Dataset Information

Nanoscale microenvironment engineering for expanding human hair follicle stem cell and revealing their plasticity.

ABSTRACT:

Background

Periodically regenerated hair follicles provide an excellent research model for studying tissue regeneration and stem cell homeostasis. Periodic activation and differentiation of hair follicle stem cells (HFSCs) fuel cyclical bouts of hair regeneration. HFSCs represent an excellent paradigm for studying tissue regeneration and somatic stem cell homeostasis. However, these crucial studies are hampered by the lack of a culture system able to stably expand human HFSCs and regulate their fate.

Results

Here, we use layer-by-layer (LbL) self-assembly with gelatin/alginate to construct a nanoscale biomimetic extracellular matrix (ECM) for an HFSC population. The LbL coating provides ECM and mechanical support for individual cells, which helps to maintain the CD200⁺α6⁺ HFSC population to a certain extent. Addition of key signal molecules (FGF-7 and VEGF-A) simulates the minimum essential components of the stem cell microenvironment, thereby effectively and stably expanding HFSCs and maintaining the CD200⁺α6⁺ HFSC population. Subsequently, BMP2 loaded to the nanocoated layer, as a slow-release signal molecule, activates BMP signaling to regulate HFSCs' fate in order to obtain a purified CD200⁺α6⁺ HFSC population.

Conclusion

This system can minimize the microenvironment of HFSCs; thus, stably amplifying HFSCs and revealing their plasticity. Our study thus provides a new tool for studies of hair follicle reconstruction and stem cell homeostasis.

SUBMITTER: Chen P

PROVIDER: S-EPMC8010974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hair regenerative medicine, a promising strategy for the treatment of hair loss, will likely involve the transplantation of autologous hair follicular stem cells (HFSCs) and dermal papilla cells (DPCs) into regions of hair loss. Cyclic hair regeneration results from the periodic partial activation of HFSCs. However, previous studies have not successfully achieved large-scale HFSC expansion in vitro without the use of feeder cells, with a lack of research focused on regulating HFSC fate for hair follicular (HF) regeneration. Hence, an emerging focus in regenerative medicine is the reconstruction of natural extracellular matrix (ECM) regulatory characteristics using biomaterials to generate cellular microenvironments for expanding stem cells and directing their fate for tissue regeneration. Methods: HFSCs were coated with gelatin and alginate using layer-by-layer (LbL) self-assembly technology to construct biomimetic ECM for HFSCs; after which transforming growth factor (TGF)-β2 was loaded into the coating layer, which served as a sustained-release signal molecule to regulate the fate of HFSCs both in vitro and in vivo. In vitro experiments (cell culture and siRNA) were employed to investigate the molecular mechanisms involved and in vivo implantation was carried out to evaluate hair induction efficiency. Results: Nanoscale biomimetic ECM was constructed for individual HFSCs, which allowed for the stable amplification of HFSCs and maintenance of their stem cell properties. TGF-β2 loading into the coating layer induced transformation of CD34+ stem cells into highly proliferating Lgr5+ stem cells, similar to the partial activation of HFSCs in HF regeneration. Thus, LbL coating and TGF-β2 loading partially reconstructed the quiescent and activated states, respectively, of stem cells during HF regeneration, thereby mimicking the microenvironment that regulates stem cell fate for tissue regeneration during HF cycling. Improved HF regeneration was achieved when the two HFSC states were co-transplanted with neonatal mouse dermal cells into nude mice. Conclusion: This study provides novel methods for the construction of stem cell microenvironments and experimental models of HF regeneration for the treatment of hair loss.

Project description:When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and β1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200- keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation-the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.