donors adds a personalized component to these studies. The high reproducibility and relatively simple protocol, enables future medium-throughput (96-well format) testing of chemicals, drugs and their potential to induce or treat diseases.
Methods and Materials
Chemicals
Rotenone and MPP+ were supplied from Sigma-Aldrich (St. Louis, Mo.). A 10 mM rotenone stock was prepared in DMSO Hybri-Max (Sigma) while MPP+ was diluted in water to a concentration of 30 mM.
iPSC Generation
CCD1079Sk (ATCC® CRL2097™), IPS IMR90 (WiCELL) and ATCCDYP0730 Human (IPS) Cells (ATCC® ACS1003™) fibroblasts were originally purchased from ATCC. All studies followed institutional IRB protocols approved by the Johns Hopkins University School of Medicine. Human fibroblasts and mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech Inc.) supplemented with 10% fetal bovine serum (FBS, HyClone) and 2 mM L-glutamine (Invitrogen). MEFs were derived from E13.5 CF-1 mouse embryos. Human iPCS cells were generated with the EBV-based vectors as previously described [75]. iPSC from other sources were used as well. Colonies of iPSCs were manually picked after 3-6 weeks for further expansion and characterization. iPSCs (passage ≤20) were cultured on irradiated MEFs in human embryonic stem cell (hESC) medium comprising D-MEM/F12 (Invitrogen), 20% Knockout Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM MEM NEAA (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), and 10 ng/mL human basic FGF (bFGF, PeproTech). Media were changed daily and iPSC lines were passaged using collagenase (Invitrogen, 1 mg/ml in D-MEM/F12 for 1 hr at 37° C.). These iPSC lines have been previously fully characterized [75].
Neuronal Progenitor Cells (NPC) Production
NPC generated followed the previous published protocol [75]. Briefly, iPSCs colonies were detached from the feeder layer with collagenase (1 mg/ml) treatment for 1 hr and suspended in EB medium, comprising of FGF-2-free hESC medium supplemented with Dorsomorphin (2 μM) and A-83 (2 μM), in non-treated polystyrene plates for 4 days with a daily medium change. After 4 days, EB medium was replaced by neural induction medium (hNPC medium) comprising of DMEM/F12, N2 supplement, NEAA, heparin (2 μg/ml) for 15 more days. The floating neurospheres were then dissociated to single cells in Accutase and plated in 175 mm flasks and were allowed to expand for 7 days. NPCs were expanded in poly-1-ornithine and laminin-coated 175 mm flask on StemPro® NSC SFM (Life Technologies). Half of the media was changed every day. Cultures were maintained at 37° C. in an atmosphere of 5% CO2. After NPC generation, iPSCs colonies were detached and NPCs were expanded in poly-1-ornithine and laminin-coated 175 mm flask in StemPro® NSC SFM (Life Technologies). Half of the media was changed every day. Cultures were maintained at 37° C. in an atmosphere of 5% CO2.
BMPS Differentiation
At 100% confluence NPCs were detached mechanically and counted. 2×106 cells per well were plated in 2 ml of medium in non-treated 6 well-plates. Cells were grown in NPC media for two days under constant gyratory shaking. Subsequently, medium was changed to differentiation medium (Neurobasal® electro Medium (Gibco) supplemented with 5% B-27® Electrophysiology (Gibco), 1% glutamax (Gibco), 0.02 μg/ml human recombinant GDNF (Gemini), 0.02 μg/ml human recombinant BDNF (Gemini)) Cultures were maintained at 37° C., 5% CO2 under constant gyratory shaking for up to 8 weeks. Differentiation medium was routinely changed every 2 days.
Size Measurement
Aggregates (n=20) from 3 independent experiments were randomly selected per time point for obtaining pictures and measuring size using SPOT software 5.0. Results were expressed as mean±SD. Cells were kept two days in NPC medium, indicated as NPC med. 2d in
RNA and miRNA Extraction
Total RNA was extracted from aggregates every week up to 8 weeks of differentiation using Tripure (Roche, Switzerland) according to Chomczynski and Sacchi (1987) [76]. The same RNA extraction method was used to isolate RNA after toxicant treatment. RNA quantity and purity was determined using NanoDrop 2000c (Thermo Scientific). One microgram of RNA was reverse-transcribed using the M-MI V Promega Reverse Transcriptase (Promega) according to the manufacturer's recommendations. For miRNA reverse-transcription 60 ng of RNA were reverse transcribed using TaqMan microRNA Reverse transcription kit in combination with miRNA specific stem-loop primers, which are a part of TaqMAn microRNA expression assay. Upto eight stem-loop primers were multiplexed in one reaction.
Quantitative RT-PCR
The expression of genes was evaluated using specific Taqman® gene expression assays (Life Technologies). miRNA expression was analyzed using TaqMAn microRNA expression assay in combination with TaqMan miRNA Reverse Transcription kit using protocol described in [77]. Table 1 shows a summary of the genes assayed. Real time RT-PCRs were performed using a 7500 Fast Real Time system machine (Applied Biosystems). Fold changes were calculated using the 2(−ΔΔCt) method [78]. β-actin and 18 s were used as a housekeeping genes for mRNA and RNU44 for miRNA. There were no statistically significant differences in expression for β-actin, 18s, and RNU44. Data were presented as mean±SD, normalized to housekeeping genes and week 0.
