Enhanced Reprogramming and Cardiac Differentiation of Human Keratinocytes Derived from Plucked Hair Follicles, Using a Single Excisable Lentivirus

Abstract

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.

Rat Embryonic Fibroblasts Improve Reprogramming of Human Keratinocytes into Induced Pluripotent Stem Cells

“…This technique needed first to be reproduced and optimized especially since there are very few further reports applying this promising technique (Fig. 1C) in a routine manner [28]. Human head hair was plucked from healthy individuals and cultivated to obtain keratinocyte outgrowth (Fig.…”

Recapitulating Long-QT Syndrome Using Induced Pluripotent Stem Cell Technology

“…Human skin fibroblasts suitable for reprogramming can be harvested by performing a skin biopsy, which is a small and relatively safe procedure that can be performed with local anesthesia. More recently, even less-invasive methods for harvesting somatic cells for reprogramming have become available due to advanced reprogramming techniques, e.g., the reprogramming of keratinocytes from plucked hair follicles [26] or the reprogramming of T lymphocytes isolated from blood [5].…”

Generation of Patient Specific Stem Cells: A Human Model System

“…Therefore, reprogramming experiments have also been performed using plucked human hair follicle keratinocytes. These iPSCs were also able to differentiate into cells from all three germ layers including cardiomyocytes (Novak et al, 2010 Table 2. Different somatic cell types that human iPSCs have been generated from…”