Autologous, Non-Invasively Available Mesenchymal Stem Cells from the Outer Root Sheath of Hair Follicle Are Obtainable by Migration from Plucked Hair Follicles and Expandable in Scalable Amounts

Abstract

Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.

The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers

“…Next to the neural and neuroectodermal/skin stem cell lineages, the mesenchymal stem cell (MSC) lineage has also been characterized and cultured from the ORS-harvested material [ 12 , 13 ]. Recently, we reported a reproducible method for culturing MSCs from plucked hair follicles and upscaling them to therapy-relevant amounts [ 14 ].…”

“…To meet the evolving requirements of personalized medicine in terms of a low-to-non-invasive approach, fewer invasive hair follicle isolation methods have also been devised and subsequently optimized. To this day, the least invasive method for obtaining cell cultures from the hair follicle has been based on explant procedure in an air-liquid interface set-up and it yielded multiple cell types [ 14 ]. The procedure has hereby bridged the gap between non-invasive ex vivo sampling and a putative in vitro culture of targeted cell types.…”

“…Furthermore, the proximal part of the plucked follicle is a major source of dermal carry-over and poses a risk of contaminating and overpopulating the entire stem cell culture with dermal fibroblasts. Therefore, the proximal part of the follicle is routinely truncated as a part of current explant procedures [ 14 ]. In the worst case of explant procedure, the entire distal part is lost, whilst the proximal part is cut off and the mid-portion is oftenall that is left to utilize.…”

“…In the worst case of explant procedure, the entire distal part is lost, whilst the proximal part is cut off and the mid-portion is oftenall that is left to utilize. The recently reported explant procedures for culturing keratinocytes, melanocytes, and mesenchymal stem cells resulted in successfully obtaining pure monocultures from 30–50 plucked follicles [ 14 , 16 , 17 ]. Nevertheless, those procedures utilized all the available ORS material of the plucked follicle, not exclusively an isolated central part, which is the only one that is reproducibly available.…”

“…Isolating the follicle mid-part for purposes of cell culture involves losing the bulge in the course of plucking and intentionally withdrawing the proximal follicle part by microdissection [ 14 ], based on observable morphological features. Further, the bulge is more precisely defined by the expression of specific topological markers rather than upon recognizing morphologically distinct areas.…”