Immunocytochemistry of the BMPS
BMPS aggregates were collected at 2, 4 and 8 weeks. BMPS were fixed in 4% paraformaldehyde for 1 hour, washed 3 times in PBS, then incubated for 1 hour in blocking solution consisting of 5% normal goat serum (NGS) in PBS with 0.4% TritonX (Sigma). BMPS were then incubated at 4° C. for 48 hours with a combination of primary antibodies (Table 2) diluted in PBS containing 3% NGS and 0.1% TritonX. BMPS were washed in PBS 3 times after which they were incubated with the appropriate fluorophore-tagged secondary antibody for 1 hour in PBS with 3% NGS at room temperature. Double immunostaining was visualized using the proper combination of secondary antibodies (e.g., goat anti-rabbit secondary antibody conjugated with Alexa 594 and goat anti-mouse secondary antibody conjugated with Alexa 488 (Molecular Probes). Nuclei were counterstained with DRAQS dye (Cell Signaling; 1:5000 in 1×PBS) or NucRed Live (Molecular Probes) for 15 minutes before mounted on slides with coverslips and Prolong Gold antifade reagent (Molecular Probes); BMPS used as negative controls for immunostaining were processed omitting the primary antibody. Images were taken using a Zeiss UV-LSM 510 confocal microscope. The experiments were performed in duplicates; at least three different fields of view were analyzed for each combination of antibodies. 3D reconstruction was done using Imaris 7.6.4 software for scientific imaging.
Automated Quantitation of Cell Types
BMPS was differentiated for 8 weeks. Randomly selected pictures from three experiments were acquired by confocal imaging and then analyzed with a custom algorithm created with the Cellomics TargetActivation (Thermo Fisher Scientific, Pittsburgh, Pa.) image-analysis software package. With this algorithm, cells were identified based on DRAQS stained nucleus and quantified oligodendrocytes and astrocytes based on staining of CNPase, NOGO1 and GFAP.
Myelination Assessment and Quantification
To calculate the percentage of axonal myelination, a semi-automated computer platform was used, termed computer-assisted evaluation of myelin formation (CEM) [82], which uses NIH Image J built-in tools as well as a Math lab processing functions. The results were generated as pixel counts and percent values. The percent of myelinated axons was calculated by dividing the pixel count for myelin by the pixel count for axons after cell body removal and multiplying by 100. For each time point at least 18 fields from at least two independent experiments were analyzed.
Electron Microscopy
BMPS aggregates were collected at 2, 4 and 8 weeks and were fixed in 2% glutaraldehyde and 4% formaldehyde in 0.1M Sodium Cacodylate buffer (EMS, electron microscopy sciences) pH 7.4 with 3% sucrose and 3 mM CaCl2). Post-fixation was done with 2% osmium for 2 hours. The BMPS aggregates were then stained en bloc with 2% uranyl acetate in distilled water for 30 min and subsequently dehydrated in graded ethanol. Embed 812 (EMS) was used as the embedding media. Thin sections (70-80 nm) were cut on a Reichert Jung Ultracut E microtome and placed on formvar coated 100 mesh copper grids. The grids were stained with uranyl acetate and followed by lead citrate. All imaging was performed on a Zeiss Libra 120 electron microscope with a Veleta (Olympus) camera.
Treatment and Cytotoxicity Assay
BMPS was exposed to different concentrations of rotenone and MPP+ for 24 and 48 hours after 4 weeks of differentiation. Rotenone working solutions were prepared in differentiation medium from 10 nM or 100 μM stocks to reach final concentrations of 0.1, 1, 10, 25 and 50 μM. DMSO was used as vehicle control. MPP+ working solutions were prepared in differentiation medium from 30 mM stocks to reach final concentrations of 10, 50, 100, 500, 1,000, 5,000 and 10,000 μM. Four independent experiments in 3 replicates were performed for each experimental condition (control and toxicant exposure for the different time points). Resazurin reduction assay was performed in order to determine cell viability after rotenone and MPP+ treatment. Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue dye that is reduced into red fluorescent resorufin by redox reactions in viable cells. 100 μl Resazurin (2 mg/ml stock) were added directly to the 6 well plates (2 ml/well). Plates were incubated for 3 h at 37° C., 5% CO2. Subsequently, 50 μl of medium were transferred from each well in triplicates to a 96-well plate and fluorescence was measured at 530 nm/590 nm (excitation/emission) using a multi-well fluorometric reader CytoFluor series 4000 (PerSeptive Biosystems, Inc). Data were presented as mean±SD. Statistical analysis was performed using Dunnett's test.
Reactive Oxygen Species Measurement
Reactive oxygen species (ROS) were measured in cell media collected 24 hours after treatment with 5 μM rotenone or 1,000 μM MPP+ using the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, San Diego, Calif.). This is a fluorescence-based assay measuring the presence of total free radicals within a sample and was used according to the manufacturer's protocol. The quenched fluorogenic dye dichlorodihydrofluorescin-DiOxyQ (DCFH-DiOxyQ) which is similar to the popular 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) is first primed with a quench removal reagent. The resulted highly reactive non-fluorescent DCFH can react with present ROS species in the cell supernatant and is then oxidized to the highly fluorescent DCF (2′,7′-dichlorodihydroxyfluorescein). At every time point, 50 μl of the cell supernatant was added to a 96-well plate in triplicates and was mixed and incubated with the DCFH-DiOxyQ for 45 minutes. The fluorescence intensity was measured with a fluorescence microplate reader at 480 nm/530 nm (excitation/emission) and was proportional to the total ROS/RNS levels within the sample.
