Described herein are methods for generating human forebrain-like endothelial cells, compositions comprising the cells, and methods of use thereof in therapy.
Abnormal migration, positioning and reduction in GABAergic interneurons during the critical prenatal developmental period results in dysfunctional cortical neuronal synchrony implicated in brain diseases such as autism, epilepsy and schizophrenia1-5, conditions awaiting more effective treatments. Cell transplantation is a powerful tool to introduce new cells with intrinsic plasticity to overcome cellular deficits and initiate repair and regeneration. To be successful, grafted cells should possess the ability to migrate and disperse through affected areas, differentiate into fully mature neurons, functionally integrate, and modulate circuitry activity in the damaged host brain. A better comprehension of the cellular and molecular mechanisms of interneuron development has led to use of their neuronal precursors in transplantation6-8. While the origin and specification of cortical GABAergic interneurons was well established9-11, mechanisms that underlined their migration was not fully understood. Our studies served to address this critical gap by showing that embryonic forebrain vascular networks are strategically positioned to provide physical support and critical guidance cues for GABAergic interneuron migration in the developing telencephalon12-14. Additionally, our work has established novel autonomous links between the periventricular vascular network and the origin of psychiatric disorders, from the earliest developmental time points13, 15. Understanding brain development thus begins with an appreciation of all of its cellular components. Deeper insights into the anatomy, origin, molecular regulation, function and dysfunction of the periventricular vascular network in the last decade12-15 was crucial for understanding its direct significance for psychiatric disorders.
Abnormalities of or reductions in GABAergic interneurons are implicated in the pathology of severe neuropsychiatric disorders, for which effective treatments are still elusive. Transplantation of human stem cell-derived interneurons is a promising cell-based therapy for treatment of these disorders. In mouse xenograft studies, human stem cell derived-interneuron precursors could differentiate in vivo, but required a prolonged time of four to seven months to migrate from the graft site and integrate with the host tissue. This poses a serious roadblock for clinical translation of this approach. For transplantation to be effective, grafted neurons should migrate to affected areas at a faster rate. Endothelial cells of the periventricular vascular network are the natural substrates for GABAergic interneurons in the developing mouse forebrain, and provide valuable guidance cues for their long-distance migration. Additionally, periventricular endothelial cells house a GABA signaling pathway with direct implications for psychiatric disease origin. As shown herein, this discovery translates into humans, with significant therapeutic implications. The present inventors generated human periventricular endothelial cells, using human pluripotent stem cell technology, and extensively characterized its molecular, cellular and functional properties. Co-culture of human periventricular endothelial cells with human interneurons significantly accelerated interneuron migration in vitro and led to faster migration and wider distribution of grafted interneurons in vivo, compared to neuron-only transplants. Furthermore, the co-transplantation strategy was able to rescue abnormal behavioral symptoms in a pre-clinical model of psychiatric disorder, within one month after transplantation. This strategy facilitates angiogenesis-mediated treatment of psychiatric disorders.
Thus, provide herein are methods for generating a population of human forebrain periventricular endothelial cells. The methods include providing a population of pluripotent stem cells: (i) culturing the population of pluripotent stem cells in a first, stem cell media comprising Wnt7a protein and gamma amino-butyric acid (GABA), for a first time period sufficient to generate mesodermal cells; (ii) culturing the mesodermal cells in a second, vascular inducing medium comprising an inhibitor of transforming growth factor beta (TGFβ) signaling, vascular endothelial growth factor-A (VEGF-A), WNT7A e.g., about and GABAe, for a time sufficient to generate endothelial-like cells;
(iii) culturing the endothelial-like cells in a third, endothelial cell culture medium, (e.g., E6 medium) comprising growth factors, e.g., VEGF-A and FGF2, and GABA, for a time sufficient for development of a population of cells comprising CD31+GABRB3+ endothelial cells, and (iv) optionally isolating CD31+GABRB3+ cells from the mixture, thereby generating a population of human forebrain periventricular endothelial cells.
In some embodiments, the population of pluripotent stem cells comprise human embryonic stem cells or induced pluripotent stem cells (iPSC); in some embodiments the cells can be identified by the presence of markers OCT4 and TRA1-60.
In some embodiments, the first, stem cell media comprises E8 media with one or more growth factors; bone morphogenetic protein 4 (BMP4); Activin A; and a small molecule activator of WNT signaling.
In some embodiments, the growth factors comprise Fibroblast growth factor 2 (FGF2) and either TGFβ or NODAL.
In some embodiments, the small molecule activator of WNT signaling is a compound listed in Table A, preferably CHIR99021.
In some embodiments, the inhibitor of TGFβ signaling is a small molecule inhibitor of TGF-β/Smad signaling pathway, preferably SB431542.
In some embodiments, the endothelial-like cells express CD31 and von In some embodiments, the growth factors in the third, endothelial cell culture medium comprise VEGF and FGF2.
In some embodiments, the cells in the population of human forebrain perivascular endothelial cells express one or more periventricular endothelial cell markers selected from the group consisting of GABRB3, GABA, NKX2.1, PAX6, and ISL1.
In some embodiments, the methods further include maintaining the purified CD31+GABRB3+ cells in endothelial cell culture medium comprising growth factors and GABA.
In some embodiments, the concentration of GABA in the first and second media is 1-10 uM, preferably 5 uM.
Also provided herein are populations of human forebrain perivascular endothelial cells generated by a method described herein, and compositions comprising the populations of human forebrain periventricular endothelial cells, in a sterile carrier. In some embodiments, the cells are frozen.
Further provided herein are methods of treating a mammal, the method comprising administering to the brain of the mammal a population of human forebrain periventricular endothelial cells generated by a method described herein, and optionally a population of neuronal cells. Also provided are populations of human forebrain periventricular endothelial cells generated be a method described herein, for use in transplantation into a mammal, e.g., in combination with a population of neuronal cells.
In some embodiments, the methods increase numbers of interneurons in the forebrain of the mammal. In some embodiments, the mammal is in need of such treatment. In some embodiments, the mammal is a human.
In some embodiments, the neuronal cells are GABAergic interneurons, e.g., calretinin+, parvalbumin+, somatostatin+ or Neuropeptide Y+ neurons.
