基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells

Understanding how defects in mechanotransduction affect cell‐to‐cell variability will add to the fundamental knowledge of human pluripotent stem cell (hPSC) culture, and may suggest new approaches for achieving a robust, reproducible, and scalable process that result in consistent product quality and yields. Here, the current state of the understanding of the fundamental mechanisms that govern the growth kinetics of hPSCs between static and dynamic cultures is reviewed, the factors causing fluctuations are identified, and culture strategies that might eliminate or minimize the occurrence of cell‐to‐cell variability arising from these fluctuations are discussed. The existing challenges in the development of hPSC expansion methods for enabling the transition from process development to large‐scale production are addressed, a mandatory step for industrial and clinical applications of hPSCs.     

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

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Human Induced Pluripotent Stem Cells on Autologous Feeders

Background

For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders.

Methodology/Principal Findings

This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation.

Conclusions/Significance

These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

综述:基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.     

Derivation of Hair-Inducing Cell from Human Pluripotent Stem Cells

Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.     

Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells

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Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.     

A Chemical Probe that Labels Human Pluripotent Stem Cells

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Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.