Controlled induction of bioengineered neuroepithelial tissue and 3D neuroepithelial tubes

Abstract

Described herein are methods, compositions, and kits for the directed differentiation of human pluripotent stem cells, neuro-mesodermal progenitors, and neural stem cells into bioengineered oval neuroepithelial tissues and bioengineered neuroepithelial tubes that contain single rosettes of polarized neuroepithelial cells and have a microscale cellular organization similar to that of the developing human neural tube in vivo.

Description

[Technical Field]

[0001] Statements concerning federally funded research or development This invention was made with government support under RD-83573701-0, authorized by the Environmental Protection Agency, NS082618, authorized by the National Institutes of Health, and 1651645, authorized by the National Science Foundation. The U.S. Government has certain rights to this invention. [Background technology]

[0002] Human pluripotent stem cells (hPSCs) have seen significant advancements using suspension embryoid culture techniques to generate in vitro models of brain, kidney, liver, prostate, and gastrulated embryo-like tissues, providing an unrestricted starting material for biomanufacturing of tissue / organ transplantation and designing a wide variety of tissue, organ, and embryo models, as well as for in vitro investigation of human development, physiology, and disease; however, their structural and compositional variations limit the use of organoids as a scalable in vitro screening platform or for tissue / organ transplantation biomanufacturing.

[0003] The brain and spinal cord are composed of the neural tube, which consists of hollow, tubular, polarized neuroepithelial cells (also known as neural stem cells). In vitro, 2D and 3D cultures of differentiating neuroepithelial cells can initiate organoid formation via the spontaneous polarization of neural rosettes, which function like slices of the neural tube. Neural stem cell differentiation protocols, as well as micropatterned in vitro culture protocols for the regulated formation of single neural rosette structures, have been previously disclosed. While such neural rosettes model transverse slices of the developing neural tube, their small size limits their use as substrates for further bioengineering; for example, their diameter is too small to generate a meaningful microfluidic gradient of cell culture factors. Thus, there is a continuing need for improved methods and compositions for the regulated induction of the formation of larger rosette tissues, which are more biomimetic neural tube morphologies and further facilitate morphogenetic bioengineering using microfluidic platforms. [Overview of the project]

[0004] This specification describes methods, compositions, and kits for the bioengineered production of larger neuroepithelial tissues and neuroepithelial tubes, which are more biomimetic neural tube morphologies and further advance morphogenesis bioengineering using a microfluidic platform.

[0005] In a first aspect, the foregoing provides an in vitro method for efficiently, reproducibly, and reliably producing bioengineered neuroepithelial tissue having a single neuroepithelial rosette core, wherein the tissue has an elliptical morphology (e.g., a long axis greater than 1 mm and a short axis of about 250-300 μm), which can be further cultured to form a 3D tubular structure having the characteristics of a neonatal neural tube from biomimetic elliptical neuroepithelial tissue. In certain embodiments, the foregoing provides a method for producing biomimetic oval neuroepithelial tissue having a single rosette structure in vitro, comprising the steps of: (a) seeding human pluripotent stem cells (hPSCs) on a micropatterned substrate that induces biomimetic neural morphogenesis of cells cultured thereon, wherein the micropatterned substrate comprises at least two cell-adherent circular regions connected by cell adhesion bridges; and (b) culturing the seeded micropatterned substrate for a first culture period of about 1 to 2 days in the presence of a pluripotency-maintaining basal medium to obtain a first cell population. (a) a step in which the pluripotency maintenance basic medium contains a Rho kinase inhibitor; (c) a step in which the finely patterned substrate is cultured in a neural differentiation basic medium under adhesive conditions for a second period of approximately 2 to 6 days, thereby obtaining biomimetic oval neuroepithelial tissue containing polarized neuroepithelial cells and having a microscale cellular composition similar to a transverse section of an in vivo developing human neural tube; and (d) a step in which the surface of the adhesive finely patterned culture is covered with a hydrogel for a second culture period of approximately 5 to 7 days, thereby releasing the oval neuroepithelial tissue from the substrate and making it 3D neuroepithelial tissue while simultaneously maintaining a solitary polarized cyclic structure similar to that of an in vivo developing human neural tube. In a particular embodiment, the above method further includes the steps of: covering the surface of the obtained bioengineered neuroepithelial tissue with a hydrogel layer; culturing the bioengineered neuroepithelial tissue for about 24 hours to thereby transform the tissue into a bioengineered neuroepithelial tube and enclosing it with a hydrogel layer; and removing the hydrogel containing the enclosed neuroepithelial tube from a micropatterned substrate to obtain a bioengineered neuroepithelial tube.

[0006] In another embodiment, the present disclosure therefore provides a microfluidic technology apparatus comprising a micropatterned substrate as described herein for obtaining neuroepithelial tissue in vitro by biotechnology. As used herein, the term “morphogen” means a molecule or mixture of molecules that induces the differentiation and / or proliferation of cells such as neural progenitor cells. In one embodiment, the morphogen provides spatial information via a concentration gradient that can influence the patterning of differentiation in neural tissue. In some embodiments, the morphogen is a diffusible protein, cytokine, or growth factor.

[0007] In another embodiment, the herein provides a kit comprising one or more components useful for obtaining in vitro biomimetic neuroepithelial tissue or bioengineered neuroepithelial tubules. The components of the kit may include one or more micropatterning substrates described herein. The kit may also include a chemically defined medium and one or more culture medium additives (e.g., growth factors, small molecule compounds, culture medium supplements). The kit may further include progenitor cells useful for seeding the micropatterning substrate and instructions for cell seeding and culture. In some embodiments, it would be convenient to include in the kit one or more agents (e.g., growth factors, small molecule agents, agents for creating morphogen gradients) useful for further differentiating one or more micropatterning substrates and neuroepithelial tissues described herein to obtain various types of nerve cells (neurons). In such embodiments, the kit may also include a microfluidic device or its components for, for example, delivering the patterning agent to the bioengineered neuroepithelial tissue in a predetermined manner. In some embodiments, the kit further comprises a hydrogel suitable for use in covering biomimetic elliptical neuroepithelial tissue to obtain bioengineered neuroepithelial tubes encapsulated by the method of the present disclosure.

[0008] In another embodiment, provided herein is a neuroepithelial tissue designed by in vitro bioengineering, obtained by one of the methods disclosed herein, comprising a single rosette of polarized neuroepithelial cells and having a microscale cellular composition similar to a transverse section of a human neural tube during in vivo development. In another embodiment, the foregoing provides a composition comprising one or more micro-patterned substrates capable of biomimetic neural morphogenesis of cells cultured thereon, wherein the micro-patterned substrates comprise cell-adhesive microscale regions connected by at least two circular cell-adhesive bridges.

[0009] In a first particular aspect, the present disclosure provides a method for producing biomimetic oval neuroepithelial tissue having a single rosette structure in vitro. The method includes: (a) seeding human pluripotent stem cells (hPSCs) on a micropatterned substrate capable of biomimetic neural morphogenesis of cells cultured thereon in the presence of a Rho kinase inhibitor, wherein the micropatterned substrate comprises at least two circular bounded regions connected by cell adhesion bridges; (b) culturing the seeded cells from step (a) on the micropatterned substrate in the presence of a pluripotency maintenance medium for a first culture period of about 1 to 2 days to obtain a first cell aggregate, wherein the pluripotency maintenance medium comprises a Rho kinase inhibitor; and (c) culturing the cells obtained in step (b) under adherent culture conditions in a neuronal differentiation basal medium for a second period of about 3 to 6 days to obtain biomimetic oval neuroepithelial tissue having a single rosette structure. The tissue contains polarized neuroepithelial cells and has a microscale cellular composition similar to that of a transverse section of a human neural tube during in vivo development.

[0010] In one embodiment of the first particular aspect, each of at least two circular bounded regions has a diameter of about 100 μm to about 300 μm. In one embodiment of the first particular aspect, the cell adhesion bridge has a length of about 25 μm to about 125 μm and a width of about 10 μm to about 50 μm. In one embodiment of the first particular aspect, the cell adhesion bridge has a length of about 50 μm to about 100 μm and a width of about 25 μm. In one embodiment of the first particular aspect, the hPSC is about 75 x 10 3 cells / cm 2 ~about 2.5x10 5 cells / cm 2 They are seeded on a finely patterned substrate at a density of approximately 165,000 cells / cm³. In one embodiment of the first particular aspect, hPSCs are seeded at approximately 165,000 cells / cm³. 2 The seeds are seeded on a finely patterned substrate at a density of . In one embodiment of a first particular aspect, the pluripotency maintenance medium is a synthetic medium comprising DMEM / F-12, ascorbic acid, sodium bicarbonate, selenium, insulin, transferrin, FGF2, and TGFβ1. In one embodiment of the first particular aspect, the pluripotency maintenance medium is E8 medium. In one embodiment of a particular part of the first aspect, the neuronal differentiation medium is a synthetic medium comprising DMEM / F-12, ascorbic acid, sodium bicarbonate, selenium, insulin, and transferrin. In one embodiment of the first particular aspect, the neuronal differentiation medium is E6 medium. In one embodiment of the first particular embodiment, the neuronal differentiation basal medium further comprises one or more FGFs, with or without a β-catenin pathway signaling activator. In one embodiment of the first particular embodiment, the FGF is FGF2, FGF8a, FGF8b, FGF8f, FGF17, or FGF18. In one embodiment of the first particular embodiment, the β-catenin pathway signaling activator is a GSK3 kinase inhibitor. In one embodiment of the first particular embodiment, the GSK3 kinase inhibitor is CHIR99021. In one embodiment of the first particular embodiment, the neuronal differentiation basal medium further comprises about 100 ng / ml to about 200 ng / ml of FGF8b and about 3 μM to about 9 μM of CHIR99021. In one embodiment of the first particular aspect, the second culture period is approximately 5 days.

[0011] In one embodiment of a particular aspect of the first part of the method, the method further comprises transiently exposing cells on a micropatterned substrate to a Wnt / β-catenin signaling activator for about 24-72 hours after plating. In one embodiment of the first particular aspect, the method further comprises exposing the seeded cells to RA and Sonic Hedgehog (SHH) or an SHH signaling agonist for about 1 to about 5 days, thereby causing the rosette structure to develop into Olig2 + It contains motor neuron progenitor cells (pMNs). In one embodiment of a particular part of the first part, the finely patterned substrate includes one or more polyethylene glycol (PEG) brushes or peptide-immobilized PEG brushes arranged in a user-defined bounded geometry.