Flow Cytometry
In order to quantify percentage of NPCs, and neurons within the aggregates, flow cytometry with NPC and neuronal markers was performed. Flow cytometry was performed according to previously published protocol [77] with some optimization steps for 3D cultures. Aggregates were washed once with PBS/1 mM EDTA and trypsinized directly in the well using TrypLE Express containing 4 units/ml DNAse for 30 min at 37° C. on the shaker. Pipetting the aggregates up and down with a 1 ml syringe and a 26G3/8 needle ensured generation of single cell suspension. Cells were counted, washed once with PBS/1 mM EDTA, fixed with 2% PFA for 20 min at 4° C., washed twice with PBS/1% BSA (wash solution I, WS I) and blocked for 30 min in blocking solution (PBS/1% BSA/0.15% saponin/10% NGS). 1×106 cells were stained for one hour at 4° C. with fluorochrome-conjugated antibodies dissolved in blocking solution (Table 3). Unstained cells as well as cells incubated with isotype controls were used as negative controls to set the gates for measurements. Cells were washed twice with PBS/1% BSA/0.15% saponin, once with PBS/1% BSA. Flow cytometry was performed using a Becton Dickinson FACSCalibur system by measuring 104 gating events per measurement. Data was analyzed using FlowJo v10 software.
Microelectrode Array (MEA) Recordings
After 8 weeks of differentiation, BMPS were plated on 48-well MEA plates previously coated with Matrigel. During two weeks spontaneous electrical activity was recorded using the ‘Maestro’ MEA platform and Axion's Integraded Studio (AXIS) software [Axion Biosystems inc.; Atlanta, US]. Each well of the 48-well MEA plate contains 16 individual microelectrodes (˜40-50 μm diameter, center-to-center spacing 350 μm) with integrated ground electrodes, resulting in a total of 768 electrodes/plate. The ‘Maestro’ MEA platform has an integrated heating system, which can be controlled by AXIS software. All recordings were performed at a constant temperature of 37° C. Prior to a twenty minutes recording, the MEA plates were placed in the Maestro MEA platform and equilibrated for five min. AXIS software was used to control heating system and monitor the recordings, which includes simultaneously sampling of the channels at 12.5 kHz/channel with a gain of 1200× and a band pass filter of 200-5000 Hz. The recordings were converted into RAW files. After a recording the RAW-files were re-recorded with AXIS to convert the data into a spike file, which includes spike timing and profile information. A variable threshold spike detector was used for the spike-file, it was set at 6 times standard deviations of the rms-noise on each channel. The spike file was later used for data analysis with NeuroExplorer® [Nex Technologies, Madison (AL), US] to convert data into Microsoft Excel files. Using the function rate histogram, a summary of the spikes of all electrodes of one plate was put into one Excel sheet. Only electrodes that recorded activity higher than 0.05 spikes/sec at least once over the time measured were included for data analysis.
Statistical Analysis
Statistical analysis was performed using GraphPad InStat 3. The Dunnett's test was applied to all the experiments shown here that compare to a control group. Statistically significant values (p<0.01) are marked with an asterisk (*). For myelination quantification at the different time points, a Kruskal-Wallis test was employed, statistical significance was considered for p values <0.05.
Human induced pluripotent stem cells (iPSCs), together with 21st century cell culture methods, have the potential to better model human physiology with applications in toxicology, disease modeling, and the study of host-pathogen interactions. Several models of the human brain have been developed recently, demonstrating cell-cell interactions of multiple cell types with physiologically relevant 3D structures. Most current models, however, lack the ability to represent the inflammatory response in the brain because they do not include a microglial cell population. Microglia, the resident immunocompetent phagocytes in the central nervous system (CNS), are not only important in the inflammatory response and pathogenesis; they also function in normal brain development, strengthen neuronal connections through synaptic pruning, and are involved in oligodendrocyte and neuronal survival. Here, we have successfully introduced a population of human microglia into 3D human iPSC-derived brain spheres (BrainSpheres, BS) through co-culturing cells of the Immortalized Human Microglia—SV40 cell line with the BS model (OS). We detected an inflammatory response to lipopolysaccharides (LPS) and flavivirus infection, which was only elicited in the model when microglial cells were present. A concentration of 20 ng/mL of LPS increased gene expression of the inflammatory cytokines interleukin-6 (IL-6), IL-10, and IL-1b, with maximum expression at 6-12 h post-exposure. Increased expression of the IL-6, IL-1b, tumor necrosis factor alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) genes was observed in μBS following infection with Zika and Dengue Virus, indicating a stronger inflammatory response in the model when microglia were present than when only astrocyte, oligodendrocyte, and neuronal populations were represented. Microglia innately develop within cerebral organoids (Nature communications)′, the findings indicate that the μBS model is more physiologically relevant and has potential applications in infectious disease and host-pathogen interactions research.
