An introduction to induced pluripotent stem cells

Recent landmark studies show that it is now possible to convert somatic cells, such as skin fibroblasts and B lymphocytes, into pluripotent stem cells that closely resemble embryonic stem cells. These induced pluripotent stem (iPS) cells can be generated without using human embryos or oocytes, thus bypassing some of the ethical issues that have limited the use of human embryonic stems (hES) cells. Additionally, they can be derived from the patient to be treated, thereby overcoming problems of immunological rejection associated with the use of allogeneic hES cell derived progenitors. Whilst these patient‐specific iPS cells have great clinical potential, their immediate utility is likely to be in drug screening and for understanding the disease process. This review discusses the promise of iPS cells as well as the challenges to their use in the clinic.     

Calcium signaling in pluripotent stem cells

Pluripotent stem cells represent a new source of biological material allowing the exploration of signaling phenomena during normal cell development and differentiation. Still, the calcium signaling pathways and intracellular calcium responses to various ligands or stress conditions have not been sufficiently explored as yet in embryonic or induced pluripotent stem cells and in their differentiated offspring. This is partly due to the special culturing conditions of these cell types, the rapid morphological and functional changes in heterogeneous cell populations during early differentiation, and methodological problems in cellular calcium measurements. In this paper, we review the currently available data in the literature on calcium signaling in pluripotent stem cells and discuss the potential shortcomings of these studies. Various assay methods are surveyed for obtaining reliable data both in undifferentiated embryonic stem cells and in specific, stem cell-derived human tissues. In this paper, we present the modulation of calcium signaling in human embryonic stem cells (hESC) and in their derivates; mesenchymal stem cell like (MSCl) cells and cardiac tissues using the fluorescent calcium indicator Fluo-4 and confocal microscopy. LPA, trypsin and angiotensin II were effective in inducing calcium signals both in HUES9 and MSCl cells. Histamine and thrombin induced calcium signal exclusively in the MSCl cells, while ATP was effective only in HUES9 cells. There was no calcium signal evoked by GABA, even at relatively high concentrations. In stem cell-derived cardiomyocytes a rapid increase in the beating rate and an increase of the calcium signal peaks could be observed after the addition of adrenaline, while verapamil led to a strong decrease in cellular calcium and stopped spontaneous contractions in a relaxed state.     

Strategies to produce hepatocytes and hepatocyte-like cells from pluripotent stem cells

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a potent source for unlimited production of hepatocytes and hepatocyte-like cells that may replace primary human hepatocytes in a variety of fields including liver cell therapy, liver tissue engineering, manufacturing bioartificial liver, modeling inherited and chronic liver diseases, drug screening and toxicity testing. Human ESCs are able to spontaneously form embryoid bodies, which then spontaneously differentiate to various tissue-specific cell lineages containing a total of 10-30% albumin-producing hepatocytes and hepatocyte-like cells. Enrichment of embryoid bodies with the definitive endoderm, from which hepatocytes arise, yields increasing the final ratio of hepatocyte population up by 50-65%. Current strategies of the directed differentiation of human ESCs (and iPSCs) to hepatocytes that reproduce liver embryogenesis by sequential stimulation of culturing ESCs with tissue-specific growth factors result in achieving the differentiation rate up to 60-80%. In the future, directed differentiation of human ESCs and iPSCs to hepatocytes should be further optimized towards generating homogeneous cultures of hepatocytes in order to avoid expensive procedures of separation and isolation of hepatocytes and hepatocyte-like cells.     

Differentiation of mesodermal cells from pluripotent stem cells

The pluripotency of embryonic stem cells has been well demonstrated by a vast variety of studies showing the induction of differentiation into desired cell types that have the potential to be used not only in basic studies but also in medical applications. The induction of mesodermal cells, especially blood cells, from embryonic stem cells is notable from the point of view of transplantation, and the methods for this induction have improved over the last few years, with more defined culture conditions in place. Concurrently, the generation of induced pluripotent stem cells from somatic cells opens the possibility of autologous transplantation. In fact, there are a growing number of reports demonstrating that several mesodermal cells can be differentiated from induced pluripotent stem cells using the same methods used for embryonic stem cells. This review summarizes recent advances in the differentiation of mesodermal cells from embryonic stem cells and induced pluripotent stem cells.     

Application of induced pluripotent stem cells to primary immunodeficiency diseases

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Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.     

Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview

Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.     

基于iPSC诱导分化具有电生理活动心肌细胞方法的研究

目的:通过诱导多潜能干细胞i PSC体外定向分化为心肌细胞的方法,已经成为干细胞和心血管领域研究的有效工具,也为临床治疗带来新希望。本文依据胚胎发育的分子调控机制,探索建立体外定向分化心肌的方法。方法:使用干细胞培养基培养i PSCs生长至80%,换成含6μmol/L Chir99021和25μg/L Activin A的基础培养基培养48h,基础培养基为含有2%的B27、64μg/L l-ascorbic acid2-phosphate的RPMI 1640培养基,换为含2μM Wnt-C59的WNT信号抑制剂培养基培养48h,最后换为维持培养基继续培养,每两天换液,约第8天可见心肌开始搏动。结果:i PSC在向成熟心肌分化时,显微镜下可观察到明显的肌丝肌节结构,具有心肌特异性标志物TNNT 2,MLC 2a的强阳性表达,并表现出自发跳动的电生理活动。结论:该方法可以成功诱导i PSC高效分化为具有电生理功能的心肌细胞。     

大鼠诱导多能干细胞向肝系细胞的诱导分化

目的本研究探讨利用诱导因子将大鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)快速高效诱导分化为肝细胞样细胞(hepatocyte-like cells,HLCs)。方法利用诱导因子将iPSCs向肝系细胞方向分化,采用免疫细胞化学染色和RT-PCR方法检测培养20 d时细胞各种肝系细胞标志表达。结果培养分化20 d的iPSCs形成肝细胞样集落,表达肝系细胞特征性标志,并且具有尿素合成和糖原贮备能力。结论利用诱导因子可有效地将大鼠iPSCs诱导分化为HLCs,有望为细胞移植治疗各种肝脏疾病提供种子细胞。     

Differentiation of pluripotent embryonic stem cells into cardiomyocytes

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies, ES cells can differentiate into derivatives of all 3 primary germ layers, including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells, have been established. During differentiation, cardiac-specific genes as well as proteins, receptors, and ion channels are expressed in a developmental continuum, which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro "gain-of-function" or "loss-of-function" genetic studies. Recently, human ES cell lines have been established that can be used to investigate cardiac development and the function of human heart cells and to determine the basic strategies of regenerative cell therapy. This review summarizes the current state of ES cell-derived cardiogenesis and provides an overview of how genomic strategies coupled with this in vitro differentiation system can be applied to cardiac research.