[0012] In one embodiment of the first particular aspect, the method further comprises the steps of: covering the surface of the neuroepithelial tissue obtained in step (c) of the first particular aspect with a hydrogel layer; and culturing the neuroepithelial tissue containing the hydrogel layer for about 24 hours, thereby converting the tissue into a bioengineered neuroepithelial tube and enclosing it with the hydrogel layer. In one embodiment of the first particular aspect, the method further includes the step of removing a hydrogel containing encapsulated bioengineered neuroepithelial tubules from a micropatterned substrate. In one embodiment of the first particular aspect, the method further includes the step of fixing and sectioning the removed hydrogel for analysis. In one embodiment, the hydrogel is Matrigel.

[0013] In a second particular aspect, the present disclosure provides neuroepithelial tissue obtained by in vitro bioengineering by the method of the first particular aspect, the tissue comprising a single rosette of polarized neuroepithelial cells and having a microscale cellular composition similar to a transverse section of a human neural tube during in vivo development.

[0014] In a third specific embodiment, the Disclosure provides an in vitro bioengineered neuroepithelial tube obtained by a method of the first specific embodiment or any embodiment thereof. The tube comprises a single rosette of polarized neuroepithelium and has a microscale cellular composition similar to that of a human neural tube during in vivo development.

[0015] In a fourth specific embodiment, the disclosure provides a composition comprising one or more micro-patterned substrates capable of directing biomimetic neural morphogenesis of cells cultured thereon. The one or more micro-patterned substrates comprise at least two circular cell adhesion microscale regions connected by cell adhesion bridges. In a fourth particular aspect, the Disclosure provides a kit comprising a composition of the fourth particular aspect, which further comprises vials of one or more progenitor cells, a pluripotent basal medium, a neural differentiation basal medium, a microfluidic device, and reagents for creating a morphogen gradient.

[0016] In a fifth specific aspect, the present disclosure provides a method for producing a bioengineered neural epithelial tube in vitro. The method comprises: (a) seeding human pluripotent stem cells (hPSCs) on a micropatterned substrate capable of bio-mimetic neuromorphogenesis of cells cultured thereon in the presence of a Rho kinase inhibitor, wherein the micropatterned substrate includes at least two circular bounded regions connected by cell adhesion bridges; (b) culturing the seeded cells from step (a) on the micropatterned substrate for a first culture period of about 1 to 2 days in the presence of a pluripotency maintenance medium to obtain first cell aggregates, wherein the pluripotency maintenance medium contains a Rho kinase inhibitor; (c) culturing the cells from step (b) under adherent culture conditions in a neural differentiation basal medium for a second period of about 3 to 6 days, whereby a bioengineered neural epithelial tissue is obtained; (d) covering the surface of the bioengineered neural epithelial tissue obtained in step (c) with a hydrogel layer; (e) culturing the bioengineered neural epithelial tissue from step (d) for about 24 hours, thereby transforming the tissue into a bioengineered neural epithelial tube and encapsulating it with the hydrogel layer; and (f) removing the hydrogel containing the encapsulated neural epithelial tube from the micropatterned substrate to obtain a bioengineered neural epithelial tube.

[0017] In a sixth specific aspect, the present disclosure provides an in vitro bioengineered neural epithelial tube obtained by the method of the fifth specific aspect. The tube includes a single rosette of polarized neural epithelium and has a microscale cell configuration similar to that of the human neural tube during in vivo development.

[0018] These and other features, objects, and advantages of the present invention will be better understood from the following description. In the description, reference is made to the accompanying drawings, which show, by way of illustration and not limitation, embodiments of the invention that form a part of the present invention. The description of the specific embodiments is not intended to limit the present invention, nor is it intended to cover all modifications, equivalents, and alternatives. Therefore, reference should be made to the claims set forth herein in detail for interpreting the scope of the present invention. Considering the following embodiments for carrying out the invention, the present invention will be better understood, and features, appearances, and advantages other than those described above will become apparent. In such embodiments for carrying out the invention, the following drawings are referred to.

Brief Description of the Drawings

[0019] [Figure 1] Computer-aided design of features presented on a polydimethylsiloxane (PDMS) stamp used for microcontact printing of custom culture substrates: (upper) circular micro-pattern arrangement forming pairs without bridges and (lower) pairs of circles connected by bridges, or micro-patterns called "nodes". The diameter of the circles varies from 150 to 250 μm and has a varying edge-to-edge distance. The bridge thickness is 25 microns. [Figure 2] (upper) Representative images of micro-patterned tissue pairs over the total number of days of culture at edge-to-edge distances of 25, 50, and 75 μm without bridges are shown (N-cadherin = dashed arrow, Pax6 = solid arrow). The graph in the middle shows the quantification of the percentage of micro-patterned tissue pairs that fused between pairs (the graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). The graph in the lower part shows the percentage of micro-patterned tissue pairs that fused and formed a single rosette polarization from all fused micro-patterned tissue pairs (the graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). For each experimental group, N = 56 technical replicates. [Figure 3](Top) Representative images of micropatterned tissue pairs over the entire culture period at edge-to-edge distances of 25, 50, 75, 100, and 125 μm with bridges (N-cadherin = dashed arrow, Pax6 = solid arrow). The middle graph shows the quantification of the percentage of micropatterned tissue fused between pairs (graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). The bottom graph shows the percentage of micropatterned tissue pairs that fused and formed a single rosette polarization from all fused micropatterned tissue pairs (graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). N=56 technical replicates per experimental group. [Figure 4] (Top) Shows a single-row micropattern design (N-cadherin = dashed arrow, Pax6 = solid arrow). The image montage in the middle of the top row shows representative images of micropatterned neuroepithelial tissue over all culture days at an edge-to-edge distance of 50 μm when bridging is present. The graph in the top row shows the quantification of the percentage of fused micropatterned tissue across all rows at edge-to-edge distances of 25, 50, 75, 100, and 125 μm when bridging is present (graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). The graph in the bottom row shows the percentage of micropatterned tissue rows that fused and formed a single rosette polarization from all fused micropatterned tissue rows (graph bars are D3, D4, D5, and D6 from left to right for each edge-to-edge distance). N=4 technical replicates per experimental group. [Figure 5] By day 5, we demonstrate that micro-patterned neural tissue fusion extends to all transverse rows on the micro-patterned array. N-cadherin = dashed arrow, Pax6 = solid arrow. [Figure 6](Top) The circular micropatterns separated by 50 μm edge-to-edge distances and bridges enable the formation of close-proximity fusion tissue with single N-cadherin + rosette polarization across all micropatterned rows, and begin to form three-dimensional tubes with apical N-cadherin (dashed arrow) expression over 5 days of culture (middle), as shown in the confocal Z-stack image (bottom). N-cadherin = dashed arrow, laminin = solid arrow, DAPI = composite arrow. [Figure 7] The experimental protocol for inducing finely patterned forebrain neuroepithelial ducts (top panel) and for covering the surface with Matrigel hydrogel for 24 hours on day 5 (bottom panel) is shown. [Figure 8] Representative cross-sectional sections of forebrain neuroepithelial tubules 24 hours after Matrigel coating are shown. The sections, in 88% of the 173 sections across all 14 tissues, show a central ring of polarized N-cadherin expression (upper panel), indicating single neuroepithelial formation, and basal deposition of laminin extracellular matrix protein (lower panel). N-cadherin = dashed arrow, laminin = solid arrow, DAPI = composite arrow. [Modes for carrying out the invention]

[0020] While the present invention may be subject to various modifications and alternative forms, exemplary embodiments are shown by the drawings and described in detail herein. However, it should be understood that the description of exemplary embodiments is not intended to limit the invention to any particular disclosure, but rather to encompass all modifications, equivalents, and alternatives that fall within the spirit and scope of the invention as defined by the appended claims. Built-in by reference All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as each individual publication, patent, and patent application is specifically and individually incorporated by reference.

[0021] In the central nervous system (CNS), the brain and spinal cord develop from a hollow tube called the neural tube, which is composed of polarized neuroepithelial cells (NECs) (also known as neural stem cells). In vitro, NECs spontaneously polarize during differentiation, forming similar neuroepithelial tube analogs, or neural rosette structures, in both two-dimensional (2D) and three-dimensional (3D) cultures. This invention provides a method for controlling neural rosette formation and thereby producing neuroepithelial tissue with an elliptical morphology and an adjustable aspect ratio. These elliptical neuroepithelial tissues routinely mimic primary neural tube formation and reproducibly form 3D neuroepithelial tubes that exhibit structural properties more closely resembling the developing neural tube than those obtained by previously reported neural organoid induction methods.

[0022] I. Definition Unless otherwise specified, all scientific and technical terms used herein have the same meaning as those commonly interpreted by a person of ordinary skill in the art to which this invention pertains. Any methods and materials similar or equivalent to those described herein may be used for carrying out or testing the invention, but preferred methods and materials are described herein. In the embodiments of the present invention and the claims of the claims, the following terms will be used according to the following definitions.