Microphysiological systems and organotypic cell culture models are increasingly being used to model human physiology in vitro because these models better mimic the in vivo situation compared to traditional monolayer cell culture systems (Marx et al., 2016; Pamies and Hartung, 2017; Smirnova et al., 2018). In addition, such models allow for the mechanistic understanding of biological processes that would be challenging to study in vivo. In particular, the human brain is a highly complex structure that cannot easily be interrogated in vivo and has proven difficult to model in vitro. Recently, the first in vitro human organotypic models of the brain have been developed, including 3D structures and heterogeneous cell populations. Many of these models seek to represent key events in human brain development and cell-cell interactions between various cell types (Kadoshima et al., 2013; Lancaster et al., 2013; Pasca et al., 2015; Pamies et al., 2017; Sandstrom et al., 2017). The basis for the majority of these models is a neural progenitor cell (NPC) population; however, microglia, arise from erythromyeloid precursor cells in the embryonic yolk sac, unlike the other cell types in the brain which arise from the neuroectoderm (Alliot et al., 1999; Ginhoux et al., 2013; Kierdorf et al., 2013; Sousa et al., 2017). Thus, because microglia cannot be derived from NPCs, many of the current in vitro models of the human brain do not include a microglial cell population.
Microglia are the resident mononuclear phagocytic cells in the central nervous system (CNS) (Ginhoux et al., 2013). These cells are both neuroprotective and immunocompetent, as they play critical roles in brain development, strengthen neuronal connections through synaptic pruning, are involved in neuronal maintenance and support, and are responsible for the inflammatory response in the brain following an injury or pathogenic infection (Kettenmann et al., 2011; Paolicelli et al., 2011; Michell-Robinson et al., 2015; Thompson and Tsirka, 2017). In response to local alterations, lesions, or pathogen invasion, microglia become active. Microglial activation involves changes in morphology, the release of multiple substances such as proinflammatory cytokines, chemokines, and reactive oxygen species, migration to affected areas, proliferation, and phagocytosis of cell debris (Kettenmann et al., 2011; Boche et al., 2013; Thompson and Tsirka, 2017). This process is also a prominent feature of inflammation in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and infectious processes (Wang et al., 2015; Thompson and Tsirka, 2017). Various models have been developed to study neurological disorders and lipopolysaccharide (LPS) has been widely used as an inducer of neuroinflammation and neurotoxicity as it has been shown to act as a potent stimulator of microglia (Gao et al., 2002; Hu et al., 2012; Olajide et al., 2013; Kempuraj et al., 2017). Additionally, inflammation related neurodegeneration induced by LPS is a common approach to study mechanisms of cellular neuroimmunology (Hu et al., 2012). Neurotropic virus infections are major pathogens in the CNS and cause inflammation (Ludlow et al., 2016). The Flavivirus genus constitutes some of the most serious human pathogens, including Japanese encephalitis (JEV), dengue (DENV), zika (ZIKV), and yellow fever (YF), all of which are can invade the central and peripheral nervous system and are regarded as neurotropic viruses (Neal, 2014). The mechanisms by which flaviviruses alter the immune and the CNS have only recently been examined, but remain largely unclear (Daep et al., 2014). The NPC model has been used in ZIKV studies to understand mechanisms that govern neuropathogenesis and immunopathogenesis in CNS infection (Dang et al., 2016; Garcez et al., 2016; Tang et al., 2016; Qian et al., 2017), but these models do not contain microglia. We previously developed a 3D brain model (BrainSpheres, BS) derived from human-induced pluripotent stem cells (iPSCs) and demonstrated that this model is reproducible with respect to size and shape and is comprised of populations of neurons, astrocytes, and oligodendrocytes (Pamies et al., 2017). In addition, the model has been applied to the study of developmental neurotoxicity (Pamies et al., 2018). In this study, we introduced a population of human microglia into the BS model and compared the inflammatory response of BS without microglia to BS co-cultured with microglia (μBS) after treatment with LPS, ZIKV, or DENY. In addition, we quantified cell death following ZIKV infection because there is evidence that ZIKV infection can impair brain growth via cell death of developing neurons.
Materials and Methods
Induced Pluripotent Stem Cell Generation
Fibroblasts (American Type Culture Collection (ATCC), Manassas, Va., United States, CCD-1079Sk ATCC® CRL-2097™) were used to generate iPSCs as previously described (Wen et al., 2014; Pamies et al., 2017). iPSCs (passage ≤20) were cultured on irradiated MEFs in human embryonic stem cell (hESC) medium comprised of D-MEM/F12 (Invitrogen, Carlsbad, Calif., United States) 20% Knockout Serum Replacement (KSR, Invitrogen, Carlsbad, Calif., United States), 2 mM L-glutamine (Invitrogen, Carlsbad, Calif., United States), 100 μM MEM NEAA (Invitrogen, Carlsbad, Calif., United States), 100 μM β-mercaptoethanol (Invitrogen, Carlsbad, Calif., United States), and 10 ng/mL human basic FGF (bFGF, PeproTech, Rocky Hill, N.J., United States). Cell culture medium was changed daily. iPSCs were passaged using collagenase (Invitrogen, Carlsbad, Calif., United States, 1 mg/mL in D-MEM/F12 for 1 h at 37° C.).
Neural Progenitor Cell Production
NPC were generated following the previously published protocol (Wen et al., 2014). NPCs were cultured with StemPro® NSC SFM media (Life Technologies, Carlsbad, Calif., United States) in 175 cm2 flasks (Thermo Scientific, Waltham, Mass., United States, Nunc™ Filter Cap EasYFlask™) coated with Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis, Mo., United States, P3655) and laminin (Sigma-Aldrich, L2020, from Engelbreth-Holm-Swarm murine sarcoma basement membrane). Half of the media was changed daily. Cultures were maintained at 37° C. with 5% CO2. NPCs were passaged by mechanical detachment with a cell scraper (Sarstedt, N.C., United States, 2-position, Blade 25,83.1830).
