Method and composition for producing stem cell-derived spinal cord GABA inhibitory neurons for use in the treatment of spinal cord injury

Abstract

Disclosed herein are in vitro methods for inducing the differentiation of human stem cells into spinal cord GABAergic interneurons and their spinal cord dorsal interneuron domain 4 (dI4) progenitor cells, and cells produced by such methods. The present disclosure also provides for the use of such cells to treat spinal cord injury.

Description

Technical Field

[0001] Cross - reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 047,697, filed Jul. 2, 2020, the entire disclosure of which is incorporated herein by reference.

[0003] Field

[0004] This disclosure relates to methods and compositions for producing spinal cord GABA - intermediate neurons differentiated from human pluripotent stem cells, particularly spinal cord dorsal intermediate neuron domain 4 (dI4) GABA - intermediate neurons, and their use for cell - based treatment of spinal cord injury.

Background Art

[0005] Introduction

[0006] Spinal cord injury (SCI) is one of the major causes of physical disability. It causes chronic motor disorders or even paralysis, and often involves complications such as muscle spasticity and pain, which further prolong the lack of walking exercise over time. Considerable research efforts have been made to develop new treatment methods for SCI. Cell transplantation has emerged as a potential strategy for repairing spinal cord injury. Many cell types, including Schwann cells, neural stem and progenitor cells, oligodendrocyte progenitor cells (OPCs), olfactory ensheathing cells (OECs), and mesenchymal stem cells (MSCs), have been evaluated for their ability to treat SCI. These cells are thought to bridge the damaged nerve cord and provide neuroprotection and myelination. The main goals of cell therapy are the restoration of walking ability and, in some cases, pain relief. Reducing muscle spasticity and neuropathic pain, the major complications of SCI, has received less attention.

[0007] Spasticity, or involuntary muscle contractions, are present in 94% of patients with cervical and thoracic SCI, and in 5% of patients with lumbar or sacral SCI. Post-SCI spasticity is caused by loss of inhibitory somatosensory interneurons, downregulation of KCC2, and increased expression of 5-HT2c receptors in spinal cord dI4, all of which lead to excessive excitation of spinal cord dI4. Therefore, reversing inhibition of the excitation imbalance in spinal cord dI4, such as through GABA inhibitory neuron transplantation, may alleviate spasticity. Methods for obtaining the necessary cells for treatment and improved SCI treatment are needed. [Overview of the project]

[0008] In one embodiment, the present disclosure relates to a method for producing a population of spinal cord dI4 progenitor cells from human stem cells. The method may comprise (a) culturing human stem cells in a culture medium comprising a Wnt signaling agonist, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor to produce a cell population comprising spinal cord neuroepithelial progenitor cells, wherein at least 95% of the resulting cell population are Sox1+ / Hoxa3+ spinal cord neuroepithelial progenitor cells; and (b) culturing the spinal cord neuroepithelial progenitor cells from step (a) in a culture medium further comprising a retinoic acid (RA) signaling agonist and an SHH signaling pathway inhibitor to produce a cell population comprising spinal cord dl4 progenitor cells, wherein at least 90% of the resulting cell population are PTF1A+ / PAX7+ / ASCL1+ spinal cord dl4 progenitor cells.

[0009] In some embodiments, the human stem cells are human pluripotent stem cells. In some embodiments, the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells. In some embodiments, the human stem cells are obtained from human embryos. In some embodiments, the Wnt signaling agonist comprises a GSK3 inhibitor selected from the group consisting of CHIR99021 and 6-bromo-iridium-3'-oxime. In some embodiments, the BMP signaling pathway inhibitor is selected from the group consisting of DMH-1, dorsomorphine, and LDN-193189. In some embodiments, the TGFβ signaling pathway inhibitor is selected from the group consisting of SB431542, SB505124, and A83-01. In some embodiments, the RA signaling agonist is selected from the group consisting of retinoic acid (RA) and EC23. In some embodiments, the SHH signaling inhibitor is selected from the group consisting of cyclopamine and SANT-1. In some embodiments, the culture medium in step (a) contains 2 μM SB431542, 2 μM DMH1, and 3 μM CHIR99021. In some embodiments, the human stem cells are cultured in the culture medium for 5 to 10 days in step (a). In some embodiments, the human stem cells are cultured in the culture medium for 7 days in step (a). In some embodiments, the culture medium in step (b) contains DMH-1, SB431542, CHIR99021, all-trans retinoic acid, and cyclopamine. In some embodiments, the culture medium in step (b) contains about 1 μM to 10 μM DMH-1, about 1 μM to 10 μM SB431542, about 1 μM to 10 μM CHIR99021, about 0.01 μM to 2 μM RA, and about 0.1 μM to 2 μM cyclopamine. In some embodiments, the culture medium of step (b) contains 2 μM SB431542, 2 μM DMH1, 3 μM CHIR99021, 0.1 μM all-trans retinoic acid, and 0.5 μM cyclopamine.In some embodiments, the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 5 to 10 days in step (b). In some embodiments, the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 7 days in step (b). In some embodiments, the spinal cord dl4 progenitor cells in step (b) express at least one gene selected from PTF1A, PAX7, and ASCL1, or a combination thereof. In some embodiments, at least a portion of the spinal cord dl4 progenitor cells in step (b) further differentiate into spinal cord GABAergic interneurons. In some embodiments, the spinal cord GABAergic interneurons express at least one gene selected from PAX2 and LHX1 / 5, or a combination thereof.

[0010] In further embodiments, the disclosure relates to a method for treating a target neurological disorder. The method may include administering to the target an effective amount of a spinal cord dI4 progenitor cell population produced by a method for producing a spinal cord dI4 progenitor cell population from human stem cells as detailed herein. In some embodiments, the neurological disorder includes spinal cord injury.

[0011] This disclosure provides other aspects and embodiments that will become apparent in connection with the following details and accompanying drawings.

[0012] This patent or application file includes at least one color drawing. A copy of the published patent or patent application, including the color drawing, will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a flowchart showing the directional differentiation of spinal cord dI4 progenitor cells and GABAergic interneurons from pluripotent stem cells. Abbreviations: PSC (pluripotent stem cell); dI4 (dorsal interneuron domain 4). [Figure 2-1]Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show the differentiation of dl4 spinal GABAergic neurons from hPSCs. Figure 2A is a schematic diagram showing the transcriptional codes of dorsal spinal progenitor cells and postmittal neurons. Figure 2B shows a scheme for the differentiation of hPSCs into spinal GABAergic neurons. SB=SB431542, CHIR=CHIR99021, RA=retinoic acid, CYC=cyclopamine. [Figure 2-2] Figures 2C to 2G show immunohistochemical staining of differentiated neural progenitor cells at D14 (Figures 2C to 2E) and postmittal neurons at D21 (Figures 2FC to 2G). [Figure 2-3] Figure 2H shows the results of RT-qPCR indicating the expression of dl4-related transcription factor genes NGN1, NGN2, LBX-1, and PAX2; dl1-3-related genes BRN3A, MATH1, TLX3, and ISL-1; dl5-6-related transcription factor genes LMX1B, WT1, TLX3, and BHLHB5; and the v1 local gene EN1. Figure 2I shows the quantification of the number of cells expressing HOXA3, PTF1A, PAX7, ASCL1, PAX2 / LHX1 / 5, and GABA in cells differentiated from hESC(H9) and iPSC(IMR-90). Scale bar: 50 μM. [Figure 3-1] Figures 3A, 3B, 3C, 3D, 3E, and 3F show patch-clamp electrophysiological recordings of differentiated cells. Figure 3A shows outward K+ and inward Na+ currents induced by electrical stimulation from -70mV to +100mV in 10mV increments. Figure 3B shows action potentials induced by current steps from -30pA to +50pA in 10pA increments. [Figure 3-2] Figures 3C to 3E show representative traces of typical firing patterns, characterized by sustained firing, delayed firing, and initial bursts. Figure 3F shows a statistical analysis of action potential amplitudes for different firing patterns. [Figure 4-1]Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, and 4J show the survival and differentiation of transplanted human cells. Figure 4A shows the experimental scheme outlining the schedule for SCI, cell transplantation, and behavioral analysis. Figure 4B shows the cavity size expressed as a percentage of the coronal section of the spinal cord in SCI (11.14% ± 1.3%), SCI + culture medium (10.95% ± 1.96%), and SCI + cell population (5.93% ± 0.79%). [Figure 4-2] Figure 4C shows the distribution of hNu and mCherry double-positive cells in the rostral spinal cord of the lesion cavity. Note that a small cavity is still present in the central part of the cross-section. Figure 4D shows the stereochemical quantification of the total number of hNu+ cells at 6 and 12 weeks post-transplant (n=6 at each time point). [Figure 4-3] Figure 4E shows the quantification of Ki67+ dividing cells in hNu+ transplanted cells and in endogenous spinal cord cells of Siamese animals at 6 and 12 weeks post-transplantation. [Figure 4-4] Figures 4F to 4H show co-immunostaining of hNu in the spinal cord 12 weeks post-transplant using NeuN (Figure 4F), DCX (Figure 4G), and GABA (Figure 4H). Figure 4I shows the quantification of NeuN+ neurons, GABA+ neurons, SOX9+ astrocytes, and MBP+ oligodendrocytes in hNu+ human cells 12 weeks post-transplant (n=6). [Figure 4-5] Figure 4J shows co-immunostaining for mCherry, GABA, and PTF1A in the graft. Scale bar: 50 μM. [Figure 5]Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, 5L, 5M, 5N, and 5O show the localization and projection of transplanted human neurons. Figure 5A shows a horizontal section cut through the transplant site and caudal (lumbar) white matter, showing the distribution of mCherry+ cells and axons. The insertion region is magnified in Figures 5B-5O. Figure 5B shows an amplification of insertion B, showing mCherry+ cells co-labeled with NeuN. Figure 5C shows a further magnification of insertion B using z-stack colocalization. Figure 5D shows an amplification of insertion D to show mCherry-positive (human) axons in contact with host NeuN+ neurons. The insertion is further magnified in Figure 5E using z-stack colocalization. Figure 5F shows a magnified view of insertion F, and Figure 5G shows a magnified view of insertion G, showing mCherry-positive axons in the lumbar white matter. A further overall cross-sectional view of the transplant site is shown, showing hNu and mCherry+ (Figure 5H), and hNu and NeuN+ (Figure 5I) human cells in the gray matter. Figure 5J shows a higher level of magnification, separating the channels of the insertion shown in Figure 5I. Figure 5K shows immunohistochemistry for hSyn and ChAT, showing numerous hSyn spots on the surface of ChAT+ spinal cord dI4. Figure 5L shows the insertion region of Figure 5K magnified in a Z section. Figure 5M is a z-stack image showing dual labeling for hSyn (green) and ChAT (red). Its insertion indicates the region of interest in relation to EM, and its arrow indicates the sampling direction of the EM section. Figure 5N shows correlated light microscopy and electron microscopy (CLEM) results, showing multiple hSyn+ terminals (green) on the surface of spinal cord dI4 (purple). Figure 5O shows a 3D reconstruction of FIB-SEM identifying hSyn on MN (EM cross-section of n=726). [Figure 6-1]Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the effects of cell transplantation on spasticity. The mean relative amplitude of the Hoffman reflex (H%) in the sham, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figure 6A) and 12 weeks (Figure 6B) post-transplant with enhanced stimulation frequencies of 0.1Hz to 5Hz is shown (n=6 in each group). The effects of H% CNO treatment in the sham surgery, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figures 6C, 6D, 6E) and 12 weeks (Figures 6F, 6G, 6H) post-transplant with enhanced stimulation frequencies of 1Hz to 5Hz are shown in Figures 6C to 6H. [Figure 6-2] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the effects of cell transplantation on spasticity. The mean relative amplitude of the Hoffman reflex (H%) in the sham, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figure 6A) and 12 weeks (Figure 6B) post-transplant with enhanced stimulation frequencies of 0.1Hz to 5Hz is shown (n=6 in each group). The effects of H% CNO treatment in the sham surgery, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figures 6C, 6D, 6E) and 12 weeks (Figures 6F, 6G, 6H) post-transplant with enhanced stimulation frequencies of 1Hz to 5Hz are shown in Figures 6C to 6H. [Figure 6-3] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the effects of cell transplantation on spasticity. The mean relative amplitude of the Hoffman reflex (H%) in the sham, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figure 6A) and 12 weeks (Figure 6B) post-transplant with enhanced stimulation frequencies of 0.1Hz to 5Hz is shown (n=6 in each group). The effects of H% CNO treatment in the sham surgery, SCI, SCI+ cell, and SCI+ culture medium groups at 6 weeks (Figures 6C, 6D, 6E) and 12 weeks (Figures 6F, 6G, 6H) post-transplant with enhanced stimulation frequencies of 1Hz to 5Hz are shown in Figures 6C to 6H. [Figure 7-1]Figures 7A, 7B, 7C, 7D, 7E, and 7F show the effect of cell transplantation on locomotor activity. Figure 7A shows the scores of the sham (black), SCI (red), SCI + cells (blue), and SCI + culture medium (green) groups. Figures 6B - 6F show the hindlimb's repetitive step time (Figure 6B), swing time (Figure 6C), repetitive step distance (Figure 6D), repetitive step frequency (Figure 6E), and locomotor speed (Figure 6F) of various groups. [Figure 7-2] Figures 7A, 7B, 7C, 7D, 7E, and 7F show the effect of cell transplantation on locomotor activity. Figure 7A shows the scores of the sham (black), SCI (red), SCI + cells (blue), and SCI + culture medium (green) groups. Figures 6B - 6F show the hindlimb's repetitive step time (Figure 6B), swing time (Figure 6C), repetitive step distance (Figure 6D), repetitive step frequency (Figure 6E), and locomotor speed (Figure 6F) of various groups. [Figure 7-3] Figures 7A, 7B, 7C, 7D, 7E, and 7F show the effect of cell transplantation on locomotor activity. Figure 7A shows the scores of the sham (black), SCI (red), SCI + cells (blue), and SCI + culture medium (green) groups. Figures 6B - 6F show the hindlimb's repetitive step time (Figure 6B), swing time (Figure 6C), repetitive step distance (Figure 6D), repetitive step frequency (Figure 6E), and locomotor speed (Figure 6F) of various groups. [Figure 8] Figures 8A and 8B show the effect of cell transplantation on pain. Figure 8A shows the mechanical withdrawal threshold of the hindlimb 6 weeks after transplantation. Figure 8B shows the mechanical withdrawal threshold of the hindlimb 12 weeks after transplantation.