[0023] As used herein, the term “human pluripotent stem cell” (hPSC) means a cell that can successively self-replicate and, under appropriate conditions, differentiate into cells of all three embryonic layers. hPSCs are SOX2 + and OCT4 +shows a gene expression profile comprising. Examples of hPSCs include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). hESCs suitable for use in the methods disclosed herein are described in Thomson et al., 1998, Science 282:1145-1147. This document is incorporated herein by reference in its entirety. As used herein, "iPS cells" means cells that are substantially genetically identical to their respective original differentiated somatic cells and exhibit characteristics similar to those of more potent cells such as ES cells described herein. Cells can be obtained by reprogramming non-pluripotent (e.g., multipotent or somatic) cells. As used herein, "pluripotency" means the ability to differentiate into cells of all three germ layers of a cell. As used herein, "neural stem cell" (NSC) is PAX6 + / SOX2 + and means a pluripotent stem cell that can differentiate into neurons or glia of the central nervous system (CNS) or peripheral nervous system (PNS). As used herein, neural epithelial cell (NEC) means a neural stem cell that is a polarized epithelial cell showing apical-basal polarity within a neural rosette structure. As used herein, the term "neural-mesodermal progenitor" (NMP) refers to the following gene expression profile: SOX2 + / OCT4 - / T + / PAX6 - and means human pluripotent stem cell-derived cells having. NMP is also called the posterior lateral epiblast. As used herein, the term "oval" means having an elongated circular shape, an oval shape or an oval-like shape that is stretched into an oval. The oval shape deviates from the ideal circular shape, and the distance from the center of the shape to the end of the shape varies (different from a circle in which the distance from the center to any position along the end remains constant). The terms "oval", "oval-shaped" or "substantially oval" are used herein in the same meaning, and it is not considered essential that the substrate, tissue, or other substance of the present disclosure has an exact oval shape. As used herein, the term “neural rosette” refers to a neuroepithelial tubule analogue containing neuroepithelial cells (NECs) that is formed when human pluripotent stem cells are differentiated into neurons in two-dimensional (2D) and three-dimensional (3D) cultures. The morphology of the neural rosette can be elucidated by the localization of apical N-cadherins along the apical-basal polarity of the constituent cell membranes. As used in this invention, "biological molecule" or "biomolecule" means a molecule having substantially biological origin. Such molecules may include non-natural components that mimic naturally occurring components, such as non-natural amino acids. As used herein, the terms “synthetic” and “designed” are used interchangeably and mean non-natural tissue materials created or modified by human hands (e.g., formed using artificial materials) or derived using such materials (e.g., devices or compositions containing designed materials). As used herein, the term “organoid” means a tissue-like structure (i.e., exhibiting the structural characteristics of a particular tissue type) that resembles a developing organ and is constructed in vitro by the addition of separate components and by the self-organization of cell types, including, but not limited to, pluripotent stem cells or neural stem cells. See, for example, Lancaster and Knoblich, Science 345(6194):1247125(2014). The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. This term applies to naturally occurring amino acids, one or more of which correspond to amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and amino acid polymers that are artificial chemical mimics of unnaturally occurring amino acid polymers. As used herein, the term “cell medium” (also referred herein as “culture medium,” “medium,” or “culture media”) is a medium for culturing cells that contains nutrients that maintain cell viability and support cell proliferation. As used herein, the terms "chemically defined culture medium" mean that the chemical structure and quantity of each medium component are specifically known or identifiable and can be individually controlled. Accordingly, a medium is not a chemically defined medium if (1) the chemical and structural properties of all medium components are unknown, (2) the medium contains any unknown amount of any component, or (3) both. As used herein, “culture medium consisting of” means a culture medium containing components that do not substantially affect the specific components and their basic properties. As used herein, “supplemented” means a culture medium containing a composition, for example, a supplementing component (e.g., retinoic acid or fibroblast growth factor (FGF)). For example, a culture medium “further supplemented” with retinoic acid (RA) or FGF contains RA or FGF, but does not mean the act of introducing RA or FGF into the culture medium. As used herein, “effective dose” means an amount of drug sufficient to induce a specific cellular effect according to the present invention. As used herein, the terms "xenogen-free" and "xeno-free" are used interchangeably and mean a substance that is free from or substantially free from heterogeneous substances or undefined components of non-human origin. As used herein, “basic neural differentiation medium” means a medium capable of promoting and supporting the differentiation of human pluripotent stem cells toward the nervous system, for example, the neuroectoderm and neuroepithelium. As described herein and in U.S. Patent Application Publication No. 2014 / 0134732, basic neural differentiation mediums may include, but are not limited to, E6 medium. This disclosure is incorporated herein by reference in its entirety. The terms “purified” and “enriched” cell populations are used herein to mean the same thing and refer to ex vivo cell populations containing a higher proportion of a particular cell type or cells having a higher specified characteristic than those observed in vivo. As used herein, “serum-free” means that the culture medium does not contain serum or serum substitutes, or is substantially free of serum or serum substitutes. For example, a substantially serum-free medium may contain less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum, and the competent culture capacity of the medium is still observed. As used herein, “substantially free” means that the culture medium or other composition or solution is free of or substantially free of a particular component. For example, “substantially free of putrescine” means that no putrescine is added to the cell medium, in addition to any putrescine present in the basic medium, e.g., DMEM / F12. Alternatively, “substantially free of putrescine” means a final putrescine concentration of 0.08 mg / l or less. As used herein, “viability” means a state free from substantial cell death. Viable pluripotent cells, if attached to the surface and without membrane disruption, do not stain with the dye propidium iodide. Short-term viability relates to the first 24 hours after seeding cells in culture. Normally, cells do not proliferate during that time. As used herein, the terms “crosslinked” or “crosslinked” mean a bond that connects one polymer chain to another, particularly to form a hydrogel. These bonds may be covalent or ionic.

[0024] The terms “about” and “approximately” generally mean an acceptable degree of error in relation to the measured quantity, taking into account the nature or precision of the measurement. A typical exemplary error is within 10%, preferably 5%, of the value or range of values. Alternatively, particularly in biological systems, the terms “about” and “approximately” may mean values ​​within a range of 10 times, preferably 5 times, and more preferably 2 times, a particular value. The numerical quantities expressed herein are approximations, and unless otherwise explicitly stated, the terms “about” and “approximately” mean that they can be estimated. As used herein, the terms “comprising,” “comprise,” and “comprised of” are synonymous with “including,” “include,” “containing,” and “contain,” and are comprehensive or open-ended and do not exclude any additional, undescribed members, elements, or steps of the method. The expressions and terms used herein are for illustrative purposes only and should not be considered limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof means that they include the items and additional items listed thereafter. The use of sequential terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not, by itself, imply any priority, prior, or temporary order in which the action of one claim element on another element is performed. Ordinal terms are used merely to distinguish claim elements as identifiers that distinguish one claim element having a specific name from another element having the same name (apart from the use of ordinal terms). When used herein and in the appended claims, the singular forms ("a," "an," and "the") include plural nouns unless otherwise explicitly stated in the context. Any reference to "or" herein is intended to include "and / or" unless otherwise specified.

[0025] II. Methods, compositions, and organoids The present invention, as described herein, discloses a micropattern design comprising multiple circular regions connected by cell adhesion bridges capable of inducing the formation of neuroepithelial tissue having a non-circular morphology and a single polarized rosette structure throughout the tissue. Furthermore, as shown in Figures 3 and 4, the creation of a micropattern having a series of connected circles enables the formation of a single neuroepithelial rosette tissue having a substantially elliptical morphology (e.g., a long axis exceeding 1 mm and a short axis of approximately 250–300 μm) that can be enlarged indefinitely. In comparison, only individual circles lacking cell adhesion bridges (see Figure 2) rarely produced fused tissue or a single rosette. Using limited conditions, in vitro organoids with higher mimetic in vivo neural tube structures and morphologies can be obtained by directing stem cell differentiation on a micropatterned substrate of a particular design. Also provided by the present invention, as described herein, are conditions for modeling human CNS patterning in in vitro 3D models for studying neurogenesis and disease in biomimetic in vitro human models and for identifying material and combination strategies for in vitro tissue engineering. Furthermore, it is shown herein that large-scale culture of such micropatterned tissues replicates primary neural tube formation-like tissue folding / fusion events for the formation of 3D neural epithelial tubes (Figure 6). This ability provides a crucial step toward the bioengineering of neural organoids with biomimetic, reproducible anatomical tissues. Similar epithelial tube formation characterizes the developmental processes of other human organs (e.g., the heart and digestive system), and thus the micropatterned culture methods described herein may be applicable to the bioengineering of other tissue / organ systems.

[0026] Accordingly, in the first aspect, the foregoing provides an in vitro method for bioengineerally producing, efficiently, reproducibly, and reliably, a neuroepithelial tube having a single neuroepithelial rosette core from biomimetic elliptic neuroepithelial tissue, the tissue having an elliptic morphology (e.g., a long axis greater than 1 mm and a short axis of about 250-300 μm), which can be further cultured to form a 3D tubular structure having the characteristics of a neonatal neural tube. As used herein, the term “biomimetic elliptic neuroepithelial tissue” means tissue comprising elongated cell aggregates or a single neuronal rosette structure produced on a micropatterned substrate of the herein disclosure, having structural similarity to a naturally occurring tissue, i.e., having the anatomical and cellular structure of an embryonic neural tube or a cross section thereof (Figure 3). The bioengineered neuroepithelial tubes produced therefrom, as shown in Figures 6 and 8, are in vitro generated (e.g., designed) cell aggregates that mimic polarized 3D neural tube cell structures, with polarized NSCs exhibiting apical N-cadherin expression and basal extracellular matrix protein deposition along the overall axis of the 3D cylindrical tissue aggregate.

[0027] In certain embodiments, the foregoing provides a method for producing biomimetic elliptic neuroepithelial tissue having an in vitro single rosette structure, comprising the steps of: seeding human pluripotent stem cells (hPSCs) on a micropatterned substrate capable of biomimetic neural morphogenesis of cells cultured thereon in the presence of a Rho kinase inhibitor, wherein the micropatterned substrate comprises at least two circular bounded regions connected by cell adhesion bridges; culturing the seeded micropatterned substrate for a first culture period of about 1 to 2 days in the presence of a pluripotency maintenance medium to obtain a first cell population, wherein the pluripotency maintenance medium comprises a Rho kinase inhibitor; and culturing the cultured micropatterned substrate under adherent culture conditions in a neural differentiation basal medium for a second period of about 2 to 6 days to obtain biomimetic elliptic neuroepithelial tissue comprising polarized neuroepithelial cells and having a microscale cellular composition similar to a transverse section of a human neural tube during in vivo development. Referring to Figure 1, in certain embodiments, the micropatterned substrate includes at least two circular bounded regions connected by cell adhesion bridges, and in some embodiments, it includes a series of interconnected circular bounded regions. The number of circular bounded regions must be at least two, but the number of series of interconnected circular bounded regions is limited only by the ability to fabricate a given arrangement on the substrate (for example, limited by the dimensions of the micropatterned substrate itself).

[0028] In some embodiments, the micropatterned substrate can be fabricated by simple microprinting of alkanethiols without graft polymer brushes and can be used in the manner disclosed herein. See Ashton et al., 2007, Stem Cells 25:2928-2935, the disclosure of which is expressly incorporated by reference herein in its entirety. In some embodiments, the micropatterned substrate comprises a predetermined (i.e., user-defined) arrangement of one or more surface-grafted poly(ethylene glycol) ("PEG") brushes, which ensures the prevention of protein adsorption and thereby prevents cell adhesion. In such embodiments, the micropatterned substrate comprises a user-defined arrangement of one or more PEG brushes grafted onto a solid support (e.g., tissue culture polystyrene, slide glass, glass, or silica substrate). Micropatterned regions that prevent cell adhesion may comprise one or more surface-grafted PEG brushes lacking all cell adhesion peptides or other portions that promote cell adhesion. Cell micropatterned regions that promote the adhesion of cells cultured thereon may comprise one or more PEG brushes having peptide-immobilized portions. In such embodiments, the arrangement of one or more peptide-immobilized PEG brushes provides a user-defined, adjustable substrate that can control cell adhesion and thereby control the morphology of adherent cell aggregates in the resulting tissue. Furthermore, the PEG brushes can be chemically modified to enable peptide insight binding, the sequences of which are derived from extracellular matrix ("ECM") proteins. For example, a micropatterned substrate may include an arrangement of PEG brushes containing peptides having one or more RGD (Arg-Gly-Asp) sequence motifs, which are also known as integrin-binding motifs that promote cell adhesion. RGD sequence motifs correspond to cell adhesion sites of numerous adherent ECM and cell surface proteins. RGD sequences are common in integrin-binding adhesion proteins such as fibronectin, collagen, and laminin.The integrin-binding activity of such adhesion proteins can be mimicked by short synthetic peptides containing RGD sequences. Therefore, to promote cell adhesion to specific regions of a finely patterned substrate, these regions may contain one or more PEG brushes bound to RGD-containing peptides. For regions that hinder cell adhesion, the use of PEG brushes lacking any cell adhesion molecules (e.g., cell linkage regions) is preferable.

