Therapeutic potential of perivascular cells from human pluripotent stem cells

Vascularization of injured tissues or artificial grafts is a major challenge in tissue engineering, stimulating a continued search for alternative sources for vasculogenic cells and the development of therapeutic strategies. Human pluripotent stem cells (hPSCs), either embryonic or induced, offer a plentiful platform for the derivation of large numbers of vasculogenic cells, as required for clinical transplantations. Various protocols for generation of vasculogenic smooth muscle cells (SMCs) from hPSCs have been described with considerably different SMC derivatives. In addition, we recently identified hPSC‐derived pericytes, which are similar to their physiological counterparts, exhibiting unique features of blood vessel‐residing perivascular cells, as well as multipotent mesenchymal precursors with therapeutic angiogenic potential. In this review we refer to methodologies for the development of a variety of perivascular cells from hPSCs with respect to developmental induction, differentiation capabilities, potency and their dual function as mesenchymal precursors. The therapeutic effect of hPSC‐derived perivascular cells in experimental models of tissue engineering and regenerative medicine are described and compared to those of their native physiological counterparts. Copyright © 2013 John Wiley & Sons, Ltd.     

The ‘sweet’ spot of cellular pluripotency: protein glycosylation in human pluripotent stem cells and its applications in regenerative medicine

Introduction: Human pluripotent stem cells (hPSCs) promise for the future of regenerative medicine. The structural and biochemical diversity associated with glycans makes them a unique type of macromolecule modification that is involved in the regulation of a vast array of biochemical events and cellular activities including pluripotency in hPSCs. The primary focus of this review article is to highlight recent advances in stem cell research from a glycobiological perspective. We also discuss how our understanding of glycans and glycosylation may help overcome barriers hindering the clinical application of hPSC-derived cells.

Areas covered: A literature survey using NCBI-PubMed and Google Scholar was performed in 2014.

Expert opinion: Regenerative medicine hopes to provide novel strategies to combat human disease and tissue injury that currently lack effective therapies. Although progress in this field is accelerating, many critical issues remain to be addressed in order for cell-based therapy to become a practical and safe treatment option. Emerging evidence suggests that protein glycosylation may significantly influence the regulation of cellular pluripotency, and that the exploitation of protein glycosylation in hPSCs and their differentiated derivatives may lead to transformative and translational discoveries for regenerative medicine. In addition, hPSCs represent a novel research platform for investigating glycosylation-related disease.     

miRNA‐1 and miRNA‐133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation

miRNA‐1 (miR‐1) and miRNA‐133a (miR‐133a) are muscle‐specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR‐1 and miR‐133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR‐1 and miR‐133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)‐mediated mesodermal and cardiac differentiation by gain‐ and loss‐of‐function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR‐1 and miR‐133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR‐1 enhanced the cardiac differentiation of P19.CL6, while miR‐133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR‐1 and miR‐133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR‐1 and miR‐133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors

Induced pluripotent stem cells (iPSCs) can provide an important source of cells for the next-generation of cell therapies in regenerative medicine, in part due to their similarity to embryonic stem cells (ESCs). Patient-specific iPSCs represent an opportunity for autologous cell therapies that are not restricted by immunological, ethical and technical obstacles. One of the technical hurdles that must be overcome before iPSCs can be clinically implemented is the scalable, reproducible production of iPSCs and their differentiated progeny. All of the iPSC lines established thus far have been generated and expanded with static tissue culture protocols, which are time-consuming and suffer from batch-to-batch variability. Alternatively, stirred suspension bioreactors propose several benefits and their homogeneous culture environment facilitates the large-scale expansion required for clinical studies at less cost. We have previously developed protocols for expanding murine and human ESCs as undifferentiated aggregates in stirred suspension bioreactors. The resulting cells were karyotypically normal, expressed pluripotency markers and could be differentiated into all three germ lineages, both in vitro and in vivo. In this study, we demonstrate that stirred suspension bioreactors yield 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro differentiation into cartilage, bone and cardiomyocytes lineages, in addition to in vivo teratoma formation, further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Stirred suspension bioreactor culture represents an efficient process for the large-scale expansion and maintenance of iPSCs, which is an important first step in their clinical application.     

多能性干细胞向红细胞定向诱导分化的研究进展简

红细胞输注是临床上治疗急性失血和严重贫血的有效手段,然而目前血源紧张,因输血引起疾病传播等问题威胁着人类的健康.因此寻求安全、可靠、充足、有效的血液来源迫不及待.多能干细胞向红细胞体外分化的研究提供了一个潜在血液替代来源的选择.大量科研工作者进行了体外造血诱导的初步探索,包括定向分化体系及其效率的优化、红细胞的脱核成熟及功能鉴定、诱导型红细胞的安全性评价等.本文根据近年来这一领域的主要进展,着重对多能干细胞体外诱导分化为成熟型功能性红细胞的诱导方法以及调控机制进行综述,并对分化过程中存在的问题与挑战以及发展前景加以讨论.     

Generation of clinical‐grade functional cardiomyocytes from human embryonic stem cells in chemically defined conditions

A highly efficient cardiac differentiation from human pluripotent stem cells (hPSCs) is achievable using existing methods, especially with the standard B27 induction system. However, bovine serum albumin (BSA), one of the essential ingredients in B27, may pose significant complications for clinical studies owing to its animal origin and potential risks of virus contamination. Furthermore, the high cost of the B27 induction system also limits the applications of hPSCs‐derived cardiomyocytes. Here, a BSA‐free and chemically defined medium has been developed for differentiating hPSCs to clinical‐grade cardiomyocytes, which generated over 80% cardiac troponin T (cTNT)‐positive cardiomyocytes with high yield. When engrafting the cardiomyocytes into the hearts of myocardial infarction model rats, the rats survived with significantly improved heart functions in Δ ejection fraction and Δ fractional shortening. Importantly, the human embryonic stem cell (hESC) line (Q‐CTS‐hESC‐2) chosen for differentiation was of a clinical‐grade maintained in defined xeno‐free conditions. Compliant with the biological safety requirements, the Q‐CTS‐hESC‐2‐derived cardiomyocytes have passed the sterility and pathogen criteria tests for clinical applications. This study reports, for the first time, the generation of clinical‐grade and functional cardiomyocytes from hPSCs where BSA‐free and chemically defined conditions were maintained throughout the whole process. This provides the possibility of future therapeutic use of clinical‐grade hPSCs‐derived cardiomyocytes in treating heart diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

诱导多潜能性干细胞的研究现状与临床应用前景

胚胎干细胞可作为细胞替代治疗中很好的供体细胞来源.但由于伦理学的原因,限制了胚胎干细胞在细胞替代治疗中的应用前景,而诱导多潜能性干细胞（induced pluripotent stem cell,iPS细胞）的出现则提供了一种替代胚胎干细胞的多潜能性细胞.因为iPS细胞的建立不需要卵细胞,也不破坏发育中的胚胎,所以iP...     

Cryopreservation of Human Stem Cells for Clinical Application: A Review

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.     

诱导性多潜能干细胞的研究进展及应用前景

背景：由于诱导性多潜能干细胞可以从患者的诸多细胞进行诱导,避开了干细胞研究的免疫排斥和伦理道德问题,且具有类似于胚胎干细胞的特征。 目的：综述诱导性多潜能干细胞研究进展及应用前景,并提出目前诱导性多潜能干细胞在诱导方法及效率、安全性、应用等方面许多亟待解决的问题。 方法：以“induced pluripotent stem cel s,efficiency,disease model,clinical application”为英文检索词,应用计算机检索Kjmed数据库中2003年1月至2013年6月发表的相关文章。纳入与诱导性多潜能干细胞研究相关的文献,排除重复性研究。 结果与结论：共检索到112篇,排除无关重复的文献,保留32篇进行综述。通过导入特定的转录因子可将成体细胞重编程为诱导性多能干细胞,转染效率低是诱导性多潜能干细胞技术的一个主要障碍。诱导性多潜能干细胞在建立疾病模型、研究疾病发病机制以及新药物的筛选和新治疗方法的探索方面有着巨大的应用前景,且诱导性多潜能干细胞也有较高的临床应用价值,但是,在诱导性多潜能干细胞应用于临床这条道路的探索过程中,还有许多亟待解决和深入探究的问题。     

Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three‐dimensional multilayered patty‐like spheroids in feeder‐free culture and maintain their pluripotency

Expansion of pluripotent stem cells in defined media devoid of animal‐derived feeder cells to generate multilayered three‐dimensional (3D) bulk preparations or spheroids, rather than two‐dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease‐modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder‐free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty‐like' spheroid structures in defined xeno‐free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial‐scale expansion and intricate organ–tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. Copyright © 2014 John Wiley & Sons, Ltd.     

Advances in stem-cell–generated transplantation therapy for Parkinson's disease

Introduction: Human pluripotent stem cells have the potential to differentiate into different cell lineages of the human body, including dopaminergic (DA) neurons. Previous studies have shown that stem-cell–derived DA neurons can improve the motor deficits of Parkinson's disease (PD) animal models. That is why current research interests focus on the development of stem-cell–derived neural cells for transplantation therapies for PD patients.

Areas covered: This review article emphasizes the safety and efficacy requirements of human pluripotent stem-cell–derived neural cells and usage of reliable preclinical animal models prior to clinical trials. The current advances and hurdles related to cell production, differentiation and transplantation are also summarized.

Expert opinion: Before entering the clinic, transplantable cell populations must be differentiated and characterized according to good manufacturing practice (GMP) regulations both in vitro and in vivo. Taking into account the rapid development of the stem-cell field and technological improvements in cell preparations and GMP facilities, we think that pluripotent stem-cell–derived DA neurons will offer a relevant cell therapy option for treatment of PD in the near future.     

体细胞重编程为干细胞的方法研究与进展

背景：克隆人类胚胎会引起伦理问题,这使科学家寻找替代的方法来逆向分化细胞为多能/全能干细胞,这个过程称为重编程。重编程的新方法是研究者关注的焦点。目的：探讨重编程技术的研究现状,并对体细胞重编程为干细胞的各种方法做一综述。方法：应用计算机检索CNKI和Pubmed数据库中1983年1月至2006年12月关于重编程的文章,在标题和摘要中以＂重编程,方法,体细胞,干细胞,分化＂或＂reprogramming,method,somatic cell,stem cell,differentiation＂为检索词进行检索。选择文章内容与重编程有关者,同一领域文献则选择近期发表或发表在权威杂志文章。最终选择17篇文献进行综述。结果与结论：一个多能干细胞重编程状态是细胞结构逐步重构,染色质表观遗传改变,转录表达和转录后调控的结果。靶细胞的重塑要求重编程有一个稳定的状态,最终可以被重新定向到特定的分化程序。有充分的证据表明,细胞鉴定可以被体外操作影响,重编程细胞的命运需要进一步体内检测。     

Generation and genetic modification of induced pluripotent stem cells

Importance of the field: The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research.

Areas covered in this review: After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs.

What the reader will gain: Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases.

Take home message: Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.     

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age‐related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC‐RPEs) and iPSCs (iPSC‐RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell‐RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC‐RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin‐111 supporting the highest pigmentation frequencies. Although iPSC‐RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin‐111. iPSCs differentiated on mouse laminin‐111 produced iPSC‐RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin‐111 resulted in high yields of functional hESC‐RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno‐free, GMP‐compliant generation of RPE from human pluripotent stem cells relevant to clinical translation. Copyright © 2012 John Wiley & Sons, Ltd.     

