WO2024173561A1 - Neural spheroids - Google Patents

Abstract

The present disclosure provides a method of preparing neural spheroids, the neural spheroid prepared by the method, and uses of the neural spheroids. In some embodiments, the present disclosure provides a method of forming neural spheroids, a three dimensional network of cells comprising neural and glial cells, where the method comprises culturing differentiated fore brain progenitor cells in a cell culture well having an ultra-low attachment surface and shaped to promote self-assembly of cells into neural spheroids.

Description

NEURAL SPHEROIDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/484,975, filed February 14, 2023, which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The present disclosure provides methods of preparing three dimensional neural spheroids, neural spheroids prepared by the method, and methods of using the neural spheroids for assessing disease processes, screening of therapeutic agents, and assessing efficacy of therapeutic agents.

BACKGROUND

[0003] Neurons and glial cells in the brain form a complex three-dimensional network, creating a microenvironment that can affect cell viability, proliferation, differentiation, protein expression, and cellcell interactions. Neural cell cultures grown on regular cell culture dishes spread out on a flat substrate to fonn a two-dimensional (2D) network, where the cells do not substantially interact and lack the three dimensional extracellular matrix present in the brain. Use of three-dimensional (3D) in vitro neural models provide an alternative that is closer to mimicking the neural structures, cellular organization, and microenvironments present in the brain. Historically, three dimensional neural cultures were developed using scaffolds, wherein complex cell compartmentalization and organization were achieved by scaffold design.

[0004] To overcome the limitations with scaffolds, neural cells have been cultured through self-assembly into spheroid structures, where the spheroids produce an extracellular matrix and the neural cells can approximate in vivo-like cell behavior. These in vitro generated three dimensional neural models generally use progenitor cells and numerous differentiation steps to form neural and glial cells assembled in a spheroid. While numerous three dimensional in vitro models of the nervous system have been generated by such methods, desirable are other straightforward methods for generating neural spheroids.

SUMMARY

[0005] The present disclosure provides a method of fonning neural spheroids, a three dimensional network of cells comprising neural and glial cells, where the method comprises culturing differentiated forebrain progenitor cells in a cell culture well having an ultra-low attachment surface and shaped to promote self-assembly of cells into neural spheroids. In some embodiments, the differentiated forebrain progenitor cells are prepared from iPSC cells by inducing the iPSC cells to differentiate into neural progenitor cells, which are then expanded and then induced to differentiate into the forebrain progenitor cells.

[0006] In some embodiments, a method of preparing a neural spheroid comprises: inducing differentiation of fibroblasts into iPSC cells; differentiating the iPSC cells to neural progenitor cells; selecting rosettes of the neural progenitor cells; expanding the neural progenitor cells from the selected rosettes of neural progenitor cells; differentiating the expanded neural progenitor cells to forebrain progenitor cells, and culturing the differentiated forebrain progenitor cells in a V or U bottom shaped cell culture well having ultra-low attachment surface to produce a neural spheroid.

[0007] In some embodiments, the iPSC cells for preparation of the neural spheroid are prepared from cells obtained from a normal, healthy subject. In some embodiments, the iPSC cells for preparation of the neural spheroids are obtained from a subject afflicted with a disease or condition of interest affecting neural cells. In some embodiments, the iPSC cells are prepared from any cell that can be induced to iPSC cells, including but not limited, fibroblasts (e.g.. skin or embry onic fibroblasts), adipose tissue cells, or hair follicle cells.

[0008] In some embodiments, the neural spheroid is prepared from cells obtained from a subject afflicted with a disease affecting neural cell function, including, among others, Fabry disease, Pompe disease, Gaucher disease, GM1 gangliosidosis, Wolman disease/CESD, mucopolysaccharidosis type I; (MP1), mucopolysaccharidosis II (MPS II), mucopolysaccharidosis type III (MPS III), Morquio A Syndrome; MPS IV, Tay-Sachs disease, acid sphingomyelinase deficiency, or Niemann-Pick disease.

[0009] In another aspect, the present disclosure provides a neural spheroid prepared by the method described herein. In some embodiments, the neural spheroid is characterized by one or more of (a) three dimensional extracellular matrix, (b) neural axonal and dendritic extensions, (c) expression of marker proteins TUJ1, GFAP. SIOOB, NEUN, TBR1. PAX6, MAP2 FOXG1. and/or NESTIN, and (d) presence of electrical activity.