In some embodiments, the cells are transplanted into the cerebral cortex or into the hippocampus of the mammal. In some embodiments, the cells are transplanted into the cingulate cortex, motor cortex, somatosensory cortex, or piriform cortex in the cerebral cortex.
In some embodiments, the subject has a neuropsychiatric or neurological disease or disorder. In some embodiments, the neuropsychiatric or neurological disease or disorder is schizophrenia, epilepsy, autism, severe depression, cortical lesions, or a neurodegenerative disease (e.g., Alzheimer's disease). In such methods, the endothelial cells generated using a method described herein can be transplanted in combination with suitable neuronal cells, e.g., neuronal cells that are missing or depleted from the brain of the subject. In some embodiments, e.g., wherein the subject has a vascular disease or disorder, cerebral ischemia or stroke, the method can include transplanting only the endothelial cells generated using a method described herein, without additional neuronal cells.
Provided herein are methods to generate human periventricular endothelial cells expressing gamma amino-butyric acid (GABA) from human Embryonic Stem Cells (e.g., as shown in
In some embodiments, the factors that induce or promote the mesodermal phenotype include, but are not limited to, Bone Morphogenetic Protein 4, Activin A and a small molecule activator of the WNT pathway. In some embodiments, the small molecule activator of the WNT pathway is CHIR99021.
In some embodiments, the factors that induce or promote the vascular phenotype include, but are not limited to, an inhibitor of the TGFβ signaling pathway and Vascular Endothelial Growth Factor-A. In some embodiments, the inhibitor of the TGFβ signaling pathway is SB431542.
In some embodiments, the factors that induce or promote a periventricular endothelial cell phenotype include, but are not limited to, Wnt7a and gamma amino-butyric acid (GABA). In some embodiments, the concentration of GABA is 5 μM.
In some embodiments, the media includes, but is not limited to, E8.
Also provided herein are methods to isolate differentiated human periventricular endothelial cells from other differentiated cells comprised of sorting said differentiated human periventricular endothelial cells from other differentiated cells by fluorescence activated cell sorting (FACS) and wherein a cell surface marker unique to embryonic periventricular endothelial cells is used to isolate said differentiated cells. In some embodiments, the cell surface marker unique to embryonic periventricular endothelial cells is CD31+GABRB3+.
Also provided herein are methods to improve human GABAergic interneuron migration comprised of providing a source of vasculature or endothelial cells for GABAergic interneurons such that said GABAergic interneurons can align and migrate along the surface of said vasculature or endothelial cells. In some embodiments, the source of vasculature or endothelial cells for GABAergic interneurons is human periventricular cells expressing GABA prepared by a method described herein.
In some embodiments, the improved GABAergic interneuron migration is an in vitro culture or microfluidic system.
In some embodiments, the improved GABAergic interneuron migration is in vivo.
Additionally, provided herein are methods to treat or repair brain damage or neurological or neuropsychiatric diseases in a patient comprised of co-transplanting in said patient a source of neuronal cells or neuronal cell precursors and a source of vasculature or endothelial cells for providing the effective guidance and migration of said transplanted neuronal cells. In some embodiments, the ratio of neuronal cells or neuronal cell precursors to vasculature or endothelial cells is 1:1. In some embodiments, the source of neuronal cells is GABAergic interneurons. In some embodiments, the source is human periventricular cells expressing GABA prepared by a method described herein. In some embodiments, the neurological or neuropsychiatric diseases include (but are not limited to) autism, epilepsy, and schizophrenia.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Due to the restricted availability of human fetal tissue for cell therapy, human pluripotent stem cell (hPSC) technology provides an unprecedented opportunity to study disease mechanisms16-22. Multiple groups have successfully derived human interneuron/interneuron progenitors from hPSCs23-26 and transplantation of interneurons/interneuron progenitors has emerged as a promising treatment option for psychiatric disorders27-32. When transplanted in mouse33 and rat34 models of epilepsy, hPSC-derived interneuron precursors, survived well, fired action potentials, formed functional synaptic connections and could reduce abnormal seizure activities. Though showing great promise, one issue that needs improvement is the migration efficiency of transplanted cells. At two weeks post transplantation, transplanted interneurons displayed minimal migration, and it was only at four to seven months post transplantation, that some migration and integration into host brain was observed23, 25, 33, 34. Therefore, the beneficial effects of interneuron graft-in-disease models were observed only several months after transplantation. This presents an obstacle for the clinical translation of interneuron-based therapy, especially for very sick or severely affected patients. Another drawback that has been described with GABA producing cell types after transplantation is their transient effects, due to reductions in GABA levels35, 36. A decrease in GABA-mediated inhibition is a critical contributing factor for hyperexcitability and seizure initiation and increased secretion of GABA, by grafted cells, is important for increasing the seizure threshold. Thus, at present, while transplantation of GABAergic interneurons represents the most promising cell-based therapeutic alternative for GABA related diseases, there are difficulties that need to be overcome.
The key fundamental discovery that pre-formed vascular networks are the natural guides for GABAergic neuronal migration from the earliest developmental time point assumes a new significance here that can serve to improve hPSC-derived GABAergic neuronal migration. The periventricular vascular network not only acts as a physical substrate for neuronal migration in the embryonic forebrain, but also has a very unique gene expression profile, unlike endothelial cells from other brain regions or organs12-15. Periventricular endothelial cells show enriched expression of cell surface marker, GABAA receptor β3 subunit (GABRB3) as opposed to pial endothelial cells or control endothelial cells prepared from midbrain and hindbrain15. Therefore, GABRB3 serves as a valuable tool to selectively sort human endothelial cells which are akin to mouse periventricular endothelial cells. Additionally we know that periventricular endothelial cells express and release GABA that promotes rapid and extensive long-distance migration of GABAergic interneurons15. Neuronal GABA cannot compensate for the roles of endothelial GABA and interneurons stall in their migration in the absence of endothelial GABA15. This close neuro-vascular interaction during embryonic brain development that is sealed by space and time is the key missing link in interneuron-based therapy. Interestingly, it has been reported that transplanted neuronal precursors align and migrate along the surface of host blood vessels37, 38 as though in search of a missing or lost counterpart. All of this fundamental knowledge provided us with strong rationale to generate human periventricular endothelial cells from human embryonic stem cells and to tap into the potential of these endothelial cells to improve human GABAergic interneuron migration in vitro and in vivo.
