ABSTRACT
We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
Subject(s)
Batch Cell Culture Techniques , Endoderm/cytology , Pluripotent Stem Cells/cytology , Tissue Engineering/methods , Animals , Antigens, Differentiation/analysis , Cell Aggregation , Cell Cycle , Cell Differentiation , Cell Division , Cell Line/cytology , Cell Line/drug effects , Coculture Techniques , Culture Media , Fibroblasts/metabolism , Hepatocytes/cytology , Humans , Mice , Pancreas/cytology , SuspensionsABSTRACT
Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.
Subject(s)
Embryonic Stem Cells/metabolism , Gene Expression Profiling , Myocardium/metabolism , Recombination, Genetic , Transcription Factors/genetics , Animals , Cell Differentiation , Cell Line , Embryonic Stem Cells/cytology , Mice , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A growing number of studies are reporting beneficial effects of the transplantation of alleged cardiac stem cells into diseased hearts after myocardial infarction. However, the mechanisms by which transplanted cells might help to promote repair of cardiac tissue are not understood and might involve processes different from the differentiation of transplanted cells into cardiomyocytes. We have compared the effects exerted by skeletal myoblasts (which are not able to form new cardiomyocytes) and ESC-derived cardiomyocytes after implantation into infarcted mouse hearts by echocardiographic follow-up and histological analysis and related these effects to the release of cardioactive cytokines. We found that both cell types led to a long-lasting improvement of left ventricle function and to an improvement of tissue architecture. Since no relevant amounts of myoblast-derived cells were present in infarcted hearts 28 days after transplantation, we investigated the release of cytokines from implanted cells both before and after transplantation into infarcted hearts. ESC-derived cardiomyocytes and myoblasts secreted substantial amounts of interleukin (IL)-1alpha, IL-6, tumor necrosis factor-beta, and oncostatin M, which strongly supported survival and protein synthesis of cultured cardiomyocytes. We postulate that the beneficial effects of the transplantation of myoblasts and cardiomyocytes on heart function and morphology only partially (if at all) depend on the integration of transplanted cells into the myocardium but do depend on the release of a complex blend of cardioactive cytokines.