Mode for Carrying Out the Invention

[0014] Detailed Description

[0015] Methods and culture media for inducing the differentiation of human stem cells into cells expressing one or more markers of spinal cord dI4 progenitor cells are described herein. Compositions of cells expressing such markers are further described herein. The spinal cord dI4 progenitor cells may be used in methods for treating neurological disorders such as spinal cord injury.

[0016] Embryologically, inhibitory neurons in the spinal cord primarily originate from PTF1A-expressing progenitor cells of the dorsal interneuron domain 4 (dI4). These dI4 progenitor cells produce both GABAergic and glycinergic neurons that participate in the motor network for regulating gait. Following this principle, strategies for efficiently differentiating human embryonic and induced pluripotent stem cells (ESCs and iPSCs) into dI4 spinal cord progenitor cells, and subsequently into functional GABAergic interneurons, are detailed herein. Upon transplantation into injured rat spinal cords, human dI4 progenitor cells generate GABAergic neurons and connect to intrinsic spinal cord dI4, and the transplanted animals exhibit alleviated spasticity, reduced neuropathic pain, and improved gait.

[0017] 1. Definition

[0018] Unless otherwise specified, all technical and scientific terms used herein are synonymous with those generally understood by those skilled in the art. In case of any conflict, this document, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in carrying out or testing the present invention. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are examples only and not limiting.

[0019] In this document, “include,” “contain,” “have,” “possess,” “can have,” “contain,” and their variants are unrestricted transitional phrases, terms, or words and do not preclude the possibility of further actions or structures. The singular forms “a,” “and,” and “the” include multiple references unless the context clearly indicates otherwise. This disclosure also contemplates other embodiments of the embodiments or elements presented herein, whether expressly stated or not, that “include,” “consist of,” and “substantially consist of.”

[0020] In the enumeration of numerical ranges in this book, each intervening number between them with the same precision is clearly considered. For example, in the range of 6 to 9, the numbers 7 and 8 are considered in addition to 6 and 9, and in the range of 6.0 to 7.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are clearly considered.

[0021] As used herein, the term “about” or “approximately” in relation to one or more values ​​of interest means a value similar to the described reference value, or a value within an acceptable margin of error for a particular value determined by a person skilled in the art (which depends in part on how the value is measured or determined, such as the limitations of the measuring system). In certain embodiments, unless otherwise stated or evident from the content, the term “about” means a range of values ​​that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the described reference value (except where such figures exceed 100% of the possible values). Alternatively, “about” may mean a standard deviation of 3 or more than 3 per implement in the art. Alternatively, the term “about” may mean within one order of magnitude of a value, preferably within five times, and more preferably within two times, as is the case with respect to biological systems or processes.

[0022] As used herein, “amino acids” refers to naturally occurring and unnaturally synthesized amino acids, as well as amino acid analogs and amino acid mimes that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. In this specification, amino acids may be referred to by either their commonly known three-letter or one-letter symbols as recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Amino acids include side chains and polypeptide backbone portions.

[0023] As used herein, “coding sequence” or “encoding nucleic acid” refers to a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that codes for a protein. The coding sequence may further include start and stop signals operably bound to a regulator, which includes a promoter and a polyadenylation signal capable of instructing expression in cells of an individual or mammal to which the nucleic acid has been administered. The regulator may include, for example, a promoter, an enhancer, a start codon, a stop codon, or a polyadenylation signal. The coding sequence may be an optimized codon.

[0024] As used herein, “complement” or “complementary” means nucleic acids, which may mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen-type base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences (where the nucleotide bases at each position are complementary when they are aligned antiparallel to each other).

[0025] The terms “control,” “reference level,” and “reference” are used interchangeably herein. The reference level may be a predetermined value or range used as a benchmark for comparative evaluation of measurement results. As used herein, “control group” refers to a control group. The predetermined level may be a cutoff value from the control group. The predetermined level may be the mean from the control group. The cutoff value (or predetermined cutoff value) can be determined by the Adaptive Index Model (AIM) method. The cutoff value (or predetermined cutoff value) can be determined from a biological sample of the patient group by receiver operating characteristic (ROC) analysis. ROC analysis, which is generally known in the biological field, is a method for determining the ability of a test to distinguish one condition from another, for example, to determine the performance of each marker in identifying patients with CRC. A description of ROC analysis is provided in PJHeagerty et al. (Biometrics 2000, 56, 337-44) (the disclosure is incorporated herein by reference in its entirety). Alternatively, the cutoff value can be determined by quartile analysis of biological samples from the patient group. For example, the cutoff value can be determined by selecting any value in the 25th to 75th percentile range, preferably the 25th, 50th, or 75th percentile, and more preferably the value corresponding to the 75th percentile. Such statistical analysis can be performed using any method in the art and can be carried out via numerous commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC.). Healthy or normal levels or ranges for target or protein activity can be defined according to standard techniques. Controls may be subjects or cells without the compositions detailed herein. Controls may be subjects or samples from subjects with a known disease state.The subjects or samples from those subjects may be healthy, diseased, diseased before treatment, diseased during treatment, diseased after treatment, or a combination of these.

[0026] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” refers to at least two nucleotides covalently linked to each other. A single-stranded description also determines the sequence of the complementary strand. Therefore, a polynucleotide also encompasses the complementary strand of a described single-stranded sequence. Many variants of a polynucleotide can be used for the same purposes as a given polynucleotide. Therefore, a polynucleotide also encompasses substantially homologous polynucleotides and their complements. A single strand provides a probe that can be hybridized to a target sequence under harsh hybridization conditions. Therefore, a polynucleotide also encompasses probes that hybridize under harsh hybridization conditions. A polynucleotide may be single-stranded or double-stranded, and may contain both double-stranded and single-stranded sequence portions. The polynucleotide may be nucleic acid, native or synthetic, DNA, both genomic DNA and cDNA, RNA, or a hybrid, and may include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The polynucleotide can be obtained by chemical synthesis or recombinant methods.

[0027] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. Polypeptides can be natural, synthetic, modified, or a combination of natural and synthetic. Examples of peptides and polypeptides include proteins, such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to the locally ordered three-dimensional structure within a polypeptide. These structures are commonly known as domains, such as enzyme domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are parts of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acid long. Exemplary domains include those with enzymatic activity or ligand-binding activity. Typical domains consist of smaller organized sections, such as beta-sheets and alpha-helix stretches. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. A "quaternary structure" refers to a three-dimensional structure formed by the non-covalent association of independent tertiary units. A "motif" is a portion of a polypeptide sequence and contains at least two amino acids. Motifs can be 2-20, 2-15, or 2-10 amino acid long. In some embodiments, motifs contain 3, 4, 5, 6, or 7 consecutive amino acids. A domain can consist of a series of motifs of the same type.