Provided herein are methods for generation of human embryonic forebrain-like endothelial cells (e.g., periventricular endothelial cells) from human embryonic stem cells; the methods include addition or GABA and WNT7A for efficient differentiation, and isolation of GABRB3+/CD31+ cell population by FACS (
The present methods can be performed using stem cells, e.g., cells from a human embryonic stem cell line (e.g., H9, H1) or embryonic stem cell-like (ESC-like) induced pluripotent stem cells (iPSCs), e.g., generated from primary cells autologous to a subject to be treated using a method described herein. In some embodiments, the stem cells express markers of pluripotency, i.e., OCT4 and TRA1-60.
Methods for generating iPSC are known in the art. In some embodiments, the methods for generating hiPSC can include obtaining a population of primary somatic cells from a subject, e.g., a subject who is afflicted with PD and in need of treatment for PD. Preferably the subject is a mammal, e.g., a human. In some embodiments, the somatic cells are fibroblasts. Fibroblasts can be obtained from connective tissue in the mammalian body, e.g., from the skin, e.g., skin from the eyelid, back of the ear, a scar (e.g., an abdominal cesarean scar), or the groin (see, e.g., Fernandes et al., Cytotechnology. 2016 March; 68(2): 223-228), e.g., using known biopsy methods. Other sources of somatic cells for hiPSC include hair keratinocytes (Raab et al., Stem Cells Int. 2014;2014:768391), blood cells, or bone marrow mesenchymal stem cells (MSCs) (Streckfuss-Bömeke et al., Eur Heart J. 2013 September; 34(33):2618-29). In some embodiments, the primary cells (e.g., fibroblasts) are exposed to (cultured in the presence of) factors sufficient to induce reprogramming to iPSC. Although other protocols for programming can be used (e.g., as known in the art or described herein), in preferred embodiments the present methods can include introducing (contacting or expressing in the cell) four transcription factors, i.e., Oct4, Sox2, Klf4, and L-Myc, known colloquially as the as Yamanaka 4 factors (Y4F). See, e.g., Takahashi and Yamanaka, Cell. 2006; 126(4):663-676; Takahashi et al., Cell. 2007; 131(5):861-872; Yu et al. Science. 2007; 318(5858):1917-1920; Park et al., Nature. 2008; 451(7175):141-146. In some embodiments, the methods also include contacting or expressing in the cell one or more miRNAs, e.g., (i) at least one miR-302 cluster member and (ii) at least one miR-200 cluster member; see US 20160298089 and Song et al., J Clin Invest. 2020;130(2):904-920.
In the first phase of the protocol, lasting about 2 days, e.g., about 44-52 hours, e.g., about 48 hours, mesodermal cell fate is induced in the stem cells by culturing them in media suitable for culture of stem cells, e.g., media comprising growth factors, E8 media (comprising insulin, selenium, transferrin, L-ascorbic acid, FGF2, and TGFβ (or NODAL) in DMEM/F12, see Chen et al., Nat Methods. 2011 May; 8(5): 424-429), supplemented with a mesoderm inducer, e.g., bone morphogenetic protein 4 (BMP4) or BMP-4/7 heterodimers, e.g., at about 2-10 ng/ml, e.g., about 5 ng/ml), Activin A (e.g., at about 15-50 ng/ml, e.g., about 25 ng/ml), and a small molecule activator of WNT pathway, e.g., a GSK3beta inhibitor (e.g., at about 0.1-5 μM, e.g., about 1 μM), e.g. as shown in Table A, as well as WNT7A protein (e.g., at about 100-1000 ng/ml, e.g., about 500 ng/ml) and a low concentration of GABA (e.g., at about 2-7.5 μM, e.g., about 5 μM).
During about days 2-5 (e.g., the subsequent 110-130 hours, e.g., about 120 hours), vascular induction is promoted by a vascular inducing medium, preferably comprising E6 medium (i.e., Dulbecco's modified Eagle's medium [DMEM]/F12, ascorbic acid, sodium bicarbonate, selenium, human transferrin, and human insulin (Lippmann et al., Stem Cells 2014; 32:1032-1042) along with inhibiting TGFβ signaling (e.g., using a small molecule inhibitor of TGF-β/Smad signaling pathway, e.g., SB431542 (e.g., at about 2-7.5 μM, e.g., about 5 μM) as known in the art or described herein) and by addition of vascular endothelial growth factor-A (VEGF-A, e.g., at about 25-75 ng/ml, e.g., about 50 ng/ml), BMP4 (e.g., at about 25-75 ng/ml, e.g., about 50 ng/ml), FGF2 (e.g., at about 50-150 ng/ml, e.g., about 100 ng/ml), WNT7A protein (e.g., at about 250-750 ng/ml, e.g., about 500 ng/ml) and a low concentration of GABA (5 μM) are also maintained in the media during phase II.
Non-limiting examples of small molecule inhibitors of TGF-β/Smad signaling pathway include SB431542; LDN-193189; Galunisertib (LY2157299); LY2109761; SB525334; A-83-01; AUDA; PD 169316; BIBF-0775; ITD-1; SB505124; Dorsomorphin (Compound C) 2HCl; Pirfenidone; GW788388; LY364947; RepSox; LDN-193189 2HCl; Sulfasalazine; K02288; SD-208; TP0427736 HCl; LDN-214117; SIS3 HCl; LY 3200882; Vactosertib; DMH1; LDN-212854; ML347; Halofuginone;
and Dorsomorphin (Compound C), all of which are commercially available. Others are known in the art.
From about days 5-7 of differentiation, the cells are maintained in E6 media comprising growth factors (e.g., VEGF-A, e.g., at about 25-75 ng/ml, e.g., about 50 ng/ml and FGF2, e.g., at about 50-150 ng/ml, e.g., about 100 ng/ml) and GABA (e.g., at about 2-7.5 μM, e.g., about 5 μM). On day 7, differentiated CD31+GABRB3+ cells (usually >60% of total differentiated cells) can be isolated, e.g., by fluorescence activated cell sorting (FACS) (see, e.g.,
Also provided herein are populations of cells derived using a method described herein. These cells functionally replicate the natural cells, in that they migrate into the brain and form vasculature that provides a migration-promoting path for co-transplanted neurons, e.g., GABAergic interneurons. The cells express one or more endothelial cell markers, i.e., CD31 and/or von Willebrand Factor (vWF) and one or more periventricular endothelial cell markers, i.e., GABRB3, GABA, NKX2.1, PAX6, and/or ISL1. They do not express pluripotent markers, e.g., OCT4 or TRA1-60.
