Hair transplantation is used routinely for the management of androgenetic alopecia, the most common cause
of hair loss. However, patients must have enough donor hair follicles (HFs) for transplant surgery. Alternative
technologies that could potentially generate HFs in vitro would be beneficial. Efforts to recapitulate HF
morphogenesis, using mouse embryonic cells or primary human adult epidermal or outer root sheath (ORS)
keratinocytes, in combination with human primary dermal papilla fibroblasts (DPs), have been largely
unsuccessful. We aimed to assess the inductive and morphogenic potential of DPs in combination with primary
human adult hair matrix cells (HMx), i.e. the cells interacting with DPs in vivo. A mixture of labelled HMx and
DP cells was injected in human scalp skin and cultured ex vivo. The injected cells proliferated for at least 3
days and were detectable in the dermis for 10 days. At day 6, HMx no longer proliferated, but did not undergo
apoptosis, presumably reflecting differentiation. We also generated in vitro self-aggregated DP spheroids that,
when co-cultured with human adult HMx, resulted in the formation of HF organoids. Labelled DP-HMx
organoids placed in human skin (in a 1 mm wound) could be tracked for 10 days in ex vivo skin organ culture.
Within the organoids, HMx cells underwent proliferation and differentiation. HMx stained positively not only for
AE13 and K85 (pre-cortical HMx keratins), but surprisingly also for K6 (ORS specific keratin, up-regulated
during wounding) in vitro and ex vivo. Inner root sheath specific keratin K71 staining was negative and hair
shaft production was absent. Longer culture period may be needed for generating fully formed HFs from HMx-
DP organoids. Although further evidence is required, our preliminary data potentially open up a new application
for regenerative medicine technology in hair loss disorders and wound healing.

Assessment of the induction and morphogenic potential of human hair matrix cells and dermal papilla fibroblasts ex vivo