Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

Induction of local immunosuppression in allogeneic cell transplantation by cell-type-specific expression of PD-L1 and CTLA4Ig.

Immune rejection has long hindered allogeneic cell transplantation therapy. Current genetic modification approaches, including direct targeting of major histocompatibility complex or constitutive expression of immune inhibitory molecules, exhibit drawbacks such as severe adverse effects or elevated tumorigenesis risks. To overcome these limitations, we introduce an innovative approach to induce cell-type-specific immune tolerance in differentiated cells. By engineering human embryonic stem cells, we ensure the exclusive production of the immune inhibitory molecules PD-L1/CTLA4Ig in differentiated cells. Using this strategy, we generated hepatocyte-like cells expressing PD-L1 and CTLA4Ig, which effectively induced local immunotolerance. This approach was evaluated in a humanized mouse model that mimics the human immune system dynamics. We thus demonstrate a robust and selective induction of immunotolerance specific to hepatocytes, improving graft survival without observed tumorigenesis. This precise immune tolerance strategy holds great promise for advancing the development of stem cell-based therapeutics in regenerative medicine.

Immunity-and-matrix-regulatory cells enhance cartilage regeneration for meniscus injuries: a phase I dose-escalation trial.

Immunity-and-matrix-regulatory cells (IMRCs) derived from human embryonic stem cells have unique abilities in modulating immunity and regulating the extracellular matrix, which could be mass-produced with stable biological properties. Despite resemblance to mesenchymal stem cells (MSCs) in terms of self-renew and tri-lineage differentiation, the ability of IMRCs to repair the meniscus and the underlying mechanism remains undetermined. Here, we showed that IMRCs demonstrated stronger immunomodulatory and pro-regenerative potential than umbilical cord MSCs when stimulated by synovial fluid from patients with meniscus injury. Following injection into the knees of rabbits with meniscal injury, IMRCs enhanced endogenous fibrocartilage regeneration. In the dose-escalating phase I clinical trial (NCT03839238) with eighteen patients recruited, we found that intra-articular IMRCs injection in patients was safe over 12 months post-grafting. Furthermore, the effective results of magnetic resonance imaging (MRI) of meniscus repair and knee functional scores suggested that 5 × 107 cells are optimal for meniscus injury treatment. In summary, we present the first report of a phase I clinical trial using IMRCs to treat meniscus injury. Our results demonstrated that intra-articular injection of IMRCs is a safe and effective therapy by providing a permissive niche for cartilage regeneration.

Safety and efficacy of human ESC-derived corneal endothelial cells for corneal endothelial dysfunction.

BACKGROUND: Research on human pluripotent stem cells (hPSCs) has shown tremendous progress in cell-based regenerative medicine. Corneal endothelial dysfunction is associated with the loss and degeneration of corneal endothelial cells (CECs), rendering cell replacement a promising therapeutic strategy. However, comprehensive preclinical assessments of hPSC-derived CECs for this cell therapy remain a challenge. RESULTS: Here we defined an adapted differentiation protocol to generate induced corneal endothelial cells (iCECs) consistently and efficiently from clinical-grade human embryonic stem cells (hESCs) with xeno-free medium and manufactured cryopreserved iCECs. Cells express high levels of typical CECs markers and exhibit transendothelial potential properties in vitro typical of iCECs. After rigorous quality control measures, cells meeting all release criteria were available for in vivo studies. We found that there was no overgrowth or tumorigenicity of grafts in immunodeficient mice. After grafting into rabbit models, the surviving iCECs ameliorated edema and recovered corneal opacity. CONCLUSIONS: Our work provides an efficient approach for generating iCECs and demonstrates the safety and efficacy of iCECs in disease modeling. Therefore, clinical-grade iCECs are a reliable source for future clinical treatment of corneal endothelial dysfunction.