人多能干细胞的冷冻与保存

背景：人多能干细胞的出现与发展是近年来生物医学研究领域的重大突破。但其在基础，临床研究中的广泛应用还有诸多限制，建立安全有效标准化的冷冻保存方案是人多能干细胞广泛应用面临的重大挑战。目的：回顾人多能干细胞冷冻领域的研究进展，探索造成冷冻损伤的原因和机制及改进方式，致力于促进新的更有效的冷冻方案形成。方法：以“人多能干细胞、人胚胎干细胞、人诱导多能干细胞、玻璃化、程序化冷冻、慢冻法、冷冻保存”为中文检索词，以“humanpluripotentstemcells，humanembryonicstemcell，humanintroducedpluripotentstemcell，vitrification，programmedcryopreservation，slow-freezing，cryopreservation”为英文检索词，应用计算机检索中国知网全文数据库、万方全文数据库、维普（viP）期刊全文数据库、PubMed数据库有关人多能干细胞冷冻保存技术的文献，排除与研究目的无关及重复文献，保留58篇文献进一步总结分析。结果与结论：了解人多能干细胞冷冻过程中造成冷冻损伤的原因和机制，是寻找高效的冻存方案的关键。需要更清晰的了解冷冻过程中损伤的原理，改进和创新低温生物技术来避免各种冷冻损伤的发生并致力于探讨可重复的，高效的，符合GMP要求的，能大规模冻人多能干细胞的方案。     

The long and winding road: stem cells for cystic fibrosis

Introduction: Cystic fibrosis (CF) is a genetic syndrome with a high mortality rate due to severe lung disease. Despite having several drugs targeting specific mutated CFTR proteins already in clinical trials, new therapies, based on stem cells, are also emerging to treat those patients.

Areas covered: The authors review the main sources of stem cells, including embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), gestational stem cells, and adult stem cells, such as mesenchymal stem cells (MSCs) in the context of CF. Furthermore, they describe the main animal and human models of lung physiology and pathology, involved in the optimization of these stem cell-applied therapies in CF.

Expert opinion: ESCs and iPSCs are emerging sources for disease modeling and drug discovery purposes. The allogeneic transplant of healthy MSCs, that acts independently to specific mutations, is under intense scrutiny due to their secretory, immunomodulatory, anti-inflammatory and anti-bacterial properties. The main challenge for future developments will be to get exogenous stem cells into the appropriate lung location, where they can regenerate endogenous stem cells and act as inflammatory modulators. The clinical application of stem cells for the treatment of CF certainly warrants further insight into pre-clinical models, including large animals, organoids, decellularized organs and lung bioengineering.     

Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data

Introduction: Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources.

Areas covered: In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases.

Expert opinion: Enhanced results have been found when combining bone marrow–derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue–derived stem cells and umbilical cord tissue–derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.     

Induced pluripotent stem cells: progress towards a biomedical application

Evaluation of: Burridge PW, Thompson S, Millrod MA et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6(4), e18293 (2011).

Some 5 years ago, Takahashi and Yamanaka first obtained induced pluripotent stem cells (iPSCs) by genetically ‘reprogramming’ adult somatic cells (fibroblasts). This breakthrough opened a new frontier in regenerative medicine, in which iPSCs might effectively replace embryonic stem cells (ESCs), with the additional advantage of permitting autologous transplant. Unfortunately, the risk of aberrant reprogramming and of the complications related to the use of transgenes in the process still hinders iPSC clinical application. Nevertheless, differentiation of iPSCs derived from patients may already provide a formidable platform for the in vitro analysis of human disease mechanisms and their modulation by drugs. Such an approach has already been validated by the finding that iPSCs obtained from patients with a specific genetic syndrome can be differentiated into cardiomyocytes retaining the gene abnormality and recapitulating, at the cell level, the syndrome functional phenotype. Unlike the use of iPSCs in regenerative therapy, their development as disease models is unencumbered by safety constraints. Whatever the intended use, the availability of reliable and reproducible methods for somatic cell reprogramming, iPSC expansion and differentiation is pivotal and remains a major challenge to date. The article by Burridge and coworkers describes a process encompassing all the phases in the preparation of precursor-derived cardiomyocytes, characterized by unprecedented efficiency and, most notably, applicable to different human precursors, including ESCs and iPSCs derived from multiple somatic cell types. Provided that the process will prove reproducible when applied by different laboratories, the contribution of Burridge and coworkers may represent a genuine leap in the development of precursor-derived technologies.     

Human embryonic stem cells: Possibilities for human cell transplantation

Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self‐renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell‐derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES‐derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES‐derived cells can be contemplated.