Microfluidic systems: A new toolbox for pluripotent stem cells

Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user-controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insignt into the implications of microfluidics on pluripotent stem cell research.     

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.     

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

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 humanPSCs 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.     

多能干细胞向雄性生殖细胞定向分化的研究进展

生殖健康是生命科学领域关注的核心之一,各种原因所致男性不育亟待解决,然而由于伦理限制等原因,缺少合适的具有人类遗传背景的研究模型开展研究。胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）均属于多能干细胞,具有多向分化潜能。一方面,可利用ESCs?/?iPSCs向生殖细胞分化的模型研究人类生殖细胞的发育规律,另一方面,在此基础上,可建立带有人类疾病遗传背景的iPSCs模型,体外诱导其向雄性生殖细胞分化,利用该模型研究男性不育的发病机制。由于精子在体内的形成遵循一定规律,体外环境中不同发育阶段的生殖细胞在不同诱导因子作用下才可稳定地往下一阶段定向分化,因此,诱导ESCs?/?iPSCs向雄性生殖细胞方向分化时,诱导因子的种类和加入时间的选择应根据生殖细胞的体内发育特征而定,并且在诱导的不同阶段循序加入,以此模拟精子在体内的形成过程,进而更好地研究男性不育的发病机制。本文将对多能干细胞向雄性生殖细胞定向分化的常用诱导因子及存在问题和展望进行综述,为相关研究的开展提供借鉴。     

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

Dendritic cells derived from pluripotent stem cells:Potential of large scale production

Human pluripotent stem cells(hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells(DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension cultureof embryoid bodies(EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Dendritic cells derived from pluripotent stem cells: Potential of large scale production简

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells (DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension culture of embryoid bodies (EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Bi-allelic gene targeting in mouse embryonic stem cells

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.     

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system

ObjectiveTo provide a standardized protocol for large‐scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC).MethodsThe hPSC were expanded and differentiated into PTEC on matrix‐coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin‐treatment was assessed to check the potential use of PTEC to model drug‐induced nephrotoxicity.ResultshPSC expansion and PTEC differentiation could be performed directly on matrix‐coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10‐fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico‐basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM‐1 after Cisplatin treatment.ConclusionWe demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix‐coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.     

Targeted genome editing in pluripotent stem cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes.     

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.     

Gene therapy,gene targeting and induced pluripotent stem cells: Applications in monogenic disease treatment

Monogenic diseases are often severe, life-threatening disorders for which lifelong palliative treatment is the only option. Over the last two decades, a number of strategies have been devised with the aim to treat these diseases with a genetic approach. Gene therapy has been under development for many years, yet suffers from the lack of an effective and safe vector for the delivery of genetic material into cells. More recently, gene targeting by homologous recombination has been proposed as a safer treatment, by specifically correcting disease-causing mutations. However, low efficiency is a major drawback. The emergence of two technologies could overcome some of these obstacles. Terminally differentiated somatic cells can be reprogrammed, using defined factors, to become induced pluripotent stem cells (iPSCs), which can undergo efficient gene mutation correction with the aid of fusion proteins known as zinc finger nucleases (ZFNs). The amalgamation of these two technologies has the potential to break through the current bottleneck in gene therapy and gene targeting.     

Gene therapy, gene targeting and induced pluripotent stem cells: Applications in monogenic disease treatment

Monogenic diseases are often severe, life-threatening disorders for which lifelong palliative treatment is the only option. Over the last two decades, a number of strategies have been devised with the aim to treat these diseases with a genetic approach. Gene therapy has been under development for many years, yet suffers from the lack of an effective and safe vector for the delivery of genetic material into cells. More recently, gene targeting by homologous recombination has been proposed as a safer treatment, by specifically correcting disease-causing mutations. However, low efficiency is a major drawback. The emergence of two technologies could overcome some of these obstacles. Terminally differentiated somatic cells can be reprogrammed, using defined factors, to become induced pluripotent stem cells (iPSCs), which can undergo efficient gene mutation correction with the aid of fusion proteins known as zinc finger nucleases (ZFNs). The amalgamation of these two technologies has the potential to break through the current bottleneck in gene therapy and gene targeting.     

Advances in genetic modification of pluripotent stem cells

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential.