Physical stimulation by REAC and BMP4/WNT-1 inhibitor synergistically enhance cardiogenic commitment in iPSCs.

It is currently known that pluripotent stem cells can be committed in vitro to the cardiac lineage by the modulation of specific signaling pathways, but it is also well known that, despite the significant increase in cardiomyocyte yield provided by the currently available conditioned media, the resulting cardiogenic commitment remains a highly variable process. Previous studies provided evidence that radio electric fields asymmetrically conveyed through the Radio Electric Asymmetric Conveyer (REAC) technology are able to commit R1 embryonic stem cells and human adipose derived stem cells toward a cardiac phenotype. The present study aimed at investigating whether the effect of physical stimulation by REAC in combination with specific chemical inductors enhance the cardiogenic potential in human induced pluripotent stem cells (iPSCs). The appearance of a cardiac-like phenotype in iPSCs cultured in the presence of a cardiogenic medium, based upon BMP4 and a WNT-inhibitor, was consistently increased by REAC treatment used only during the early fate differentiation for the first 72 hours. REAC-exposed iPSCs exhibited an upregulation in the expression of specific cardiogenic transcripts and morphologically in the number of beating clusters, as compared to cells cultured in the cardiogenic medium alone. Our results indicate that physical modulation of cellular dynamics provided by the REAC offers an affordable strategy to mimic iPSC cardiac-like fates in the presence of a cardiogenic milieu.

Generation of CD34 Fluorescent Reporter Human Induced Pluripotent Stem Cells for Monitoring Hematopoietic Differentiation.

Hematopoietic stem and progenitor cells (HSPCs) derived from human induced pluripotent stem cells (hiPSCs) hold great promise for disease modeling, drug screens, and eventually cell therapy approaches. During in vitro differentiation of hiPSCs into hematoendothelial progenitors, the emergence of CD34-positive cells indicates a critical step of lineage specification. To facilitate the monitoring of hematopoietic differentiation of hiPSCs, we established fluorescent reporter cells for the stem and progenitor cell marker CD34. An IRES-GFP (internal ribosome entry site green fluorescent protein) construct was introduced by CRISPR/Cas9 into the 3' untranslated region of one endogenous CD34 allele. Single-cell clones were generated after excision of the floxed puromycin resistance cassette by Cre recombination and correct insertion was confirmed by genotyping polymerase chain reaction and Southern blot. To validate their functionality, the reporter hiPSCs were in vitro differentiated toward CD34+ cells using the STEMdiff Hematopoietic Kit combined with short-term inhibition of GSK3 (glycogen synthase kinase 3). All cells expressing nuclear GFP were positive for cell surface CD34, thus allowing the direct monitoring of the differentiation of hiPSCs into CD34+ cells either by flow cytometry or confocal microscopy. After fluorescence-activated cell sorting, cells displaying high GFP expression exhibited increased colony-forming potential in the MethoCult colony-forming unit assays as compared with CD34+ cells obtained by magnetic-activated cell sorting. In summary, we have generated functional CD34 GFP reporter hiPSCs, which not only permit label-free separation of HSPCs, but also tracing of the emergence and fate of CD34+ progenitors at the single-cell level.

Establishment of keratinocyte cell lines from human hair follicles.

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.