[0028] As used herein, “sample” or “test sample” may mean any sample in which the presence and / or level of a target is to be detected or determined, or any sample containing components of cells or culture media as detailed herein. Examples of samples include liquids, solutions, emulsions, or suspensions. Examples of samples include medical samples. Examples of samples include any biological fluid or tissue, e.g., blood, whole blood, blood fractions, e.g., plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage fluid, vomit, fecal matter, lung tissue, peripheral blood mononuclear cells, whole leukocytes, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, bile, digestive fluids, skin, or combinations thereof. In some embodiments, the sample includes aliquots. In other embodiments, the sample includes biological fluids. The sample may be obtained by any means known in the art. The sample may be used directly as obtained from the patient, or it may be pretreated by means of, for example, filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, or addition of reagents, to modify the characteristics of the sample in any way as discussed herein or otherwise known in the art.

[0029] When used herein, “Subject” and “Patient” interchangeably refer to any vertebrate, including, but not limited to, mammals for which the compositions or methods described herein are desired or required. The subject may be human or non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal may be a non-primate such as, for example, a cow, a pig, a camel, a llama, a hedgehog, an anteater, a platypus, an elephant, an alpaca, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, and a mouse. The mammal may be a primate such as a human. The mammal may be a non-human primate such as, for example, a monkey, a crab-eating macaque, a rhesus macaque, a chimpanzee, a gorilla, an orangutan, and a gibbon. The subject may be of any age or developmental stage, such as an adult, adolescent, child (e.g., ages 0-2, 2-4, 2-6, or 6-12), or infant (e.g., age 0-1). The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may receive other forms of treatment.

[0030] "Substantially identical" may mean that the first and second amino acid or polynucleotide sequences represent at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 amino acids.

[0031] When “treatment” or “treating” or “treatment” refers to the protection of an object from a disease, it means preventing, suppressing, overcoming, mitigating, improving or inhibiting the progression of the disease, or completely eliminating the disease. Treatment may be carried out in acute or chronic form. The term also refers to reducing the severity of any disease or symptoms associated with such disease before suffering from the disease occurs. Treatment may result in a reduction of the onset, frequency, severity, and / or persistence of the symptoms of the disease. Prevention of disease includes administering the composition of the present invention to an object before the onset of the disease. Suppression of disease includes administering the composition of the present invention to an object after the induction of disease but before the clinical manifestation of the disease. Suppression or improvement of disease includes administering the composition of the present invention to an object after the clinical manifestation of the disease.

[0032] With respect to polynucleotides, the term “variant” as used herein means (i) a part or fragment of a referenced nucleotide sequence; (ii) a complement of a referenced nucleotide sequence or a part thereof; (iii) a nucleic acid substantially homologous to a referenced nucleic acid or its complement; or (iv) a nucleic acid, its complement, or a sequence substantially homologous thereto that is hybridized with a referenced nucleic acid under severe conditions.

[0033] Atypical molecules relate to peptides or polypeptides that have different amino acid sequences due to amino acid insertions, deletions, or analogous substitutions, but retain at least one biological activity. Atypical molecules may also refer to proteins having an amino acid sequence substantially homologous to a reference protein that has at least one biologically active amino acid sequence. Typical examples of "biological activity" include the ability to bind to specific antibodies or polypeptides, or the ability to catalyze reactions. Atypical molecules can mean functional fragments. Atypical molecules can also mean multiple copies of a polypeptide. These multiple copies may be tandem or cleaved by linkers. Analogous substitutions of amino acids, for example, the substitution of an amino acid with another amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally recognized in the art as involving minute changes. These minute changes can, as is common in the art, be identified, for one thing, by considering the hydrophobicity and hydrophilicity indicators of the amino acids (Kyte et al., J. Mol. Biol. 1982, 157: 105-132). The hydrophobicity and hydrophilicity indices of amino acids are based on considerations of their hydrophobicity and changeability. It is well known in the art that amino acids with similar hydrophobicity and hydrophilicity indices can be substituted for each other while still maintaining protein function. In one embodiment, amino acids with hydrophobicity and hydrophilicity indices of ±2 are substituted for each other. The hydrophilicity of amino acids can also be used to represent substitutions that will result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in peptides allows for the calculation of the maximum local mean hydrophilicity of the peptide. Substitutions can be made between amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity and hydrophilicity values ​​of amino acids are influenced by the specific side chains of those amino acids. As observed, amino acid substitutions corresponding to biological function are understood to depend on the relative similarity of these amino acids, and in particular on their side chains, as expressed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. For example, any terminology and techniques used relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well known and commonly used in the art. The meaning and scope of such terms shall be clear; any definitions provided herein shall precede any dictionary or external definitions, even if there is an event of potential ambiguity. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms.

[0035] 2. Method for generating spinal cord dI4 progenitor cells

[0036] A method for producing spinal cord dI4 progenitor cells is provided herein. As used herein, the term “spinal cord dI4 progenitor cells” refers to progenitor or precursor cells produced from the spinal cord dorsal interneuron domain 4. The spinal cord dI4 progenitor cells have a gene expression pattern including the expression of at least one of PTF1A, PAX7, or ASCL1, or a combination thereof. The spinal cord dI4 progenitor cells may subsequently produce spinal cord GABAergic interneurons. The spinal cord GABAergic interneurons have a gene expression pattern including the expression of at least one of PAX2 and LHX1 / 5, or a combination thereof. The expression of genes detailed herein, such as PTF1A, PAX7, ASCL1, PAX2, and LHX1 / 5, can be detected and / or their levels can be determined by any suitable means known in the art. For example, expression can be measured by PCR such as RT-PCR, detection of a labeled probe hybridized to the protein-coding mRNA, or detection of an antibody that binds to the protein expressed from the gene. Examples of suitable and commercially available antibodies and primer sequences are detailed in Tables 1 and 2 of Example 1.

[0037] a. Generation of spinal cord neuroepithelial progenitor cells in the first culture medium.

[0038] To produce spinal cord dI4 progenitor cells, the first step of the method may include producing a population of neuroepithelial cells from human stem cells. The human stem cells may be human pluripotent stem cells. The human stem cells may be human embryonic stem cells (hESCs) and / or human induced pluripotent stem cells (hiPSCs). The human stem cells may be obtained from human embryos. The human stem cells may be cultured in culture medium for a period of time sufficient to induce differentiation of the human stem cells into neuroepithelial cells. The human stem cells may be cultured in culture medium for 5 to 10 days, 5 to 7 days, 7 to 10 days, or 6 to 8 days. The human stem cells may be cultured in culture medium for at least 5 days, at least 6 days, at least 7 days, less than 8 days, less than 9 days, or less than 10 days. The human stem cells may be cultured in culture medium for 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the human stem cells are cultured in a culture medium for 7 days to produce neuroepithelial cells.

[0039] The cell population derived from human stem cells includes neuroepithelial cells. Neuroepithelial cells are also known as neural stem cells, and the terms “neuroepithelial cells” and “neural stem cells” are used interchangeably herein. The neuroepithelial cells may be spinal neuroepithelial progenitor cells. The neuroepithelial cells may be caudal neuroepithelial progenitor cells. The spinal neuroepithelial progenitor cells may express Sox1, or Hoxa3, or a combination thereof. The spinal neuroepithelial progenitor cells may express Sox1 and Hoxa3. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the cell population derived from human stem cells are Sox1+ / Hoxa3+ spinal neuroepithelial progenitor cells. As detailed previously, the expression of genes such as Sox1 and Hoxa3 may be detected and / or leveled by any preferred means known in the art. For example, expression can be measured by PCR such as RT-PCR, detection of a labeled probe hybridized to the protein-coding mRNA, or detection of an antibody that binds to the protein expressed from the gene. Examples of suitable and commercially available antibodies and primer sequences are detailed in Tables 1 and 2 of Example 1.

[0040] A culture medium for producing cell populations containing neuroepithelial cells from human stem cells contains several small molecules or other chemicals or components that promote the differentiation of human stem cells into neuroepithelial cells. In some cases, such a culture medium is called a differentiation culture medium. The culture medium may also contain agonists of the standard Wnt signaling pathway, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, and inhibitors of the activin / nodal / TGFβ signaling pathway.

[0041] The Wingless-INT (Wnt) signaling pathway agonist may include a GSK3 inhibitor. The GSK3 inhibitor may include CHIR99021, or the Wnt / β-catenin signaling agonist 6-bromo-iridium-3'-oxime ("BIO"; Meijer et al., Chem. Biol. 2003, 10, 1255-1266), or a combination thereof. In some embodiments, the GSK3 inhibitor includes CHIR99021. By inhibiting GSK3, CHIR99021 activates the standard Wnt signaling pathway. CHIR99021 has been reported to inhibit the differentiation of mouse and human embryonic stem cells (ESCs) through Wnt signaling (Wray and Hartmann, Trends in Cell Biology 2012, 22, 159-168). CHIR99021 can be obtained commercially from, for example, Selleckchem (Houston, TX; CAS No. 252917-06-9; catalog number S1263). In some embodiments, the GSK3 inhibitor comprises the Wnt / β-catenin signaling agonist 6-bromo-iridium-3'-oxime ("BIO"). For example, 6-bromo-iridium-3'-oxime is available commercially from Selleckchem (Houston, TX; catalog number S7198). GSK3 inhibitors such as those described herein are available from commercial chemical vendors (e.g., Selleckchem, Houston, TX; Tocris Bioscience, Bristol, UK). The culture medium may contain a Wnt signaling pathway agonist in an amount of about 1 μM to about 10 μM, about 0.5 μM to about 15 μM, about 2 μM to about 8 μM, or about 2 μM to about 5 μM. The culture medium may contain a Wnt signaling pathway agonist in an amount of at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM.The culture medium may contain Wnt signaling pathway agonists in amounts of less than approximately 10 μM, less than approximately 9 μM, less than approximately 8 μM, less than approximately 7 μM, less than approximately 6 μM, less than approximately 5 μM, less than approximately 4 μM, less than approximately 3 μM, or less than approximately 2 μM.

[0042] BMP signaling pathway inhibitors may include DMH-1, dorsomorphine, or LDN-193189, or a combination thereof. DMH-1 interferes with BMP signaling by inhibiting activin receptor-like kinase (ALK2). Dorsomorphine and LDN-193189 affect Smad-dependent and Smad-independent BMP signaling induced by BMP2, BMP6, or GDF5, respectively. For example, DMH-1 is commercially available from Selleckchem (Houston, TX; catalog number S7146). For example, dorsomorphine is commercially available from Selleckchem (Houston, TX; catalog number S7306). For example, LDN-193189 is commercially available from Selleckchem (Houston, TX; catalog number S2618). The culture medium may contain a BMP signaling pathway inhibitor in an amount of about 1 μM to about 10 μM, about 0.5 μM to about 15 μM, about 2 μM to about 8 μM, or about 2 μM to about 5 μM. The culture medium may contain a BMP signaling pathway inhibitor in an amount of at least about 0.5 μM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM. The culture medium may contain a BMP signaling pathway inhibitor in an amount of less than about 10 μM, less than about 9 μM, less than about 8 μM, less than about 7 μM, less than about 6 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, or less than about 1 μM.

