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. 2016 Nov 10:4:e2695.
doi: 10.7717/peerj.2695. eCollection 2016.

Induced pluripotent stem cells from human hair follicle keratinocytes as a potential source for in vitro hair follicle cloning

Sheng Jye Lim  1 , Shu Cheow Ho  1 , Pooi Ling Mok  2 , Kian Lee Tan  1 , Alan H K Ong  1 , Seng Chiew Gan  1
Affiliations

Induced pluripotent stem cells from human hair follicle keratinocytes as a potential source for in vitro hair follicle cloning

Sheng Jye Lim et al. PeerJ. .

Abstract

Background: Human hair follicles are important for the renewal of new hairs and their development. The generation of induced pluripotent stem cells (iPSCs) from hair follicles is easy due to its accessibility and availability. The pluripotent cells derived from hair follicles not only have a higher tendency to re-differentiate into hair follicles, but are also more suited for growth in hair scalp tissue microenvironment.

Methods: In this study, human hair follicular keratinocytes were used to generate iPSCs, which were then further differentiated in vitro into keratinocytes. The derived iPSCs were characterised by using immunofluorescence staining, flow cytometry, and reverse-transcription PCR to check for its pluripotency markers expression.

Results: The iPSC clones expressed pluripotency markers such as TRA-1-60, TRA-1-81, SSEA4, OCT4, SOX2, NANOG, LEFTY, and GABRB. The well-formed three germ layers were observed during differentiation using iPSCs derived from hair follicles. The successful formation of keratioctyes from iPSCs was confirmed by the expression of cytokeratin 14 marker.

Discussion: Hair follicles represent a valuable keratinocytes source for in vitro hair cloning for use in treating hair balding or grafting in burn patients. Our significant findings in this report proved that hair follicles could be used to produce pluripotent stem cells and suggested that the genetic and micro-environmental elements of hair follicles might trigger higher and more efficient hair follicles re-differentiation.

Keywords: HHFK; Induced pluripotent stem cell; Keratinocytes; Re-differentiation.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. The timeline showed the HHFK reprogramming process.
The timeline includes the process from culturing of the parental cells before initiation of the reprogramming process to picking of each iPSC clones for further expansion.
Figure 2
Figure 2. Reprogramming of Human Hair Follicular Keratinocytes (HHFKs) into iPSC and characterization of iPSC.
(A) (Parental cells, HHFK) The image of parental cells, HHFKs was before infection with viruses. (Live Staining of iPSC before selection) The iPSC colonies formed showed positive live-staining (red) for SSEA4, before selection. (iPSC before selection) The image showed a single iPSC clone (in red circle) before selection for further culture. (iPSC P2 on feeder & iPSC P11 on feeder) Image of iPSC, at passage 2 and 11, cultured on feeder layer. (Feeder-free iPSC P47) Image of iPSC colony which was grew in feeder-free system at passage 47. Magnification: 100×. (B) Immunofluorescence staining of pluripotent markers for iPSCs and hESC. The pluripotency markers include OCT4, SOX2, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Magnification: 100×. (C) The expression of pluripotency markers by iPSC clones, hESC and HHFK using RT-PCR. The pluripotent genes which were being analysed include SOX2, KLF4, C-MYC, NANOG, LEFTY, and GABRB. The housekeeping gene being used was GAPDH. (D) Comparison of expression of pluripotency markers in HHFKderived iPSCs and parental HHFK.
Figure 3
Figure 3. Differentiation of iPSC into three germ layers.
Formation of embryoid bodies before the differentiation process (A). The mesoderm lineage was stained against BRACHYURY (B), ectoderm was stained against MAP2 (C), and endoderm lineage was stained against SOX17 (D). Magnification: 100×.
Figure 4
Figure 4. Characterisation of differentiated cells using IF staining and RT-PCR.
The differentiated cells were stained against cytokeratin 14 marker. The differentiated cells showed expression for CK14 marker (A). Magnification: 100×. The differentiated cells from iPSCs and hESC showed the expression of cytokeratin 14. (Lane 1) Clone 1, (Lane 2) Clone 2, (Lane 3) Clone 3, (Lane 4) hESC, (Lane 5) HHFK, and (Lane 6) Negative Control (B).
See this image and copyright information in PMC

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