Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition

Abstract

Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7-10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications.

Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site

“…Until now, minicircles have been employed for transgene expression in MSCs in two reported studies [18,19]. Daneshvar et al transfected human umbilical cord (hUC)-MSCs with minicircles, which generated 18% GFP þ MSCs [19]. It is speculated that the difference of transfection efficiency originates from a different MSC source (hUC vs hAT and hBM); in our experiments, the hAT-and hBM-MSCs also showed different transfection efficiencies.…”

“…In the case of minicircle, the cationic liposome method also seemed to be also inefficient although it showed higher transfection efficiency compared with only plasmid. Until now, minicircles have been employed for transgene expression in MSCs in two reported studies [18,19]. Daneshvar et al transfected human umbilical cord (hUC)-MSCs with minicircles, which generated 18% GFP þ MSCs [19].…”

“…Especially, genetically engineered Escherichia coli capable of inducible in vivo recombination and following the enzymatic degradation of bacterial backbone made a great improvement in minicircle production by reducing time and labor to similar levels as those of routine plasmid DNA preparation [16]. Minicircles have been employed for various purposes including transgene expression, gene therapy, DNA vaccine, and the generation of induced pluripotent stem cells [11,12,17]; however, there have been only a few studies that used minicircles for transfection of MSCs [18,19]. In these studies, minicircles were delivered to MSCs by the conventional cationic liposome method or nucleofection technology, but the resulting transfection efficiencies were low (less than 35%).…”

Advances in Non-Viral DNA Vectors for Gene Therapy

“…More recently, Daneshvar et al created iPSCs from umbilical cord mesenchymal stem cells using minicircles with the same four reprogramming factors, but without the need for a layer of feeder cells supplying additional nutrients and reprogramming factors [ 147 ]. Fernandes and Chari engineered neural stem cells to secrete brain-derived neurotrophic factor (BDNF) using magnetic nanoparticles in conjunction with minicircle or plasmid DNA [ 148 ].…”

Dedifferentiation of human uterine polyp stem cells into embryo-like cells during inducing pluripotency

“…Reprogramming of fibroblasts and mesenchymal stem cells (MSCs) is a promising strategy for stem cell biology and regenerative medicine. It can be induced by nuclear transfer, cell fusion, or forced transcription factors expression 1 , 2 . Reprogramming factors (protein, RNAs and small molecules) enables somatic cells to reprogram 3 , 4 .…”