[0010] In some embodiments, the neural spheroids provide a platform for studying behavior and interactions of neural and glial cells in a three-dimensional microenvironment, particularly in diseases or conditions affecting the nervous system. In some further embodiments, the neural spheroid is used for screening of candidate therapeutic agents; assessing effectiveness of a therapeutic agent (e.g., enzyme replacement therapy); and assessing effectiveness of gene therapy (e.g., tropism of viral gene therapy vectors, expression in neural and/or glial cells, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 provides a photograph of neural spheroids assayed for CNS marker expression by immunofluorescence. Neural spheroid were prepared from normal cells (GM23279, left column) or GM1 gangliosidosis cells (GM05653, right column) shown at day 32 post-differentiation. Top row: TUJ1 in red shows the presence of neuronal axons and GFAP shown in green confirms the presence of astrocytes. Second row: Red MAP2 expression confirms neuronal dendrites. Third row: SIOOB in green is a pan-glial cell marker. Fourth row: TBR1 in red confirms a cortical neuron identity. Fifth row: PAX6 expression in green shows the presence on neural progenitor cells. Scale bar = 500 pm. GM23279 cells were obtained from the Coriell Institute for Medical Research.

[0012] FIG. 2 provides a photograph of neural spheroids transduced with various AAVs carrying the gene encoding green fluorescent protein (GFP) at day 25 of differentiation. Transduction and recovery was carried out at 1E+5 MOI per vector over the course of one week. Spheroids were then fixed and stained for GFP and MAP2, which marks neurons. Scale bar = 500 pm.

[0013] FIG 3 provides a photograph of neural spheroids assayed for p-galactosidase activity using a fluorescence enzyme activity kit (Panel A). Scale bar = 500 pm. Color scale shown on right. Age matched neural spheroids were stained by immunofluorescence for GM1 ganglioside (Panel B).

DETAILED DESCRIPTION

[0014] Tire present disclosure provides a method of generating neural spheroids and neural spheroids prepared by the method. In some embodiments, the neural spheroids provide an in vitro system for studying diseases affecting central nervous system cells, examining toxicity of agents, screening of candidate therapeutic agents, and examining efficacy of therapeutic agents.

[0015] In one aspect, the present disclosure provides a method of preparing neural spheroids that have a three-dimensional network of neural cells. In some embodiments, a method of preparing a neural spheroid comprises at least the step of culturing differentiated forebrain progenitor cells in a V or U shaped cell culture well, where the culture well has an ultra-low attachment surface. The cells are cultured until a three-dimensional network of neural cells are formed. The ultra-low attachment surface does not allow cell to attach, thereby promoting cells to grow together and self-assemble. It is to be understood that the culture well shape is not limited to a U or V shaped well and can be any shape that promotes self-assembly of cells.

[0016] In some embodiments, the ultra-low attachment surface is achieved by coating the cell culture surface with a hydrophilic hydrogel, such as poly-2 -hydroxyethyl methacrylate (polyHEMA) or with agar (see, e.g., Gao et al.. Stem Cell Res Ther. 2018, 9:243). Culture plates with ultra-low attachment surfaces are commercially available (e.g., Coming, USA).

[0017] In some embodiments, the neural spheroid expresses one or more markers of extracellular matrix, and/or one or more markers of neural and/or glial cells, as further described below. In some embodiments, the three dimensional network of neuronal cells are characterized by axonal and dendritic extensions. In some embodiments, the neural cells in the neural spheroid are electrically active.

[0018] In some embodiments, the forebrain progenitor cells are cultured for at least 8 days or more, at least 10 days or more, at least 20 days or more, at least 30 days or more, or at least 60 days in the culture well to allow grow th and self-assembly of the forebrain progenitor cells.

[0019] In some embodiments, the forebrain progenitor cells are prepared by differentiating neural progenitor cells to forebrain progenitor cells. Methods of differentiating neural progenitor cells to forebrain progenitor cells are described in. among others, Bell et al.. Bio Protoc. 2019 9(5): e3188 and Yuan et al., Sci Rep 5: 18550. In some embodiments, the differentiation of neural progenitor cells to forebrain progenitor cells are induced by the addition of factors that promote differentiation (see, e.g., Bell ct al., Bio Protoc., 2019, 9(5): c3188; incorporated by reference herein). Kits for generation of forebrain neurons are also available commercially, e.g., STEMdiff™ Forebrain Neuron Differentiation Kit.