[0029] In some embodiments, the finely patterned substrates are obtained by the synthesis protocol described by Knight et al., 2015, Chem.Commun. 51:5238-5241. This document is incorporated herein by reference as if it were described in its entirety. As described therein, the finely patterned substrates may be azido-functionalized poly(ethylene glycol) methacrylate (PEGMA) grafted substrates. These can undergo a 1,3-dipolar cycloaddition "click" reaction with peptides bound to highly strained molecules such as dibenzocyclooctyne (DBCO). Other peptide-immobilized PEG brushes are described by Sha et al., 2013, Biomacromolecules 14(9):3294-3303. This document is incorporated herein by reference as if it were described in its entirety. As described therein, PEG brushes exhibit a dual orthogonal chemistry (i.e., azide and acetylene groups) for ligand (e.g., peptide) immobilization via a versatile copper-free click reaction, which is useful for surface modification of insights in cell cultures, thereby useful for spatiotemporal control of adherent tissue morphology.

[0030] In some embodiments, the micropatterned substrate comprises one or more PEG brushes or poly(ethylene glycol) methacrylate (PEGMA)-azide brushes throughout, except for an array of interconnecting circles having diameters of approximately 100 μm, 200 μm, 250 μm, 300 μm, or greater, and these circles are connected by cell adhesion bridges. In such embodiments, when coated with extracellular matrix proteins and seeded with hPSCs, NMPs, or NSCs, the cells adhere within the brush-free regions. Upon further culture, the cells differentiate into molded tissue, which is confined by surrounding the inactive PEG or PEGMA-azide brushes. Conveniently, the micropatterned substrates provided herein can be chemically modified to present multiple cell adhesion peptides arranged according to user-defined, tunable spatial parameters. User-defined parameters include spacing, diameter (sometimes referred to herein as "width"), height (sometimes referred to herein as "length"), and the number of cell adhesion peptides per unit surface area (also referred herein as "cell adhesion peptide surface area density").

[0031] Accordingly, provided herein are micropatterned substrates comprising regions that promote the adhesion of cells cultured thereon, and these regions are designed (i.e., configured) to have a bounded geometry that promotes the differentiation and morphogenesis of neural mesoderm precursors (NMPs), neural stem cells (NSCs), or human pluripotent stem cells (hPSCs) cultured on or recruited to the matrix. As shown herein, the bounded geometry of a micropatterned substrate comprising at least two circular bounded regions connected by cell adhesion bridges can be used to control macroscale structuring and form single neuroepithelial rosette tissues having an elliptical morphology during cell differentiation and morphogenesis of hPSCs, NSCs, or NMPs on a culture substrate. The bounded geometric shapes may be various two-dimensional (2D) shapes (e.g., regular or irregular shapes) having dimensions defined by the shape (e.g., diameter, width, length, etc.), but for a finely patterned substrate, it is preferable to include a predetermined two-dimensional pattern of at least two circular regions of various dimensions (e.g., diameters of 36 μm, 100 μm, 490 μm, 4.8 mm, and 12.6 mm; typically 150–300 μm) separated from each other by a predetermined distance and connected by bridges of the same cell-adhesive material. In a particular embodiment, the bounded geometric shapes of each of the at least two circles have diameters of about 100 μm, 150 μm, 180 μm, 200 μm, about 300 μm, or about 400 μm (e.g., about 100, 150, 200, 250, 300, 350, 400 μm (including the values ​​at both ends)). In some embodiments, circles or other shapes with diameters of less than 100 μm, and in other embodiments, approximately 50 μm (e.g., 50, 60, 70, 80, 90, or 100 μm (including the values ​​at both ends)) can be used. However, proliferating cells grown on smaller fine-patterned surfaces (i.e., surfaces with a diameter of less than 100 μm) tend to detach from the surface as cell aggregates or spheres. Therefore, a lower limit can be set for the dimensions of the fine-patterned surface as a practical constraint.

[0032] With respect to the spacing between at least two circular bounded regions (spacing due to cell adhesion bridges), the regions are preferably spaced about 25 μm to about 125 μm (for example, about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 μm (including the values ​​at both ends)), and more preferably spaced about 50 μm to about 100 μm. Therefore, the length of the cell adhesion bridge connecting at least two circular bounded regions is preferably about 10 μm to about 100 μm (for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μm (including the values ​​at both ends)), more preferably about 20 μm to about 60 μm.

[0033] In some embodiments, the finely patterned substrate includes, for example, a series of predetermined two-dimensional patterns with multiple interconnecting circles, each having substantially the same diameter, and each of any two adjacent circles sharing substantially the same inter-edge spacing. While the diameter of each circle and the spacing between circles may vary, the inventors have concluded that a particular fine pattern having inter-edge spacings between interconnecting circles of about 25 μm to about 125 μm, more preferably about 25 μm to about 50 μm (Figures 3 and 4), is a favorable arrangement. With respect to the width of the cell adhesion bridge connecting at least two circular bounded regions, the bridge width is conveniently about 10 μm to about 100 μm, more specifically 25 μm. The arrangement of the cell adhesion bridges may vary, but the inventors have concluded that it is advantageous to use bridges located in the central part. The arrangement of the cell adhesion bridges does not need to be at the exact center point of each pair of circles in order to fall within the scope of this disclosure.

[0034] Any suitable means for fabricating a micropatterned substrate can be used. In some embodiments, the micropatterned substrate can be fabricated by manually depositing peptide-immobilized PEG brushes and cell adhesion peptides onto a solid support. In other embodiments, the micropatterned substrate is fabricated using automated (e.g., robotic) techniques, microcontact printing, microfluidic etching, or deposition of various materials. In other embodiments, photolithography-based microfabrication techniques can be used. For example, photolithography-based microfabrication techniques can be used to fabricate templates or molds for micrometer-level patterning onto substrates (e.g., glass slides, glass coverslips, or elastomer polymers (e.g., polydimethylsiloxane (PDMS))) of cell adhesion-blocking materials (e.g., non-adhesive agar) coated with cell adhesion materials. In this method, a micropatterned substrate containing distinct adhesive and non-adhesive domains can be obtained by the method disclosed herein.

[0035] As is known in the art, the micropatterned substrates disclosed herein can be used in any culture system, including static (e.g., tissue culture plates) and fluid flow reactor systems (e.g., microfluidic devices). Such microfluidic devices are useful for rapid drug screening when low flow rates and small amounts of reagent are required. Microfluidic devices are also useful for in vitro exposure of the micropatterned substrate to one or more gradients of transcription factors or other factors that can direct or influence neural differentiation (e.g., drugs that generate morphogen gradients for morphogenesis patterning of neural tissue). In another embodiment, the disclosure therefore provides a microfluidic technology apparatus comprising the micropatterned substrates described herein for obtaining neuroepithelial tissue in vitro by biotechnology. As used herein, the term “morphogen” means a molecule or mixture of molecules that induces the differentiation and / or proliferation of cells such as neural progenitor cells. In one embodiment, the morphogen provides spatial information via a concentration gradient that can influence the patterning of differentiation in neural tissue. In some embodiments, the morphogen is a diffusible protein, cytokine, or growth factor.

[0036] The resulting bioengineered neuroepithelial tissue may have a nanoscale or microscale cellular structure similar to that of the corresponding in vivo developing organ. As used herein, the term “microscale cellular structure” means that the structural composition of the cellular components of the biomimetic 3D organoid (also known as “cellular structure”) is similar to that of the corresponding in vivo tissue at the microscale level and is on the order of less than 1000 μm, more preferably less than 100 μm.

[0037] Any suitable progenitor cells can be seeded onto a micropatterned substrate. For example, pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can be used. Suitable progenitor cells for bioengineered neuroepithelial tubules are those capable of forming polarized neural rosette structures (e.g., polarized staining with laminin and N-cadherin), and include, but are not limited to, hPSCs, neural stem cells (NSCs), or neuronal mesoderm precursors (NMPs).

[0038] Referring to Figures 7 and 8, in some embodiments, it would be advantageous for the methods described herein to further include a hydrogel material covering the surface of the resulting biomimetic oval neuroepithelial tissue. In this configuration, the biomimetic oval neuroepithelial tissue detaches from the micropatterned substrate as a three-dimensional structure encapsulated in the hydrogel. The hydrogel-encapsulated bioengineeric neuroepithelial tubes are useful for a variety of applications, including, for example, bioengineered neural organoids having a 3D structure similar to an in vivo neural tube during development. The hydrogel-encapsulated bioengineeric neuroepithelial tubes are applicable to further development and analysis, including, but not limited to, expansion bioreactor culture, fixation, sectioning, immunohistochemical examination (see Figure 8), imaging, and insight hybridization. In some embodiments, the surface coating method includes coating the biomimetic oval neuroepithelial tissue obtained by the method of the present disclosure with a hydrogel layer; culturing the tissue including the superimposed hydrogel layer for about 20 to 52 hours (e.g., about 20, 22, 24, 26, 28, 48, 50, or 52 hours), thereby encapsulating the biomimetic oval neuroepithelial tissue in the hydrogel layer to obtain stable biotechnically engineered neuroepithelial tubes. In some embodiments, the hydrogel containing the encapsulated biotechnically engineered neuroepithelial tubes is removed from the micropatterned substrate for subsequent use or analysis. While the present disclosure exemplifies the use of Matrigel for hydrogel surface coating, it will be understood that any flexible hydrogel, including known compositional substitutes for commercially available Matrigel substrates, may be used, but is not limited to these examples.

