An Experimental Approach to the Generation of Human Embryonic Stem Cells Equivalents

Recently, particular attention has been paid to the human embryonic stem cells (hESC) in the context of their potential application in regenerative medicine; however, ethical concerns prevent their clinical application. Induction of pluripotency in somatic cells seems to be a good alternative for hESC recruitment regarding its potential use in tissue regeneration, disease modeling, and drug screening. Since Yamanaka’s team in 2006 restored pluripotent state of somatic cells for the first time, a significant progress has been made in the area of induced pluripotent stem cells (iPSC) generation. Here, we review the current state of knowledge in the issue of techniques applied to establish iPSC. Somatic cell nuclear transfer, cell fusion, cell extracts reprogramming, and techniques of direct reprogramming are described. Retroviral and lentiviral transduction are depicted as ways of cell reprogramming with the use of integrating vectors. Contrary to them, adenoviruses, plasmids, single multiprotein expression vectors, and PiggyBac transposition systems are examples of non-integrative vectors used in iPSC generation protocols. Furthermore, reprogramming with the delivery of specific proteins, miRNA or small chemical compounds are presented. Finally, the changes occurring during the reprogramming process are described. It is concluded that subject to some limitations iPSC could become equivalents for hESC in regenerative medicine.     

Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

Anatomical templates for tissue (re)generation and beyond

Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4. Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as “cellular agriculture.” iPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely “cultured meat,” a product that improves animal welfare and encourages the consumption of healthier meat along with environmental preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species.     

Application of biomaterials to advance induced pluripotent stem cell research and therapy

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.     

综述: 诱导重编程过程中的表观遗传重塑

多能干细胞(pluripotent stem cell,PSC)是一类具有自我更新能力和多向分化潜能的细胞,具有广泛的临床应用前景．诱导性多功能干细胞(induced pluripotent stem cell,iPS cell)的获得,解决了传统方式中的细胞来源和伦理学等问题,从理论研究和应用上实现了体细胞重编程的重大突破,也为疾病发生机制研究、药物筛选、个性化药物选择、细胞治疗和再生医学等研究创造了难得的机会,从而开启了多能干细胞应用的新纪元．iPS过程中有很多问题尚未得到解决,尤其是诱导重编程的分子机制方面,这也是近年来干细胞领域研究的热点．其中如何实现表观遗传的重编程被认为是亟待解决的核心问题之一．本文结合我们的研究,主要介绍诱导重编程领域表观遗传修饰重塑机制的研究进展,并展望未来研究中大规模信息整合分析的重要性．     

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.     

Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.     

Generating pluripotent stem cells: Differential epigenetic changes during cellular reprogramming

Pluripotent stem cells hold enomous potential for therapuetic applications in tissue replacement therapy. Reprogramming somatic cells from a patient donor to generate pluripotent stem cells involves both ethical concerns inherent in the use of embryonic and oocyte-derived stem cells, as well as issues of histocompatibility. Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)-derived by ectopic expression of four reprogramming factors in donor somatic cells-are superior in terms of ethical use, histocompatibility, and derivation method. However, iPSC also show genetic and epigenetic differences that limit their differentiation potential, functionality, safety, and potential clinical utility. Here, we discuss the unique characteristics of iPSC and approaches that are being taken to overcome these limitations.     

诱导性多能干细胞向神经细胞分化的研究进展

诱导性多能干细胞(induced pluripotent stem cell,iPS cell)是通过转染外源特定的基因组合来诱导成体细胞重编程为类似于胚胎干细胞的一种多潜能干细胞,iPS细胞与胚胎干细胞不仅在形态上相似,而且在功能方面几乎相同.另外,iPS细胞的诞生克服了胚胎干细胞在临床应用时涉及的移植免疫排斥与伦理道德问题,因此具有重要的临床应用价值.目前iPS在治疗中枢神经系统性疾病方面的研究已取得很大进展,包括iPS细胞向神经细胞诱导分化方法的改进、分化机理的探索以及iPS细胞分化来源神经细胞在神经系统疾病模型中治疗作用的研究等.从iPS细胞的创建及特点、iPS细胞向神经细胞分化的诱导方法及研究新进展方面予以综述.     

Derivation of iPSCs after Culture of Human Dental Pulp Cells under Defined Conditions

Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.     

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.     

皮毛之道：以表皮器官为研究模型探讨再生生物学与再生医学的基础概念

再生医学是一个具有巨大潜力的新兴医学领域。该文以此为方向讨论了再生医学研究中的三个关键问题,并以非神经外胚层器官的干细胞行为为例做进一步的探讨。第一,如何获取干细胞,介绍了包括胚胎干细胞、组织干细胞和诱导性多能干细胞的获得途径,以及若干组织细胞重编程的成功范例;第二,如何将干细胞转化为组织和器官,这需要了解干细胞分化以及形态发生的机制,并以羽毛的形态发生为模型,引入了千细胞拓扑生物学的概念以及干细胞微环境调控塑造器官形态的机制;第三,如何将干细胞及其转化产物置于患者体内,并以鼠毛生长周期波为例,阐明了宏观环境因素如何调控干细胞的活性：最后,还分析了在器官发生中干细胞的自组织对于新生毛发组织工程的重要意义。该文的许多原则不仅限于皮肤,同时也适用于其它体内器官。通过对生物再生的过程的基础研究,我们可以受到生物再生之道的启发,逐渐理解组织修复及再生的机制,并提高分子和细胞水平上的干细胞操作技术,希望在不久的将来将干细胞研究成果应用于临床医学。     

Identification of spectral modifications occurring during reprogramming of somatic cells

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.     

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.     

Reprogramming somatic cells to pluripotency: A fresh look at Yamanaka’s model

In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (C_d, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field.     

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.