News induced pluripotent stem cells (iPSCs)

February 2011
The selection of fully reprogrammed induced pluripotent stem cells (iPSCs) is one of the technical issues that remains to be resolved before these cells can be used for clinical applications. Problems to be discussed are the limited understanding of the reprogramming mechanism and the criterion that investigators use to select the fully reprogrammed iPSCs.
In this article, Chen, HF. and colleagues reported that two surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin), can be used for identification and isolation of mouse iPSCs. Furthermore, they observed that the conventional mouse embryonic stem cell (mESC) markers (AP and SSEA1)  are present throughout the entire process of reprogramming, while the EpCAM and E-cadherin only in the final stages (activation of Nanog and endogenous Oct4).  ˃ Read More  

In this article, the authors showed that microRNAs (miRNAs) are the important elements of cell control system implicated in iPSCs generation. Firstly, they proved the miRNAs function directly in iPSCs induction process. For this, they used lentiviral shRNA vectors to stably knock-down Ago2, Dicer and Drosha mouse factors, that are involved in miRNA biogenesis. Secondly, they also identified three clusters of miRNAs, miR-17~92, miR-106b~25 and miR-106a~363, which are highly induced in early stage of reprogramming. Finally, their analysis showed that Tgfbr2 and p21 are directly targeted by these miRNA and these interactions influence iPSCs induction process. ˃ Read More      

January 2011

This article shows a facile method of generating integration-free human induced pluripotent stem cells (iPSCs) from blood mononuclear cells (MNCs), that will accelerate their use in research and clinical application. For expressing the 5 mouse reprogramming factors (Oct4, Sox2, Klf4, c-Myc and Lin28) an improved EBNA1/OriP plasmid was used. The plasmid consisted of EBNA1/OriP-based pCEP episomal vector (Invitrogen), cDNAs for 5 mouse reprogramming factors, the synthetic CAG promoter, cDNA for the SV40 large T antigen and the expression cassette p53-shRNA. Cord blood mononuclear cells (CB MNCs) and peripheral blood mononuclear cells (PB MNCs) were used for transfection. Efficiency - up to 1000 iPSCs colonies per 2 million transfected (CB MNCs) and the duration of obtaining iPSCs - 14 days. ˃ Read More   

For distinguish successfully reprogrammed iPSCs from partially reprogrammed colony, the researchers used different tests to identify the reactivation of endogenous pluripotency-associated genes (Fbxo15, Nanog, Oct4), the cell markers (TRA-1-81, TRA-1-60, AP, SSEA1) and in situ live cell imaging. Furthermore, for clinical applications of human pluripotent stem cells (hPSCs) the genomic stability is an important requirement.
The authors reported a higher frequency of numerical chromosomal modifications (aneuploidies) and subchromosomal copy number variations (CNV) in hPSCs compared to nonpluripotent samples performing high-resolution SNP (single nucleotide polymorphism) analysis. They observed: large regions of CNV in hESCs and hiPSCs (trysomies, deletions, duplications), smaller regions of CNV in  hESCs and hiPSCs (duplications and deletions on chromosomes 12, 20), duplications of pseudogenes of pluripotency-associated genes (NANOG, Oct4, NANOGP1), dynamic changes in genomic structure in hPSCs (eg trysomy 15 between passage 33 and passage 88) and genomic aberration during reprogramming, passage and differentiation of hPSCs. Results obtained show the dynamic nature of genomic abnormalities in hPSCs transformations and hence the need for genomic monitoring of these cells simultaneously with the identification of their differentiations.   ˃ Read More