Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix简

Neural cells differentiated from pluripotent stem cells (PSCs), including both embryonic stem cells and induced pluripotent stem cells, provide a powerful tool for drug screening, disease modeling and regenerative medicine. High-purity oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) have been derived from PSCs recently due to the advancements in understanding the developmental signaling pathways. Extracellular matrices (ECM) have been shown to play important roles in regulating the survival, proliferation, and differentiation of neural cells. To improve the function and maturation of the derived neural cells from PSCs, understanding the effects of ECM over the course of neural differentiation of PSCs is critical. During neural differentiation of PSCs, the cells are sensitive to the properties of natural or synthetic ECMs, including biochemical composition, biomechanical properties, and structural/topographical features. This review summarizes recent advances in neural differentiation of human PSCs into OPCs and NPCs, focusing on the role of ECM in modulating the composition and function of the differentiated cells. Especially, the importance of using three-dimensional ECM scaffolds to simulate the in vivo microenvironment for neural differentiation of PSCs is highlighted. Future perspectives including the immediate applications of PSC-derived neural cells in drug screening and disease modeling are also discussed.     

Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.     

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.     

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.     

人多能干细胞来源的肾定向分化平台的构建

肾是一种重要的人体器官,具有多种生理功能。然而,全球范围内约有10%的人口患有肾疾病。因此,建立一种接近人体肾的结构与功能的模型进行肾疾病的研究是十分必要的。多能干细胞体外定向诱导分化技术的兴起,为再生医学和精准医学领域注入了新的动力。本研究通过在体外条件下模拟体内肾发育的过程,将人多能干细胞包括胚胎干细胞和诱导多能干细胞,通过体外定向诱导分化形成肾的祖细胞,进而建立肾的结构与功能单位：肾元。该研究通过激活WNT信号通路,同时抑制TGF-β信号通路,将人多能干细胞从多能态定向诱导至原条阶段。之后通过细胞自分化的能力使其发育至中间中胚层,再通过激活FGF信号通路,将其分化至肾祖细胞阶段。流式细胞检测结果显示,肾祖细胞占总细胞数的51.5%～61.9%。通过免疫荧光检测发现：分化得到的结构中包含肾小球足细胞、近端小管、远端小管等肾组织结构。该研究建立的肾体外分化方法,具有稳定性好、分化效率高、重复性好的特点。为研究人类肾的早期发育机制,肾疾病模型构建,以及药物筛选提供了一种新的方法。     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.     

An Engineered N-Cadherin Substrate for Differentiation,Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell—material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.     

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.     

Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.     

Process engineering of stem cell metabolism for large scale expansion and differentiation in bioreactors

Mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs) emerge as promising tools for tissue engineering, cell therapy, and drug screening. Their potential use in clinical applications requires the efficient production of differentiated cells at large scale. Glucose, amino acid, and oxygen metabolism play a key role in MSC and PSC expansion and differentiation. This review summarizes recent advances in the understanding of stem cell metabolism for reprogramming, self-renewal, and lineage commitment. From the reported data, efficient expansion of stem cells has been found to rely on glycolysis, while during differentiation stem cells shift their metabolic pathway to oxidative phosphorylation. During reprogramming, the reverse metabolic shift from oxidative phosphorylation to glycolysis has been observed. As a consequence, the demands for glucose and oxygen vary upon different phases of stem cell production. Accurate understanding of stem cell metabolism is critical for the rational design of culture parameters such as oxygen tension and feeding regime in bioreactors towards efficient integrated reprogramming, expansion, and differentiation processes at large scale.     

Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes

Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC(PSA-NCAM+)) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC(PSA-NCAM+) extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80～98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC(PSA-NCAM+) showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPC(PSA-NCAM+) differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC(PSA-NCAM+) compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.     

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.     

There is More to a Lipid than just Being a Fat: Sphingolipid-Guided Differentiation of Oligodendroglial Lineage from Embryonic Stem Cells

Dr. Robert K. Yu’s research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.     

Neural induction of porcine‐induced pluripotent stem cells and further differentiation using glioblastoma‐cultured medium

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine‐induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma‐ and Mad‐related protein (SMAD) inhibitors, a TGF‐β inhibitor and BMP4 inhibitor. The dSMADi‐derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid‐treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma‐cultured medium including injury‐related cytokines treated porcine iPSC‐NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.