Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: Improving the safety of iPS cell transplantation to myocardium

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P > 0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.     

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.     

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。     

Evaluation of antiangiogenic activity of azumamides by the in vitro vascular organization model using mouse induced pluripotent stem (iPS) cells

Evaluation of antiangiogenic activity of marine sponge derived azumamides by the in vitro vascular organization model using mouse induced pluripotent stem (iPS) cells was carried out. Azumamide E (5) strongly inhibited in vitro angiogenesis from iPS cells at 1.9 μM while azumamide A (1) showed only weak inhibition at 19 μM. These results were well correlated with HDAC inhibitory activity of these compounds, revealing the prospect of azumamides as the probe molecules useful for stem cell chemical biology.     

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。     

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.     

The therapeutic potential of stem cells

In recent years, there has been an explosion of interest in stem cells, not just within the scientific and medical communities but also among politicians, religious groups and ethicists. Here, we summarize the different types of stem cells that have been described: their origins in embryonic and adult tissues and their differentiation potential in vivo and in culture. We review some current clinical applications of stem cells, highlighting the problems encountered when going from proof-of-principle in the laboratory to widespread clinical practice. While some of the key genetic and epigenetic factors that determine stem cell properties have been identified, there is still much to be learned about how these factors interact. There is a growing realization of the importance of environmental factors in regulating stem cell behaviour and this is being explored by imaging stem cells in vivo and recreating artificial niches in vitro. New therapies, based on stem cell transplantation or endogenous stem cells, are emerging areas, as is drug discovery based on patient-specific pluripotent cells and cancer stem cells. What makes stem cell research so exciting is its tremendous potential to benefit human health and the opportunities for interdisciplinary research that it presents.     

Comparative study of myocytes from normal and mdx mice iPS cells

Recently, induced pluripotent stem cells (iPS cells) have been derived from various techniques and show great potential for therapy of human diseases. Furthermore, the iPS technique can be used to provide cell models to explore pathological mechanisms of many human diseases in vitro, such as Duchenne muscular dystrophy (DMD), which is a severe recessive X-linked form of muscular dystrophy without effective treatment. In this study, we try to determine whether there are different characteristics of myocytes from mdx iPS cells and C57BL/10 iPS cells. Our results showed that both of mdx and C57BL/10 cells could be induced into iPS cells in vitro, whereas colony-forming ability of mdx iPS cells was much weaker than that of C57BL/10 iPS cells. Meanwhile, mdx iPS cells could be induced to differentiate into myocytes, whereas their differentiation efficiency was much lower than that of C57BL/10 iPS cells. And, the number of apoptotic cells in differentiated myocytes from mdx iPS cells was significantly higher than that from C57BL/10 iPS cells. More importantly, treatment of a pan-caspase inhibitor (Z-VAD) produced a significant decrease in apoptotic cells. This study might add some insight to the biology study of dystrophin gene.     

慢病毒直接诱导形成猪iPS细胞无需限定因子

为了证实慢病毒对细胞具有遗传修饰和重编程作用,在本实验中使用慢病毒感染猪胎儿成纤维细胞.结果显示:慢病毒介导的EGFP在猪胎儿成纤维细胞中稳定和高效表达,使用添加LIF和bFGF的细胞培养液,部分猪的胎儿成纤维细胞逐渐改变原有的纤维状形态,形成圆形的细胞,细胞逐步增殖形成细胞集落,细胞集落边界清晰,在饲养层上细胞集落生长迅速,具有稳定的生长性能和正常核型,细胞碱性磷酸酶染色为阳性,表达干细胞特有的标记Oct4、Nanog和SSEA1,在体外能够形成拟胚体,在体内分化形成包含三个生殖层的畸胎瘤.作为核移植的供体细胞,克隆胚的卵裂率为53.33%、桑椹胚率为9.03%、囊胚率为2.07%、孵化囊胚的总细胞数为26.5,在桑椹胚率和囊胚率方面显著低于猪普通胎儿成纤维细胞核移植克隆胚的发育能力(P<0.05).结果证实慢病毒能够直接使猪的胎儿成纤维细胞转变成iPS细胞,因此慢病毒将成为一种理想的材料和工具用于细胞的遗传修饰和细胞重构等方面的研究.     