[0039] In some embodiments, fixing or freezing the hydrogel containing the removed biotechnically engineered neuroepithelial tubules may be advantageous for tissue or microscopic observation. For example, the hydrogel containing the removed biotechnically engineered neuroepithelial tubules can be fixed in formalin or paraformaldehyde for sectioning using a standard method. If necessary, the tissue can be removed and cleared to facilitate imaging using light-based microscopy. In particular, light-sheet imaging and scanning electron microscopy (SEM) are useful for detecting and analyzing polarization in biotechnically engineered neuroepithelial tubules. In exemplary embodiments, a confocal or light-sheet microscope can reveal the distribution of cell types throughout the biotechnically engineered neuroepithelial tubules prepared according to the method of the present invention. In some embodiments, a three-dimensional assembly of images obtained by a confocal or light-sheet microscope is used to analyze the distribution and composition of various cells and structures within them.

[0040] In some embodiments, neural stem cells for use in the methods provided herein are obtained by induced differentiation of human pluripotent stem cells (hPSCs). Suitable pluripotent cells for use herein include human embryonic stem cells (hESCs) and human iPSCs. These cells express Oct-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 and appear as compact colonies with a high nucleus-to-cytoplasmic ratio and significant nucleoli. ESCs are commercially available from sources such as the WiCell Research Institute (Madison, Wis.). As used herein, “induced pluripotent stem cells” or “iPS cells” means pluripotent cells or populations of pluripotent cells that can mutate to their respective original differentiated somatic cells, can mutate to a particular set of potency determinants, and can mutate to the culture conditions used to isolate them, but nevertheless are substantially genetically identical to their respective differentiated original somatic cells and exhibit properties similar to more potent cells, such as the ESCs described herein. See, for example, Yu et al., 2007, Science 318:1917-1920. This disclosure is incorporated in its entirety by reference.

[0041] Induced pluripotent stem cells (iPS cells) exhibit morphological characteristics (e.g., round shape, large nucleolus, and small cytoplasm) and growth characteristics (e.g., doubling time of approximately 17-18 hours) similar to ESCs. In addition, iPS cells express pluripotency-specific markers (e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, or Tra-1-81, but not SSEA-1). However, induced pluripotent stem cells are not directly induced from embryos. As used herein, "not directly induced from embryos" means that the starting cell type for producing iPS cells is a non-pluripotent cell such as a pluripotent cell, or a terminally differentiated cell such as a somatic cell obtained from a postnatal individual.

[0042] Human iPS cells can be used according to the methods described herein to obtain primitive macrophages and microglia cells having genetic complements for a specific human subject. For example, it may be advantageous to obtain biomimetic oval neuroepithelial tissue exhibiting one or more specific phenotypes associated with or arising from a specific disease or disorder in a specific mammalian subject. In such embodiments, iPS cells are obtained by reprogramming somatic cells of a specific human subject according to methods known in the art. See, for example, Yu et al., 2009, Science 324:797-801; Chen et al., 2011, Nat. Methods 8(5):424-9; Ebert et al., 2009, Nature 457:277-80; Howden et al., 2011, Proc. Natl. Acad. Sci. USA 108(16):6537-42. Each of these references is incorporated herein by reference.

[0043] Prior to seeding, hPSCs (e.g., hESCs or hiPSCs) can be cultured in the absence of a feeder layer (e.g., a fibroblast layer) on a substrate suitable for hPSC proliferation, such as Matrigel®, vitronectin, vitronectin fragments, or vitronectin peptides, or Synthemax®. In certain embodiments, hPSCs are subcultured at least once to at least about five times in the absence of a feeder layer. Suitable subculturing and maintenance media for hPSCs include, but are not limited to, mTeSR® and E8® media, available from Thermal Fisher / Life Technologies Inc. as Essential 8 or from Stem Cell Technologies as TeSR-E8. In some embodiments, hPSCs are maintained and subcultured under xeno-free conditions, and the cell medium is a synthetic medium such as E8 or mTeSR, but the cells are maintained on a completely clear xeno-free substrate such as vitronectin or Synthemax® (or another type of self-coating substrate). In specific embodiments, hPSCs are maintained and subcultured in E8 medium on a self-coating substrate such as vitronectin, vitronectin fragment, or vitronectin peptide or Synthemax®.

[0044] In certain embodiments, pluripotent stem cells cultured in an adherent monolayer in a culture plate are subcultured on a micropatterned surface, then cultured in a pluripotency maintenance medium of a distinct composition for a first culture period of about 1-2 days, and subsequently cultured in a medium of a distinct composition that supports the differentiation of human pluripotent stem cells into neural stem cells for a second culture period of 3-6 days, thereby obtaining PAX6-positive neural stem cells. Once seeded, hPSCs can be cultured in a differentiation medium suitable for differentiation into organoids. Suitable medium conditions for in vitro differentiation of hPSCs or other progenitor cells into organoid tissues are known in the art. For example, the medium is sufficient to promote in vitro self-organization and spontaneous morphogenesis of hPSCs into designed neuroepithelial tubule organoids. Suitable media include, for example, neural differentiation mediums. Preferably, the hydrogel on which the cells are seeded is cultured for a sufficient time, for example, at least 4 days, at least 6 days, at least 8 days, or at least 16 days, to form a 3D organoid structure. As used herein, “neural differentiation medium” means a medium capable of promoting and supporting the differentiation of human pluripotent stem cells toward the nervous system, for example, neuroectoderm and neuroepithelium. As described herein and in U.S. Patent Application Publication 2014 / 0134732, basic neural differentiation media include, but are not limited to, E6 medium. This patent application publication is incorporated herein by reference in its entirety. In some embodiments, the neuronal differentiation medium used in the neuronal differentiation method is "E4" medium, which is basically a basic medium (e.g., DMEM / F12 or a similar basic medium described herein) containing water, salt, amino acids, vitamins, a carbon source, and buffers, as well as selenium and insulin. If necessary, the neuronal differentiation medium may also contain ascorbate (the medium is also referred to herein as "E5" medium). In some embodiments, the neuronal differentiation medium used in the neuronal differentiation method is "E6" medium, which is basically carbonate-buffered E5 medium + transferrin.

[0045] As used herein, the terms “E6 medium” and “E6” are used interchangeably and refer to a synthetic medium containing or based on DF3S supplemented with insulin (20 μg / mL) and / or transferrin (10.67 ng / mL). The medium can be prepared based on the formula in Chen et al., 2011, Nature Methods. 8(4), 424-429. This document is incorporated herein by reference in its entirety. Similar media are available from Thermal Fisher / Life Technologies Inc. as Essential 6 or from Stem Cell Technologies as TeSR-E6. As used herein, the terms “E8 medium” and “E8” are used interchangeably and refer to a synthetic medium containing or based on DF3S supplemented with insulin (20 μg / mL) and / or transferrin (10.67 ng / mL), human FGF2 (100 ng / mL), and human TGFβ1 (transforming growth factor β1) (1.75 ng / mL). The medium can be prepared based on the formula in Chen et al., 2011, ibid. Alternatively, the medium is also available from Thermal Fisher / Life Technologies Inc. as Essential 8, or from Stem Cell Technologies as TeSR-E8.

[0046] In other embodiments, the useful medium contains at least the same components as the neural differentiation medium described above, but the medium is substantially free of TGFβ superfamily agonists (e.g., Nodal), albumin, and at least one putrescine and progesterone. Fibroblast growth factor (e.g., FGF2) may also be included in the medium as needed. In another embodiment, the medium does not contain fibroblast growth factor. In some embodiments, a retinoic acid receptor agonist is also included to promote neural differentiation into specific nervous systems, depending on the concentration of the retinoid used. Exemplary types of suitable retinoic acid receptor agonists are retinoids and retinoid analogs, including: all-trans retinoic acid (ATRA), retinyl acetate, EC23 (4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl)benzoic acid; CAS number: 104561-41-3), BMS453 (4-[(1E)-2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl]benzoic acid; CAS number: 166977-43-10), and fenretinide (N-(4-hydroxyphenyl)retinamide; C AS number: 65646-68-6), AM580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]benzoic acid; CAS number: 102121-60-8), Tazarotene (6-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)ethynyl]-3-pyridinecarboxylate ethyl ester; CAS number: 118292-40-3), and TTNPB (4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; CAS number: 71441-28-6).Other exemplary retinoic acid receptor agonists available include: AC261066 (4-[4-(2-butoxyethoxy-)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid; CAS number: 870773-76-5), AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid; CAS number: 59662-49-6), adapalene (6-(4-methoxy-3-tricyclo[3.3.1.13,7]deca-1-ylphenyl)-2-naphthalenecarboxylic acid; CAS number :106685-40-9), AM80 (4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)amino]carbonyl]benzoic acid; CAS number: 94497-51-5), BMS753 (4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl)carbonyl]amino]benzoic acid; CAS number: 215307-86-1), BMS961 (3-fluoro-4-[[ 2-Hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)acetyl]amino]benzoic acid; CAS number: 185629-22-5), CD1530(4-(6-hydroxy-7-tricyclo[3.3.1.13,7]deca-1-yl-2-naphthalenyl)benzoic acid; CAS number: 107430-66-0), CD2314(5-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2 (-anthracenyl)-3-thiophenecarboxylic acid; CAS number: 170355-37-0), CD437(6-(4-hydroxy-3-tricyclo[3.3.1.13,7]deca-1-ylphenyl)-2-naphthalenecarboxylic acid; CAS number: 125316-60-1), and Ch55(4-[(1E)-3-[3,5-bis(1,1-dimethylethyl)phenyl]-3-oxo-1-propenyl]benzoic acid; CAS number: 110368-33-7). In some embodiments, the concentration of the retinoic acid receptor agonist (e.g., all-trans retinoic acid (ATRA)) is about 0.1 μM to about 1.0 μM.A preferred concentration of retinoic acid receptor agonist is in the range of about 0.1 μM to about 20 μM, for example, about 0.2 μM, 0.3 μM, 0.5 μM, 1.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 5 μM, 7 μM, 10 μM, 12 μM, 15 μM, 17 μM, or another ATRA concentration in the range of about 0.1 μM to about 20 μM. In some embodiments, the ATRA concentration is about 3.0 μM.

[0047] Guidelines for the induction and differentiation of pluripotent stem cells into neural stem cells can be found in U.S. Patent Application No. 13 / 795,485, entitled "Simplified Compositions and Methods of Generating Neural Stem Cells from Human Pluripotent Stem Cells," U.S. Patent Application No. 14 / 496,796, entitled "Compositions and Methods for Precise Patterning of Posterior Neuroectoderm from Human Pluripotent Stem Cells," and U.S. Patent Application No. 16 / 044,236, entitled "Methods And Culture Substrates For Controlled Induction Of Biomimetic Neural Tissues Comprising Singular Rosette Structures." The contents of each of these patent applications are incorporated herein by reference in their entirety.