BrainSpheres Differentiation
The production of BS was published previously (Pamies et al., 2017). Briefly, to produce BS, NPCs were detached mechanically with a cell scraper (Sarstedt, 2-position, Blade 25, 83.1830), re-pipetted for disaggregation, and counted using the Countess Automated Cell Counter (Invitrogen, Carlsbad, Calif., United States). 2×106 cells per well were plated in non-treated Falcon™ Polystyrene 6-well plates (Corning, Corning, N.Y., United States). Cells were grown in differentiation medium [Neurobasal® electro Medium (Gibco, Gaithersburg, Md., United States)] supplemented with 2% B-27® Electrophysiology (Gibco), 1% GlutaMAX (Gibco), 0.01 μg/mL human recombinant GDNF (Gemini, Woodland, Calif., United States), and 0.01 μg/mL human recombinant BDNF (Gemini). Cultures were maintained at 37° C. in an atmosphere of 5% CO2 under constant gyratory shaking (88 rpm) for 7 weeks. Differentiation medium was changed every 2 days.
Microglia Culture
Immortalized Human Microglia—SV40 (Applied Biological Materials Inc., Richmond, BC, Canada) were grown in non-treated 75 cm2 flasks (Thermo Scientific, Waltham, Mass., United States, Nunc™ Filter Cap EasYFlask™). Cells were expanded using Prigrow III medium (Applied Biological Materials Inc.) supplemented with 10% Fetal Bovine Serum (Gibco, Gaithersburg, Md., United States, Certified, Heat Inactivated, United States Origin) and 1% Penicillin-Streptomycin (Gibco, 10,000 U/mL). Microglia were passaged by mechanical detachment using a cell scraper (Sarstedt, 2-position, Blade 25, 83.1830). Cultures were maintained at 37° C. in an atmosphere of 5% CO2 immortalized Human Microglia—SV40 cell line was declared mycoplasma free by a mycoplasma testing using a PCR based MycoDtect kit from Greiner Bio-One (Genetic Resource Core Facility is a core resource of the Johns Hopkins School of Medicine, Institute of Genetic Medicine).
Formation of BS with Microglia (pBS)
The density of microglia added to the aggregates was studied previously and the optimal number of cells was chosen to avoid the formation of microglia-only spheres and to allow enough microglia attachment to aggregates. BS were differentiated for 7 weeks at which time, 3×105 microglia/well were added to the BS suspension. Upon addition of microglia, plates were kept in static conditions in the incubator at 37° C. with 5% CO2 for 24 h, with manual shaking of the plates every 6 h. This allowed for the attachment of the microglia to the surface of the BS while avoiding BS agglomeration. Microglia which did not attach to BS during this time attached to the bottom of the well. We then carefully removed aggregates from the well and transferred them to a new plate. μBS aggregates were then washed twice with PBS, and new differentiation media was added. After the incorporation of microglia, μBS were maintained in culture for one more week.
Histology and Immunohistochemistry
BS with and without incorporated microglia were fixed in 4% formalin/PBS. After fixation, spheres were carefully embedded into low-melting agarose and the solidified mixture was processed into paraffin blocks using an ASP300S dehydration machine (Leica, Wetzlar, Germany) and an EG1160 tissue embedding system (Leica, Wetzlar, Germany) Sections (2 μm) were stained with hematoxylin-eosin (H&E) or processed for immunohistochemistry as follows: After dewaxing and inactivation of endogenous peroxidases (PBS/3% hydrogen peroxide), antibody specific antigen retrieval was performed using the Ventana Benchmark XT machine (Ventana, Tucson, Ariz., United States). Sections were blocked (PBS/10% FCS) and afterward incubated with the primary antibodies TMEM119 (1:100; Sigma), Mertk (1:100; R&D), Ax1 (1:100; LSBio), CD11b (1:2,000; Abcam), and P2ry12 (1:100; Sigma). For double staining of microglia and neurons, microglia were detected with TMEM119 and visualized with DAB in brown, whereas neurons were stained with NeuN (1:50; Millipore) and incubated with anti-mouse AP-coupled secondary antibody and developed in red. Bound primary antibodies were detected with secondary antibodies using Histofine Simple Stain MAX PO immune-enzyme polymer (Nichirei Biosciences) and stained with 3,3′-Diaminobenzidine (DAB) substrate using the ultraView Universal DAB Detection Kit (Ventana). Tissues were counterstained with hematoxylin. Representative images were taken with a Leica DMD108 digital microscope.
Virus Propagation and Titering
Vero (ATCC, Manassas, Va., United States, ATCC® CCL81™) and BHK-21 (ATCC®, C-13 ATCC CCL-10) cell lines were cultured in D-MEM (Gibco) containing 10% Fetal Bovine Serum (Gibco, Certified, Heat Inactivated, United States Origin), 2 mM L-glutamine (Invitrogen), Penicillin-Streptomycin (Gibco 10,000 U/mL), and 10 mM Hepes buffer (Gibco) in an incubator at 37° C. with 5% CO2. Vero and BHK-21 cells were passaged 3 times a week. To passage Vero and BHK-21 cells, the cells were incubated with 0.05% trypsin (Gibco) in 1×PBS (Gibco) at 37° C. with 5% CO2 for 5 min, resuspended in 10 mL D-MEM, and centrifuged at 1200 rpm for 8 min. The supernatant is then removed and the cells were resuspended in fresh media and transferred to a new flask.