The general notion that it is not important which kind of human vasculature is used or that their purpose is to merely improve tissue survival and metabolic exchange is incorrect. Endothelial cells from different organs are not exclusively homogenous, responding to the metabolic demands of cell populations. Cell autonomous programs within CNS endothelial cells dictate complex aspects of their vascular function and specific neurovascular interactions12-15 that can be re-capitulated only by developing isogenic vasculature to induce precise structural organization. In that respect, human periventricular endothelial cells generated using a method as described herein have significant potential as a source of cells for transplantation, e.g., into the forebrain, and can be used for human brain development modeling and disease.
The human periventricular endothelial cells generated using the present methods provide a migration promoting corridor that help human GABAergic interneurons migrate long distances within shorter periods of time to integrate themselves with the host tissue, thereby providing greater significance for faster repair of brain damage. As shown herein, when transplanted into adult striatum, both endothelial cells and interneurons dispersed significantly and migrated both tangentially and long distance into the cerebral cortex, recapitulating the embryonic situation. Thus the embryonic stem cell-derived periventricular endothelial cells can be used in brain repair strategies, e.g., targeting different regions of the forebrain (e.g., as shown in
Thus for example, these cells can be used alone for induction of forebrain specific angiogenesis, for treatment of forebrain vascular diseases, cerebral ischemia or stroke, by transplantation into or near an affected area of the brain. Thus in cases where the vasculature is abnormal and there is reduced blood flow in the brain, cells generated using a method described herein can be used for transplantation treatment strategies.
Also provided herein is a co-transplantation strategy with both endothelial and neuronal cell types, with significant benefits for brain repair. The identification of the molecular components involved in forebrain GABAergic interneuron development triggered the efficient generation of GABAergic neuronal populations based on ES cell engineering (see, e.g., Kim et al., Stem Cells. 2014; 32:1789-804; Maroof et al., Cell Stem Cell. 2013; 12:559-72; Nicholas et al., Cell Stem Cell. 2013; 12:573-86). However, these GABAergic interneurons have been used in transplantation without their natural substrate or guide, causing them to stall at transplantation sites with an inability to migrate into regions that require new neurons.
In some embodiments, the human forebrain endothelial cells generated using a method described herein can be used in co-transplantation protocols with GABAergic interneurons or subtypes (i.e., neurons that are calretinin+, parvalbumin+, somatostatin+ or Neuropeptide Y+) and with glutamatergic projection neurons, or with precursors thereof. Methods for generating these neurons are known in the art, see, e.g., Kim et al., Stem Cells. 2014; 32:1789-804; Maroof et al., Cell Stem Cell. 2013; 12:559-72; Nicholas et al., Cell Stem Cell. 2013; 12:573-86. The cells can be transplanted into the appropriate brain region in the cerebral cortex (e.g., in the cingular, motor, somatosensory, piriform) or hippocampus. In preferred embodiments, the human periventricular endothelial cells are generated from autologous (patient-derived) iPSCs. As shown herein, forebrain endothelial cell therapy improved behavioral function in an animal model. Gabrb3ECKO mice, a model of psychiatric disorder with partial loss of endothelial cell-secreted GABA in the embryonic telencephalon, have reductions in both blood vessels and GABAergic interneurons; therefore, transplantation of interneurons only did not rescue the abnormal behavioral symptoms. Co-transplantation of both the human periventricular endothelial cells and GABAergic interneurons resulted in behavioral rescue.
Methods for transplantation of cells into the brain are known in the art. In some embodiments, the cells are administered by being implanted directly into or near the affected area of the subject's brain, e.g., unilaterally or bilaterally, e.g., into one or more of the appropriate brain regions in the cerebral cortex (e.g., in the cingular, motor, somatosensory, piriform) or hippocampus, e.g., using magnetic resonance imaging-guided stereotactic surgery. See, e.g., Garitaonandia et al., Stem Cells Dev. 2018 Jul. 15; 27(14):951-957; Kikuchi et al., Nature 548: 592-596 (31 Aug. 2017); MOrizane et al., Nature Communications 8:385 (2017); Sonntag et al., Prog Neurobiol. 2018 September; 168:1-20.
Although the present methods exemplify humans, the methods can also be used in other mammals, e.g., primates and non-primate veterinary subjects including cats, dogs, and horses.
Human periventricular endothelial cells generated using the present methods can be used in brain organoid technology. Lack of blood vessels within growing brain organoids limits their application, both with respect to disease modeling and in the context of clinical transplantation. The current technique available for vascularization of organoids involves complex in vivo grafting of organoids'. The present methods for co-culture with periventricular endothelial cells can be used to improve forebrain organoid development, compartmentalization and structure as well as minimize organoid to organoid growth and variability. Exemplary methods to prepare brain organoids are known in the art, see, e.g., Mansour et al., Nat Biotechnol. 2018; 36:432-41; Kelava and Lancaster, Dev. Biol. 420, 199-209 (2016); Lancaster et al., Nature 501, 373-379 (2013); Paca et al., Nat. Methods 12, 671-678 (2015); Renner et al., EMBO J. 36, 1316-1329 (2017); Yin et al., Cell Stem Cell 18, 25-38 (2016); Giandomenico and Lancaster, Curr. Opin. Cell Biol. 44, 36-43 (2017); Schwarz et al., Proc. Natl. Acad. Sci. USA 112, 12516-12521 (2015). In some embodiments, the organoids can be developed from iPSCs obtained from patients to provide patient specific or disease-specific models, which can provide insights into disease etiology and pathogenesis.
The following materials and methods were used in the Examples below.