Human ES and iPS cells as cell sources for the treatment of Parkinson's disease: Current state and problems

Cell therapy using human embryonic stem cells (hESCs) is a promising therapeutic option for Parkinson's disease (PD), an incurable neurodegenerative disease. A prerequisite for clinical application of hESCs for PD is an efficient and strict differentiation of hESCs into midbrain dopamine (mDA) neuron‐like cells, which would be directly translated into high effectiveness of the therapy with minimum risk of undesirable side effects. Due to fruitful efforts from many laboratories, a variety of strategies for improving efficiency of dopaminergic differentiation from hESCs have been developed, mostly by optimizing culture conditions, genetic modification, and modulating intracellular signaling pathways. The rapid advances in the fields of dopaminergic differentiation of hESCs, prevention of tumor formation, and establishment of safe human induced pluripotent stem cells (hiPSCs) would open the door to highly effective, tumor‐free, and immune rejection‐free cell therapy for PD in the near future. J. Cell. Biochem. 109: 292–301, 2010. © 2009 Wiley‐Liss, Inc.     

Neural crest stem cells: discovery, properties and potential for therapy

Neural crest (NC) cells are a migratory cell population synonymous with vertebrate evolution. They generate a wide variety of cell and tissue types during embryonic and adult development including cartilage and bone, connective tissue, pigment and endocrine cells as well as neurons and glia amongst many others. Such incredible lineage potential combined with a limited capacity for self-renewal, which persists even into adult life, demonstrates that NC cells bear the key hallmarks of stem and progenitor cells. In this review, we describe the identification, characterization and isolation of NC stem and progenitor cells from different tissues in both embryo and adult organisms. We discuss their specific properties and their potential application in cell-based tissue and disease-specific repair.     

Hepatocyte differentiation of human induced pluripotent stem cells is modulated by stearoyl‐CoA desaturase 1 activity

Stearoyl‐CoA desaturase 1 (SCD1) plays important roles in organ development, glucose tolerance, insulin sensitivity, and cancer. Here, we examined the role of SCD1 for the differentiation of human induced pluripotent stem (hiPS) cells to liver cells by using drug inhibition and biochemical experiments. hiPS cells cultured in a pro‐hepatic medium were exposed to an SCD1 inhibitor at various stages throughout differentiation. Liver‐specific markers, specifically α‐fetoprotein, albumin and urea in conditioned medium, and hepatocyte nuclear factor 4α (HNF4α) and cytochrome P450 7A1 (CYP7A1) gene expressions and triglyceride in cellular extracts were analyzed at various development stages. Measures of hepatocyte‐specific function and triglyceride accumulation in later stages were strongly inhibited a minimum of −29% (P < 0.05) by SCD1 inhibitor in the early stage of hepatic differentiation and effectively reversed (>30%, P < 0.01) by the addition of oleate. The results were also reproducible with human primary mononuclear cells (hPMN). SCD1 inhibitor had no significant effect on liver‐specific markers when it was added in the hepatic maturation stage. However, it strikingly led to higher albumin (1.6‐fold, P = 0.03) and urea (1.9‐fold, P = 0.02) production, and HNF4α (1.9‐fold, P = 0.02) and CYP7A1 (1.3‐fold, P = 0.03) expression upon incubation during the lineage‐commitment stage. Hepatic differentiation from cultured hiPS cells is sensitive to SCD1 inhibition and this sensitivity is affected by the stage of cellular differentiation. Notably, findings also indicate that this notion can be extended to hPMN. The requirement for SCD1 activity in functional differentiation of hepatocytes may have relevance for human liver disease and metabolic dysregulation.     

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.