[0020] In some embodiments, the neural progenitor cells are prepared from iPSC cells (induced pluripotent stem cells) by differentiating the iPSC cells to neural progenitor cells. Neural progenitor cells are generally cells that are multipotent cells capable of differentiating into various neuronal lineages, including neuronal and glial cell types. In some embodiments, differentiation of iPSC cells to neural progenitor cells is achieved by culturing iPSC cells in presence of dual SMAD inhibitors, e.g., Noggin and SB431542 (Chambers et al., Nat Biotechnol., 2009, 27(3): 275-280).

[0021] In some embodiments, the iPSC cells selected for preparing neural progenitor cells express pluripotency markers, such as SSEA, OCT4, TRA-1-60. and NANOG. Other pluripotency markers that can be used for selection of iPSC cells include DNMT3b, EST2, and ZFP42. Presence of the pluripotency markers can be done by antibody detection or qPCR.

[0022] In some embodiments, the iPSC cells are obtained from healthy human volunteers or mammals of interest. In some embodiments, the iPSC cells are prepared from cells obtained from a subject afflicted with a disease or condition affecting the nervous system. In some embodiments, iPSC cells are prepared from fibroblasts obtained from a subject. In some embodiments, fibroblasts from a subject are cultured in the presence of a mixture of factors Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28 to produce iPSC cells (see, e.g., Ghaedi et aL, Methods Mol Biol., 2019; 1576:55-92; Rivera et al.. Current Protocols in Stem Cell Biology, 2020, 54:el 17). In some embodiments, at least the factors Oct4/Sox2/Klf4, which can be provided by viral transduction, are used to reprogram a fibroblast into iPSC cells. In some embodiments, other cells, such as adipose tissue cells or hair follicle cells, may be used for the production of iPSC cells. In some embodiments, the fibroblasts, when used for preparation of iPSC cells, can be skin fibroblasts or embryonic fibroblasts.

[0023] In some embodiments, the cells (e.g., fibroblasts) for preparing the iPSC cells, and thus the neural spheroids, can be obtained from subjects afflicted with a disease affecting nervous system cells. including, among others, Fabry disease, Pompe disease, Gaucher disease, Tay-Sachs disease, MPS III or Sanfilippo syndrome, acid sphingomyelinase deficiency, Niemann-Pick disease type C, and neuronal ceroid lipofuscinoses.

[0024] In some embodiments, a method of preparing a neural spheroid comprises at least the steps of differentiating neural progenitor cells to forebrain progenitor cells, and culturing the differentiated forebrain progenitor cells in V or U a bottom shaped culture well with ultra-low attachment surface to produce a neural spheroid.

[0025] In some embodiments, a method of preparing a neural spheroid comprises: inducing differentiation of fibroblasts into iPSC cells; differentiating the iPSC cells to neural progenitor cells; selecting rosettes of the neural progenitor cells; expanding the neural progenitor cells from the selected rosettes of neural progenitor cells; differentiating tire expanded neural progenitor cells to forebrain progenitor cells, and culturing the differentiated forebrain progenitor cells in a V or U bottom shaped cell culture well having ultra-low attachment surface to produce a neural spheroid.

[0026] In some embodiments, the present disclosure also provides a neural spheroid prepared by the methods described herein. In some embodiments, the neural spheroid is characterized by one or more of the following: (a) three dimensional extracellular matrix, (b) axonal and dendritic extensions, (c) expression of marker proteins TUJ1, GFAP, SIOOB, NEUN, TBR1, PAX6, MAP2 FOXG1, and/or NESTIN, and (d) presence of electrical activity. TUJ1 is a marker for neuronal axons; GFAP is a marker for astrocytes and glial cells; SIOOB is a pan-glial cell marker; NEUN is a marker for neurons; TBR1 is a marker for cortical neurons; PAX6 is a marker for neural progenitor cells; MAP2 is a marker for neuronal dendrites; FOXG1 is a marker for forebrain cell differentiation; and NESTIN is a marker of neural stem/progenitor cells. In some embodiments, the extracellular matrix is characterized by the presence of one or more extracellular matrix marker, e.g., ECMI, ECM2, ECM3, ECM4, ECM5, ECMG. ECM7, ECM8. and/or ECM9. In some further embodiments, the neural spheroid is characterized by presence of neurotransmitters. In some embodiments, the neural spheroid is provided in an array.

