Initial Colony Morphology-Based Selection for iPS Cells Derived from Adult Fibroblasts Is Substantially Improved by Temporary UTF1-Based Selection

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells. Recently, selection of fully reprogrammed cells was achieved based on colony morphology reminiscent of embryonic stem (ES) cells. The maintenance of pluripotency was analysed.Clonal murine iPS cell line TiB7-4, which was derived from adult fibroblasts, was analysed for maintenance of pluripotency. Colony morphology, expression of pluripotency factors and stage specific embryonic antigen 1 (SSEA1) were analysed by real time PCR and flow cytometry. We found the iPS cell line TiB7-4 and its subclones to be rather diverse and exhibiting a tendency towards spontaneous differentiation and loss of pluripotency independent of their initial colony morphology. In contrast an undifferentiated transcription factor 1 (UTF1) promoter-driven G418 (Neo) resistance significantly improved the quality of these iPS cells. After selection with UTF-Neo for two weeks iPS subclones could be stably maintained for at least 40 passages in culture and differentiate into all three germ layers. As control, a construct expressing G418 resistance under the control of the ubiquitously active SV40 early promoter formed subclones with different colony morphology. Some of these subclones could be cultured for at least 12 passages without loosing their pluripotency, but loss of pluripotency eventually occured in an unpredictable manner and was independent of the subclones' initial morphology and SSEA1 expression. A UTF-Neo-based selection of a whole population of TiB7-4 without further subcloning resulted in the generation of cultures with up to 99% SSEA1 positive cells under stringent selection conditions.Our data indicate that temporary selection using a genetic UTF1-based system can generate homogenous pluripotent iPS cells that can be maintained without permanent selection pressure

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

<p>Abstract</p> <p>Background</p> <p>Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.</p> <p>Results</p> <p>Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.</p> <p>Conclusions</p> <p>These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.</p

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

Microarray analysis reveals that the specific pattern of gene expression in cardiomyocytes derived from embryonic stem cells reflects the biological, physiological and functional processes occurring in mature cardiomyocytes

Effects of physicochemical properties of polyacrylamide (PAA) and (polydimethylsiloxane) PDMS on cardiac cell behavior

In vitro cell culture is commonly applied in laboratories around the world. Cultured cells are either of primary origin or established cell lines. Such transformed cell lines are increasingly replaced by pluripotent stem cell derived organotypic cells with more physiological properties. The quality of the culture conditions and matrix environment is of considerable importance in this regard. In fact, mechanical cues of the extracellular matrix have substantial effects on the cellular physiology. This is especially true if contractile cells such as cardiomyocytes are cultured. Therefore, elastic biomaterials have been introduced as scaffolds in 2D and 3D culture models for different cell types, cardiac cells among them. In this review, key aspects of cell-matrix interaction are highlighted with focus on cardiomyocytes and chemical properties as well as strengths and potential pitfalls in using two commonly applied polymers for soft matrix engineering, polyacrylamide (PAA) and polydimethylsiloxane (PDMS) are discussed

Functional Expression and Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels (HCN) in Mouse iPS Cell-derived Cardiomyocytes after UTF1-Neo Selection

Background/Aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and I-f currents as well as functional hormonal regulation and sensitivity to If channel blockers. Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes. Copyright (C) 2014 S. Karger AG, Base