Two strains of ZIKV (ATCC, VR-1838™, Uganda 1947, ZIKV-UG, and Brazil 2015, ZIKV-BR) were propagated in Vero cells and Dengue Virus type 1 (ATCC, VR-1586™, DENV-1) was propagated in BHK-21 cells. ZIKV-BR was isolated from a febrile non-pregnant Brazilian woman with a rash in Paraiba, Brazil in 2015 and kindly provided by Professor Pedro Vasconcelos from the Instituto Evandro Chagas, Belem, State of Para, Brazil (Pompon et al., 2017). ZIKV-UG was isolated in 1947 from a rhesus macaque exposed to mosquitos in Uganda. The Viral stocks were prepared by infecting Vero or BHK-21 cells at 80-90% confluency in 25 cm2 flasks with 2 mL of virus dilution in OptiPro™ SFM media (Gibco) at a multiplicity of 0.1. The cells were incubated for 6 h at 37° C. with 5% CO2, then the supernatant was removed and the cell lines were washed two times with 1×PBS and replaced with 10 mL of D-MEM. The infected cells were further incubated for 5 days, and then the supernatants were collected and transferred to a 75 cm2 flask with respective cell lines at 90% confluence. The ZIKV-UG, ZIKVBR, and DENV-1 were collected after 7 days post-infection (p.i.), clarified by centrifugation at 500×g for 10 min, and filtered through a 0.22-μm membrane. All viral stocks were stored in 1 mL aliquots at −80° C. Virus titers used in the assays were determined by double-overlay plaque assay of Vero and BHK-21 cells to ZIKV and DENV, respectively, as previously described (Baer and Kehn-Hall, 2014).
Lipopolysaccharide Treatment
After 8 weeks of differentiation, 2 μl from a 10 μg/mL stock of LPS were added to 2 mL media containing ether the microglia, BS or μBS, obtaining a final concentration of 20 ng/mL. Samples were collected 3, 6, 12, and 24 h after the exposure. Time 0 refers to samples before LPS exposure. Samples were collected for qPCRs.
Viral Infection
Immortalized Human Microglia—SV40 were plated in 24-well plates at a density of 2.5×105 cells per well. The BS and μBS or microglia from one well were transferred to a 24-well-plate (and divided into triplicates) before infecting with ZIKV-UG, ZIKVBR, and DENV-1. A MOI of 0.1 for 6 h at 37° C. with 5% CO2 was used. Three wells of non-infected BS, μBS, and microglia were used as control (MOCK). The cells were washed 3 times with 500 μl PBS, and then 250 μl of supernatant were collected for each condition as time zero of infection. The PBS was totally removed and BS, μBS or microglia were re-suspended in fresh complete media. BS and μBS were then transferred back to a 6-well plate containing media. The plates containing the infected and uninfected cells were incubated at 37° C. with 5% CO2 for 24 to 72 h. The supernatant was collected at different times points 24, 48, and 72 h post infection (p.i.) to check viral load. The cells were collected at 48 and 72 h p.i. to analyze gene expression, Annexin-V, and cell cycle (3 technical replicates). One well of BS, μBS and microglia were used for confocal microscope analysis. All work with infectious ZIKV was performed in an approved BSL-3 facility.
Analysis of Cell Viability and Cytotoxicity Using Flow Cytometry
Apoptosis in microglia cells, BS, and μBS was assayed using the Muse Annexin V and Dead Cell kit (Millipore, Billerica, Mass., United States) according to the user guide and the manufacturer's instructions. After 72 h incubation at 37° C. in 5% CO2, microglia cells for all conditions were harvested through mechanical detachment with a cell scraper (Sarstedt, Blade 25, 83.1830) in 500 μl of the PBS, and centrifuged for 5 min at 2000 RPM. Two aggregates of BS and μBS were collected using a 1 mL micropipette. The pellet was resuspended in 100 μL of the PBS with 1% FBS, mixed with 100 μL of the Muse Annexin V and Dead Cell Reagent at room temperature. Tubes were mixed using a vortex for 10 s. Samples were incubated for 20 min at room temperature and protected from light. The percentages of apoptotic cells were analyzed by flow cytometry using Muse Cell Analyzer (Millipore, United States) system and values were expressed as mean of apoptotic cells relative to mock with error bars representing the SD. All values are expressed as mean±SD (n=3). Statistical significance (defined as P-value <0.05) was evaluated using multiple t-test to compare control and infected or treated cells (Graph Prism 7 Software, San Diego, Calif., United States).