[0048] Suitable progenitor cells and culture conditions capable of forming cardiac or intestinal epithelial ducts are intentionally designed. For example, cardiac progenitor cells or intestinal epithelial progenitor cells can be seeded on a micropatterned substrate, and a supported hydrogel can be placed on top of it, as described above.

[0049] Bioengineered neuroepithelial tubes obtained by the method of the present invention have a microscale cellular composition similar to that of the corresponding in vivo developing organ. For example, in the case of bioengineered neuroepithelial tubes, the biomimetic organoid has a single, closely spaced, polarized neuronal rosette structure (as shown, e.g., by laminin and N-cadherin polarizing staining within the neuroepithelial tube) that extends along 75% of the organoid's length in some embodiments. In some embodiments, the closely spaced polarized neuronal rosette structure extends along at least 75%, 80%, 90%, or 99% of the organoid's length. As used herein, the term “polarized” means a cell having a bipolar (or tripolar or more) morphology in which certain cellular components are heterogeneously distributed between two or more poles of the cell (e.g., apical and basal poles). In some embodiments, polarized cells are neuroepithelial cells that exhibit apical-basal polarity with respect to N-cadherin expression. For example, the presence of apical polarity in N-cadherin foci is a surrogate marker for neuronal rosette formation. Polarized neuroepithelial tissue is characterized by the presence of coherent cadherin ring structures (formed by apical localization of N-cadherins) directed toward the center of laminin, a basement membrane protein that surrounds the neuroepithelial tubules on the outer surface and is in contact with the hydrogel. Images in Figure 8 show various immunostained cross sections of bioengineered neuroepithelial tubules prepared according to the methods of this disclosure. The basal green fluorescence is an artifact of the tissue section / staining and is not the same as the apical N-cadherin staining within the center of each section.

[0050] The methods provided herein are in vitro methods for efficiently and reliably producing biomimetic oval neuroepithelial tissue and bioengineered neuroepithelial tubes having a single rosette throughout the elongated tissue (as can be seen in the cross-section of the tube), wherein the designed biomimetic oval neuroepithelial tissue exhibits a microscale cellular composition (i.e., cellular structure) similar to that of a developing human neuroepithelial tube. The term “biomimetic” as used in relation to the designed neuroepithelial tissue preferably means a single elongated tissue having an oval tubular or substantially tubular structure and exhibiting similarity to the anatomical and cellular structure of an in vivo embryonic neuroepithelial tube (e.g., a neuronal rosette-like cross-sectional structure) or a cross-sectional section thereof. The biomimetic oval neuroepithelial tissue described herein is an in vitro generated (e.g., designed) tissue or tube that mimics the cellular structure of a polarized nerve, where the polarized NSC exhibits apical N-cadherin expression and basal extracellular matrix protein deposition of an embryonic neural tube. As used herein, the term “substantially tubular” means that the structure as a whole has a tubular arrangement, but does not need to be a perfect cylinder. In some embodiments, the structure has a tubular arrangement with an elliptical or circular cross-sectional shape as a whole.

[0051] In some embodiments, the methods disclosed herein preferably exhibit a polarized rosette-like cross-sectional structure, where at least 60% of the cells in the polarized rosette structure are Pax6 + / N-Cadherin + By generating neuroepithelial tissue organoids using bioengineering, we found that over 75% of the bioengineered 3D neuroepithelial tissue exhibits a single, closely spaced rosette structure comparable to developing human neuroepithelial tubules.

[0052] The expression (or absence thereof) of numerous cell type-related markers can be used to characterize the differentiation of hPSCs or NMPs into neural stem cells during the implementation of the methods described herein. For example, the expression of several markers associated with the pluripotency of hPSCs is decreased during the differentiation of hPSCs into neural stem cells. Such pluripotency markers include Oct4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Neural mesoderm precursors (NMPs) have the following expression profile: SOX2 + / OCT4 - / T + / PAX6 - During the differentiation of NMPs into neural stem cells or neuronal cell types, the expression of these NMP markers, as well as other mesoderm or endoderm-related markers, such as T(braculi) and SOX17, similarly decreases or disappears over time. Conversely, the expression of neural stem cell-related markers increases during differentiation. Suitable markers for neural stem cells and neuronal differentiation (at the RNA or protein level) include, but are not limited to, PAX6, SOX2, nestin, N-cadherin, and SOX1.

[0053] In some embodiments, the differentiation methods provided herein further include exposing cultured cells (e.g., cultured hPSCs) to a transient increase or "boost" of Wnt / β-catenin signaling by transiently exposing the cultured hPSC cells to a Wnt / β-catenin signaling agonist about 72 hours after culture, as described, for example, in U.S. Patent Application No. 14 / 496,796 (which is incorporated herein by reference as if it were described in its entirety). In some embodiments, cultured NMP cells are exposed to a Wnt / β-catenin signaling "boost" about 72 hours after culture (i.e., about 72 hours after seeding on a micropatterned substrate). As will be understood by those skilled in the art, Wnt / β-catenin signaling can be activated by modulating the function of one or more proteins involved in the Wnt / β-catenin signaling pathway that increase β-catenin expression levels or activity, TCF and LEF expression levels, or β-catenin / TCF / LEF-induced transcriptional activity. In some embodiments, activation of Wnt / β-catenin signaling is achieved by inhibiting Gsk3 phosphotransferase activity or Gsk3 binding interactions. While not theoretically bound, inhibition of Gsk3 phosphorylation of β-catenin is thought to suppress tonic degradation of β-catenin, thereby increasing β-catenin levels and activity and promoting differentiation of pluripotent stem cells. Gsk3 inhibition can be achieved in various ways, including, but not limited to, Gsk3 phosphotransferase activity, RNA interference knockdown of Gsk3, and providing small molecules that inhibit the overexpression of dominant-negative forms of Gsk3. Dominant-negative forms of Gsk3 are known in the art, for example, in Hagen et al., 2002, J. Biol. Chem., 277:23330-23335, which describes Gsk3 including the R96A mutation. This literature is incorporated herein by reference in its entirety.

[0054] In some embodiments, Gsk3 is inhibited by contacting cells with Gsk3 phosphotransferase or a small molecule that inhibits Gsk3 binding interactions. Suitable small molecule Gsk3 inhibitors include, but are not limited to, CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-azakemporone, bis-7-indolylmaleimide, and any combination thereof. In some embodiments, CHIR99021, CHIR98014, and BIO-acetoxime are used to inhibit Gsk3 in pluripotent stem cells in the differentiation methods described herein. In one embodiment, the small molecule Gsk3 inhibitor is CHIR99021 at concentrations ranging from about 3 μM to about 12 μM, for example, about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, and 12 μM, or CHIR99021 at other concentrations ranging from about 3 μM to about 12 μM. In another embodiment, the small molecule Gsk3 inhibitor is CHIR98014 at concentrations ranging from about 0.1 μM to about 1 μM, for example, about 0.1 μM, about 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, and 0.9 μM, or CHIR98014 at other concentrations ranging from about 0.1 μM to about 1 μM. In another embodiment, the small molecule Gsk3 inhibitor is BIO-acetoxime at concentrations ranging from about 0.1 μM to about 1 μM, for example, about 0.1 μM, about 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, and 0.9 μM, and other concentrations of BIO-acetoxime in the range of about 0.1 μM to about 1 μM.

[0055] In other embodiments, Gsk3 activity is inhibited by RNA interference knockdown of Gsk3. For example, Gsk3 expression levels can be knocked down using commercially available siRNA against Gsk3, e.g., SignalSilence® GSK-3α / β siRNA (catalog #6301 from Cell Signaling Technology®, Danvers, MA), or retroviral vectors with inducible expression cassettes for Gsk3, e.g., the commercially available Tet-inducible retroviral RNA interference (RNAi) system from Clontech (Mountain View, CA, catalog no. 630926), or the cumate-induction system from Systems Biosciences, Inc. (Mountain View, CA), e.g., the SparQ® system, catalog no. QM200PA-2.

[0056] In other embodiments, the Wnt / β-catenin signaling pathway is activated by overexpressed β-catenin itself, for example, human β-catenin (exemplary nucleotide and amino acid sequences can be found in Genbank acceptance numbers: X87838 and CAA61107.1, respectively). In one embodiment, β-catenin overexpression is achieved using inductive expression, for example, any of the aforementioned inductive expressions. Alternatively, a stabilized isoform of constitutively active β-catenin is used, which includes, for example, point mutations S33A, S37A, T41A, and S45A, as described in Baba et al., 2005, Immunity 23(6):599-609. This document is incorporated herein by reference in its entirety.

[0057] In yet another embodiment, activation of the Wnt / β-catenin signaling pathway in pluripotent stem cells is achieved by contacting the cells with a drug that disrupts the interaction between β-catenin and axin, a member of the β-catenin disruption complex. Disruption of the axin / β-catenin interaction allows β-catenin to avoid degradation by the disruption complex, thereby increasing net β-catenin levels and promoting β-catenin signaling. For example, the axin / β-catenin interaction can be disrupted in pluripotent cells by contacting the cells with the compound 5-(furan-2-yl)-N-(3-(1H-imidazole-1-yl)propyl)-1,2-oxazole-3-carboxamide ("SKL2001") (commercially available from EMD Millipore as catalog number 681667). The effective concentrations of SKL2001 that activate Wnt / β-catenin signaling are in the range of approximately 10 μM to 100 μM, which are concentrations of approximately 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or other concentrations of SKL2001 in the range of approximately 10 μM to 100 μM.

[0058] Any suitable method can be used to confirm the homogeneity of the biomimetic oval neuroepithelial tissue provided herein or the presence or absence of specific components. Suitable methods for detecting the presence or absence of biological markers are well known in the art and are not limited to, but include immunohistochemical testing, qRT-PCR, RNA sequencing, etc., for evaluating gene expression at the RNA level. In some embodiments, cell types or biomolecules within the biomimetic oval neuroepithelial tissue are detected and identified using methods such as immunohistochemical testing. For example, whole organoids or parts thereof can be stained by immunohistochemical testing for specific differentiation markers. In some embodiments, it would be convenient to perform dual-labeled immunofluorescence to evaluate the relative expression of individual marker proteins or to detect multiple progenitor cells or differentiated cell types within a construct. Suitable primary and secondary antibodies are known and available to those working in the art. In addition, gene expression profiles of the biomimetic oval neuroepithelial tissue of the present invention can be obtained using microarray technology or nucleic acid sequencing (e.g., RNA sequencing). Biological markers for neuroepithelial cells include, for example, Pax6 and N-cadherins. Quantitative methods for evaluating marker expression at the protein level are also known in the art. For example, flow cytometry can be used to determine the fraction of cells in a given cell population that express or do not express the biological marker of interest.