Endothelial cell differentiation and culture: H9 cells (WiCell, Madison, WI) were maintained in E8 media (Thermo Fisher Scientific) on Matrigel (BD Biosciences)-coated plates and passaged once a week with 0.5 mM EDTA (Thermo Fisher Scientific) in PBS. On day 0 of differentiation, H9 cells were dissociated with Accutase (Sigma), and plated at a density of 105 cells/cm2 on Matrigel-coated plates. Cells were cultured for two days in E8 medium supplemented with BMP4 (5 ng/ml, Peprotech), Activin (25 ng/ml, Peprotech), CHIR 99021 (1 μM,) WNT7A (500 ng/ml, Peprotech), and GABA (5 μM, Sigma). Rock inhibitor, Y-27632 (1004, Selleck Chemicals) was added for first 24 hours to improve cell survival. On day 2, cells were switched to vascular inducing medium composed of E6 medium (Thermo Fisher Scientific) containing BMP4 (50 ng/ml), SB431542 (5 μM, Cayman Chemicals), GABA (5 μM), WNT7A (500 ng/ml), FGF2 (100 ng/ml, Peprotech) and VEGF-A (50 ng/ml, Peprotech). On day 5, cells were split at 1:6 ratio using Accutase and plated on Matrigel-coated plates in periventricular endothelial cell (PVEC) medium consisting of E6 with VEGF-A (50 ng/ml), FGF2 (100 ng/ml) and GABA (5 μM). On day 7, periventricular endothelial cells were isolated from the mixed population by fluorescence-activated cell sorting (FACS) and seeded on Matrigel-coated plates in periventricular endothelial cells medium at a density of 6×105 cells/cm2. For routine culturing, periventricular endothelial cells were dissociated using Accutase, and seeded on Matrigel-coated plates at a density of 6×105 cells/cm2 (high density seeding) or 1.2×105 cells/cm2 (low density seeding), with medium change every alternate day. Periventricular endothelial cells were cryopreserved after at least one passaging in freezing medium composed of 90% periventricular endothelial cells medium and 10% DMSO. As a source of neurons, we used human iPSC-derived GABAergic neurons from Cellular Dynamics (Madison, WI, Cat #R1013). As control for microarray analysis and for cell migration assays, we used human-iPSC derived endothelial cells from Cellular Dynamics (Madison WI, Cat #R1022), whose gene expression profile is similar to human aortic endothelial cells (HAECs; https://fujifilmedi.com/assets/CDI130925MIPTEC04.pdf). The human GABAergic neurons and human HAEC-like endothelial cells were cultured using the manufacturer's protocol. Cell Lines were routinely tested for mycoplasma contamination using a Mycoplasma Detection Kit (InvivoGen, San Diego, CA). Cells used in this study were verified to be mycoplasma free before undertaking any experiment with them.
Periventricular endothelial cell isolation by fluorescence activated cell sorting (FACS): On day 7 of differentiation, cells were dissociated with Accutase and filtered through a 35 μm nylon mesh cell strainer cap to obtain a single cell suspension. Cell suspension was washed with ice-cold FACS buffer (2% FCS and 0.1% NaN3 in PBS) and incubated with Fcγ blocker (BD Biosciences Pharmingen, 1 μg/ml) for 30 min. Cells were then washed in ice-cold FACS buffer and stained with PE/Cy7 anti-human CD31 antibody (BioLegend, Cat #303117) and anti-human GABRB3 antibody (Creative Diagnostics, Cat #DCABH-10376) conjugated with APC using an antibody conjugation kit (Abeam) for 1 hr. Cells were washed in ice-cold FACS buffer and CD31+GABRB3+ endothelial cells were isolated by fluorescence-activated cell sorting using a BD FACS Aria-II flow cytometer (BD Biosciences, San Jose, CA).
Gene expression profile analysis: Total RNA was extracted using the RNeasy plus minikit (Qiagen) according to manufacturer's instruction. RNA quality was determined using nanodrop and an Agilent 2100 bioanalyzer. Microarray hybridization was performed using the Human Gene Array 2.0ST gene chip (Affymetrix) at the Boston University Microarray and Sequencing Resource (BUMSR) Core, Boston, MA. Principal component analysis (PCA) was performed after normalizing gene-level expression values from CEL files of Affymetrix human gene 2.0 ST arrays by using the implementation of the Robust Multiarray Average (RMA) in the Affymetrix transcriptome analysis console (TAC) (v4.0.1, Applied Biosystem, Foster City, CA, USA). For exploratory group analysis, a Volcano plot, and a hierarchical clustering heatmap using TAC software were created after curating with a threshold parameter, 1.5-fold expression, P<0.05, FDRq<0.1. Relative Log Expression (RLE) and Normalized Unsealed Standard Error (NUSE) using the affyPLM package (version 1.34.0) and differential expression were assessed using the moderated (empirical Bayesian) t-test implemented in the limma package (v 3.14.4)44. Heatmap visualization was performed using Morpheus (Broad Institute, Boston, MA, USA). Violin plot visualization for expression level comparison of samples was generated with the log2 expression value using GraphPad Prism v8.0 (GraphPad Software, La Jolla California USA). The gene ontology for gene enrichment study, was performed in three GO TERM annotation categories by using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 with modified Fisher's exact test45 and was visualized using GraphPad Prism software.
Tube formation assay: For the Matrigel-based tube formation assay, 105 periventricular endothelial cells (at passage number P2 or P3) were suspended in 400 ul of periventricular endothelial cells medium and seeded in one well of 24-well culture plates precoated with growth factor-reduced Matrigel (BD Biosciences). Cells formed tubular structures within 24 hours which were imaged using light microscope. The ability of periventricular endothelial cells to form tubes in 3D was assessed using the Fibrin Gel In Vitro Angiogenesis Assay Kit (Chemicon). 5×105 cells per ml of medium were seeded in one well of a 24-well plate coated with a fibrin matrix as described by the manufacturer. After 24 hours, cells were covered with a second layer of fibrin and fresh medium added. Capillary tube networks formed inside the fibrin gel within two days and were imaged using a light microscope.
Sprouting assay: Sprouting of periventricular endothelial cells was observed using a fibrin gel bead assay. Briefly, 106 periventricular endothelial cells (at passage P2 or P3) were coated onto 2500 Cytodex beads and allowed to attach overnight. Next day, beads were embedded in fibrin gels in 24-well culture plates (500 beads/ml), and human primary lung fibroblasts (ATCC) were plated on top of the gel (20,000 fibroblasts/well). Budding and sprouting of endothelial cells from the beads were observed from day 2 onwards and lumen formation was visible from day 4.