Flow Cytometry Analysis of Cell Cycle
Microglia cells, BS and μBS (infected and uninfected) were cultivated in 6-well plates and incubated for 48 h. Microglia cells for all conditions were harvested through mechanical detachment with a cell scraper (Sarstedt, Blade 25, 83.1830) in 500 μl of the PBS, and centrifuged for 5 min at 2000 RPM. Two aggregates of BS and μBS were collected using a 1 mL micropipette. Obtained pellets were fixed with 70% ethanol. The cells were kept in −20° C. overnight. After ethanol removal, cells were suspended in 250 μL PBS and centrifuged for 5 min at 2000 RPM. Cell pellets were suspended in 200 μL of Muse Cell Cycle Reagent and were incubated for 30 min at room temperature and protected from light. The cell suspension was transferred to a 1.5 mL microcentrifuge tube prior to analysis on Muse Cell Analyzer. Cell cycle was assessed by fluorescence-activated cell analysis using a Muse Cell Analyzer (Merck, Millipore, United States). All values are expressed as mean±SEM (n=3). Statistical significance (defined as P-value <0.05) was evaluated using multiple t-test to compare control and infected or treated cells with LPS (Graph Prism 7 Software).
Viral RNA Extraction
Viral RNA for the real-time RT-PCR (qPCR) assays was purified from the culture supernatant of the uninfected and infected microglia, BS and μBS. The viral RNA was extracted from 240 μl of culture supernatant by using the QlAamp MinElute virus spin kit (Qiagen, Valencia, Calif., United States) according to the manufacturer's instructions, with the exception of the elution volume, which was 65 μl.
DENV-1 and ZIKV qPCR
RNA isolated from the supernatant after DENV-1 and ZIKV infections were used in qPCR independently. The MOCK samples were used as negative controls in both qPCRs. The QuantiTect One-Step RT-PCR kit (Qiagen, Hilden, Germany) was used with a 25 μl reaction mixture under the following conditions: 5 μl of kit master mixture (including Taq polymerase and RT enzyme), 1.25 μl of 10 μM of each primer, 0.5 μl of 10 μM of probe, 7 μl of RNA-free-water (Mol Bio grade, Hamburg, Germany), and 10 μl of the extracted sample. Each amplification run contained negative control (NC) of each experiment (MOCK); non-template control (NTC), and positive control (PC). The non-template control consisted of blank reagent and water. For the positive control, nucleic acid extracted from each virus stock were used after dilution 1:1000 to avoid cross-contamination. The protocol used to ZIKV and DENV-1 qPCR was the same conditions except primers and probes (Table 4) and cycling thermal. The DENV-1 qPCR was done as described previously (Johnson et al., 2005) with some modification; a single cycle of reverse transcription for 30 min at 50° C., 15 min at 95° C. for reverse transcriptase (RT) inactivation, and DNA polymerase activation followed by 45 amplification cycles of 15 s at 95° C. and 1 min 55° C. (annealing-extension step). The following thermal profile was used to ZIKV qPCR a single cycle of reverse transcription for 30 min at 50° C., 15 min at 95° C. for RT inactivation, and DNA polymerase activation followed by 40 amplification cycles of 15 s at 95° C. and 1 min 60° C. (annealing-extension step). For RNA standards, RNA was isolated from purified, titered stock of ZIKV-UG or DENV-1. RNA yield was quantified by spectrometry and the data was used to calculate genomes/μl. ZIKV-UG and DENY-1 RNA was serially diluted 1:10 in 8 points of dilution for standard curve (107 to 101). The standard curve was amplified in duplicate using primers and conditions described above for each specific qPCR. The number of infectious viral RNA transcripts detected was calculated by generating a standard curve from 10-fold dilutions of RNA isolated.
Analysis of Confocal Microscopy
After 72 h exposure BS and μBS were collected and fixed with PFA (4%) for 1 h at room temperature. Subsequently, BS, and μBS were washed twice with PBS (1×) and incubated with blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at 4° C. for 1 h. The BS were washed with washing solution (1% BSA, 0.15% saponin in PBS), and incubated overnight at 4° C. with primary antibodies: Zika NS1 (OWL, 55788, 1:200, ZIKV) anti-NF200 (Sigma Aldrich, N4142, 1:200, neurofilament for all neurons), and IBA1 (WAKO, Richmond, Va., United States, 019-19741, 1:200, microglia) diluted in blocking solution. The next day the BS and μBS were washed twice with washing solution and incubated with the secondary antibody 568-Alexa anti-rabbit and 488-Alexa anti-mouse both 1:200 diluted in blocking solution for 24 h at 4° C. and protected from light. Thereafter, BS and μBS were washed twice again with washing solution. Nuclei were stained with Hoechst 33342, diluted 1:10,000 in PBS for 1 h. The BS were transferred to microscope glass slides and mounted with “Immuno mount” and imaged with a confocal microscope Zeiss LSM 510 Confocal III (Zeiss) with a 20× objective.
Cellular RNA Isolation and qPCR
Total RNA was extracted from microglia, BS and μBS 72 h post infection (p.i.) using RNAeasy Mini Kit (Qiagen, Hilden, Germany). RNA quantity and purity was determined using a NanoDrop 2000c (Thermo Scientific). 1 μg of RNA was reverse-transcribed using the MLV Promega RT (Promega) according to the manufacturer's recommendations. The expression of genes was evaluated using specific TaqMan® Gene Expression Assays (Life Technologies). Table 5 shows a summary of the assayed genes. qPCR was performed using a 7500 Fast Real Time system machine (Applied Biosystems). Fold changes were calculated using the 2 (−ΔΔCt) method (Livak and Schmittgen, 2001). β-actin and 18s were used as housekeeping genes for mRNA. Data are presented as mean±SD, normalized to housekeeping genes and MOCK.