[0027] In some embodiments, the neural spheroids prepared by the methods are derived from cells of a subject having a disease affecting central nervous system cells. In some embodiments, the neural spheroid have disease characteristics of Fabry disease, Pompe disease, Gaucher disease, Tay-Sachs disease, MPS III or Sanfilippo syndrome, acid sphingomyelinase deficiency, Niemann-Pick disease type C, or neuronal ceroid lipofuscinoses.

[0028] In some embodiments, the neural spheroids are used for, among others, study of disease processes; screening of candidate therapeutic agents; testing effectiveness of a therapeutic agent (e.g., enzyme replacement therapy): and assessing effectiveness of gene therapy (e.g.. tropism of viral gene therapy vectors, expression in neural and/or glial cells, etc.).

[0029] In some embodiments, the neural spheroid is derived from cells obtained from a subject with a disease or condition, and neural spheroid is assessed for disease phenotype. In some embodiments, the disease or condition is Fabr ' disease, Pompe disease, Gaucher disease, GM1 gangliosidosis, Wolman discasc/CESD, mucopolysaccharidosis type I; (MP1), mucopolysaccharidosis II (MPS II), mucopolysaccharidosis type III (MPS III). Morquio A Syndrome; MPS IV, Tay-Sachs disease, acid sphingomyelinase deficiency, or Niemann-Pick disease. By way of example and not limitation, the disease condition assessed in the neural spheroid in Pompe disease includes, among others, acid alphaglucosidase A activity, glycogen accumulation, increased lysosomal staining, and lipid buildup. In some embodiments, the disease condition assessed in the neural spheroid in Fabry' disease include, among others, alpha-galactosidasc A activity, globotriaosylccramidc levels, and presence of neurotransmitters (e.g., acetylcholine). In some embodiments, the disease condition assessed in the neural spheroid in Gaucher disease includes, among others, glucocerebrosidase activity, glucosylceramide levels, and aggregation of a-synuclein. In some embodiments, the disease condition assessed in the neural spheroid in GM1 gangliosidosis includes, among others, beta-galactosidase activity, levels of GM1 ganglioside, presence of activated glia (esp., microglia), and levels of dopaminergic, glutamatergic, and cholinergic neurons. The disease conditions of other diseases are known such that the neural spheroids prepared from a subject with the disease can be assessed for the known cellular phenotypes.

[0030] In some embodiments, the neural spheroids are used to screen for therapeutic agents for treating diseases affecting nervous system cells. In some embodiments, a method of screening therapeutic agents comprises contacting a neural spheroid prepared by the method disclosed herein with a candidate therapeutic agent, and determining effect of the candidate therapeutic agent on one or more disease characteristics in the neural spheroid, wherein the neural spheroid is prepared from cells obtained from a subject afflicted with the disease. In some embodiments, the therapeutic agent is a small molecule compound, an RNA therapeutic (e.g., shRNA or anti-sense RNA), or a gene therapy therapeutic agent. In some embodiments, the neural spheroid used in the screening of candidate therapeutic agent is derived from cells obtained from a subject with Fabry disease, Pompe disease, Gaucher disease, GM1 gangliosidosis, Wolman disease/CESD, mucopolysaccharidosis type I; (MP1), mucopolysaccharidosis II (MPS II), mucopolysaccharidosis type III (MPS III), Morquio A Syndrome: MPS IV, Tay-Sachs disease, acid sphingomyelinase deficiency, or Niemann-Pick disease.

[0031] In some embodiments, the neural spheroid is used to assess the effectiveness of enzyme replacement therapy for disease associated with deficiencies in an enzyme or protein that affect neural cells. In some embodiments, the neural spheroid is used to assess uptake of exogenous enzyme, and/or effectiveness of the exogenous enzyme. In some embodiments, a method of assessing replacement enzyme therapy comprises contacting a neural spheroid prepared by the method disclosed herein with an exogenous enzyme, and assessing uptake of the exogenous enzyme, and/or activity of the exogenous enzyme in the neural and/or glial cells of the neural spheroid. In some embodiments, the neural spheroid is prepared from normal healthy cells. In some embodiments, the neural spheroid is derived from disease cells or cells obtained from a subject afflicted with the disease of interest. In some embodiments, the exogenous or replacement enzyme is, among others, glucocerebrosidase (Gaucher disease), acid alphaglucosidase (Pompe disease), alpha-galactosidase A (Fabry disease), beta-galactosidase (GM1 gangliosidosis), lysosomal acid lipase (Wolman disease/CESD). alpha-L-iduronidase (mucopolysaccharidosis type 1; MP1). iduronate-2-sulfatase (mucopolysaccharidosis II: MPS II), N- sulfoglucosamine sulfohydrolase (Mucopolysaccharidosis type III: MPS III), N-acetylgalactosamine-6- sulfatase (Morquio A Syndrome; MPS IV), and glycosaminoglycan N-acetylgalactosamine 4-sulfatase (MPS VI).

[0032] In some embodiments, the neural spheroid is used to assess the presence of neutralizing antibodies to the exogenous enzyme. In some embodiments, the exogenous enzyme is treated with scrum or antibodies prepared from a subject, the enzyme-antibody complexes removed, e.g., via affinity separation (Protein A coated beads), and the resulting exogenous enzyme composition examined for uptake by and activity levels in a neural spheroid.

[0033] In some embodiments, the neural spheroids are used to study delivery and effectiveness of gene therapy vectors. In some embodiments, the neural spheroid is used to assess tropism of gene therapy viral vectors to neural and glial cells in the neural spheroids. In some embodiments, the method comprises contacting a neural spheroid with a gene therapy vector, and assessing presence or delivery of the gene therapy vector in cells of the neural spheroid. In some embodiments, the presence of gene therapy vectors can be determined by qPCR or expression of a reporter transgene (e.g., fluorescent proteins, such as GFP or EGFP). In some embodiments, the gene therapy viral vector comprises adeno-associated viral vector (e.g., based on AAVi, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13), lentiviral vectors (e.g., based on human immunodeficiency virus (HIV), feline immunodeficiency virus, or equine infectious anemia virus), adenoviral vectors, retroviral vectors, or herpes simplex viral vectors. In some embodiments, the gene therapy delivery vehicle is a non-viral delivery vehicle, where a gene therapy vector polynucleotide is formulated in, among others, liposomes, cationic polymers, dendrimers, or conjugated to cell -penetrating peptides.

[0034] In some embodiments, following contacting with the gene therapy vector, the neural spheroid is accessed for expression of a transgene of interest. In some embodiments, the neural spheroid for assessing transgene expression is obtained from a normal or healthy subject. In some embodiments, the neural spheroid for assessing transgene expression is obtained from a subject with the disease or condition targeted by the gene therapy. In some embodiments, the transgene comprises a recombinant polynucleotide encoding, for example, glucocerebrosidase, acid alpha-glucosidase, alpha-galactosidase A, beta-galactosidase, lysosomal acid lipase, alpha-L-iduronidase, iduronate-2 -sulfatase, N-sulfoglucosamine sulfohydrolase, N-acetylgalactosamine-6-sulfatase, and glycosaminoglycan N-acetylgalactosamine 4- sulfatase. Other transgenes for use with the neural spheroids including, among others, non-coding RNA (e.g., shRNA/siRNA, miRNA, anti-sense RNA, etc.), structural proteins, signaling proteins, and polypeptide ligands.

EXAMPLES

[0035] Tire following Examples, including experiments and results achieved, arc provided for illustrative purposes only and are not to be construed as limiting the present invention.

Example 1

Preparation of Neural Spheroid

[0036] iPSC cultures were maintained under standard conditions and grown in mTeSR plus with antibiotics on Matrigel coated culture ware. Tire iPSC cultured cells are passaged with standard methods.

[0037] The iPSC cells were differentiated into neural progenitors using StemCell Technologies STEMdiff™ SMADi Neural Induction Kit (Embryoid body-based option; Stem Cell Technologies). Antibiotics/antimycotics w ere included in the culture medium.