Long-distance migration assay: In preparation for migration assays, two-well silicone culture inserts (from ibidi GmbH, Cat #80209) were converted into one-well inserts by cutting with a sharp, sterile blade. Individual one-well inserts were placed in the middle of a 35 mm dish coated with Poly-Ornithine and Laminin. The boundary of the insert was marked on the back of each dish with a thin marker. For this assay, periventricular endothelial cells, control endothelial cells and endothelial cells derived without GABA and WNT7A were used at passage number P2 or P3, while GABA interneurons cultured for six weeks were used. 104 periventricular endothelial cells or endothelial cells derived without GABA and WNT7A were seeded in each insert in periventricular endothelial cell medium without GABA. Same number of GABAergic interneurons or control endothelial cells were seeded per insert in their respective manufacturer's recommended medium. The insert was removed after 48 hours and cells cultured for five days. After 5 days, cells were fixed and fluorescently labeled with an anti-human CD31 antibody (for endothelial cells) or an anti-human β-Tubulin antibody (for neurons) and DAPI. The distance between each cell body and the edge of the insert- boundary was measured using ImageJ.
Co-culture migration assay: Periventricular endothelial cells or control endothelial cells at passage number P2 or P3, and GABA interneurons cultured for six-weeks were used for this assay. 3×104 GABAergic interneurons and 3×104 periventricular endothelial cells were co-suspended in 70 ul of co-culture medium (50% PVEC medium without GABA and 50% GABA neuron maintenance medium from Cellular Dynamics) and seeded in a one-well insert (prepared as described above) in a poly-Ornithine/Laminin coated 35 mm dish. As control, 3×104 GABAergic interneurons only or 3×104 GABAergic interneurons and the same number of control endothelial cells were co-seeded. Inserts were removed after 2 days, and co-culture was maintained for five days. After 5 days, cells were fixed and double-labeled with anti-human CD31 and anti-human β-Tubulin antibodies. Neuronal migration was assessed by measuring the distance travelled by β-Tubulin+ neurons from the day 0 mark using ImageJ software.
Chemo-attractivity assay: For this assay periventricular endothelial cells at passage number P2 or P3, control endothelial cells at passage P2, and GABA interneurons cultured for six weeks were used. Three-well culture inserts (ibidi GmbH, Cat #80369) were placed in the center of poly-Ornithine/laminin coated 35 mm dish. 3×104 GABA neurons were seeded in the center well. Equal number (104 cells) of periventricular endothelial cells and control endothelial cells were seeded in the two side wells. The inner edge of the neuronal well was demarcated at the back of the dish using a thin sharpie. Inserts were removed one day post-seeding, and cells were fixed after 36 hours. Cells were double labeled with anti-human CD31 and anti-human β-Tubulin antibodies and imaged. The chemo-attractive response of β-Tubulin+ neurons towards endothelial cells in each experiment were imaged and quantified using a scoring scheme modified from Won et al.13.
Migration assays with chemicals: To assess the roles of GABA or SDF-1/CXCL12 signaling on migration of human periventricular endothelial cells, 104 cells were seeded in one well insert and allowed to migrate in the presence of respective agonist or antagonist in periventricular endothelial cell medium (without GABA). After five days, cells were fixed, stained with anti-human CD31 antibody (Millipore), imaged and the distance migrated was calculated using ImageJ. To examine the effect of endothelial GABA or endothelial SDF-1/CXCL12 signaling on interneuron migration, human periventricular endothelial cells were seeded in 35 mm dish (105 cells/cm2) and incubated with respective chemical for a period of 48 hours. 3×104 GABAergic interneurons were seeded on top of the periventricular endothelial cells using a one-well insert, and allowed to migrate over the pre-incubated periventricular endothelial cells for 2 days. Migration of neurons was assayed by staining with anti-human β-Tubulin antibody (Biolegend) and imaged. The concentration of chemicals used in the assays are as follows: muscimol (Sigma) 100 uM, BMI (Sigma) 100 uM, AMD3100 (Sigma) 50 μM, recombinant human SDF-1α (Peprotech) 40 nM. The chemicals were kept at −20° as concentrated stock solutions and diluted on the day of the experiment.
Animals: Adult NOD-SCID mice (8 weeks old) were purchased from Charles River Laboratories, MA. Tie2-cre mice and Gabrb3 floxed (Gabrb3fl/fl) mice were obtained from Jackson Labs. The Tie2-cre transgene is known for uniform expression of cre-recombinase in endothelial cells during embryogenesis and adulthood14, 15. To selectively delete Gabrb3 in endothelial cells, Tie2-cre transgenic mice (males) were crossed to Gabrb3fl/fl mice (females) to generate Tie2-cre; Gabrb3fl/+ mice (males). These were further crossed with Gabrb3fl/fl mice (females) to obtain the Gabrb3 conditional knock-out (Tie2-cre; Gabrb3fl/fl mice). Animal experiments were in full compliance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the McLean Institutional Animal Care Committee (IACUC).
Stereotaxic surgery and cell transplantation: NOD-SCID mice, Gabrb3fl/fl mice and Gabrb3ECKO mice were housed on a 12 h light/12 h dark cycle and had free access to food and water throughout the study. 8 weeks old mice were used for all transplantations. Cells for transplantation were suspended in transplantation medium composed of DMEM/F-12 with no phenol red (Thermo Fisher Scientific), BDNF (10 ng/ml, Peprotech), GDNF (10 ng/ml, Peprotech), Rock-Inhibitor (10 uM), and Boc-Asp(OMe) fluoromethyl ketone (20 uM, Cayman Chemicals). For co-transplantation, periventricular endothelial cells or control endothelial cells derived without WNT7A and GABA (at passage P2 or P3; 50,000 cells/ul) and GABAergic interneurons (at 6 weeks of differentiation; 50,000 cells/ul) were suspended in a 1:1 ratio. For interneuron-only or endothelial cell-only transplants, cells were suspended at a concentration of 50,000 cells/ul. Before surgery, mice were anesthetized with 4% isoflurane, and kept under 2% isoflurane gas throughout the procedure. All microinjections were performed through a pulled borosilicate glass pipette with a long, gently tapering shank using an UMP microsyringe pump (World Precision Instruments) and a Kopf stereotaxic frame (Kopf Instruments, CA). 1 μL of cell solution were injected at a rate of 0.125 μL/min. After each injection, the microcannula remained in position for 5 min before withdrawing slowly to avoid back-flow of cells. Injection coordinates were: for striatum-bregma: 0.49 mm, ventral: −3.0 mm, lateral: −1.8 mm; for neocortex-bregma: 0.49 mm, ventral: 1.8 mm, lateral: 2.0 mm. Transplanted Gabrb3ECKO mice received subcutaneous injections of cyclosporine (35 ul of 50 mg/ml stock, Perrigo), beginning two days before surgery, and continuing every day until mice were sacrificed. Transplanted mice were terminally anesthetized with a Ketamine/Xylazine cocktail (100 mg/kg and 10 mg/kg, respectively) and perfused intracardially with cold 4% formaldehyde for cryo-processing and immunohistochemistry (IHC), or with a zinc fixative (BD Biosciences Pharmingen) for paraffin histology and IHC.
