Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient‐specific embryonic‐like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding factors that control differentiation of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their differentiation toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into embryonic‐like state by transduction with defined factors (Oct3/4, Sox2, c‐Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC‐like state. Treatment of iPSCs‐derived embryoid bodies (EBs) with transforming growth factor beta 1 (TGF‐β1) in the presence of retinoic acid enhanced generation of MSC‐like cells. The MSCs‐like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs‐like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF‐β1 deposited more calcium in vitro than cells derived without TGF‐β1 treatment. Taken together, the data demonstrate that iPSC give rise to MSCs‐like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC‐derived EBs with TGF‐β1 may be an approach for directing iPSC toward MSC‐like state. J. Cell. Biochem. 109: 643–652, 2010. © 2009 Wiley‐Liss, Inc.     

iPS细胞的遗传安全性

自2006年Takahashi和Yamanaka首次成功地从小鼠成纤维细胞诱导得到诱导多能性干细胞(Induced pluripotent stem cells,iPS细胞)以来,iPS细胞由于其潜在的广阔应用前景而迅速成为干细胞研究领域的新热点;与此同时,iPS细胞的遗传安全性也越来越多地受到人们的关注。文章将对iPS细胞遗传安全性的研究进展进行综述,分析造成iPS细胞遗传不稳定的可能原因,希望可以促进对iPS细胞诱导条件的优化,获得遗传上较为安全的iPS细胞。     

iPS细胞的遗传安全性

自2006年Takahashi和Yamanaka首次成功地从小鼠成纤维细胞诱导得到诱导多能性干细胞(Induced pluripotent stem cells, iPS细胞)以来, iPS细胞由于其潜在的广阔应用前景而迅速成为干细胞研究领域的新热点; 与此同时, iPS细胞的遗传安全性也越来越多地受到人们的关注。文章将对iPS细胞遗传安全性的研究进展进行综述, 分析造成iPS细胞遗传不稳定的可能原因, 希望可以促进对iPS细胞诱导条件的优化, 获得遗传上较为安全的iPS细胞。     

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.)     

The applications of induced pluripotent stem (iPS) cells in drug development

The introduction of induced pluripotent stem (iPS) cells has been a milestone in the field of regenerative medicine and drug discovery. iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy. Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease, Alzheimer’s disease, Huntington disease, ischemic heart disease, diabetes and so on. Moreover, iPS cells derived from patients suffering from unique incurable diseases can be developed into patient- and disease-specific cell lines. These cells can be used as an effective approach to study the mechanisms of diseases, providing useful tools for drug discovery, development and evaluation. The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening. Furthermore, iPS cells can be applied in the field of toxicological and pharmacokinetics tests. This review focuses on the applications of iPS cells in the field of pharmaceutical industry.     

Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was, for the first time, to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined, and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC, Osx, and Runx2 genes, both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group, histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering, by promoting the formation of new cementum, alveolar bone, and normal periodontal ligament. J. Cell. Physiol. 226: 150–157, 2010. © 2010 Wiley‐Liss, Inc.     

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs)

Introduction

(1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory.

Sources of data

We reviewed the literature related to reprogramming, pluripotency and fetal stem cells.

Areas of agreement

(1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both.

Areas of controversy

(1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The “level” of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined.

Growing points

Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.     

Potentialities of induced pluripotent stem (iPS) cells for treatment of diseases

Induced pluripotent stem (iPS) cell research has been growing a new height throughout the world due to its potentialities in medical applications. We can explore several therapeutic applications through the iPS cell research. In this review, we have first discussed the development of iPS cells, reprogramming factors, and effectiveness of iPS cells. Then we have emphasized the potential applications of iPS cells in pharmaceutical and medical sectors, such as, study of cellular mechanisms for spectrum of disease entities, disease-specific iPS cell lines for drugs discovery and development, toxicological studies of drugs development, personalized medicine, and regenerative medicine.     

Genomic analysis of induced pluripotent stem (iPS) cells: routes to reprogramming

The phenomenal proliferation of scientific studies into the nature of induced pluripotent stem (iPS) cells following publication of the findings of Takahashi and Yamanaka little more than 2 years ago, have significantly expanded our understanding of cellular mechanisms relating to cell lineage, differentiation, and proliferation. While the full potential of iPS cell lineages for both scientific tool and therapeutic applications is as yet unclear, findings from several lines of investigation suggests that multipotential and terminally differentiated cells from an array of cell types are competent to undergo epigenetic reprogramming to a pluripotential state. The nature of this pluripotential state appears to be similar to, but not identical with that previously described for embryonic stem (ES) cells. Understanding the nature of this induced reprogrammed state will be critical to determining the full potential of iPS cells. Recently, this issue has been examined through an integrated analysis of the genome in fully and partially reprogrammed iPS cell lineages. These results provide a window onto the temporal components of reprogramming and suggest mechanisms by which the efficacy of reprogramming can be enhanced.     

诱导多能干细胞(iPS)的诱导培养与鉴定

通过外源转录调控因子的诱导,使成体细胞重编程为胚胎干细胞(ES细胞)样的多能细胞,这种细胞称为诱导多能干细胞(iPS细胞),这一方法被称为iPS技术。目前,iPS技术已先后在小鼠、人、猕猴、大鼠和猪中成功应用,建立了相应的iPS细胞系,并获得了iPS细胞嵌合小鼠和四倍体克隆小鼠。尽管iPS与ES细胞在形态和生长特性上有许多相同之处,但iPS细胞的建立需要较独特的诱导培养体系和鉴定方法。以下结合近年来iPS技术的发展和本实验室的相关研究,对iPS细胞的建立和培养体系的优化进行了深入探讨。     

Current concepts in reprogramming somatic cells to pluripotent state

Recently considerable interests have been roused in nuclear reprogramming by somatic cell nuclear transfer using an egg cytoplasm and/or by other means, such as fusion, cell extracts treatment and genes transfections. However, the very mechanism of reprogramming still remains elusive. Epigenetic modifications, which play a significant role in normal mammalian development in vivo is also involved in the process of reprogramming in vitro. The latter shares some of the other features observed in nuclear reprogramming in vivo. In this review, we discuss the main epigenetic changes involved in nuclear reprogramming and currently available approaches to achieve nuclear reprogramming in vitro and its future prospects.     

Reprogramming the pluripotent cell cycle: restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed to possess stem cell like properties. However, the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. hES cells are characterized by an abbreviated G1 phase (～ 2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis [5-bromo-2'-deoxy-uridine (BrdU) incorporation], cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (～ 2.5 h) and an abbreviated cell cycle (16-18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 1.5-2 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency.     

FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development

The infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. Here we describe that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. We show that collar cells are composed of Fgf3(+) SOX3(+) proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10(+) anterior-dorsal infundibular cells. Together, our study shows that a subset of anterior floor plate-like cells gives rise to Fgf3(+) SOX3(+) progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth.     

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.