Human iPS Cell-Derived Cell Aggregates Exhibited Dermal Papilla Cell Properties in in vitro Three-Dimensional Assemblage Mimicking Hair Follicle Structures.

Masahiro Fukuyama,Aki Tsukashima,Momoko Kimishima,Manabu Ohyama,Yoshimi Yamazaki,Hideyuki Okano

doi:10.3389/fcell.2021.590333

Masahiro Fukuyama, Aki Tsukashima + Show 4 more

Open Access

https://doi.org/10.3389/fcell.2021.590333

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Abstract

Current approaches for human hair follicle (HF) regeneration mostly adopt cell-autonomous tissue reassembly in a permissive murine intracorporeal environment. This, together with the limitation in human-derived trichogenic starting materials, potentially hinders the bioengineering of human HF structures, especially for the drug discovery and treatment of hair loss disorders. In this study, we attempted to reproduce the anatomical relationship between an epithelial main body and the dermal papilla (DP) within HF in vitro by three-dimensionally assembling columnarly molded human keratinocytes (KCs) and the aggregates of DP cells and evaluated how HF characteristics were reproduced in the constructs. The replaceability of human-induced pluripotent stem cell (hiPSC)-derived DP substitutes was assessed using the aforementioned reconstruction assay. Human DP cell aggregates were embedded into Matrigel as a cluster. Subsequently, highly condensed human KCs were cylindrically injected onto DP spheroids. After 2-week culture, the structures visually mimicking HFs were obtained. KC-DP constructs partially reproduced HF microanatomy and demonstrated differential keratin (KRT) expression pattern in HFs: KRT14 in the outermost part and KRT13, KRT17, and KRT40, respectively, in the inner portion of the main body. KC-DP constructs tended to upregulate HF-related genes, KRT25, KRT33A, KRT82, WNT5A, and LEF1. Next, DP substitutes were prepared by exposing hiPSC-derived mesenchymal cells to retinoic acid and subsequently to WNT, BMP, and FGF signal activators, followed by cell aggregation. The resultant hiPSC-derived DP substitutes (iDPs) were combined with KCs in the invented assay. KC-iDP constructs morphologically resemble KC-DP constructs and analogously mimicked KRT expression pattern in HF. iDP in the constructs expressed DP-related markers, such as vimentin and versican. Intriguingly, KC-iDP constructs more intensely expressed KRT33A, KRT82, and LEF1, which were stepwisely upregulated by the addition of WNT ligand and the mixture of WNT, SHH, and EDA signaling activators, supporting the idea that iDP exhibited biological properties analogous to DP cell aggregates in the constructs in vitro. These preliminary findings suggested the possibility of regenerating DP equivalents with in vitro hair-inductive capacity using hiPSC-derived cell composites, which potentially reduce the necessity of human tissue-derived trichogenic cell subset and eventually allow xeno-free bioengineering of human HFs.

Highlights

The hair follicle (HF) is a multifunctional mammalian skin appendage, providing a physical barrier against external insults, facilitating thermoregulation, and transmitting tactile sense (Stenn and Paus, 2001; Schneider et al, 2009; Nagao et al, 2012; Zimmerman et al, 2014)
The HF is a cylindric structure composed of the epithelial main body consisting of keratinocytes (KCs) including stem cells and the mesenchymal structures; the dermal papilla (DP), a specialized hair-inductive dermal cell aggregate located at the bottom of the HF, and the dermal sheath (DS) surrounding the main body (Yang and Cotsarelis, 2010; Ohyama, 2019)
After DP aggregates and condensed KCs were placed in Matrigel to reproduce their anatomical locations in HFs, individual cell components were in close contact with each other but clearly distinguishable (Figure 1A)

Summary

Introduction

The hair follicle (HF) is a multifunctional mammalian skin appendage, providing a physical barrier against external insults, facilitating thermoregulation, and transmitting tactile sense (Stenn and Paus, 2001; Schneider et al, 2009; Nagao et al, 2012; Zimmerman et al, 2014). The HF continuously self-renews throughout life via the hair cycle (Stenn and Paus, 2001). Both HF morphogenesis and regeneration are enabled by well-orchestrated epithelial– mesenchymal interactions (EMIs) via biological signaling pathways, such as WNT, BMP, Sonic hedgehog (SHH), and Ectodysplasin A (EDA) pathways (St-Jacques et al, 1998; Millar, 2002; Zhang et al, 2009; Mikkola, 2011; Sennett and Rendl, 2012; Ohyama, 2019). To achieve successful HF regeneration, sufficient folliculogenic EMIs need to be elicited and maintained between receptive KCs and inductive DP (Millar, 2002; Sennett and Rendl, 2012; Ohyama, 2019)

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