From Hair to iPSCs—A Guide on How to Reprogram Keratinocytes and Why

Abstract

Keratinocytes, as a primary somatic cell source, offer exceptional advantages compared to fibroblasts, which are commonly used for reprogramming. Keratinocytes can beat fibroblasts in reprogramming efficiency and reprogramming time and, in addition, can be easily and non-invasively harvested from human hair roots. However, there is still much to know about acquiring keratinocytes and maintaining them in cell culture. In this article, we want to offer readers the profound knowledge that we have gained since our initial use of keratinocytes for reprogramming more than 10 years ago. Here, all hints and tricks, from plucking the hair roots to growing and maintaining keratinocytes, are described in detail. Additionally, an overview of the currently used reprogramming methods, viral and non-viral, is included, with a special focus on their applicability to keratinocytes. This overview is intended to provide a brief but comprehensive insight into the field of keratinocytes and their use for reprogramming into induced pluripotent stem cells (iPSCs).

Generating iPSCs with a High-Efficient, Non-Invasive Method—An Improved Way to Cultivate Keratinocytes from Plucked Hair for Reprogramming

“…Lentiviral reprogramming is a well-established technique to deliver the reprogramming factors to the host cell [ 22 ]. It has been shown that the lentiviral transfection of keratinocytes shows a very high reprogramming efficiency [ 13 ]. KGM2 is used for reprogramming since its media composition is comparable with the EpiLife medium.…”

“…This method has been previously described in detail [ 13 ]. In short: HFs were obtained from seven different, healthy donors ( Table S1 in Supplementary Materials ).…”

“…Deviations from the standard protocol are explicitly mentioned in the results section. The standard protocol has been previously published [ 13 ]. A summary of the described methods: after the outgrowth of the first keratinocytes, the medium was changed from MEFCM to the EpiLife medium + HKGS (ThermoFisher, Waltham, MA, USA) supplement according to the manufacturer’s protocol.…”

“…Cells were also directly used, termed “fresh cells”, for further reprogramming using two different approaches: the incubation of keratinocytes with viral particles for 48 h with or without an additional centrifugation step. The lentiviral transduction has been previously described in detail [ 13 ]. In short: lentiviral particles were obtained from transfected Lenti-X 293T cells for 4 h in a serum-free medium using 8 µg lentiviral pRRL.PPT.SF.hOKSMco.idTom.pre FRT plasmid [ 14 ] together with 5.5 µg psPAX2 and 2 µg pDM2.6 plasmid using polyethylenimine (Sigma Aldrich, St. Louis, MO, USA).…”

Advancing Stem Cell Technologies and Applications: A Special Collection from the PluriCore Network in the German Stem Cell Network (GSCN)

“…Two articles provide novel information on technologies to reprogram cells to induced pluripotent stem cells (iPSC). The starting material in one of these articles, by Klingenstein and colleagues (Klingenstein, Klingenstein, Kleger, & Liebau, 2020), are keratinocytes from hair samples. These cells have the advantage of being easily accessible, and can even be acquired from different areas in the world without special transport requirements.…”