Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells
TL;DR: The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line, and found that iPS cells differentiate into functional cardiomyocytes.
Abstract: Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription p...
Citations
More filters
TL;DR: It is concluded that human iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.
Abstract: Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines due to residual transgene expression. Proliferation of iPS and ES cell derived-cardiomyocytes based on BrdU labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to β-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.
1,327 citations
TL;DR: It is found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts.
1,114 citations
Cites background from "Generation of Functional Murine Car..."
...Several recent studies have demonstrated that miPSC and hiPSCs can give rise to cardiomyocytes in culture (Kuzmenkin et al., 2009; Mauritz et al., 2008; Narazaki et al., 2008; Pfannkuche et al., 2009; Zhang et al., 2009)....
[...]
TL;DR: In this article, isolated embryonic cardiomyocytes cultured on a series of flexible substrates were found to be optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating.
Abstract: Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A `cysteine shotgun9 method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.
833 citations
Cites background from "Generation of Functional Murine Car..."
...The results may be relevant to differences in the beating of embryoid bodies generated by embryonic stem cells (ESC) versus induced pluripotent stem (iPS) cells, with the latter appearing to beat less (Mauritz et al., 2008)....
[...]
TL;DR: The generation of human iPSCs from cord blood (CB) as a juvenescent cell source is reported, showing characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes.
489 citations
Cites methods from "Generation of Functional Murine Car..."
...PCR and qPCR were performed as previously described (Mauritz et al., 2008)....
[...]
TL;DR: Results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells.
Abstract: Human induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells, and can proliferate intensively and differentiate into a variety of cell types However, the hepatic differentiation of human iPS cells has not yet been reported In this report, human iPS cells were induced to differentiate into hepatic cells by a stepwise protocol The expression of liver cell markers and liver-related functions of the human iPS cell-derived cells were monitored and compared with that of differentiated human ES cells and primary human hepatocytes Approximately 60% of the differentiated human iPS cells at day 7 expressed hepatic markers alpha fetoprotein and Alb The differentiated cells at day 21 exhibited liver cell functions including albumin Asecretion, glycogen synthesis, urea production and inducible cytochrome P450 activity The expression of hepatic markers and liver-related functions of the iPS cell-derived hepatic cells were comparable to that of the human ES cell-derived hepatic cells These results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells
481 citations
References
More filters
TL;DR: Induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions is demonstrated and iPS cells, designated iPS, exhibit the morphology and growth properties of ES cells and express ES cell marker genes.
23,959 citations
TL;DR: It is demonstrated that iPS cells can be generated from adult human fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc.
18,175 citations
TL;DR: This article showed that OCT4, SOX2, NANOG, and LIN28 factors are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells.
Abstract: Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.
9,836 citations
TL;DR: It is indicated that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.
Abstract: Myocardial infarction leads to loss of tissue and impairment of cardiac performance The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time1 Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation2,3,4,5; these events promote structural and functional repair6,7,8 This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein9 by fluorescence-activated cell sorting on the basis of c-kit expression10 Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells The developing tissue comprised proliferating myocytes and vascular structures Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease
5,331 citations
TL;DR: iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.
Abstract: We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application
4,371 citations