Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.     

慢病毒介导的绿色荧光蛋白报告基因对人多能干细胞的生物风险评估研究

摘要 目的：探讨慢病毒介导的绿色荧光蛋白（GFP）标记人诱导多能干细胞（hiPSC）是否影响其细胞生物学特性,为多角度评价生物学风险提供实验基础。方法：慢病毒感染hiPSC后24小时,通过抗性基因表达筛选成功标记GFP的hiPSC。利用流式细胞分析法（FACS）检测GFP阳性的细胞比例。通过碱性磷酸酶染色检验干细胞的多能性,并通过免疫荧光染色检测多能性标记基因OCT4,NANOG,SOX2,SSEA4的表达情况。体外拟胚体分化实验检测GFP标记的hiPSC分化为不同胚层细胞的能力。结果：慢病毒感染不仅可以成功hiPSC标记上GFP,而且抗性基因表达筛选使GFP阳性细胞比例从37.5%提高到97.4%。AP染色和多能性标记基因的免疫染色证明标记后的细胞能维持多能性。体外分化实验显示感染后hiPSC可以形成拟胚体并实现三个胚层细胞共存。结论：慢病毒能够高效的标记hiPSC,并且不影响其多能性和拟胚体形成能力,可以用于后续的分化和细胞示踪研究。     

Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic‐ (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC‐ and hiPSC‐derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC‐derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.     

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich''s ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich''s ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.     

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.     

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryoproper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endoand meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor 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.     

hiPSC‐derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine

Mesenchymal stem cells (MSCs) are being assessed for ameliorating the severity of graft‐versus‐host disease, autoimmune conditions, musculoskeletal injuries and cardiovascular diseases. While most of these clinical therapeutic applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor remains limited. The utility of MSCs derived from human‐induced pluripotent stem cells (hiPSCs) has been shown in recent pre‐clinical studies. Since adult MSCs have limited capability regarding proliferation, the quantum of bioactive factor secretion and immunomodulation ability may be constrained. Hence, the alternate source of MSCs is being considered to replace the commonly used adult tissue‐derived MSCs. The MSCs have been obtained from various adult and foetal tissues. The hiPSC‐derived MSCs (iMSCs) are transpiring as an attractive source of MSCs because during reprogramming process, cells undergo rejuvination, exhibiting better cellular vitality such as survival, proliferation and differentiations potentials. The autologous iMSCs could be considered as an inexhaustible source of MSCs that could be used to meet the unmet clinical needs. Human‐induced PSC‐derived MSCs are reported to be superior when compared to the adult MSCs regarding cell proliferation, immunomodulation, cytokines profiles, microenvironment modulating exosomes and bioactive paracrine factors secretion. Strategies such as derivation and propagation of iMSCs in chemically defined culture conditions and use of footprint‐free safer reprogramming strategies have contributed towards the development of clinically relevant cell types. In this review, the role of iPSC‐derived mesenchymal stromal cells (iMSCs) as an alternate source of therapeutically active MSCs has been described. Additionally, we also describe the role of iMSCs in regenerative medical applications, the necessary strategies, and the regulatory policies that have to be enforced to render iMSC's effectiveness in translational medicine.     

Novel strategy to improve hepatocyte differentiation stability through synchronized behavior-driven mechanical memory of iPSCs

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.     

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.     

猪诱导性多能干细胞技术的研究进展及应用前景

猪作为实验材料,具有由于来源方便、基因序列与人类的相近及其在畜牧业中的重要地位等优势,成为国内外研究的热点,但是猪的胚胎干细胞(Embryonic stem cells,ESC)建系方面的研究进展缓慢。诱导性多能干细胞(induced pluripotent stem cells,iPSC)技术的诞生,开创了体细胞重编程的全新方法。猪iPSC体系的建立将为家畜ESC体系的建立奠定基础,同时也对提高猪转基因克隆的效率,高效育种和保种,乃至生物医学领域均产生深远的影响。文章综述了iPSC技术的主要进展,重点阐述了猪iPSC技术的现状及其在生物医学和畜牧业中的应用前景,以期为从事该领域研究的科研人员提供参考。     

Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells

PurposeTo compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs).ResultsGeneration of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle‐like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single‐cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP‐derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time‐consuming due to an intrinsically faster timing of retinal differentiation.ConclusionsRetinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS) cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs) are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cell...     

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.     

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.