[0038] Rosette selection w as performed according to the SMADi kit, and the selected cells expanded to prepare neural progenitor cell (NP) cultures with neural progenitor media from Stem Cell Technologies. [0039] The cultured neural progenitor cells were then differentiated into forebrain progenitors using STEMdiff™ Forebrain Neuron Differentiation Kit (Stem Cell Technologies). Antibiotics/antimycotics were included in the culture medium.

[0040] The forebrain progenitor cells were the placed into a Coming Ultra-low attachment 96-well spheroid plate. Cell number was approximately that suggested in the protocol (~2E4 cells). As in the previous step, antibiotics/antimycotics were included in the cell culture medium.

[0041] Tire forebrain progenitor cells were cultured using STEMdiff™ Forebrain Neuron Maturation Kit (Stem Cell Technologies) for 20 days in presence of antibiotics/antimycotics to allow maturation into neurospheres.

Claims

CLAIMS What is claimed is:

1. A method of preparing a neural spheroid, comprising culturing differentiated forebrain progenitor cells in a V or U bottom shaped cell culture well with an ultra-low attachment surface to produce a neural spheroid.

2. The method of claim 1 , wherein the differentiated forebrain progenitor cells are prepared by differentiating neural progenitor cells to forebrain progenitor cells.

3. The method of claim 2, wherein tire neural progenitor cells are prepared from iPSC cells by differentiating the iPSC cells to neural progenitor cells.

4. Tire method of claim 3, wherein the differentiation of iPSC cells is to neural progenitor cells.

5. Tire method of claim 4, wherein tire iPSC cells are differentiated to neural progenitor cells by dual SMAD inhibition.

6. A method of preparing a neural organoid or neural spheroid, comprising: differentiating neural progenitor cells to forebrain progenitor cells; and culturing differentiated forebrain progenitor cells in V or U bottom shaped culture with ultra-low attachment surface to produce a neural spheroid.

7. A method of preparing a neural spheroid, comprising: inducing differentiation of fibroblasts into iPSC cells; inducing the iPSC cells to differentiate into neural progenitor cells; selecting rosettes of the neural progenitor cells; expanding the neural progenitor cells from the selected rosettes of neural progenitor cells; inducing the expanded neural progenitor cells to differentiate into forebrain progenitor cells; and culturing the differentiated forebrain progenitor cells in a V or U botom shaped cell culture well with ultra-low atachment surface to produce a neural spheroid.

8. The method of any one of claims 1-7, further comprising culturing the neural spheroid to mature tire neural spheroid.

9. The method of claim 8, wherein the culturing is for more than 8 days.

10. Tire method of claim 9, wherein the culturing is for 20 days or more.

11. Tire method of any one of claims 1-10, wherein the V or U botom shaped cell culture well is coated with a hydrogel.

12. The method of claim 11, wherein the hydrogel is a hydrophilic neutral hydrogel.

13. The method of claim 11, wherein the hydrogel is polyHEMA.

14. The method of any one of claims 1-13, wherein the iPSC cells are derived from a subject with a disease affecting neurons.

15. The method of claim 14, wherein the disease is Fabry disease, GM 1 -gangliosidosis, Gaucher disease, Metachromatic Leukodystrophy, or GM2 -gangliosidosis.

16. The method of claim 14 or 15, wherein the iPSC cells are derived from fibroblasts obtained from a subject afflicted with the disease affecting neurons.

17. A neural spheroid prepared by the method of any one of claims 1-16.

18. The neural spheroid of claim 17, wherein the neural spheroid expresses one or more of marker proteins TUJ1. GFAP, SIOOB, NEUN, TBR1, PAX6, MAP2 F0XG1, AND NESTIN.

19. The neural spheroid of claim 17 or 18, wherein the neural spheroid is characterized by astrocytes and/or neurons with projections and axons, respectively.

20. A method of using the neural spheroid of any one of claims 17-19 for disease modeling.

21. The method of claim 20, wherein the disease modeling is for Fabry disease, Pompe disease, GM 1 -gangliosidosis, GM2-gangliosidosis, or Gaucher disease.

22. Tire method of claim 20 for assessing tropism of gene therapy viral vectors.

23. Tire method of claim 22, wherein the gene therapy viral vector is AAV, adenovirus, lentivirus, or retrovirus.

24. Tire method of claim 20, wherein the neural spheroid is for assessing neural function.