Abnormal chromatin remodeling caused by ARID1A deletion leads to malformation of the dentate gyrus.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be a tumor suppressor. Recently, loss-of-function of ARID1A gene has been shown to cause intellectual disability. Here we generate Arid1a conditional knockout mice and investigate Arid1a function in the hippocampus. Disruption of Arid1a in mouse forebrain significantly decreases neural stem/progenitor cells (NSPCs) proliferation and differentiation to neurons within the dentate gyrus (DG), increasing perinatal and postnatal apoptosis, leading to reduced hippocampus size. Moreover, we perform single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and reveal that Arid1a is necessary for the maintenance of the DG progenitor pool and survival of post-mitotic neurons. Transcriptome and ChIP-seq analysis data demonstrate that ARID1A specifically regulates Prox1 by altering the levels of histone modifications. Overexpression of downstream target Prox1 can rescue proliferation and differentiation defects of NSPCs caused by Arid1a deletion. Overall, our results demonstrate a critical role for Arid1a in the development of the hippocampus and may also provide insight into the genetic basis of intellectual disabilities such as Coffin-Siris syndrome, which is caused by germ-line mutations or microduplication of Arid1a.

Endothelial Arid1a deletion disrupts the balance among angiogenesis, neurogenesis and gliogenesis in the developing brain.

The vascular system and the neural system processes occur simultaneously, the interaction among them is fundamental to the normal development of the central nervous system. Arid1a (AT-rich interaction domain 1A), which encodes an epigenetic subunit of the SWI/SNF chromatin-remodelling complex, is associated with promoter-mediated gene regulation and histone modification. However, the molecular mechanism of the interaction between cerebrovascular and neural progenitor cells (NPCs) remains unclear. To generate Arid1acKO-Tie2 mice, Arid1afl/fl mice were hybridized with Tie2-Cre mice. The Angiogenesis, neurogenesis and gliogenesis were studied by immunofluorescence staining and Western blotting. RNA-seq, RT-PCR, Western blotting, CO-IP and rescue experiments were performed to dissect the molecular mechanisms of Arid1a regulates fate determination of NPCs. We found that the absence of Arid1a results in increased the density of blood vessels, delayed neurogenesis and decreased gliogenesis, even after birth. Mechanistically, the deletion of Arid1a in endothelial cells causes a significant increase in H3k27ac and the secretion of maternal protein 2 (MATN2). In addition, matn2 alters the AKT/SMAD4 signalling pathway through its interaction with the NPCs receptor EGFR, leading to the decrease of SMAD4. SMAD complex further mediates the expression of downstream targets, thereby promoting neurogenesis and inhibiting gliogenesis. This study suggests that endothelial Arid1a tightly controls fate determination of NPCs by regulating the AKT-SMAD signalling pathway.

Neuronal cell-based medicines from pluripotent stem cells: Development, production, and preclinical assessment.

Brain degeneration and damage is difficult to cure due to the limited endogenous repair capability of the central nervous system. Furthermore, drug development for treatment of diseases of the central nervous system remains a major challenge. However, it now appears that using human pluripotent stem cell-derived neural cells to replace degenerating cells provides a promising cell-based medicine for rejuvenation of brain function. Accordingly, a large number of studies have carried out preclinical assessments, which have involved different neural cell types in several neurological diseases. Recent advances in animal models identify the transplantation of neural derivatives from pluripotent stem cells as a promising path toward the clinical application of cell therapies [Stem Cells Transl Med 2019;8:681-693; Drug Discov Today 2019;24:992-999; Nat Med 2019;25:1045-1053]. Some groups are moving toward clinical testing in humans. However, the difficulty in selection of valuable critical quality criteria for cell products and the lack of functional assays that could indicate suitability for clinical effect continue to hinder neural cell-based medicine development [Biologicals 2019;59:68-71]. In this review, we summarize the current status of preclinical studies progress in this area and outline the biological characteristics of neural cells that have been used in new developing clinical studies. We also discuss the requirements for translation of stem cell-derived neural cells in examples of stem cell-based clinical therapy.

Infusion of hESC derived Immunity-and-matrix regulatory cells improves cognitive ability in early-stage AD mice.

