Abstract
The dermal papilla (DP) is a specialised mesenchymal component of the hair follicle (HF) that plays key roles in HF morphogenesis and regeneration. Current technical difficulties in preparing trichogenic human DP cells could be overcome by the use of highly proliferative and plastic human induced pluripotent stem cells (hiPSCs). In this study, hiPSCs were differentiated into induced mesenchymal cells (iMCs) with a bone marrow stromal cell phenotype. A highly proliferative and plastic LNGFR(+)THY-1(+) subset of iMCs was subsequently programmed using retinoic acid and DP cell activating culture medium to acquire DP properties. The resultant cells (induced DP-substituting cells [iDPSCs]) exhibited up-regulated DP markers, interacted with human keratinocytes to up-regulate HF related genes, and when co-grafted with human keratinocytes in vivo gave rise to fibre structures with a hair cuticle-like coat resembling the hair shaft, as confirmed by scanning electron microscope analysis. Furthermore, iDPSCs responded to the clinically used hair growth reagent, minoxidil sulfate, to up-regulate DP genes, further supporting that they were capable of, at least in part, reproducing DP properties. Thus, LNGFR(+)THY-1(+) iMCs may provide material for HF bioengineering and drug screening for hair diseases.
Highlights
Efficient isolation and in vitro expansion capable of maintaining their intrinsic properties has not yet been fully established[7,16]
We established a novel protocol involving embryoid body (EB) formation, two days of floating culture, subsequent seeding onto a humanised substrate, and culture in mesenchymal stem cell (MSC) serum-free medium (MSC-SFM) containing PDGF, TGF-β, and FGF, reported to promote MSC proliferation and maintenance[28]
Resultant Human induced pluripotent stem cells (hiPSCs)-derived cells could be maintained by passage (>passage 4) onto humanised substrate in MSC-SFM or onto plastic culture vessels in human MSC medium (Fig. 1a,b and Supplementary Materials and Methods)
Summary
Efficient isolation and in vitro expansion capable of maintaining their intrinsic properties has not yet been fully established[7,16]. A subset of human bone marrow-derived cells marked by high levels of LNGFR (CD271), THY-1 (CD90) and VCAM-1 (CD106) expression was found to exhibit properties of multipotent bone marrow stromal cells[18,19] including rapid colony expansion, robust multilineage differentiation and self-renewal potency[19]. These cells show minimal expression of P16INK4a in vitro, indicating genetic stability and resistance to cellular senescence, clearly demonstrating the advantage of using this subset for the generation of specific dermal cell subpopulations, including DP cells. This study supports the potential use of hiPSCs for the generation of tissue-inductive mesenchyme with the capacity for crosstalk with cells of epithelial lineage
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