[0059] The structure of the designed neuroepithelial tissue and tubules obtained according to the methods of this disclosure can be analyzed using any suitable method. In certain embodiments, the analytical methods are useful for detecting and identifying the presence of nerve cells (neurons) and neuronal supporting cells (e.g., glia) in the bioengineered neuroepithelial tissue or tubules. For example, confocal microscopy and other microscope-based imaging methods can be used. In such embodiments, confocal microscopy can be used to obtain confocal images of biomimetic oval neuroepithelial tissue or bioengineereric tubules fluorescently labeled to detect N-cadherin expression, as described in the following examples, which can collect multiple images of cross-sections of biomimetic oval neuroepithelial tissue or bioengineereric tubules treated with detectably labeled antibodies or dyes that have specificity for various cell types present at specific developmental stages of the human neural tube (Figures 3, 6, and 9). For example, an exemplary protocol for detecting and analyzing biomimetic oval neuroepithelial tissue structure includes scanning through biomimetic oval neuroepithelial tissue or bioengineered neuroepithelial tubes in a user-defined incremental or step-by-step manner using a 60X objective lens at 1024x1024 pixels to acquire one or more (e.g., a series of images) confocal images, and analyzing the large number of acquired confocal images using, for example, a machine learning program for image classification to detect cell types present in or adjacent to the cell structure.

[0060] Biomimetic oval neuroepithelial tissues designed from induced pluripotent stem cells enable, for example, the modeling of drug responses in 3D structures that replicate neurogenesis in individuals with a specific genetic background or detectable phenotype. Therefore, target-specific human iPS cell-derived biomimetic oval neuroepithelial tissues are useful for identifying genetic factors and epigenetic influences that contribute to the variable effects of known or unknown drugs on neurogenesis / neurodifferentiation.

[0061] Patient-specific somatic cells for reprogramming into induced pluripotent stem cells can be obtained or isolated from the target tissue of interest by biopsy or other tissue sampling methods. In some embodiments, the target-specific cells are manipulated in vitro before use in the neuroepithelial tissue construct of the present invention. For example, the target-specific cells may be proliferated, differentiated, genetically modified, exposed to polypeptides, nucleic acids or other factors, cryopreserved, or otherwise modified before seeding onto a micropatterned substrate to obtain the bioengineered neuroepithelial tissue described herein.

[0062] Standardizing culture conditions through the use of synthetic media minimizes the potential for lot-to-lot or batch-to-batch variability in the substances to which cells are exposed in the cell culture. Therefore, the effects of various differentiation factors are more predictable when added to cells and tissues cultured under known compositional conditions. As used herein, the term “serum-free” means cell cultures that are free from or substantially free from serum obtained from animal (e.g., fetal bovine) blood. Generally, culturing cells or tissues in the absence of animal-derived substances (i.e., under conditions free from heterogeneous substances) reduces or eliminates the possibility of interspecies viral or prion transmission.

[0063] Applications of the biomimetic oval neuroepithelial tissue provided herein include, but are not limited to, in vitro screening of drugs for applications that modulate in vivo neural tube formation or CNS development. For example, in vitro-generated biomimetic oval neuroepithelial tissue can be used for high-throughput screening of candidate drugs. The standardized and reproducible production of biomimetic oval neuroepithelial tissue can provide a revolutionary experimental paradigm for conducting personalized neuroscience tests. For example, neuroepithelial organoid tissues are useful for testing the effects of gene mutations on development and the function of the entire human CNS, conducting personalized neuroscience tests using induced pluripotent stem cell (iPS cell)-derived neural stem cells, and evaluating the neurotoxicity or other effects of various drugs on neurodevelopment. In some embodiments, the designed neuroepithelial tissues of the present invention are useful for drug discovery and development, including screening for metabolic stability, drug-drug interactions, toxicity, and infectious diseases. Exemplary test agents include, but are not limited to, infectious pathogens, proteins, peptides, antibodies, small molecules, oligonucleotides, polynucleotides, peptide mimetic compounds, cytotoxic agents, pharmaceuticals, and xenobiotics (e.g., environmental toxins, chemical / biological weapons agents, natural compounds, and dietary supplements).

[0064] In some embodiments, the biomimetic oval neuroepithelial tissue described herein can be screened to identify reagents that modulate neural tube development and human CNS development. The screening method may include, or be based thereon, contacting the test agent with the bioengineered neuroepithelial tissue derived in vitro; and detecting the bioengineered effect of the agent on the neuroepithelial tissue (e.g., an effect that interferes with or alters the development of the biomimetic oval neuroepithelial tissue or the differentiation of neuronal cell types within the biomimetic oval neuroepithelial tissue). In some embodiments, the screening method includes screening candidate compounds to identify test agents that promote human CNS development. In other embodiments, candidate compounds can be screened for toxicity to human neuronal cell types or tissues. In some embodiments, detection includes detecting at least one positive or negative effect of the agent on the morphology or lifespan of such cells and tissues, thereby identifying agents that extend or shorten the lifespan of human neuronal cell types or tissues, or agents that have a positive or negative effect on the morphology of human neuronal cell types or tissues, as having an effect on development in human neuroepithelial tubes or nerve tissues. In some embodiments, detection includes, but is not limited to, performing methods including RNA sequencing, gene expression profiling, transcriptome analysis, cell proliferation assays, metabolome analysis, reporter or sensor detection, protein expression profiling, Förster resonance energy transfer (FRET), metabolic profiling, and microdialysis. In some embodiments, drugs can be screened for their effects on gene expression, and detection may include assaying differential gene expression compared to uncontacted biomimetic oval neuroepithelial tissue. In addition, in some embodiments, the biomimetic oval neuroepithelial tissue of this disclosure is suitable as a direct graft for tissue regeneration and repair.

[0065] In exemplary embodiments, detecting and / or measuring positive or negative changes in the expression levels of at least one gene after exposure (e.g., contact) to one or more biomimetic oval neuroepithelial tissues of a test compound includes, for example, whole transcriptome analysis using RNA sequencing. In such embodiments, gene expression is calculated using, for example, LightCycle, RSEM (RNA-Seq with maximal expected value), Excel, and data processing software programs such as Prism. See Stewart et al., PLoS Comput. Biol. 9:e1002936 (2013). This document is incorporated herein by reference in its entirety. Where appropriate, statistical comparisons can be performed using ANOVA analysis, analysis of variance with Bonferroni correction, or two-tailed Student's t-test, where a value of P<0.05 indicates significantity. RNA or proteins can be isolated from the neural construct using any suitable method. For example, total RNA can be isolated and reverse transcribed to obtain cDNA for base H determination. The test compounds can be dissolved in a solvent such as dimethyl sulfoxide (DMSO) before contact with one or more bioengineered neuroepithelial tissues provided herein. In some embodiments, drug identification involves analyzing positive or negative changes in bioactivity, including, but not limited to, gene expression, protein expression, cell viability, and cell proliferation, of the bioengineered neuroepithelial tissue. For example, gene expression profiles can be analyzed using microarrays before, during, or after contact with multiple test compounds in the bioengineered neuroepithelial tissue. In some embodiments, the methods of this disclosure further include additional analyses such as metabolic assays and protein expression profiling.

[0066] In another embodiment, the foregoing provides a cell culture substrate comprising one or more components useful for obtaining in vitro biomimetic elliptical neuroepithelial tissue or bioengineered neuroepithelial tubules. The components of the cell culture substrate may include one or more micropatterned substrates described herein. The cell culture substrate may be a tissue culture dish, a well plate (e.g., a multiwell plate), a microfluidic device, or any other substrate suitable for culturing cells on one or more micropatterned substrates to obtain in vitro biomimetic neuroepithelial tissue.

[0067] In another embodiment, provided herein is a kit comprising one or more components useful for obtaining in vitro biomimetic neuroepithelial tissue or bioengineered neuroepithelial tubules. The components of the kit may include one or more micropatterning substrates described herein. The kit may also include a synthetic culture medium and one or more culture medium additives (e.g., growth factors, small molecule compounds, culture medium supplements). The kit may further include progenitor cells useful for seeding the micropatterning substrate and instructions for cell seeding and culture. In some embodiments, it would be convenient to include one or more micropatterning substrates described herein and one or more agents useful for differentiating the neuroepithelial tissue to obtain various types of nerve cells (neurons) (e.g., growth factors, small molecule agents, agents for creating morphogen gradients) in the kit. In such embodiments, the kit may also include a microfluidic device or its components for, for example, delivering the patterning agent to the bioengineered neuroepithelial tissue in a predetermined manner. In some embodiments, the kit further includes a hydrogel used to cover biomimetic elliptical neuroepithelial tissue to obtain bioengineered neuroepithelial tubes encapsulated by the method of the present disclosure. The present invention will be fully understood by considering the following non-limiting embodiments.

[0068] Examples This example demonstrates the in vitro fabrication of biomimetic elliptical neuroepithelial tissue containing a single polarized rosette structure along the length of the tissue (see Figures 3, 4, 5, and 6). As shown in Figure 8, a cross-section of the bioengineered neuroepithelial tubule fabricated therefrom revealed a well-organized single neuronal rosette of polarized N-cadherin-expressing cells along the length of the tissue.

[0069] Materials and methods Fabrication of Micropatterned Array Substrates: Micropatterned array cell culture substrates were fabricated using a combination of methods known in the art (e.g., Knight et al., 2015, Chemical Communications 51:5238-241; Sha et al., 2013, Biomacromolecules 14:3294-3303, these publications are incorporated herein by reference in their entirety). A polydimethylsiloxane (PDMS) stamp with a post and microwell mechanism array was produced as a relief mold on a silicon wafer (Figure 1). The wafer was designed in AutoCAD and purchased from FlowJEM (available at flowjem.com). The PDMS stamp was coated with ω-mercaptoundecylbromoisobutyrate (2 mM in 100% ethanol), dried under inert gas, and then brought into equiangle contact with a coverslip coated with 180 nm gold (Au) on 30 nm titanium (Ti). Next, the finely patterned slides were incubated in 100% ethanol for 10 minutes, dried under nitrogen, and transferred to a Schlenk flask under reduced pressure. A solution of poly(ethylene glycol) methyl ether methacrylate (PEG-MEMA) macromonomer (Sigma Aldrich) with water, methanol (obtained from Thermo Fisher), copper(II) bromide (Sigma Aldrich), and 2',2'-bipyridine (Sigma Aldrich) was degassed and transferred to a reaction flask. Surface-initiated atom transfer radical polymerization (SI-ATRP) of the PEG polymer was initiated by adding L-ascorbic acid (Sigma Aldrich) in deionized water to the reaction flask. ATRP was continued at room temperature for 16 hours to produce finely patterned PEG brushes. Polymerization was terminated by adding air, followed by rinsing with ethanol and water, and then drying in an inert gas. In a sterile hood, the substrates were rinsed five times with sterile PBS (Thermo Fisher) and transferred to individual wells of a 12-well tissue culture polystyrene (TCPS) plate, where they were made cell-adherent by adsorption of 0.083 mg / mL of Matrigel (WiCell) in DMEM / F-12 (Thermo Fisher) via overnight incubation at 37°C (Figure 2).