[0043] Transforming growth factor β (TGFβ) signaling pathway inhibitors may include SB431542, SB505124, or A83-01, or a combination thereof. In some cases, the TGFβ / activin / nodal signaling inhibitor includes SB431542, which inhibits activin receptor-like kinases 4, 5, and 7 (ALK4, ALK5, and ALK7). By inhibiting activin receptor-like kinases 4, 5, and 7, SB431542 inhibits TGFβ / activin / nodal signaling. SB505124 and A83-01 are other small molecule inhibitors of activin receptor-like kinase 5 (ALK5) (also known as transforming growth factor-α1 receptor kinase) that can also be used to inhibit TGFβ / activin / nodal signaling. SB431542 can be purchased from any of several commercial chemical vendors (e.g., Tocris Bioscience, Bristol, UK; Sigma-Aldrich, St. Louis, MO). For example, SB431542 is commercially available from Selleckchem (Houston, TX; catalog number S1067). For example, SB505124 is commercially available from Selleckchem (Houston, TX; catalog number S2186). For example, A83-01 is commercially available from Selleckchem (Houston, TX; catalog number S7692). The culture medium may contain TGFβ signaling pathway inhibitors in amounts of approximately 1 μM to 10 μM, approximately 0.5 μM to 15 μM, approximately 2 μM to 8 μM, or approximately 2 μM to 5 μM. The culture medium may contain a TGFβ signaling inhibitor in an amount of at least about 0.5 μM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM. The culture medium may also contain a TGFβ signaling pathway inhibitor in an amount of less than about 10 μM, less than about 9 μM, less than about 8 μM, less than about 7 μM, less than about 6 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than 2 μM, or less than about 1 μM.

[0044] In exemplary embodiments, a culture medium for producing a cell population containing neuroepithelial cells from human stem cells comprises the GSK3 inhibitor CHIR99021, the BMP signaling inhibitor DMH-1, and the TGFβ signaling inhibitor SB431542. In some embodiments, the culture medium comprises about 1 μM to about 10 μM of CHIR99021, about 1 μM to about 10 μM of DMH-1, and about 1 μM to about 10 μM of SB431542. In some embodiments, the culture medium comprises 2 μM of SB431542, 2 μM of DMH-1, and 3 μM of CHIR99021.

[0045] In some embodiments, the culture medium for producing a cell population containing neuroepithelial cells from a human stem cell composition comprises a culture medium supplemented with a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, and / or a TGFβ signaling pathway inhibitor, as detailed herein. A conventional culture medium to which a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, and / or a TGFβ signaling pathway inhibitor may be added may be a neuronal culture medium. For example, the culture medium may be Neurobasal® culture medium (Life Technologies, Carlsbad, CA). In some cases, the culture medium comprises DMEM / F12, 1:1 Neurobasal culture medium, 1 × N2 neuronal supplement (Gibco, Dublin, Ireland), 1 × B27 neuronal supplement (Gibco, Dublin, Ireland), and 1 mM ascorbic acid.

[0046] Any suitable culture method may be used in conjunction with the culture media detailed herein. For example, adherent culture methods may be used. Adherent culture (or “colony culture”) allows for direct visualization of neural differentiation, including neuroepithelial induction and the formation of neural tube-like rosettes during the migration of neuroepithelial cells. Adherent / colony culture allows for preparation for the removal of non-neuronal colonies and promotes subsequent neural differentiation. In some cases, suspension culture may be used to isolate pluripotent cells from mouse embryonic fibroblast (MEF) supporting cells or to purify neuroepithelial cells.

[0047] b. Generation of spinal cord dl4 progenitor cells in a second culture medium

[0048] A method for producing spinal cord dI4 progenitor cells further includes inducing differentiation of neuroepithelial cells (e.g., the neuroepithelial cells produced as detailed above) into spinal cord dI4 progenitor cells. Neuroepithelial cells, such as spinal cord neuroepithelial progenitor cells, may be cultured in culture medium for a period of time sufficient to produce a cell population containing spinal cord dI4 progenitor cells. For example, neuroepithelial cells, such as spinal cord neuroepithelial progenitor cells, may be cultured in culture medium for a period of time sufficient to induce expression of a spinal cord dI4 progenitor cell marker (e.g., PTF1A). The neuroepithelial cells may be cultured in culture medium for 5–10 days, 5–7 days, 7–10 days, or 6–8 days. The human stem cells may be cultured in culture medium for at least 5 days, at least 6 days, at least 7 days, less than 8 days, less than 9 days, or less than 10 days. The neuroepithelial cells may be cultured in culture medium for 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the neuroepithelial cells are cultured in culture medium for 7 days to produce neuroepithelial cells for generating spinal cord dI4 progenitor cells.

[0049] Spinal cord dl4 progenitor cells may be identified based on PTF1A+ expression. The bHLH transcription factor PTF1A serves as a distinctive marker for spinal cord dl4 progenitor cells. The fate of dl4 progenitor cells is PTF1A-dependent, and loss of this gene results in the loss of all GABAergic dorsal neurons and respecification to dl5 fate (Henke R. et al., Development 2009, 136, 2945-2954; Meredith DM et al., J. Neurosci. 2009, 29, 11139-11148). Spinal cord dl4 progenitor cells may express PTF1A, PAX7, ASCL1, or any combination thereof. In some embodiments, spinal cord dl4 progenitor cells express PTF1A, PAX7, and ASCL1. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the produced cell population are PTF1A+ / PAX7+ / ASCL1+ spinal cord dl4 progenitor cells. As detailed previously, the expression of genes such as PTF1A, PAX7, and ASCL1 may be detected and / or leveled by any suitable means known in the art. For example, expression can be measured by PCR such as RT-PCR, detection of a labeled probe hybridized to the protein-coding mRNA, or detection of an antibody that binds to the protein expressed from the gene. Examples of suitable and commercially available antibodies and primer sequences are detailed in Tables 1 and 2 of Example 1.

[0050] In some embodiments, neuroepithelial cells, such as spinal neuroepithelial progenitor cells, are cultured in a culture medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor, as described in detail above, and further comprising a sonic hedgehog (SHH) signaling pathway inhibitor and a retinoic acid (RA) signaling pathway agonist.

[0051] The sonic hedgehog (SHH) signaling inhibitor may include, for example, cyclopamine. Cyclopamine inhibits SHH signaling by directly targeting Smoothened ("Smo"), which inhibits components of the SHH signaling pathway. Other small molecule inhibitors of Smo, such as SANT-1, can be used to inhibit SHH signaling. SHH acts in a stepwise manner to establish different neural progenitor cell populations (Briscoe et al., Semin. Cell Dev. Biol. 1999, 10, 353-362). Cyclopamine is available from several commercial chemical vendors (e.g., Tocris Bioscience, Bristol, UK; Selleckchem, Houston, TX). For example, cyclopamine is commercially available from Selleckchem (Houston, TX; CAS No. 4449-51-8; catalog number S1146). SANT-1 is available commercially, for example, from Selleckchem (Houston, TX; CAS No. 304909-07-7; catalog number S7092). The culture medium may contain a sonic hedgehog (SHH) signaling inhibitor in amounts of approximately 0.1 μM to 2 μM, approximately 0.05 μM to 5 μM, approximately 0.1 μM to 1.0 μM, or approximately 0.05 μM to 3 μM. The culture medium contains at least about 0.05 μM, at least about 0.1 μM, at least about 0.15 μM, at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 0.35 μM, at least about 0.4 μM, at least about 0.45 μM, at least about 0.5 μM, at least about 0.55 μM, at least about 0.6 μM, at least about 0.65 μM, at least about 0.7 μM, at least about The mixture may contain a sonic hedgehog (SHH) signaling inhibitor in an amount of 0.75 μM, at least about 0.8 μM, at least about 0.85 μM, at least about 0.9 μM, at least about 1.0 μM, at least about 1.5 μM, at least about 2.0 μM, at least about 2.5 μM, at least about 3.0 μM, at least about 3.5 μM, at least about 4.0 μM, at least about 4.5 μM, or at least about 5.0 μM.The culture medium may contain a sonic hedgehog (SHH) signaling inhibitor in an amount of less than approximately 5.0 μM, less than approximately 4.5 μM, less than approximately 4.0 μM, less than approximately 3.5 μM, less than approximately 3.0 μM, less than approximately 2.5 μM, less than approximately 2.0 μM, less than approximately 1.5 μM, less than approximately 1.0 μM, less than approximately 0.9 μM, less than approximately 0.8 μM, less than approximately 0.7 μM, less than approximately 0.6 μM, less than approximately 0.5 μM, less than approximately 0.4 μM, less than approximately 0.3 μM, or less than approximately 0.2 μM.

[0052] Examples of retinoic acid (RA) signaling agonists include retinoic acid (RA), EC23, or combinations thereof. In some embodiments, the RA is all-trans retinoic acid. RA activates RA signaling by binding to the retinoic acid receptor (RAR), a nuclear hormone receptor, which is necessary for the clarification of spinal cord dI4 progenitor cells (Novitch et al., Neuron 2003, 40, 81-95). RA is available from several commercial chemical vendors (e.g., Tocris Bioscience, Bristol, UK; Sigma-Aldrich, St. Louis, MO). For example, RA is commercially available from Tocris Bioscience (Bristol, UK; Cat. No. 0695). For example, EC23 is commercially available from Tocris Bioscience (Bristol, UK; Cat. No. 4011). The culture medium is The culture medium may contain retinoic acid (RA) agonists in amounts of approximately 0.01 μM to approximately 2 μM, approximately 0.005 μM to approximately 5 μM, approximately 0.01 μM to approximately 1.0 μM, or approximately 0.05 μM to approximately 3 μM. The culture medium may contain at least approximately 0.005 μM, at least approximately 0.01 μM, at least approximately 0.02 μM, at least approximately 0.03 μM, at least approximately 0.04 μM, at least approximately 0.05 μM, at least approximately 0.06 μM, at least approximately 0.07 μM, at least approximately 0.08 μM, at least approximately 0.09 μM, at least approximately 0.1 μM, at least approximately 0.2 μM, at least approximately 0.3 μM, at least approximately 0.4 μM, at least approximately 0.5 μM, at least approximately The mixture may contain retinoic acid (RA) signaling agonists in amounts of 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1.0 μM, at least about 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, or at least 2.0 μM. The culture medium may contain retinoic acid (RA) signaling agonists in amounts less than approximately 2.0 μM, less than approximately 1.5 μM, less than approximately 1.0 μM, less than approximately 0.9 μM, less than approximately 0.8 μM, less than approximately 0.7 μM, less than approximately 0.6 μM, less than approximately 0.5 μM, less than approximately 0.4 μM, less than approximately 0.3 μM, less than approximately 0.2 μM, less than approximately 0.1 μM, less than approximately 0.09 μM, less than approximately 0.08 μM, less than approximately 0.07 μM, less than approximately 0.06 μM, less than approximately 0.05 μM, less than approximately 0.04 μM, less than approximately 0.03 μM, or less than approximately 0.02 μM.