Immunohistochemistry (IHC): For frozen section IHC, PFA-perfused brains were post-fixed in cold 4% formaldehyde for 48 hours, cryo-protected in a sucrose gradient, flash frozen in dry ice, and cryo-sectioned into 40 μm coronal sections. For immunostaining, sections were washed once with PBS, blocked in FBS containing 0.5% Triton X100 for 1 hour, and incubated with the primary antibody overnight at 4° C. The following day, slides were washed six times with PBS at room temperature, incubated with secondary antibodies (Alexa-568 and Alexa-488, 1:400) for 2 hours at room temperature, washed with PBS six times, and mounted onto slides with a DAPI-containing mounting medium (Vectashield). For paraffin IHC, brains were post fixed in Zinc fixative (BD Biosciences Pharmingen) for 48 hours, dehydrated in an alcohol gradient (70%, 80%, 95%, 100%), cleared in Xylene, embedded in paraffin wax, and sectioned into 8 um coronal sections. Prior to immunostaining of paraffin sections, tissue was deparaffinized and antigen retrieval was performed in a pH 9 solution (DAKO) at 96° C. Primary antibodies used for IHC were as follows: anti-human CD31 (1:100, Biolegend), anti-human vWF (1:100, Sigma), anti-human nuclei (1:100, Rockland), anti-human mitochondria (1:100, Millipore), anti-human β-TUBULIN (1:2000, Biolegend), anti-GABA (1:1000, Sigma), anti-GABRB3 (1:200, Sigma), anti-Caspase (1:50, Millipore), anti-Claudin5 (1:200, Sigma), anti-human Ki67 (1:50, Thermo Fisher Scientific), anti- ZO-1 (1:400, Thermo Fisher Scientific), isolectin B4 (1:50, Sigma), anti-NKX2.1 (1:250, Abcam), anti-OCT4 (1:200, SCBT), ant-TRA1-60 (1:200, Millipore). The secondary antibodies used were Alexa-594 and Alexa-488 (1:400, Thermo Fisher Scientific).
Immunocytochemistry: Cells were grown to 70-80% confluency on coverslips, fixed in 4% PFA for 15 minutes at room temperature, blocked in blocking solution (PBS supplemented with 1% Bovine Serum and 0.25% Triton X) for 1 hour at room temperature, and incubated with the primary antibodies overnight in 4° C. Next day, cells were stained with a secondary antibody (Alexa-594 or Alexa-488, 1:500, Thermo Fisher Scientific) for 2 hours at room temperature and mounted using ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). The primary antibodies were same as that for IHC and used at same dilutions (mentioned above), except for anti-human CD31 which was purchased from Millipore and used at a dilution of 1:100.
H and E staining: Brains were post fixed in a Zinc fixative (BD Biosciences Pharmingen) for 48 hours, dehydrated in an alcohol gradient (70%, 80%, 95%, 100%), cleared in Xylene, embedded in paraffin wax, and sectioned into 8 um coronal sections. Briefly, slides were deparaffinized in Xylene, hydrated in ethanol gradient (100%, 95%, 70%), rinsed in tap water, stained with Hematoxylin for 1 minute, rinsed again in tap water, stained in Eosin for 30 secs, dehydrated in an ethanol series (70%, 95%, 100%) followed by xylene, and mounted onto glass slides in permount (Sigma).
Microscopic analysis and cell counting: Twenty sections from each brain were used for IHC and histology experiments. All low and high magnification images were obtained with a FSX100 microscope (Olympus). Counting of human nuclei+ cells in each type of transplant was performed using ImageJ. The number of transplanted neurons that had migrated into the cerebral cortex was obtained by counting human nuclei+ and β-tubulin+ cells using the ImageJ software. Blinding was performed during cell counting analysis.
Behavioral experiments: All behavioral tests were done during the light phase of the light/dark cycle. Before behavioral testing, mice were acclimatized to the testing room for 1 hour. Behavioral assays were performed according to established protocols referenced here: nest building with shredded paper46, self-grooming47, light-dark box48, tail suspension test49, and three-chamber social interaction test50. Both males and females were used for all behavioral assays. Experimenters scoring behaviors were blinded to the genotypes. Results were analyzed using Student's t-test, and one-way ANOVA. Data is presented as mean±standard deviation. Values of p<0.05 were considered statistically significant.
ELISA: ELISA was used to detect and compare GABA levels released from human interneurons-only, human periventricular endothelial cells-only and periventricular endothelial cells-neuron co-culture. Periventricular endothelial cells-only and interneuron-only cultures were prepared by seeding cells in a 12 well plate at a density of 105 cells/cm2. The endothelial and interneuron co-culture was prepared by co-seeding both cells at a 1:1 ratio with a final density of 105 cells/cm2. Supernatants from cells were collected after 96 hours and stored at −80° C. GABA concentrations were quantitatively determined by competitive ELISA according to the manufacturers' protocol (GABA Research ELISA kits, Labor Diagnostica Nord, Germany), and absorbance was measured using a multiplate microplate fluorescence reader (Molecular Devices, CA) at 450 nm.
Quantitative real-time PCR: Total RNA was prepared using the RNeasy plus minikit (Qiagen). cDNA from total RNA was generated using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific) as per manufacturer's protocol. PCR reactions were run on a CFX96 Touch Real Time PCR (Bio-Rad) with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). Primers for qPCR were obtained from Thermo Fisher Scientific. The relative expression level for each gene was normalized to that of GAPDH gene and subsequent fold changes were determined according to published methodology51.
