Abstract
The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.
Highlights
Adult stem cells have an edge over somatic cells in terms of being an efficient starting cell source for reprogramming[1]
Comparative gene expression analysis between human embryonic stem cells and natal dental pulp stem cells (nDPSC) revealed that while the expression pattern of 17 genes associated with pluripotency were similar (See Fig. 1c), the core pluripotency factors such as POU5F1, SOX2 and NANOG were not highly upregulated in nDPSC (See Fig. 1c)
IL6ST, KLF4, COMMD3, NR5A2, RUNX2 and TBX3 genes were highly expressed in nDPSC as compared to other genes. 2−∆∆CT formula was used to calculate the fold change and human embryonic stem cells (hESC) was used as calibrator
Summary
Adult stem cells have an edge over somatic cells in terms of being an efficient starting cell source for reprogramming[1] This is because they have relatively fast cell cycle kinetics, plasticity and endogenously express a subset of pluripotency factors, albeit at lower levels compared to embryonic stem cells. The recently described Sendai virus platform for human induced pluripotent stem cell (hiPSC) generation is an ideal vector system for delivering reprogramming factors because of the following reasons: (i) the virus replicates in the form of negative-sense single-stranded RNA in the cytoplasm of infected cells and it does not integrate into the host genome[21]; (ii) it efficiently delivers exogenous genes in a wide spectrum of host cell species and tissues[22]; (iii) it shows low cytotoxicity upon infection[23,24]; and (iv) allows high-level expression of the delivered exogenous genes[25]
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