[0053] In exemplary embodiments, a culture medium for producing spinal cord dI4 progenitor cells from spinal cord neuroepithelial progenitor cells comprises the Wnt signaling agonist CHIR99021, the BMP signaling inhibitor DMH-1, the TGFβ signaling inhibitor SB431542, the SHH signaling inhibitor cyclopamine, and retinoic acid. In some cases, the culture medium contains about 1 μM to about 10 μM of CHIR99021, about 1 μM to about 10 μM of DMH-1, about 1 μM to about 10 μM of SB431542, about 0.1 μM to about 2 μM of cyclopamine, and about 0.01 μM to about 2 μM of RA. In some embodiments, the culture medium contains 2 μM of SB431542, 2 μM of DMH-1, 3 μM of CHIR99021, 0.1 μM of all-trans retinoic acid, and 0.5 μM of cyclopamine.

[0054] Any suitable culture method may be used in conjunction with the culture media detailed herein. For example, an adherent culture method may be used.

[0055] In some embodiments, at least a portion of spinal cord dl4 progenitor cells further differentiate into spinal cord GABAergic interneurons. In some cases, the method further comprises culturing spinal cord dl4 progenitor cells in culture medium to produce GABAergic interneurons. Spinal cord GABAergic interneurons may express at least one gene selected from PAX2 and LHX1 / 5, or a combination thereof. For example, spinal cord dl4 progenitor cells may be cultured in neuronal differentiation culture medium to produce PAX2+, LHX1 / 5+ GABAergic interneurons. Lhx1 and Lhx5 maintain the inhibitory neurotransmitter status of interneurons in the dorsal spinal cord (Pillai A. et al., Development 2007, 134, 357-366). As detailed previously, the expression of genes such as PAX2 and LHX1 / 5 may be detected and / or leveled by any suitable means known in the art. For example, expression can be measured by PCR such as RT-PCR, detection of a labeled probe hybridized to the mRNA encoding the protein, or detection of an antibody that binds to the protein expressed from the gene. Examples of suitable and commercially available antibodies and primer sequences are detailed in Tables 1 and 2 of Example 1. Spinal cord dI4 progenitor cells may be cultured in neuronal differentiation culture medium for 5–10 days, 5–7 days, 7–10 days, or 6–8 days. Spinal cord dI4 progenitor cells may be cultured in neuronal differentiation culture medium for at least 5 days, at least 6 days, at least 7 days, less than 8 days, less than 9 days, or less than 10 days. Spinal cord dI4 progenitor cells may be cultured in neuronal differentiation culture medium for 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, spinal cord dI4 progenitor cells are cultured in neuronal differentiation culture medium for about one week. In some embodiments, spinal cord dI4 progenitor cells are cultured in neuronal differentiation culture medium for 7 days.

[0056] 3. Methods for treating neurological disorders

[0057] Methods for treating the target neurological disorders are further provided herein. In vitro differentiated cells expressing one or more markers of spinal cord GABAergic neurons, or such spinal cord dl4 progenitor cells as detailed herein (also referred to as “stem cell-derived spinal cord dl4 progenitor cells”), can be used to treat the neurological disorders. The method may include administering an effective amount of stem cell-derived spinal cord dl4 progenitor cells as detailed herein to a subject suffering from the neurological disorder. In some embodiments, the stem cell-derived spinal cord dl4 progenitor cells are differentiated stem cell-derived spinal cord dl4 progenitor cells. In some embodiments, a portion of the spinal cord dl4 progenitor cells were further differentiated into spinal cord GABAergic interneurons before administration. In some embodiments, a portion of the spinal cord dl4 progenitor cells were further differentiated into spinal cord GABAergic interneurons after administration. The method may include administering an effective amount of spinal cord GABAergic interneurons as detailed herein to a subject suffering from the neurological disorder. The administered or transplanted cells may exhibit therapeutic effects in alleviating spasticity, reducing neuropathic pain, and / or improving walking ability in the subject.

[0058] An unrestricted example of neurological disorders is spinal cord injury. In some embodiments, the neurological disorder includes spinal cord injury. Spinal cord injury (SCI) is a spinal cord injury that results in loss of function such as mobility and / or touch. Common causes of spinal cord injury are traumatic injuries (such as car accidents, shootings, or falls) or diseases (such as polio, spina bifida, or Friedreich's ataxia). Treatment of SCI may result in spinal nerve regeneration, recovery of neurological function and movement, or a combination thereof. Spinal cord injuries can include both acute and chronic spinal cord injuries. The term "acute injury" refers to an injury that has occurred recently. For example, an acute injury may have occurred very recently, within an hour or less, within a day or less, within a week or less, or within two weeks or less. The term "chronic injury" refers to an injury that has persisted for a period of time. For example, a chronic injury may have occurred more than two weeks ago, more than three weeks ago, more than two months ago, or more than three months ago. The term “injury” generally refers to damage to the membrane of nerve cells, such that the nerve membrane’s ability to separate the salt-containing gel inside (cytoplasm) from the salt-containing fluid (extracellular fluid) surrounding them is impaired. Because the types of salts in these two fluid divisions are vastly different, the exchange of ions and water caused by injury leads to a decrease in the nerve’s ability to generate and propagate nerve impulses—and even cell death. SCI can also refer to damage that directly or indirectly affects the normal functioning of the central nervous system (CNS). Such SCI may be structural, physical, or mechanical damage, such as bruising, compression, or stretching of nerve fibers, and may be caused by physical effects. Alternatively, the cell membrane may be damaged or impaired due to disease, chemical imbalance, or physiological dysfunction, such as blood oxygen deficiency (e.g., stroke), aneurysm, or reperfusion. Examples of CNS injuries include, but are not limited to, damage to retinal ganglion cells, traumatic brain injury, stroke-related injury, cerebral aneurysm-related injury, spinal cord injury, monoplegia, paraplegia, bilateral paraplegia, hemiplegia and quadriplegia, neuronal proliferation disorders, or neuropathic pain syndromes.Injuries to the spinal cord in mammals can disrupt the connections between spinal nerves. Such injuries interrupt the flow of impulses in the affected nerve pathways, resulting in impairments of both sensory and motor function. Injuries to the spinal cord can result from compression or other contusions, sprains, or transections of the spinal cord. A transection of the spinal cord, also referred to herein as a “cross-section,” may be a complete or incomplete transection.

[0059] Spinal neuroepithelial progenitor cells and / or spinal dI4 progenitor cells, or pharmaceutical compositions containing them, may be administered to subjects. Such compositions can be administered in dosages and techniques known to medical professionals, taking into account factors such as the age, sex, weight, and condition of the specific subject, as well as the route of administration. The cells or compositions containing them currently disclosed may be administered to subjects by a variety of routes, including oral, parenteral, sublingual, transdermal, transrectal, transmucosal, topical, intranasal, vaginal, and inhalation methods, via oral administration, intrapleural, intravenous, intra-arterial, intraperitoneal, subcutaneous, intradermal, epithelial, intramuscular, intranasal, subarachnoid, intracerebral, and intra-articular or combinations thereof.

[0060] The currently disclosed stem cell-derived spinal cord dI4 progenitor cells may be administered or provided to a subject systemically or directly to treat or prevent neurological disorders. In certain embodiments, the currently disclosed stem cell-derived spinal cord dI4 progenitor cells are directly injected into the organ of interest (e.g., the central nervous system (CNS) or peripheral nervous system (PNS)). In certain embodiments, the currently disclosed stem cell-derived spinal cord dI4 progenitor cells are directly injected into the spinal cord. Spinal cord neuroepithelial progenitor cells and / or spinal cord dI4 progenitor cells, or pharmaceutical compositions containing them, may be injected into the brain or other components of the central nervous system.

[0061] The pharmaceutical composition comprising spinal nerve epithelial progenitor cells and / or spinal dI4 progenitor cells as described above may be formulated according to standard techniques well known to those skilled in the pharmaceutical art. The pharmaceutical composition may be formulated according to the method of administration to be used. The pharmaceutical composition may contain pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients may be functional molecules as vehicles, adjuvants, carriers, or diluents. The term "pharmaceutically acceptable carrier" may refer to any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, capsule encapsulant, or formulation aid. Examples of pharmaceutically acceptable carriers include diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, antioxidants, preservatives, flow enhancers, solvents, suspending agents, wetting agents, surfactants, emollients, propellants, humectants, powders, pH adjusters, and combinations thereof.

[0062] Stem cell-derived spinal cord dI4 progenitor cells can be administered by any physiologically acceptable vehicle. Pharmaceutical compositions comprising spinal cord dI4 progenitor cells and a pharmaceutically acceptable vehicle are also provided. Spinal cord dI4 progenitor cells and pharmaceutical compositions comprising the cells can be administered by local injection, orthotopic (OT) injection, systemic administration, intravenous injection, or parenteral administration. In certain embodiments, spinal cord dI4 progenitor cells are administered by orthotopic (OT) injection to subjects suffering from neurological disorders.

[0063] A pharmaceutical composition comprising stem cell-derived spinal cord dI4 progenitor cells and said cells may be conveniently provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid formulations may be easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions may be more convenient to administer, particularly by injection. Viscous compositions may be formulated within an appropriate viscosity range to provide a longer period of contact with specific tissues. The liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. A sterile injection solution may be prepared by incorporating the composition of the currently disclosed content, for example, the composition comprising the currently disclosed stem cell-derived progenitor cells in the required amount of a suitable solvent together with various amounts of other components as desired. Such compositions may be mixtures with suitable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions may be freeze-dried. Depending on the route of administration and the desired preparation, the compositions may contain auxiliary agents such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents, or viscosity-enhancing additives, preservatives, flavorings, and colorings. Standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985 (incorporated herein by reference) may be referenced to prepare suitable preparations without unnecessary experimentation.

[0064] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Sustained absorption of the injectable pharmaceutical form may be achieved by the use of absorption-delaying agents, such as aluminum monostearate or gelatin. However, according to the currently disclosed information, any vehicle, diluent, or additive used must be compatible with the currently disclosed stem cell-derived progenitor cells.

[0065] If desired, the viscosity of the composition can be maintained at a selected level by using a pharmaceutically acceptable thickener. Methylcellulose is used because it is readily and economically available and easy to work with. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The amount may be used to achieve the desired viscosity. The selection of a suitable carrier and other additives depends on the appropriate route of administration and the properties of the specific dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated in a solution, suspension, gel, or other liquid form, e.g., sustained-release or liquid-filled form).

