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Abstract
Induced pluripotent stem cells (iPSCs) indeed hold great potential as a basis for cell-based therapies for multiple degenerative diseases. It involves the administration of transcription factors in order to transduce multiple terminally differentiated cell types into pluripotent embryonic-like stem cells. These cells can overcome the issues of immune rejection, as the cells are autologous, and also eliminate any ethical issues regarding the use of human embryos. Objective: Here we present a onetime transfection repro-gramming protocol for peripheral blood mononucleocytes (PBMNCs) Methods: PBMNCs were cultured in conditions favoring hematopoietic progenitor population. Three plasmids encoding five reprogramming factors (Oct3/4, Sox2, Klf4, L-Myc and Lin28) in addition to EBNA1, an enhancer of transfection efficiency and episomal plasmids expression were applied to reprogram the isolated cells. Results: A single transfection of this plasmid combination was sufficient to generate pluripotent markers positive iPSC colonies. Colonies resembling human ES-like cells were observed as early as 20 days after episomal plasmid neucleofection. These colonies stained positively for alkaline phosphatase. Moreover, the generated iPSCs expressed stem cell markers TRA-1-60, SSEA4, NANOG and OCT3/4 as determined by immunocytochemistry. These results demonstrated that iPSC could be effectively generated from PBMNCs using episomal plasmid vectors encoding OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC and LIN28. Conclusion: Finally, it could be concluded that the method described in this research provides a promising approach for generating clinical grade induced pluripotent stem cells from highly accessible source of adult somatic cells.
Keywords: Induced pluripotent stem cells, peripheral blood mononucleocytes, reprogramming, neucleofection
1.Introduction
Since its discovery in 2007, induced pluripotent stem cells (iPSCs) research has evolved exponentially offering promising insights to the future of regenerative medicine. Fibroblast derived from skin biopsy, was the first source of somatic cells described for generating iPSCs via reprograming [1].Human iPSCs have since been reported to be generated from a broad array of cell types, including blood cells [1-5]. Peripheral blood mononucleocytes (PMNCs) offer a more convenient and abundant cell source than many other somatic sources. PBMNCs are generally comprised of a mixture of cells that include; T cells, B cells and myeloid cells in addition to a fraction of hematopoietic progenitor cells. However, blood mononucleocytes, are considered closer to embryonic stem cells (ESCs) than fibroblasts especially when cultured in conditions favoring the...