OBJECTIVES: In this study, we administered immunity-and-matrix regulatory cells (IMRCs) via tail vein (IV) and intracerebroventricular (ICV) injection to 3-month-old 5×FAD transgenic mice to assess the effects of IMRC transplantation on the behaviour and pathology of early-stage Alzheimer's disease (AD). MATERIALS AND METHODS: Clinical-grade human embryonic stem cell (hESC)-derived IMRCs were produced under good manufacturing practice (GMP) conditions. Three-month-old 5×FAD mice were administered IMRCs via IV and ICV injection. After 3 months, the mice were subjected to behavioural tests and electrophysiological analysis to evaluate their cognitive function, memory ability and synaptic plasticity. The effect of IMRCs on amyloid-beta (Aß)-related pathology was detected by thioflavin-S staining and Western blot. Quantitative real-time PCR, ELISA and immunostaining were used to confirm that IMRCs inhibit neuroinflammation. RNA-seq analysis was performed to measure changes in gene expression and perform a pathway analysis in response to IMRC treatment. RESULTS: IMRC administration via tail vein injection significantly ameliorated cognitive deficits in early-stage AD (5×FAD) mice. However, no significant change was observed in the characteristic pathology of AD in the ICV group. Plaque analysis revealed that IMRCs did not influence either plaque deposition or BACE1 expression. In addition, IMRCs inhibited inflammatory responses and reduced microglial activation in vivo. CONCLUSIONS: We have shown that peripheral administration of IMRCs can ameliorate AD pathology and associated cognitive deficits.

Low expression of miR-142-3p promotes intervertebral disk degeneration.

BACKGROUND: Intervertebral disk degeneration (IDD) is a degenerative disease characterized by cytoplasm loss and extracellular matrix degradation. Numerous evidence reported that miRNAs participated in IDD development. Nevertheless, the function of miR-142-3p in IDD development remains unknown. This study mainly explored the potential role and function of miR-142-3p in IDD development. METHODS: One percent fetal bovine serum was used to induce the degeneration of ATDC5 cells, and miR-142-3p level was examined by qRT-PCR. Then, miR-142-3p mimic/inhibitor and its corresponding negative control were transfected into ATDC5 normal and degenerative cells. Viability, migration, invasion, apoptosis, cycle, Bax, Bcl-2, P62, and Beclin1 expression levels were assessed using CCK8, wound healing assay, annexin V-FITC/PI staining, western blot, and qRT-PCR, respectively. RESULTS: The results revealed that the expression levels of MMP13, ADAMTS5, MMP3, and Col-X were increased as well as the expression levels of SOX-9 and Col-II were reduced in ATDC5 degenerative cells, indicating the degeneration model was constructed. We observed that miR-142-3p was decreased in ATDC5 degenerative cells and its suppression could promote ATDC5 cell degeneration. However, miR-142-3p overexpression could reverse the cell viability inhibition, as well as apoptosis and autophagy enhancement in ATDC5 degenerative cells. CONCLUSIONS: Our results proved that miR-142-3p may play an important role in disk degeneration. Further animal study is needed to illustrate the role of the miR-142-3p in IDD development.

Stem Cell Therapy for Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases caused by specific degeneration and loss of dopamine neurons in substantia nigra of the midbrain. PD is clinically characterized by motor dysfunctions and non-motor symptoms. Even though the dopamine replacement can improve the motor symptoms of PD, it cannot stop the neural degeneration and disease progression. Electrical deep brain stimulation (DBS) to the specific brain areas can improve the symptoms, but it eventually loses the effectiveness. Stem cell transplantation provides an exciting potential for the treatment of PD. Current available cell sources include neural stem cells (NSCs) from fetal brain tissues, human embryonic stem cells (hESCs) isolated from blastocyst, and induced pluripotent stem cells (iPSCs) reprogrammed from the somatic cells such as the fibroblasts and blood cells. Here, we summarize the research advance in experimental and clinical studies to transplant these cells into animal models and clinical patients, and specifically highlight the studies to use hESCs /iPSCs-derived dopaminergic precursor cells and dopamine neurons for the treatment of PD, at last propose future challenges for developing clinical-grade dopaminergic cells for treating the PD.

Immunity-and-matrix-regulatory cells derived from human embryonic stem cells safely and effectively treat mouse lung injury and fibrosis.

