Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

BACKGROUND: Eccrine sweat gland (SG) plays a crucial role in thermoregulation but exhibits very limited regenerative potential. Although SG lineage-restricted niches dominate SG morphogenesis and benefit SG regeneration, rebuilding niches in vivo is challenging for stem cell therapeutic applications. Hence, we attempted to screen and tune the critical niche-responding genes that dually respond to both biochemical and structural cues, which might be a promising strategy for SG regeneration. METHODS: An artificial SG lineage-restricted niche consisting of mouse plantar dermis homogenates (i.e. biochemical cues) and 3D architecture (i.e. structural cues) was built in vitro by using an extrusion-based 3D bioprinting approach. Mouse bone marrow-derived mesenchymal stem cells (MSCs) were then differentiated into the induced SG cells in the artificial SG lineage-restricted niche. To decouple biochemical cues from structural cues, the transcriptional changes aroused by pure biochemical cues, pure structural cues and synergistic effects of both cues were analyzed pairwise, respectively. Notably, only niche-dual-responding genes that are differentially expressed in response to both biochemical and structural cues and participate in switching MSC fates towards SG lineage were screened out. Validations in vitro and in vivo were respectively conducted by inhibiting or activating the candidate niche-dual-responding gene(s) to explore the consequent effects on SG differentiation. RESULTS: Notch4 is one of the niche-dual-responding genes that enhanced MSC stemness and promoted SG differentiation in 3D-printed matrix in vitro. Furthermore, inhibiting Notch4 specifically reduced keratin 19-positive epidermal stem cells and keratin 14-positive SG progenitor cells, thus further delaying embryonic SG morphogenesis in vivo. CONCLUSIONS: Notch4 not only participates in mouse MSC-induced SG differentiation in vitro but is also implicated in mouse eccrine SG morphogenesis in vivo.