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Redirecting differentiation of mammary progenitor cells by 3D bioprinted sweat gland microenvironment.

ABSTRACT: Background:Mammary progenitor cells (MPCs) maintain their reproductive potency through life, and their specific microenvironments exert a deterministic control over these cells. MPCs provides one kind of ideal tools for studying engineered microenvironmental influence because of its accessibility and continually undergoes postnatal developmental changes. The aim of our study is to explore the critical role of the engineered sweat gland (SG) microenvironment in reprogramming MPCs into functional SG cells. Methods:We have utilized a three-dimensional (3D) SG microenvironment composed of gelatin-alginate hydrogels and components from mouse SG extracellular matrix (SG-ECM) proteins to reroute the differentiation of MPCs to study the functions of this microenvironment. MPCs were encapsulated into the artificial SG microenvironment and were printed into a 3D cell-laden construct. The expression of specific markers at the protein and gene levels was detected after cultured 14 days. Results:Compared with the control group, immunofluorescence and gene expression assay demonstrated that MPCs encapsulated in the bioprinted 3D-SG microenvironment could significantly express the functional marker of mouse SG, sodium/potassium channel protein ATP1a1, and tend to express the specific marker of luminal epithelial cells, keratin-8. When the Shh pathway is inhibited, the expression of SG-associated proteins in MPCs under the same induction environment is significantly reduced. Conclusions:Our evidence proved the ability of differentiated mouse MPCs to regenerate SG cells by engineered SG microenvironment in vitro and Shh pathway was found to be correlated with the changes in the differentiation. These results provide insights into regeneration of damaged SG by MPCs and the role of the engineered microenvironment in reprogramming cell fate.

SUBMITTER: Wang R

PROVIDER: S-EPMC6755689 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Redirecting differentiation of mammary progenitor cells by 3D bioprinted sweat gland microenvironment.

Wang Rui R Wang Yihui Y Yao Bin B Hu Tian T Li Zhao Z Liu Yufan Y Cui Xiaoli X Cheng Liuhanghang L Song Wei W Huang Sha S Fu Xiaobing X

Burns & trauma 20190923

<h4>Background</h4>Mammary progenitor cells (MPCs) maintain their reproductive potency through life, and their specific microenvironments exert a deterministic control over these cells. MPCs provides one kind of ideal tools for studying engineered microenvironmental influence because of its accessibility and continually undergoes postnatal developmental changes. The aim of our study is to explore the critical role of the engineered sweat gland (SG) microenvironment in reprogramming MPCs into fun ...[more]

PMID: 31559316

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Project description:The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. STATEMENT OF SIGNIFICANCE: Factors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland's ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.

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.

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.

Dataset Information

Redirecting differentiation of mammary progenitor cells by 3D bioprinted sweat gland microenvironment.

Publications

Redirecting differentiation of mammary progenitor cells by 3D bioprinted sweat gland microenvironment.

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