[0070] Results demonstrating the generation of single fused nerve rosette tissue and neuroepithelial tissue, including neuroepithelial tubules, are described in detail below and in corresponding Figures 2–8, as provided below. Generation of finely patterned forebrain neuroepithelial tissue: WA09(H9)hESCs were obtained from WiCell, verified by WiCell as karyotypically normal, and negative for mycoplasma. All pluripotent strains were maintained in Essential 8 medium (E8) on Matrigel-coated plates and passaged 10 times using Versine (Thermo Fisher) in the usual manner. NEC induction from hPSCs was performed according to the E6 protocol (Lippmann et al., 2014, Stem Cells 32:1032-1042; this document is incorporated herein by reference in its entirety). To generate finely patterned tissue from hPSCs, hPSC cultures at approximately 85% concentration were rinsed with PBS, dissociated with actase at 37°C for 5 minutes, and collected by centrifugation at 1000 rpm for 5 minutes (Figures 2-3). Next, unicellular hPSCs were suspended in E8 medium with 10 μM ROCK inhibitor, and 165,000 cells / cm³ were placed on a fine patterning substrate in 2 mL of medium per well of a 12-well plate. 2 Seeds were seeded. The following day, the medium was replaced with 2 mL of E6 medium, and thereafter, 50% of the medium was changed daily (Figure 4). By day 5, micropatterned neural tissue fusion extended to all transverse rows on the micropatterned array (Figure 5). Circular micropatterns separated by 50 μm edge-to-edge distances and bridges were single N-cadherins across all transverse rows of the micropatterned array. + The cells formed adjacent fusion tissue with rosette polarization and began to form three-dimensional tubes with apical N-cadherin expression over the 5-day culture period (Figure 6).

[0071] Matrigel encapsulation of finely patterned forebrain neuroepithelial tissue: At the time of tissue fusion and rosette alignment (day 5 or 6), 750 μL of neuroepithelial tissue medium was removed using a P1000 micropipette and replaced with 750 μL of Matrigel at 4°C (Figure 7). The Matrigel was then incubated at 37°C for 20 minutes to induce gelation (Figure 7). After gelation, 1 mL of E6 medium was pipetted into each well and returned to the incubator. The following day, the Matrigel layer was detached from the tissue culture plate wall using sterile forceps (Figure 8). Fresh E6 medium was pipetted beneath the gel to separate the Matrigel layer from the finely patterned substrate (Figure 8). The substrate was then carefully removed with tweezers and discarded. 50% E6 medium replacement was performed daily thereafter until the target experimental time (Figure 8).

[0072] The present invention has been described in relation to what is considered to be the most practical and specific embodiment to date. However, the present invention is presented illustratively and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will understand that the present invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as described in the appended claims.

Claims

1. A method for producing biomimetic elliptical neuroepithelial tissue having a single rosette structure in vitro, (a) A step of seeding human pluripotent stem cells (hPSCs) on a finely patterned substrate capable of biomimetic neural morphogenesis of cells cultured thereon in the presence of a Rho kinase inhibitor, wherein the finely patterned substrate includes at least two circular bounded regions connected by cell adhesion bridges, (b) A step of culturing the seeded cells from step (a) on the fine patterned substrate in the presence of a pluripotency maintenance medium for a first culture period of about 1 to 2 days to obtain a first cell aggregate, wherein the pluripotency maintenance medium contains a Rho kinase inhibitor, (c) A step of culturing the cells obtained in step (b) in a basic neuronal differentiation medium for a second culture period of about 3 to about 6 days under adherent culture conditions, A method characterized by having a biomimetic elliptical neuroepithelial tissue having a single rosette structure, wherein the tissue contains polarized neuroepithelial cells and has a microscale cellular composition similar to a transverse section of an in vivo-developed human neural tube.

2. The method according to claim 1, wherein each of the at least two circular bounded regions has a diameter of about 100 μm to about 300 μm.

3. The method according to claim 1, wherein the cell adhesion bridge has a length of about 25 μm to about 125 μm and a width of about 10 μm to about 50 μm.

4. The method according to claim 1, wherein the cell adhesion bridge has a length of about 50 μm to about 100 μm and a width of about 25 μm.

5. The aforementioned hPSC is approximately 75 x 10 3 cells / cm 2 ~Approx. 2.5x10 5 cells / cm 2 The method according to claim 1, wherein the seeds are sown on the finely patterned substrate at a density of .

6. hPSCs: approximately 165,000 cells / cm² 2 The method according to claim 5, wherein the seeds are sown on the finely patterned substrate at a density of .

7. The method according to claim 1, wherein the pluripotency maintenance medium is a synthetic medium containing DMEM / F-12, ascorbic acid, sodium bicarbonate, selenium, insulin, transferrin, FGF2, and TGFβ1.

8. The method according to claim 1, wherein the pluripotency maintenance medium is E8 medium.

9. The method according to claim 1, wherein the basic neuronal differentiation medium is a synthetic medium comprising DMEM / F-12, ascorbic acid, sodium bicarbonate, selenium, insulin, and transferrin.

10. The method according to claim 1, wherein the basic neuronal differentiation medium is E6 medium.

11. The method according to claim 10, wherein the basic neuronal differentiation medium contains or does not contain a β-catenin pathway signaling activator, and further contains one or more FGFs.

12. The method according to claim 11, wherein the FGF is FGF2, FGF8a, FGF8b, FGF8f, FGF17, or FGF18.

13. The method according to claim 11, wherein the activator of the β-catenin pathway signaling is a GSK3 kinase inhibitor.

14. The method according to claim 13, wherein the GSK3 kinase inhibitor is CHIR99021.

15. The method according to claim 9, wherein the basic neuronal differentiation medium further comprises about 100 ng / ml to about 200 ng / ml of FGF8b and about 3 μM to about 9 μM of CHIR99021.

16. The method according to claim 1, wherein the second culture period is approximately 5 days.

17. The method according to claim 1, further comprising transiently exposing cells on the finely patterned substrate to a Wnt / β-catenin signaling activator about 24 to 72 hours after plating on the finely patterned substrate.

18. The method further comprises seeding cells onto the finely patterned substrate for approximately 24 to 72 hours, and then exposing the seeded cells to RA and Sonic Hedgehog (SHH) or an SHH signaling agonist for approximately 1 to 5 days, thereby causing the rosette structure to develop Olig2 + The method according to claim 1, comprising motor neuron progenitor cells (pMNs).

19. The method according to claim 1, wherein the finely patterned substrate comprises one or more polyethylene glycol (PEG) brushes or peptide-immobilized PEG brushes arranged in a user-defined bounded geometry.

20. A step of covering the surface of the neuroepithelial tissue obtained in step (c) of claim 1 with a hydrogel layer, and The method according to claim 1, further comprising the steps of culturing the neuroepithelial tissue containing the hydrogel layer for about 24 hours, thereby transforming the tissue into a bio-engineered neuroepithelial tube and enclosing it with the hydrogel layer.

21. The method according to claim 20, further comprising the step of removing the hydrogel containing the encapsulated bioengineered neuroepithelial tubule from the micropatterned substrate.

22. The method according to claim 21, further comprising the step of fixing and sectioning the extracted hydrogel for analysis.

23. The method according to claim 20, wherein the hydrogel is Matrigel.

24. Neuroepithelial tissue obtained by in vitro biotechnology according to the method of claim 1, wherein the tissue comprises a single rosette of polarized neuroepithelial cells and has a microscale cellular structure similar to a transverse section of a human neural tube during in vivo development.

25. A neuroepithelial tube obtained by in vitro bioengineering according to the method of claim 20, comprising a single rosette of polarized neuroepithelium and having a microscale cellular structure similar to that of a human neural tube during in vivo development.

26. A cell culture substrate comprising one or more finely patterned substrates capable of instructing the biomimetic neuroepithelial tissue morphogenesis of cells cultured thereon, wherein the one or more finely patterned substrates include at least two circular cell-adherent microscale regions connected by cell adhesion bridges.

27. A kit comprising the cell culture substrate described in claim 26, further comprising one or more of the following: a vial of progenitor cells, a pluripotent basal medium, a basal neuronal differentiation medium, a microfluidic device, and a reagent for creating a morphogen gradient.

28. A method for producing neuroepithelial tubules by biotechnology in vitro, (a) A step of seeding human pluripotent stem cells (hPSCs) on a finely patterned substrate capable of biomimetic neural morphogenesis of cells cultured thereon in the presence of a Rho kinase inhibitor, wherein the finely patterned substrate includes at least two circular bounded regions connected by cell adhesion bridges, (b) A step of culturing the seeded cells from step (a) on the fine patterned substrate in the presence of a pluripotency maintenance medium for a first culture period of about 1 to 2 days to obtain a first cell aggregate, wherein the pluripotency maintenance medium contains a Rho kinase inhibitor, (c) The cells obtained in step (b) are cultured in a basic neuronal differentiation medium for a second culture period of approximately 3 to 6 days under adherent culture conditions, thereby obtaining neuroepithelial tissue by biotechnology. (d) A step of covering the surface of the bio-engineered neuroepithelial tissue obtained in step (c) with a hydrogel layer, (e) A step of culturing the bio-engineered neuroepithelial tissue from step (d) for approximately 24 hours, thereby transforming the tissue into a bio-engineered neuroepithelial tube, and enclosing it with the hydrogel layer, (f) A step of removing the hydrogel containing the enclosed neuroepithelial tube from the fine patterned substrate to obtain a neuroepithelial tube by biotechnology, A method characterized by including the following.

29. An in vitro bioengineeral neuroepithelial tube obtained by the method of claim 28, comprising a single rosette of polarized neuroepithelium and having a microscale cellular structure similar to that of an in vivo-developed human neural tube.

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