[0066] Those skilled in the art will recognize that the components of the composition must be selected to be chemically inert and that they do not affect the viability or efficacy of spinal cord dI4 progenitor cells. This will not pose any problem to those skilled in the art of chemical and pharmaceutical principles, or any problem can be easily avoided by referring to standard texts from this disclosure and the documents referenced herein or by simple experiments (without requiring any additional experiments).

[0067] In certain non-limiting embodiments, the spinal cord dI4 progenitor cells described herein are included in a composition further comprising a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that facilitates tissue regeneration when the cells are implanted or transplanted into a subject. In certain non-limiting embodiments, the biocompatible scaffold comprises polysaccharides including extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels (see, for example, U.S. Patent Application Publications 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of which are incorporated herein by reference in whole). In certain embodiments, the composition further comprises growth factors that promote the maturation of cells implanted into or transplanted into spinal GABAergic interneurons.

[0068] One consideration regarding the therapeutic use of spinal cord dI4 progenitor cells is the quantity of cells required to achieve optimal efficacy. Optimal efficacy includes, but is not limited to, regeneration of the CNS and / or PNS regions in the neurologically impaired subject, and / or improved function of the subject's CNS and / or PNS.

[0069] An effective dose (or therapeutically effective dose) is the amount sufficient to produce a beneficial or desired clinical outcome from the treatment. An effective dose may be administered to a patient in one or more doses. In terms of treatment, an effective dose is sufficient to mitigate, improve, stabilize, overcome or delay the progression of neurological disorders, or otherwise reduce the pathological outcomes of the neurological disorders. The effective dose is usually determined by a physician on a case-by-case basis and is within the realm of the skills of those skilled in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective dose. These factors include the patient's age, sex, and weight; the condition being treated; the severity of the condition; and the morphology and effective concentration of the cells being administered.

[0070] In certain embodiments, the effective amount of spinal cord dI4 progenitor cells is sufficient for regeneration of the CNS and / or PNS regions of a subject suffering from neurological impairment. In certain embodiments, the effective amount of spinal cord dI4 progenitor cells is sufficient to improve the function of the subject's spinal cord, such as alleviating spasticity, reducing neuropathic pain, and improving gait, for example, the improved function may be about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the spinal cord function of a healthy human.

[0071] The amount of cells administered varies depending on the subject being treated. The precise quantification of what constitutes an effective dose may be based on individual factors for each subject, including their body shape, age, sex, weight, and the specific condition of the subject. The dosage can be readily determined by those skilled in the art from this disclosure and knowledge in the art.

[0072] Unless otherwise specified, all technical and scientific terms used in this document have the same meaning as those generally understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, and such preferred methods and materials are described below.

[0073] Various exemplary embodiments of the compositions and methods of the present invention are described herein in the following non-limiting examples. These examples are provided solely for illustrative purposes and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above description and the following examples and will fall within the scope of the appended claims. [Examples]

[0074] 4. Examples

[0075] Example 1

[0076] Efficient generation of spinal cord dI4 progenitor cells from human ESCs in 2 weeks

[0077] To induce neuroepithelial cell characteristics from human pluripotent stem cells, a dual TGFβ / BMP inhibition approach was applied to monolayer cultured human embryonic stem cells (see Chambers et al., Nature Biotech. 2009, 27, 275-280). Figure 1 is a flowchart showing the directional differentiation of spinal cord dI4 progenitor cells and GABAergic interneurons from pluripotent stem cells. The small molecule SB431542 selectively inhibits activin receptor-like kinases ALK4 / 5 / 7, thereby facilitating TGFβ signaling. The small molecule DMH-1 selectively inhibits BMP receptor kinase ALK2, thereby suppressing BMP signaling. Human embryonic stem cells (hESCs) were treated with cell cultures containing 2 μM DMH-1 and 2 μM SB431542 for one week. The cell culture medium was a neurobasal medium containing DMEM / F12, 1:1 Neurobasal medium, 1×N2 neuronal supplement (Gibco, Dublin, Ireland), 1×B27 neuronal supplement (Gibco, Dublin, Ireland), and 1 mM ascorbic acid (with 2 μM DMH-1 and 2 μM SB431542). The cells were cultured under normal conditions at 37°C with 5% CO2. Next, the treated hESCs were differentiated into a population containing approximately 85% SOX1+ neuroepithelial cells, but also into a population containing other cell lineages due to spontaneous ESC differentiation, as the dual TGFβ / BMP inhibitors SB431542 and DMH-1 could not prevent all spontaneous differentiation into other cell lineages, especially when the ESC colonies were small. We maintained the ESC status during culture using a small molecule that inhibits glycogen synthase kinase-3 (CHIR99021). The antibodies used in these experiments are detailed in Table 1. The primers used for PCR are detailed in Table 2. [Table 1-1] [Table 1-2]

[0078] [Table 2]

[0079] To further improve neuronal properties, small molecules inhibiting glycogen synthase kinase-3 (CHIR99021) were applied to cell cultures in combination with DMH-1 and SB431542. GSK3 negatively modulates WNT signaling, which promotes the self-renewal of ESCs and neural progenitor cells. When exposed to these three molecules for approximately 7 days, hESCs not only produced a purer population of SOX1+ neuroepithelial cells (e.g., at least 95% of the cells in the total population were SOX1+ neuroepithelial cells), but also generated more than 2.5 times the number of neuroepithelial cells. However, CDS ("CDS" represents CHIR99021, DMH-1, and SB431542)-induced neuroepithelial cells showed caudal identity, as demonstrated by staining for HOXA3. In contrast, DS-treated (where "DS" represents DMH-1 and SB431542) neuroepithelial cells showed rostral identity, as demonstrated by staining for OTX2.

[0080] Next, we compared the efficiency of spinal cord dI4 progenitor cell generation from these two populations of neuroepithelial cells. Furthermore, after 7 days of treatment with 0.1 μM retinoic acid (RA) and 0.5 μM cyclopamine (a small molecule for inhibiting SHH signaling), over 90% of PTF1A+ spinal cord dI4 progenitor cells were induced from CDS-treated neuroepithelial cells, compared to only 60% from DS-treated neuroepithelial cells. These data suggest that... Contacting stem cells with a three-molecule cocktail of CDS (CHIR99021, DMH-1, and SB431542) or a cocktail of compounds that affect the Wnt pathway, BMP pathway, and activin / nodal signaling pathway, respectively, suggests an efficient approach to inducing spinal cord dI4 progenitor cells from pluripotent stem cells.

[0081] Example 2

[0082] Human spinal cord dI4 progenitor cells reduce spasticity, pain, and improve gait in rats with spinal cord injury.

[0083] We efficiently generated spinal GABAergic interneurons from hPSCs.

[0084] We further investigated the spinal cord dI4 progenitor cells generated by Example 1. Spinal cord inhibitory neurons originated from the dl4 domain of the developing spinal cord (Figure 2A) (Lai et al. Development 2016, 143, 3434-3448). We devised a protocol for the initial induction of neuroepithelium from hESCs (Du et al. Nat. Commun. 2015, 6, 662), and then formed these neuroepithelium progenitor cells in the dI4 domain of the spinal cord (Figures 2A and 2B). In the presence of SB431542 (2 μM), DMH1 (2 μM), and CHIR99021 (3 μM) for 7 days, hESCs were differentiated into a nearly homogeneous population of SOX1-expressing neuroepithelium derived from rosette morphology. From days 7 to 14, in the presence of retinoic acid (RA, 0.1 μM), cyclopamine (0.5 μM), and a Sonic Hedgehog antagonist, neuroepithelium was differentiated into dorsal spinal progenitor cells, as evidenced by the expression of the homeodomain transcription factors HOXA3 and HOXB4, which are expressed in the hindbrain and spinal cord, in over 90% of differentiated cells. Dorsal-ventral marker analysis showed that over 90% of day 14 cells were PTF1A+ (Figures 2C and 2I), and 90% of these cells were positive for the dorsal transcription factors PAX7 and ASCL1 (Figures 2D, 2E, and 2I). PTF1A, PAX7, and PAX2, along with LHX1 / 5+, were relatively limited to dl4 progenitor cells in the spinal cord (Figure 2A). Accordingly, no cells were found to be positive for OLIG2, ventral markers, or FOXG1, forebrain markers. RT-PCR analysis revealed that these cells expressed additional dl4-related dorsal transcription factor genes, including LBX-1 and PAX2, but expressed relatively low levels or no expression of dorsal dI1-3 related genes BRN3A, MATH1, TLX3, and ISL-1; dI5-6 related transcription factor genes LMX1B, WT1, TLX3, and BHLHB5; and the V local gene EN1 (Figure 2H). These results indicated that the majority of the differentiated cells were dorsal spinal cord dI4 progenitor cells.

[0085] Following the regression of morphogenetic agents, spinal cord progenitor cells were further differentiated into neurons by culturing them in a neuronal culture medium containing 1:1 DMEM / F12, Neurobasal culture medium, 1×N2 neuronal supplement (Gibco, Dublin, Ireland), 1×B27 neuronal supplement (Gibco, Dublin, Ireland), and 1 mM ascorbic acid, and also with the neuronutrients BDNF, GDNF, and TGFb. By day 21, the majority of cells (93.2% ± 3.5%) were process-carrying neurons, as revealed by positive immunostaining for NF-200 in over 90% of GABA+ cells. Many of the GABA neurons retained expression of PAX2 and LHX1 / 5 (Figures 2F and 2G). Since dI4 progenitor cells can also produce glycine neurons, we examined the cultures and found no cells positive for GlyT1+ even after 50 days post-differentiation. Immunostaining for VGluT2+ showed 2.5% ± 0.8% of cells positive, suggesting a relatively low proportion of glutamatergic neurons under this differentiation scheme. Therefore, the differentiated cells were mainly spinal GABAergic interneurons.

[0086] To validate the differentiation scheme for application to other cell lines, we differentiated iPSCs (IMR-90 strain) into spinal GABAergic neurons using the same protocol. Approximately 95% ± 1.82% of the differentiated cells were positive for GABA, similar to those from H9ESCs (Figure 2I). Thus, both human ESCs and iPSCs were efficiently directed into spinal somatosensory GABAergic neurons carrying the distinctive features of the dI4 region.

[0087] Spinal GABA neurons have a unique firing pattern.

[0088] In rodents, spinal somatosensory neurons exhibit a unique firing pattern, mostly showing a sustained firing pattern. Sustained firing typically occurs without presynaptic input and is often characterized by stable action potential firing at a constant frequency. Patch-clamp electrophysiological recordings of differentiated cells at day 40 revealed that outward K+ and inward Na+ currents were induced in most cells by electrical stimulation of -70mV to +100mV in 10mV increments (Figure 3A). Action potentials were induced by current steps of -30pA to +50pA in 10pA increments (Figure 3B). Many neurons also exhibited spontaneous action potentials (Figures 3C-3E). Of the 30 patched neurons, 16 showed a sustained firing pattern with a frequency of 1.6Hz ± 0.29Hz, similar to that of dorsal horn GABA neurons in mice (Figure 3C). Six neurons exhibited a delayed firing pattern (Figure 3D), and several showed an early burst (2 out of 30, Figure 3E). The amplitudes of sustained firing and delayed firing were smaller than those of early bursts (Figure 3F). Therefore, the majority of neurons generated in vitro had the distinctive firing pattern of spinal inhibitory neurons.