Lung injury and fibrosis represent the most significant outcomes of severe and acute lung disorders, including COVID-19. However, there are still no effective drugs to treat lung injury and fibrosis. In this study, we report the generation of clinical-grade human embryonic stem cells (hESCs)-derived immunity- and matrix-regulatory cells (IMRCs) produced under good manufacturing practice requirements, that can treat lung injury and fibrosis in vivo. We generate IMRCs by sequentially differentiating hESCs with serum-free reagents. IMRCs possess a unique gene expression profile distinct from that of umbilical cord mesenchymal stem cells (UCMSCs), such as higher expression levels of proliferative, immunomodulatory and anti-fibrotic genes. Moreover, intravenous delivery of IMRCs inhibits both pulmonary inflammation and fibrosis in mouse models of lung injury, and significantly improves the survival rate of the recipient mice in a dose-dependent manner, likely through paracrine regulatory mechanisms. IMRCs are superior to both primary UCMSCs and the FDA-approved drug pirfenidone, with an excellent efficacy and safety profile in mice and monkeys. In light of public health crises involving pneumonia, acute lung injury and acute respiratory distress syndrome, our findings suggest that IMRCs are ready for clinical trials on lung disorders.

Generation of qualified clinical-grade functional hepatocytes from human embryonic stem cells in chemically defined conditions.

Hepatocytes have been successfully generated from human pluripotent stem cells (hPSCs). However, the cost-effective and clinical-grade generation of hepatocytes from hPSCs still need to be improved. In this study, we reported the production of functional hepatocytes from clinical-grade human embryonic stem cells (hESCs) under good manufacturing practice (GMP) requirements. We sequentially generated primitive streak (PS), definitive endoderm (DE), hepatoblasts and hepatocyte-like cells (HLCs) from hESCs in the different stages with completely defined reagents. During hepatoblast differentiation, dimethyl sulfoxide (DMSO), transferrin, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Vc-Mg), insulin, and sodium selenite were used instead of cytokines and FBS/KOSR. Then, hepatoblasts were differentiated into HLCs that had a typical hepatocyte morphology and possessed characteristics of mature hepatocytes, such as metabolic-related gene expression, albumin secretion, fat accumulation, glycogen storage, and inducible cytochrome P450 activity in vitro. HLCs integrated into the livers of Tet-uPA Rag2-/- Il2rg-/- (URG) mice, which partially recovered after transplantation. Furthermore, a series of biosafety-related experiments were performed to ensure future clinical applications. In conclusion, we developed a chemically defined system to generate qualified clinical-grade HLCs from hESCs under GMP conditions. HLCs have been proven to be safe and effective for treating liver failure. This efficient platform could facilitate the treatment of liver diseases using hESC-derived HLCs transplantation.

Reactive Amphiphilic Conjugated Polymers for Inhibiting Amyloid ß Assembly.

Protein misfolding and aberrant aggregations are associated with multiple prevalent and intractable diseases. Inhibition of amyloid assembly is a promising strategy for the treatment of amyloidosis. Reported here is the design and synthesis of a reactive conjugated polymer, a poly(p-phenylene vinylene) derivative, functionalized with p-nitrophenyl esters (PPV-NP) and it inhibits the assembly of amyloid proteins, degrades preformed fibrils, and reduces the cytotoxicity of amyloid aggregations in living cells. PPV-NP is attached to the proteins through hydrophobic interactions and irreversible covalent linkage. PPV-NP also exhibited the capacity to eliminate Aß plaques in brain slices in ex vivo assays. This work represents an innovative attempt to inhibit protein pathogenic aggregates, and may offer insights into the development of therapeutic strategies for amyloidosis.

Zinc finger E-box-binding homeobox 1 (ZEB1) is required for neural differentiation of human embryonic stem cells.

Human pluripotent stem cells hold great promise for improving regenerative medicine. However, a risk for tumor formation and difficulties in generating large amounts of subtype derivatives remain the major obstacles for clinical applications of stem cells. Here, we discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is highly expressed upon differentiation of human embryonic stem cells (hESCs) into neuronal precursors. CRISPR/Cas9-mediated ZEB1 depletion did not impede neural fate commitment, but prevented hESC-derived neural precursors from differentiating into neurons, indicating that ZEB1 is required for neuronal differentiation. ZEB1 overexpression not only expedited neural differentiation and neuronal maturation, which ensured safer neural cell transplantation, but also facilitated the generation of excitatory cortical neurons, which were valuable for managing certain neurological disorders, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Our study provides useful information on how human neural cells are generated, which may help in forming strategies for developing and improving replacement therapies for treating patients with neurological diseases.