Results
Incorporation of SV40-Immortalized Human Microglial Cells Into a 3D BS Model
Microglia cells were added to the BS at 7 weeks of differentiation (
is immortalized and proliferation takes place, we were only able to maintain (IBS (BS with microglia) for 1 week after incorporation for our experiments Immunohistochemistry of (IBS demonstrated they were positive for microglia markers such as TMEM19, Mertk, Ax1 (
Presence of Microglia in BS Alters Gene Expression of Cytokines in Response to Inflammatory Stimuli
Once microglial cells were successfully incorporated into BS, we evaluated whether the μIBS model would then respond to inflammatory stimuli in a distinct manner to BS. Microglial culture, BS, and μIBS were exposed to 20 ng/mL LPS, which is a potent activator of inflammatory pathways in myeloid cells (Fujihara et al., 2003). Cells were collected at 0, 3, 6, 12, and 24 h post-treatment (p.t.) and levels of CCL2, TNF-α, IL-1b, IL-6, and IL-10 RNA were quantitated by qPCR. In the absence of microglia, BS responded poorly to LPS (
The μBS Support Replication of Dengue 1 (DENV-1) and Zika Viruses (ZIKV)
Organoids have been previously used as models for virus pathogenesis (Gabriel et al., 2017; Salick et al., 2017; Watanabe et al., 2017). To evaluate whether the incorporation of microglial cells into BS would interfere with viral replication, microglia, BS, and μBS were infected with DENY-1 and two distinct strains of ZIKV (ZIKV-UG and ZIKV-BR).
The Presence of Microglia Alters the Cytokine Gene Expression in Response to Flavivirus Infection
As part of the innate immune response, microglial cells respond to viral infection by secreting inflammatory cytokines. To determine whether the addition of microglial cells to BS modulates the immune response against flavivirus infection, the levels of intracellular TNF-α, CCL2, IL-1b, and IL-6 mRNA were measured in sole microglia, BS, and μBS 72 h after exposure to DENY-1, ZIKV-UG, and ZIKV-BR. After DENY-1 infection, cytokine expression was observed mostly in microglia and μBS, but not BS. Interestingly, the secretion pattern varied between the analytes (
3D brain models have been increasingly used in the last years, primarily in ZIKV studies (Qian et al., 2016, 2017; Wells et al., 2016; Salick et al., 2017; Watanabe et al., 2017; Zhou et al., 2017). These studies, however, have mainly focused on neurogenesis. The relevance of other brain populations such as microglia has been highlighted, indicating that these tissue-resident brain macrophages are key players in brain homeostasis, development, and diseases (Derecki et al., 2014; Salter and Stevens, 2017). Microglia incorporation into 3D models has not been achieved previously. In this study, we have successfully introduced microglia into our 3D brain spheroids, and demonstrated they alter the response to viral infection. Two techniques were compared to establish the protocol: in the first approach, human microglia immortalized cells were added on top of the NPCs before the formation of the spheres, and then differentiated in 3D over 8 weeks (
Flavivirus (DENY-1, ZIKV-BR, and ZIKV-UG) infection induced different responses depending of the model used (microglia, BS or μBS). Flavivirus infection resulted in produced a higher number of RNA virus copies in microglia cells (
Flavivirus infection in μBS and BS showed differences in the expression profile of the genes studied. After infection of μBS by ZIKV-BR and ZIKV-UG, upregulation of TNF-α, IL6, IL1b, and CCL2 was observed when compared with BS (
The findings indicate that the microglia-driven inflammatory response to ZIKV infection may be exacerbated by endogenous signaling molecules such as viral proteins that can contribute to the pathological damage of neurons. Based on these findings, the activated microglia in μBS play a potential role in enhancing the expression of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and TNF-α in response to ZIKV-BR infection.
In conclusion, the incorporation of human microglia in BS produced an inflammatory response following LPS or flavivirus exposure. This has not been studied previously in a 3D in vitro brain model which includes a population of microglial cells. Both exposures produced changes in cell cycle and Annexin V analysis in presence of microglia, indicating that the microglia-driven inflammatory response induces cytotoxicity leading to a decrease in cell viability. Without being bound by theory, microglia derived from iPSC-derived microglia can represent an improvement of this model to limit proliferation and extend the co-culture period. As microglia occupy a central position in the defense and maintenance of the CNS, a 3D in vitro model including microglia can be fundamental for studies of the brain. Moreover, various anti-inflammatory drugs have been identified in treating microglia-mediated neuroinflammation in the CNS (Baby et al., 2014). Without wishing to be bound by theory, the disclosed model can serve as a potential tool for the screening of therapeutic targets in neurological disorders and recovery from brain injury. In conclusion, the findings indicate that the μBS model has potential applications as a physiologically relevant model to study infectious disease, host-pathogen interactions, and neuro-inflammation.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a continuation application, filed under 35 U.S.C. § 120, of U.S. application Ser. No. 16/077,411, filed on Aug. 10, 2018, which is a national stage application, filed under 35 U.S.C. § 371, of International Stage Application No. PCT/US2017/017464, filed on Feb. 10, 2017, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/294,112, filed Feb. 11, 2016, all of which are incorporated herein by reference in their entireties.
The invention was made with government support under the following grant awarded by the National Institute of Health (NIH): U18TR000547. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62294112 | Feb 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16077411 | Aug 2018 | US |
| Child | 16700750 | US |
