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Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration.

ABSTRACT: Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)-like matrix to direct the conversion of MSC into functional SGs and facilitated SGs recovery in mice. By extracellular matrix differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator for SG specification. Our findings showed that Hmox1 could respond to the 3D structure activation and also be involved in MSC differentiation. Using inhibition and activation assay, CTHRC1 and Hmox1 synergistically boosted SG gene expression profile. Together, these findings indicated that biochemical and structural cues served as two critical impacts of 3D-printed matrix on MSC fate decision into the glandular lineage and functional SG recovery.

SUBMITTER: Yao B

PROVIDER: S-EPMC7056319 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration.

Yao Bin B Wang Rui R Wang Yihui Y Zhang Yijie Y Hu Tian T Song Wei W Li Zhao Z Huang Sha S Fu Xiaobing X

Science advances 20200304 10

Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)-like matrix to direct the conversion of MSC into functiona ...[more]

PMID: 32181358

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Project description:BackgroundEccrine 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.MethodsAn 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.ResultsNotch4 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.ConclusionsNotch4 not only participates in mouse MSC-induced SG differentiation in vitro but is also implicated in mouse eccrine SG morphogenesis in vivo.

Project description:Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

Dataset Information

Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration.

Publications

Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration.

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