[0089] Human transplantation of spinal cord dI4 progenitor cells in spinal cord injury

[0090] Spinal GABAergic neurons play a role in regulating spinal dI4 excitability by synaptic transmission with spinal dI4. In SCI, the loss of these GABAergic neurons is the primary pathological basis underlying spasticity (excessive excitation of spinal dI4). To determine whether our in vitro-generated spinal GABAergic neurons could alleviate spasticity and improve gait in SCI, we created a rat SCI model in the T11 / 12 spinal cord by dropping a 10g impactor from a height of 25mm onto the exposed spinal cord after laminectomy in the T10 vertebra. Rats subjected to the impact exhibited bilateral hindlimb spasms and tail fluttering, and the injured spinal cord appeared bloated. This SCI model allowed for the evaluation of spasticity. Animals that underwent laminectomy alone without impact to the spinal cord served as a control.

[0091] One week after the injury, we administered 10 units of (3 μL of culture medium consisting of basic neuronal culture medium supplemented with B27) to the spinal cord at the T10 / T11 boundary. 6 hESC-derived dI4 progenitor cells were transplanted (Figure 4A). Eighty rats were randomly assigned to four groups: a sham group (laminectomy without SCI), an SCI group, an SCI group with cell transplantation, and an SCI group with culture medium injection.

[0092] Animals were histologically analyzed 6 and 12 weeks after transplantation. The injured spinal cord showed atrophy at the site of injury. HE staining revealed cavities in the spinal cord injury with destroyed tissue. Cavity size was 5.93% ± 0.79% in spinal cord sections of the SCI+ cell group, but 11.14% ± 1.3% and 10.95% ± 1.96% in the SCI group and SCI+ culture medium group at 12 weeks, respectively (Figure 4B, SCI+ cells vs. SCI, p=0.02, SCI+ cells vs. SCI+ culture medium, p=0.03). Reactive microglia identified by IBA1+, and astrocytes revealed by staining for GFAP, materializing them compared to cells far from the injury site, and processing them to show giant cells, surrounded the cavities. Thus, our spinal cord injury model showed pathological changes similar to those previously reported.

[0093] Transplanted human cells, identified by positive staining for hNu and mCherry, were present in all transplanted animals (Figure 4C). Three-dimensional analysis showed a total of 0.89 ± 0.04 × 10⁻¹⁴ cells at 6 weeks post-transplantation. 6 , and 1.06 ± 0.08 × 10 at 12 weeks post-transplant 6The hNu+ cells were shown (Figure 4D). Immunostaining for Ki67 showed that 1.98% ± 0.04% of the hNu+ transplanted cells were positive at 6 weeks, and 0.72% ± 0.09% remained positive up to 3 months post-transplant (Figure 4E). The percentage of Ki67+ cells in the spinal cord of Siamese animals was 0.65 ± 0.03% (Figure 4E). These results indicate that the transplanted human dI4 cells survived and their proliferation rate decreased to a level similar to that of endogenous cells.

[0094] Analysis of (hNu+) cells showed that transplanted cells spread to the injured area and its surroundings, mainly in the gray matter (Figure 4C). At 6 weeks, 47.71% of transplanted cells were positive for NeuN. The remainder were positive for DCX, but no cells were positive for SOX9 (related to astrocytes) or myelin basic protein (MBP, related to oligodendrocytes). Up to 12 weeks after transplantation, 98% ± 1.7% of transplanted cells were positive for NeuN, while human cells were not DCX positive (Figures 4F and 4G), 3.47% ± 0.93% were positive for SOX9, and no MBP-positive cells were present.

[0095] Simultaneous immunohistochemical staining for neuronal cell subtype markers showed that 92.8% ± 4.73% of human cells (hNu+, Stem121+) were positive for GABA (Figure 4H, Figure 4I). Further analysis of transplanted human cells showed that 95.5% ± 2.01% of human GABA was PTF1A-positive (Figure 4J), and 51% ± 4.02% were positive for LHX1 / 5 and dI4 domain neuron markers. These results indicate that transplanted dI4 progenitor cells differentiated primarily into GABAergic neurons.

[0096] The transplanted human neurons form synaptic connections with the host's spinal cord dI4.

[0097] Somatosensory GABAergic neurons synapse with spinal cord dI4 to regulate spinal cord dI4 activity and, therefore, regulate muscle tension. Immunostaining for mCherry in horizontal sections (Figures 5A-5G) and transverse sections (Figures 5H-5L) showed that transplanted human neurons and their axons were present not only at the transplant site (T10 / 11) but also in the lumbar spinal cord. The majority of mCherry+ / hNu+ transplanted cells were located in and around the gray matter at the transplant site (Figures 4C, 5H-5L). This was also observed in longitudinal sections cut through the gray matter. Almost all mCherry+ or hNu+ cells were positive for NeuN (Figures 5B, 5C, 5H, 5J), and some were located near host spinal cord dI4, indicated by large cells and NeuN+ but not by hNu- (Figure 5J). mCherry+ human axons were better observed in longitudinal sections and had migrated mostly to the white matter of the lumbar spinal cord (Figures 5A–5G) and slightly to the gray matter. These results indicate that the transplanted cells migrated around the site of injury, matured, and their axons migrated to the lumbar spinal cord, mainly along the white matter.

[0098] Immunostaining for human-specific synaptophysin (hSyn) and ChAT revealed numerous hSyn spots on the surface of ChAT+ spinal cord dI4, confirmed by z-stack (0.5 μm step size) under super-resolution confocal analysis (Figure 5 K, Figure 5 L). These (many NeuN+ but mCherry- and mCherry+ and hSyn+ spots on host neurons) were also observed in horizontal sections. To further confirm the legitimacy of synaptic connections between transplanted human cells and endogenous rat spinal cord dI4, we performed correlative optical and electron microscopy studies in the area of ​​interest (Figure 5 M). As shown, multiple synaptic junction terminals were present on the surface of spinal cord dI4 (Figure 5 M, Figure 5 N). Reconstruction of a series of EM sections revealed numerous synaptic buttons on the surface of spinal cord dI4 (Figure 5 O). Thus, transplanted human neurons formed synaptic junctions with host spinal cord dI4.

[0099] Spinal inhibitory neuron transplantation reduces spasticity.

[0100] Spasticity was assessed by measuring the amplitude of the Hoffmann reflex (H reflex), which evaluates the function of the spinal cord inhibitory system (Jolivalt et al. Pain 2008, 140, 48-57; Kakinohana et al. Neuroscience 2006, 141, 1569-1583). In patients or animals with SCI, the amplitude of the H reflex is attenuated when the stimulus frequency reaches or exceeds 1 Hz and the response is impaired. As predicted, the mean relative amplitude (H%) of the H reflex was significantly lower in sham-operated rats when the stimulus frequency was augmented from 0.1 Hz to 5 Hz (100%±8.76%, 80.82%±8.24%, 46.06%±9.66%, 29.61%±9.00%, and 10.45%±4.27% for 0.1, 0.5, 1, 2, and 5 Hz, respectively). All other groups with SCI did not show a significant decline in the H reflex at 6 weeks post-transplant (Figure 6A). However, when the stimulation frequency reached 5 Hz, the cell-transplanted animals showed a reduction in the H reflex (36.35% ± 7.46%), whereas the culture medium injection group did not (58.05% ± 5.81%) (Figure 6A). This suggests that GABA neuron transplantation alleviated spasticity at 6 weeks post-transplant only when the stimulation was strong.

[0101] At 12 weeks post-transplantation, the cell transplantation group showed a reduction in H-reflex similar to that of Siamese surgical animals, while the SCI group and the culture medium injection group showed no clear changes even with enhanced stimulation frequencies of 0.1–5 Hz (Figure 6B). These results suggest that GABAergic interneuron transplantation alleviated late spasticity.

[0102] The reduction of spasticity by nerve cell transplantation can be attributed to multiple factors, ranging from nutritional support to inflammation regulation and neural circuit repair, as suggested by measurements of pain and gait. Using the DREADD-expressing nerve cells we transplanted, we asked whether the therapeutic effect depended on the activity of the transplanted cells. As previously shown, spasticity, measured by the mean relative amplitude of the H reflex, showed no clear change at 6 weeks post-transplant (Figure 6A). Interestingly, following CNO injection (3 mg / kg), H% decreased to 47% ± 4.12% with 2 Hz stimulation and to 28.21% ± 5.63% with 5 Hz stimulation, but not at 1 Hz (Figures 6C, 6D, and 6E). At 12 weeks post-transplant, CNO caused a reduction in H% of 59.99% ± 8.28% to 26.05% ± 5.64% even at 1 Hz (p=0.0475, Figure 6F). Similar changes were observed at 2 Hz (50.85%±8.86%~17.57%±4.98%, p=0.0286, Figure 6G) and 5 Hz (40.31%±8.13%~9.09%±1.52%, p=0.0346, Figure 6H). As controls, animals that underwent sham surgery, SCI, and SCI injected with culture medium did not show changes corresponding to CNO treatment (Figures 6C~6H). These results suggest that transplanted GABAergic neurons reduced spasticity by modulating spinal cord dI4 activity.

[0103] Transplantation of spinal inhibitory neurons improves walking movement.

[0104] Spasticity affects walking and locomotion. We measured weekly walking motion using open-field BBB scoring (Basso et al. Exp. Neurol. 1996, 139, 244-256) and unbiased treadscan analysis at 12 weeks (Chen et al. J. Clin. Invest. 2015, 125, 1033-1042; Ma et al. Cell Stem Cell 2012, 10, 455-464; Qian et al. Stem Cell Reports 2017, 8, 843-855). BBB scores decreased with injury, then recovered, and remained stable for 2-3 weeks post-injury (Figure 7A), suggesting stable spastic constriction (SCI). Animals with culture medium injections did not show a clear difference from those with SCI. Animals that underwent cell transplantation showed improvement in BBB scores (SCI+ cells vs. SCI+ medium, P<0.0001) and improved walking ability 8–12 weeks after transplantation.

[0105] Because the available assays for evaluating spasticity are limited, we performed an unbiased Treadscan to measure gait with the intention of identifying additional parameters for predicting spasticity. Many parameters, including limb stride time, swing time, stride distance, stride frequency, and gait speed, may be used to measure gait and locomotion. Stide distance is measured as the average anterior-posterior distance between two consecutive heel strike markers on the same side. Stide frequency (stride steps / second) is defined as the reciprocal of the interval between heel strikes on the same foot. Gait speed is the average of all instantaneous gait speeds during stride steps.