A Chemical Recipe for Generation of Clinical-Grade Striatal Neurons from hESCs.

Differentiation of human pluripotent stem cells (hPSCs) into striatal medium spiny neurons (MSNs) promises a cell-based therapy for Huntington's disease. However, clinical-grade MSNs remain unavailable. Here, we developed a chemical recipe named XLSBA to generate clinical-grade MSNs from embryonic stem cells (ESCs). We introduced the γ-secretase inhibitor DAPT into the recipe to accelerate neural differentiation, and replaced protein components with small molecules. Using this optimized protocol we could efficiently direct regular human ESCs (hESCs) as well as clinical-grade hESCs to lateral ganglionic eminence (LGE)-like progenitors and striatal MSNs within less than half of the time than previous protocols (within 14 days and 21 days, respectively). These striatal cells expressed appropriate MSN markers and electrophysiologically acted like authentic MSNs. Upon transplantation into brains of neonatal mice or mouse model of Huntington's disease, they exhibited sufficient safety and reasonable efficacy. Therefore, this quick and highly efficient derivation of MSNs offers unprecedented access to clinical application.

Human Clinical-Grade Parthenogenetic ESC-Derived Dopaminergic Neurons Recover Locomotive Defects of Nonhuman Primate Models of Parkinson's Disease.

Clinical application of stem cell derivatives requires clinical-grade cells and sufficient preclinical proof of safety and efficacy, preferably in primates. We previously successfully established a clinical-grade human parthenogenetic embryonic stem cell (hPESC) line, but the suitability of its subtype-specific progenies for therapy is not clear. Here, we compared the function of clinical-grade hPESC-derived midbrain dopaminergic (DA) neurons in two canonical protocols in a primate Parkinson's disease (PD) model. We found that the grafts did not form tumors and produced variable but apparent behavioral improvement for at least 24 months in most monkeys in both groups. In addition, a slight DA increase in the striatum correlates with significant functional improvement. These results demonstrated that clinical-grade hPESCs can serve as a reliable source of cells for PD treatment. Our proof-of-concept findings provide preclinical data for China's first ESC-based phase I/IIa clinical study of PD (ClinicalTrials.gov number NCT03119636).

Precise immune tolerance for hPSC derivatives in clinical application.

Human pluripotent stem cells (hPSCs) promise a foreseeing future for regeneration medicine and cell replacement therapy with their abilities to produce almost any types of somatic cells of the body. The complicated immunogenicity of hPSC derivatives and context dependent responses in variable transplantations greatly hurdle the practical application of hPSCs in clinic. Especially for applications of hPSCs, induction of immune tolerance at the same time increases the risks of tumorigenesis. Over the past few years, thanks to the progress in immunology and practices in organ transplantation, endeavors on exploring strategies to induce long term protection of allogeneic transplants have shed light on overcoming this barrier. Novel genetic engineering techniques also allow to precisely cradle the immune response of transplantation. Here we reviewed the current understanding on immunogenicity, and efforts have been attempted on inducing immune tolerance for hPSC derivatives, with extra focus on modifying the graft cells. We also glimpse on employing cutting-edge genome editing technologies for this purpose, which will potentially endow hPSC derivatives with the nature of wide spectrum drugs for therapy.

A single mutation in the prM protein of Zika virus contributes to fetal microcephaly.

Zika virus (ZIKV) has evolved into a global health threat because of its unexpected causal link to microcephaly. Phylogenetic analysis reveals that contemporary epidemic strains have accumulated multiple substitutions from their Asian ancestor. Here we show that a single serine-to-asparagine substitution [Ser139→Asn139 (S139N)] in the viral polyprotein substantially increased ZIKV infectivity in both human and mouse neural progenitor cells (NPCs) and led to more severe microcephaly in the mouse fetus, as well as higher mortality rates in neonatal mice. Evolutionary analysis indicates that the S139N substitution arose before the 2013 outbreak in French Polynesia and has been stably maintained during subsequent spread to the Americas. This functional adaption makes ZIKV more virulent to human NPCs, thus contributing to the increased incidence of microcephaly in recent ZIKV epidemics.