Morphometric prediction of mesenchymal stromal cell-like immunosuppressive capacity of human hair follicle dermal sheath cup cells: an implication for regenerative medicine in hair loss diseases

Abstract

Background Human hair follicle dermal sheath cup cells (DSCCs) hold promise as a cell source of regenerative medicine treatment for hair loss owing to their ability to secrete growth factors and/or signal pathway activators. The therapeutic effect of autologous DSCCs transplantation for male/female pattern hair loss (PHL) was demonstrated in a phase III equivalent clinical study. Intralesional inflammation has been implicated in the pathophysiology of various hair loss diseases, including PHL. As DSCCs possess mesenchymal stem/stromal cell (MSC)-like properties and MSCs are immunosuppressive, we investigated whether they exhibit immunoregulatory capabilities comparable to MSCs and developed an in vitro morphometric assay to predict this capability. Methods DSCCs were isolated via microdissection and propagated in vitro. Their conformity to MSC criteria was assessed based on cell surface antigen expression and differentiation potential. Furthermore, immunoregulatory capabilities were assessed by co-culturing DSCCs with anti-CD3/28 antibody-stimulated peripheral blood mononuclear cells (PBMCs) and examining the suppression mechanisms through pharmacological intervention. Multiple lots of DSCCs derived from various donors and manufacturing conditions were cultured and analyzed by phase-contrast microscopy to obtain their morphometric profiles. Parameters correlating with the expression levels of immunomodulatory factors were used to create a predictive model. Additional DSCC lots were manufactured to validate the predictive model. Results Similar to MSCs, cell differentiation assays revealed that DSCCs exhibited multipotency, and they did not express co-stimulatory molecules in response to immunogenic stimuli, suggesting low immunogenicity. Moreover, co-culture experiments with allogeneic PBMCs revealed that DSCCs reduced T cell proliferation (from 78 to 5%) and

1. Introduction

Development and regeneration of hair follicles (HFs) depend on the well-orchestrated interactions between hair-inductive mesenchymal and receptive epithelial components [1]. The dermal sheath (DS) is a connective tissue structure that envelops the main body of the hair follicle. It consists of multiple layers of cylindrical epithelial structures, and studies have suggested that it contains cell populations with hair-inducing activity [2]. The cells located at the proximal cup-shaped end of the HF are known as DS cup (DSC) cells (DSCCs), which can be obtained by microdissection and subsequent in vitro propagation [3]. DSCCs have garnered attention for their potential contribution to HF regeneration. When transplanted into mouse ears, murine DSCCs were incorporated into the dermal papilla (DP), a specialized mesenchymal component that regulates hair follicle development and regeneration, and promoted hair growth [4]. Implanted allogeneic DSC aggregates yielded new HFs containing DSC-derived cells in humans [5]. Moreover, injected human DSCCs were shown to migrate into human HF structures reconstituted in immunodeficient mice [6]. In recent Phase IIb and III equivalent studies, autologous DSCC injection improved hair density and cumulative hair diameter in male/female pattern hair loss (PHL) [3, 7]. These results indicate the HF regenerative potential of this cell population.

Mesenchymal stromal/stem cells (MSCs) have attracted interest in the field of regenerative medicine because of their low immunogenicity and intrinsic immunosuppressive capacity in allogeneic transplantation [8, 9]. MSCs have been used for the treatment of graft-versus-host disease and intractable fistulas associated with Crohn's disease [10, 11], and for the treatment of alopecia areata, an autoimmune hair loss disease [12]. A major limitation to developing effective MSC-based immunosuppressive therapies is that they exhibit notably different immunosuppressive capacities depending on the donors and culture conditions. Several approaches have been useful for the assessment of immunosuppressive capacity of MSCs in vitro [13, 14]; however, their reliability and versatility remain elusive.