[0106] After SCI, compared to the sham surgery group (Figures 7B-7E), hindlimb double-step time and swing time increased (Figures 7B, 7C), while double-step distance and double-step frequency decreased (Figures 7D, 7E). As a result, walking speed became slower (Figure 7F). Animals that received cell transplantation but not culture medium injection recovered these parameters, and their walking speed was faster than those that received culture medium injection (Figures 7B-7F), suggesting that cell transplantation reduced spasticity and improved gait.

[0107] Transplantation of spinal inhibitory neurons reduces neuropathic pain.

[0108] Neuropathic pain was evaluated by measuring mechanical avoidance of the hind limbs in response to stimulation using an Electronic Von Frey Anesthesiometer 2450 (IITC Life Sciences Inc). At 6 weeks (Figure 8A), animals with SCI showed a significant decrease in mechanical avoidance threshold compared to those that underwent Siamese surgery (laminectomy only, no SCI: 36.39±1.07g vs. 54.50±0.20g, p<0.0001), and the stem cell transplantation group showed an enhanced mechanical avoidance threshold compared to the SCI+ medium group (42.87±1.90g in SCI+ cells vs. 37.47±0.75g in SCI+ medium, p=0.0084). A similar trend was observed at 12 weeks post-transplantation (Figure 8B). Therefore, transplantation of spinal GABAergic interneurons reduced neuropathic pain in the SCI model.

[0109] ***

[0110] The above descriptions of specific embodiments fully illustrate the general nature of the invention, which allows others to readily modify and / or adapt such specific embodiments to various uses without excessive experimentation and without departing from the general concept of the disclosure, by applying knowledge within the scope of the skill in the art. Accordingly, such adapted and modified forms shall be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that the words or terms used herein are for illustrative purposes only and not for limitation, and as a result, the words or terms used herein should be interpreted by those skilled in the art in light of the teachings and guidance provided herein.

[0111] The breadth and scope of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims and equivalents.

[0112] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, and / or other document is individually indicated as being incorporated by reference for all purposes.

[0113] For the sake of comprehensiveness, various aspects of the present invention are described by the following numbered clauses:

[0114] Article 1.

[0115] A method for producing a population of spinal cord dI4 progenitor cells from human stem human stem cells, comprising: (a) culturing human stem cells in a culture medium containing a Wnt signaling agonist, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor to produce a cell population containing spinal cord neuroepithelial progenitor cells, wherein at least 95% of the produced cell population are Sox1+ / Hoxa3+ spinal cord neuroepithelial progenitor cells; and (b) culturing the spinal cord neuroepithelial progenitor cells from step (a) in a culture medium further containing a retinoic acid (RA) signaling agonist and an SHH signaling pathway inhibitor to produce a cell population containing spinal cord dl4 progenitor cells, wherein at least 90% of the produced cell population are PTF1A+ / PAX7+ / ASCL1+ spinal cord dl4 progenitor cells.

[0116] Article 2.

[0117] The method according to Clause 1, wherein the human stem cells are human pluripotent stem cells.

[0118] Article 3.

[0119] The method according to Clause 2, wherein the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0120] Article 4.

[0121] The method according to any one of the claims 1 to 3, wherein the human stem cells are obtained from a human embryo.

[0122] Article 5.

[0123] The method according to any one of claims 1 to 4, wherein the Wnt signaling agonist comprises a GSK3 inhibitor selected from the group consisting of CHIR99021 and 6-bromo-iridium-3'-oxime.

[0124] Article 6.

[0125] The method according to any one of claims 1 to 5, wherein the BMP signaling pathway inhibitor is selected from the group consisting of DMH-1, dolsomorphine, and LDN-193189.

[0126] Article 7.

[0127] The method according to any one of claims 1 to 6, wherein the TGFβ signaling pathway inhibitor is selected from the group consisting of SB431542, SB505124, and A83-01.

[0128] Article 8.

[0129] The method according to any one of claims 1 to 7, wherein the RA signaling agonist is selected from the group consisting of retinoic acid (RA) and EC23.

[0130] Article 9.

[0131] The method according to any one of claims 1 to 8, wherein the SHH signaling inhibitor is selected from the group consisting of cyclopamine and SANT-1.

[0132] Article 10.

[0133] The method according to any one of claims 1 to 9, wherein the culture medium of step (a) comprises 2 μM SB431542, 2 μM DMH1, and 3 μM CHIR99021.

[0134] Article 11.

[0135] The method according to any one of the claims 1 to 10, wherein the human stem cells are cultured in the culture medium for 5 to 10 days in step (a).

[0136] Article 12.

[0137] The method according to clause 11, wherein the human stem cells are cultured in the culture medium for 7 days in step (a).

[0138] Article 13.

[0139] The method according to any one of the claims 1 to 12, wherein the culture medium of step (b) comprises DMH-1, SB431542, CHIR99021, all-trans retinoic acid, and cyclopamine.

[0140] Article 14.

[0141] The method according to any one of claims 1 to 13, wherein the culture medium of step (b) comprises approximately 1 μM to 10 μM of DMH-1, approximately 1 μM to 10 μM of SB431542, approximately 1 μM to 10 μM of CHIR99021, approximately 0.01 μM to 2 μM of RA, and approximately 0.1 μM to 2 μM of cyclopamine.

[0142] Article 15.

[0143] The method according to any one of claims 1 to 14, wherein the culture medium of step (b) comprises 2 μM SB431542, 2 μM DMH1, 3 μM CHIR99021, 0.1 μM all-trans retinoic acid, and 0.5 μM cyclopamine.

[0144] Article 16.

[0145] The method according to any one of claims 1 to 15, wherein the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 5 to 10 days in step (b).

[0146] Article 17.

[0147] The method according to clause 16, wherein the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 7 days in step (b).

[0148] Article 18.

[0149] The method according to any one of the claims 1 to 17, wherein the spinal cord dl4 progenitor cells of step (b) express at least one gene selected from PTF1A, PAX7, and ASCL1, or a combination thereof.

[0150] Article 19.

[0151] The method according to any one of the claims 1 to 18, wherein at least a portion of the spinal cord dl4 progenitor cells of step (b) further differentiate into spinal cord GABAergic interneurons.

[0152] Article 20.

[0153] The method according to clause 19, wherein the spinal cord GABA interneurons express at least one gene selected from PAX2 and LHX1 / 5, or a combination thereof.

[0154] Article 21.

[0155] A method for treating a target neurological disorder, comprising administering to the target an effective amount of a spinal cord dI4 progenitor cell population produced by the method described in any one of the paragraphs 1 to 20.

[0156] Article 22.

[0157] The method according to Clause 21, wherein the nerve disorder includes spinal cord injury.

Claims

1. A method for producing a population of spinal cord dI4 progenitor cells from human stem cells, the following: (a) Human stem cells are cultured in a culture medium containing a Wnt signaling agonist, a BMP signaling pathway inhibitor, and a TGFβ signaling pathway inhibitor to produce a cell population including spinal nerve epithelial progenitor cells, wherein at least 95% of the produced cell population are Sox1+ / Hoxa3+ spinal nerve epithelial progenitor cells; and (b) The spinal cord neuroepithelial progenitor cells from step (a) are cultured in a culture medium further containing a retinoic acid (RA) signaling agonist and an SHH signaling inhibitor to produce a cell population containing spinal cord dI4 progenitor cells, wherein at least 90% of the produced cell population are PTF1A+ / PAX7+ / ASCL1+ spinal cord dI4 progenitor cells. A method that includes this.

2. The method according to claim 1, wherein the human stem cells are human pluripotent stem cells.

3. The method according to claim 2, wherein the human pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

4. The method according to any one of claims 1 to 3, wherein the human stem cells are obtained from a human embryo.

5. The method according to any one of claims 1 to 4, wherein the Wnt signaling agonist comprises a GSK3 inhibitor selected from the group consisting of CHIR99021 and 6-bromo-iridium-3'-oxime.

6. The method according to any one of claims 1 to 5, wherein the BMP signaling pathway inhibitor is selected from the group consisting of DMH-1, dolsomorphine, and LDN-193189.

7. The method according to any one of claims 1 to 6, wherein the TGFβ signaling pathway inhibitor is selected from the group consisting of SB431542, SB505124, and A83-01.

8. The method according to any one of claims 1 to 7, wherein the RA signaling agonist is selected from the group consisting of retinoic acid (RA) and EC23.

9. The method according to any one of claims 1 to 8, wherein the SHH signaling inhibitor is selected from the group consisting of cyclopamine and SANT-1.

10. The method according to any one of claims 1 to 9, wherein the culture medium in step (a) comprises 2 μM SB431542, 2 μM DMH1, and 3 μM CHIR99021.

11. The method according to any one of claims 1 to 10, wherein the human stem cells are cultured in the culture medium for 5 to 10 days in step (a).

12. The method according to claim 11, wherein the human stem cells are cultured in the culture medium for 7 days in step (a).

13. The method according to any one of claims 1 to 12, wherein the culture medium of step (b) comprises DMH-1, SB431542, CHIR99021, all-trans retinoic acid, and cyclopamine.

14. The method according to any one of claims 1 to 13, wherein the culture medium of step (b) comprises 1 μM to 10 μM of DMH-1, 1 μM to 10 μM of SB431542, 1 μM to 10 μM of CHIR99021, 0.01 μM to 2 μM of RA, and 0.1 μM to 2 μM of cyclopamine.

15. The method according to any one of claims 1 to 14, wherein the culture medium of step (b) comprises 2 μM SB431542, 2 μM DMH1, 3 μM CHIR99021, 0.1 μM all-trans retinoic acid, and 0.5 μM cyclopamine.

16. The method according to any one of claims 1 to 15, wherein the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 5 to 10 days in step (b).

17. The method according to claim 16, wherein the spinal cord neuroepithelial progenitor cells are cultured in the culture medium for 7 days in step (b).

18. The method according to any one of claims 1 to 17, wherein the spinal cord dI4 progenitor cells of step (b) express at least one gene selected from PTF1A, PAX7, and ASCL1, or a combination thereof.

19. The method according to any one of claims 1 to 18, wherein at least a portion of the spinal cord dI4 progenitor cells of step (b) further differentiate into spinal cord GABAergic interneurons.

20. The method according to claim 19, wherein the spinal cord GABA interneurons express at least one gene selected from PAX2 and LHX1 / 5, or a combination thereof.

21. A pharmaceutical composition for treating a target neurological disorder, comprising an effective amount of a spinal cord dI4 progenitor cell population produced by the method described in any one of claims 1 to 20.

22. The pharmaceutical composition according to claim 21, wherein the nerve disorder includes spinal cord injury.

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