Western Blot: Cell lysates were prepared in standard radioimmunoprecipitation (RIPA) buffer containing 1× protease Inhibitor cocktails, and protein was quantified using Bio-Rad protein quantification assay. Cell lysates were then separated in a 12% gel and immunoblotted onto a nitrocellulose (NC) membrane using the NOVEX SDS electrophoresis unit. Membrane was blocked for 1 hour at RT using the Odyssey blocking buffer (Licor), and incubated with primary antibodies; anti-CD31 (Thermo Fisher) and anti-VE-cadherin (Sigma) and anti-GAPDH (Proteintech) overnight at 4° C. Next day, membranes were washed and incubated with secondary antibodies, anti-mouse IRD 800 and anti-rabbit IRD 680 for 1 hour at RT in dark. Fluorescent signal was detected using Licor detection system and data analyzed using the lmageLite studio software.
Statistical analysis: All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla CA). Power analysis was performed using Java Applets for Power and Sample size. Simple random sampling was used. Sample sizes assigned to the same experimental groups were equal. The exact sample size (n) for each experimental group is reported in individual figure legends. For in vitro assays, the number of cells examined, is mentioned in respective methods sections. For histology and IHC experiments, twenty sections from each brain were used for each experimental group. Uniform penetration of antibodies or stains throughout the section was ascertained and quality of the staining in each digital section was examined. Only those sections which showed uniform labeling were included in further analysis. Blinding was used during cell counting analysis and behavioral experiments. No data were excluded. Statistical significance of differences between groups was analyzed by two-tailed Student's t test (Prism; GraphPad software) and has been noted in individual figure legends. Significance was reported at p<0.05.
Brain endothelial cells have been derived from human pluripotent stem cells previously39-42, but their analysis, gene expression studies and functions have largely focused on the blood-brain barrier attributes. Generation of embryonic forebrain specific periventricular endothelial cells has not been reported so far. Periventricular endothelial cells show enrichment in genes controlling neurogenesis, neuronal migration, chemotaxis, and axon guidance and can be distinguished from other endothelial cells by their specific transcription factor expression, signaling molecules and extracellular receptors12-15. Indeed, this specific population of endothelial cells houses a novel GABA signaling pathway that is distinct from the classical neuronal GABA signaling pathway in the embryonic forebrain. Vasculature is autonomous with respect to anatomy, patterning, gene expression, developmental regulation and function in different organs and/or regions at different time points. Developing strategies to generate embryonic forebrain-like vasculature will augment its region-specific vessel growth and function after transplantation and will be important for clinical applications. Based on our knowledge of gene expression of mouse periventricular endothelial cells of the embryonic forebrain, we first established a differentiation strategy to generate human periventricular endothelial cells from human ES cell lines (
To test the efficacy and specificity of our differentiation protocol, we performed microarray analyses of H9 ES cells, human periventricular endothelial cells (
We further assessed the expression of periventricular endothelial cell markers in purified human periventricular endothelial cell cultures by immunocytochemistry. All H1-derived periventricular endothelial cells in culture (100%) expressed endothelial cell markers—CD31, (
Next, we performed cell migration assays to test the long-distance migratory potential of human periventricular endothelial cells and its role in guiding human interneuron migration (
Next, we investigated whether human periventricular endothelial cells could facilitate human interneuron migration in vivo. To this end, we transplanted human periventricular endothelial cells along with human interneurons in a ratio of 1:1 into the striatum of adult NOD-SCID mice, on each side of the brain (
GABA secreted from mouse periventricular endothelial cells plays both autocrine and paracrine roles. It enhances the migration of periventricular endothelial cells, triggers angiogenesis, and promotes robust migration of interneurons13, 15. We next studied whether GABA signaling from human periventricular endothelial cells also performed these functions (
In order to test the functional significance of co-transplantation of human periventricular endothelial cells with interneurons, when compared to interneuron-only transplants, we used a well-established pre-clinical model of psychiatric disorder—the Gabrb3 endothelial cell conditional knockout model (Gabrb3ECKO mice), in which there are both vascular and GABAergic interneuron deficits in the cingulate, motor, and somatosensory cortex15. The developmental dysfunction of endothelial GABAA receptors and reduction in endothelial GABA levels significantly impaired angiogenesis and GABAergic interneuron migration in the embryonic brain that persisted in the adult brain (
Subsequently, we evaluated the impact of the cell transplantation and migration on behavioral function in adult mice of interneuron-only transplanted group, interneuron+control endothelial cell transplanted group and interneuron+periventricular endothelial cell co-transplanted group, one month after transplantation. We compared the results with two groups, Gabrb3ECKO mice and Gabrb3fl/fl mice with sham surgery (
To confirm that the rescue of behavioral deficits in interneuron+periventricular endothelial cell co-transplanted Gabrb3ECKO mice was due to the dispersion of GABAergic interneurons facilitated by periventricular endothelial cells, and not due to GABA released by periventricular endothelial cells only, we assessed behavioral function of periventricular endothelial cells-only transplanted Gabrb3ECKO mice. Though there was widespread distribution of human periventricular endothelial cells in the cortex, these mice continued to show behavioral deficits that were comparable to the Gabrb3ECKO mice. Periventricular endothelial cell—only transplanted mice continued to show behavioral deficits that was comparable to Gabrb3ECKO mice and interneurons only—Gabrb3ECKOmice. They had poor nest building ability, long immobility time in tail suspension test, high self-grooming time, high exploration time in dark in light-dark box test assay, and poor social interaction ability.
Together, these behavioral assays show that co-transplantation of periventricular endothelial cells and interneurons rescue behavioral deficits of Gabrb3ECKO mice within one month of transplantation, as opposed to interneuron-only, periventricular endothelial cell-only or interneuron+control endothelial cell transplants that do not show a rescue effect during the same duration.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/923,512, filed on 19 Oct. 2019. The entire contents of the foregoing are incorporated herein by reference.
This invention was made with Government support under Grant Nos. MH110438 and NS100808 awarded by the National Institutes of Health. The Government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/056366 | 10/19/2020 | WO |
Number | Date | Country | |
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62923512 | Oct 2019 | US |