Physoxia reduces the negative impact of in vitro culture conditions on DP cells
DP cell cultures are typically established under normoxia, rapidly losing their native phenotype and intrinsic properties [28,29], including their key self-aggregation capacity [30,31]. Moreover, they also have a short lifespan [32] in culture, which is accompanied by morphological changes such as shifting from a small polygonal morphology to a spindle-like shape [33,34], before acquiring an enlarged morphology [18]. Therefore, we investigated if those changes also occurred under physoxia to understand how the O2 level impacts DP cells phenotype in culture. DP cells under physoxia depicted a polygonal and less spindle-like shape and higher nuclei-to-cytoplasm ratio, as demonstrated by the significant decrease in the cells’ area, perimeter and major axis length in comparison to normoxia (Fig. 1a-d). Physoxia also significantly decreased the percentage of senescent DP cells in culture (Fig. 1e) and improved their aggregative capacity (Fig. 1f). Moreover, it enhanced cell proliferation, albeit the DNA amount at day 3 was similar in normoxia and physoxia (Fig. 1g). Interestingly, an opposite effect was observed regarding collagenous (COL, Fig. 1h) and non-collagenous (NCOL, Fig. 1i) proteins secretion under physoxia, beneficial only after 3 days in culture. Altogether, these results suggest that physoxia promotes a healthier state in cultured DP cells, which featured characteristics typically associated with low passage cells.
Physoxia enhances hMel migration, tyrosinase activity and proliferation within short culture times
Although hMel are normally cultured under normoxia, there are indications that their proliferation and tyrosinase activity are favoured under lower oxygen tensions [23]. We found that physoxia significantly increased both hMel migration (Fig. 2a) and tyrosinase activity (Fig. 2b), although this last effect was not sustained along with the culture. Similarly, significantly higher DNA levels were observed for hMel cultured under physoxia at day 3 of culture (Fig. 2c), suggesting an improved proliferative capacity. This effect was lost with the culture time, despite the high number of Ki67-positive cells (Fig. 2d). Physoxia did not seem to affect hMel morphology (Fig. 2e). Collectively, these results indicate that physoxia supports hMel functional features better than normoxia but only for short culture periods.
DP cell and hMel response to physoxia depends on their type of interaction
Although residing in close vicinity in the hair bulb and having their functions coupled to anagen [35,36], little is known about how human DP cells and hMel interact and potentially affect each other’s functionality. Knowing that physoxia individually improved hMel and DP functional features after 3 days in culture, we then explored its effect when these cells were indirectly co-cultured (Fig. 3a). The co-culture with DP cells did not add to the increased hMel proliferation induced by physoxia (Fig. 3b), in opposition to normoxia. Like for proliferation, co-culture with DP cells under physoxia did not affect hMel tyrosinase activity, contrarily to normoxia, which promoted a recovery from the negative effect of the co-culture medium (Fig. 3c).
Regarding DP cells, they proliferated significantly more under physoxia than in normoxia but only when cultured in their conventional medium. Thus, the higher DNA amount detected in co-culture might be due to the medium used. This is also sustained by the results similar to the control condition, both under physoxia and normoxia (Fig. 3d). The amount of active alkaline phosphatase (ALP) cells in the co-culture was not affected by physoxia but, as for proliferation, the medium used led to a significant increase of this inductive marker. In the presence of hMel, a significant decrease of DP cells active ALP was observed under physoxia, but not in normoxia (Fig. 3e).
In the HF, hMel and DP cells are separated only by a thin and permeable basal lamina [2]. Therefore, we sought to investigate if physoxia effects were different than those observed in the indirect co-cultures, assuming a direct interaction between hMel and DP cells. When hMel were cultured with DP spheroids, they organized themselves around the spheroid in a biomimetic fashion, displaying a polarized positioning over one-half of the DP spheroid, independently of the oxygen level (Fig .3f). Highly-stable aggregates with similar DNA content were obtained (Fig. 3g). Interestingly, both cell types phenotype was improved under physoxia, as demonstrated by a significant increase on hMel tyrosinase activity (Fig. 3h) and by the higher amount of active ALP in DP cells (Fig. 3i), respectively their main functional markers. Moreover, COL and NCOL proteins production by aggregates cultured in physoxia was significantly higher than in normoxia (Fig. 3j). Physoxia benefits hMel and DP cells functionality when both cells types are directly contacting. Interestingly, hMel response to physoxia does not seem to be indirectly affected by DP cells, while hMel signalling appears to have an impact on DP cells ALP activity.
