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.

中文翻译：

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Notch4 参与 3D 打印基质中间充质干细胞诱导的分化，并与小汗腺形态发生有关

背景 小汗腺（SG）在体温调节中发挥着至关重要的作用，但其再生潜力非常有限。尽管 SG 谱系限制的生态位主导着 SG 形态发生并有利于 SG 再生，但体内重建生态位对于干细胞治疗应用来说具有挑战性。因此，我们尝试筛选和调整对生化和结构线索双重响应的关键生态位响应基因，这可能是 SG 再生的一个有前途的策略。方法通过使用基于挤出的3D生物打印方法，在体外构建由小鼠足底真皮匀浆（即生化线索）和3D架构（即结构线索）组成的人工SG谱系限制生态位。然后，小鼠骨髓来源的间充质干细胞 (MSC) 在人工 SG 谱系限制的微环境中分化为诱导的 SG 细胞。为了将生化线索与结构线索分离，分别对纯生化线索、纯结构线索和两种线索的协同效应引起的转录变化进行了两两分析。值得注意的是，只有那些响应生化和结构线索而差异表达并参与将 MSC 命运转变为 SG 谱系的生态位双重响应基因被筛选出来。通过抑制或激活候选生态位双响应基因分别进行体外和体内验证，以探讨对 SG 分化的后续影响。结果Notch4是体外3D打印基质中增强MSC干性并促进SG分化的生态位双重响应基因之一。此外，抑制Notch4可特异性减少角蛋白19阳性表皮干细胞和角蛋白14阳性SG祖细胞，从而进一步延迟体内胚胎SG形态发生。结论 Notch4不仅在体外参与小鼠MSC诱导的SG分化，而且在体内参与小鼠小汗腺SG的形态发生。