ROS generation due to hMel and DP cells interaction does not directly correlate with DP cells functionality
During hair growth and pigmentation the bulb is a ROS-enriched environment [26], therefore in addition to the functionality of hMel and DP cells, we addressed the involvement of ROS in their response. The production of ROS by hMel was significantly lower under physoxia, although this effect was significant only for the co-cultures (Fig. 4a). Moreover, hMel in co-culture produced significantly more ROS than in the control conditions, regardless of the oxygen level. Physoxia also led to a reduction of the ROS levels in DP cells in comparison to normoxia, independently of the culture conditions (Fig. 4b). The indirect co-culture with hMel under physoxia also resulted in significantly higher amounts of ROS than in control conditions (Fig. 4b). Surprisingly, when cells were directly cultured, ROS production in physoxia was significantly higher than in normoxia (Fig. 4c), the opposite of what was observed in indirect co-cultures.
Considering that in indirect (Fig. 3e, 4b) or direct (Fig. 3i, 4c) co-cultures the effect of physoxia over the amount of active ALP and ROS followed a common trend, we then investigated if there was a correlation between these responses. For that, DP cells were treated with hydrogen peroxide (H2O2) to increased oxidative stress, with the ROS inhibitor N-acetyl cysteine (NAC) or both. Treatment with the exogenous ROS (H2O2) led to a significant decrease in the amount of DNA (Fig. 4d) but surprisingly, it did not affect DP cells ROS intracellular levels (Fig. 4e) while a significant decrease of active ALP was observed (Fig. 4f). Moreover, NAC pre-treatment before H2O2 addition further enhanced ROS production in comparison to H2O2 alone, but it also reduced the H2O2 effect on the amount of active ALP. Although it is not clear the mechanism by which H2O2 decreases ALP activity in DP cells, these results suggest that it is not directly correlated with an increase in ROS levels.
Physoxia and 3D co-culture supports DP cells and hMel native phenotype
To further explore both physoxia and hMel influence on DP cells hair regenerative potential, we looked at the production of a known promoter [37,38] or inhibitor factor [39] of hair induction, respectively VEGF and BMP2. In physoxia, the amount of VEGF released by DP cells was significantly higher than in normoxia independently of the culture condition (Fig. 5a). The co-culture medium negatively impacted VEGF release by DP cells, which was not overcome with the co-culture with hMel. In opposition, the amount of BMP2 in physoxia was significantly lower than in normoxia, for both co-cultures and conventional DP culture medium (Fig. 5b). As this effect was not seen in the control established with the co-culture medium, it suggests that hMel presence was essential for the observed result.
Considering hMel-DP cell aggregates 3D-architectural resemblance with the HF bulb and the observed advantages regarding their functionality under physoxia, we then investigated the rescue of cell’s native-like phenotype under these conditions. The expression profile of different DP and hMel markers in the 3D-aggregates (Fig. 5c) showed that the cells display a phenotype and organization identical to the native HF (Fig. 5c, upper panel). The identification of the hMel markers tyrosinase, PMEL and MelanA, and vimentin and S100 also expressed by DP cells, showed clear compartmentalization between the cell types. Moreover, Ki67 immunolabelling confirmed a low number of proliferative cells within both cellular compartments (Fig. 5c). Noteworthy, tyrosinase expression in the cellular aggregates was higher than in conventionally cultured hMel (Fig. S1). Moreover, the in vivo predominant V2-isoform of versican [40], a DP inductive marker typically absent in 2D-cultured cells (Fig. S1) was weakly expressed in the cellular aggregates, confirming what was previously described for DP spheroids [28].
Overall, these results indicate that under physoxia DP cells inductive secretome is promoted which, in conjunction with 3D-culture conditions, allows the recovery of critical melanocytes and DP cells native markers.