Single-cell sequencing reveals PDFGRα+ stromal cell subpopulations that promote proacinar differentiation in embryonic salivary gland organoids
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE181430 | GEO | 2022/03/01
REPOSITORIES: GEO
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Project description:Single-cell sequencing reveals PDFGRα+ stromal cell subpopulations that promote proacinar differentiation in embryonic salivary gland organoids
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Project description:Human cardiac-derived c-kit+ stromal cells (CSCs) have demonstrated efficacy in preclinical trials for the treatment of heart failure and myocardial dysfunction. Unfortunately, large variability in patient outcomes and cell populations remains a problem. Previous research has demonstrated that the reparative capacity of CSCs may be linked to the age of the cells: CSCs derived from neonate patients increase cardiac function and reduce fibrosis. However, age-dependent differences between CSC populations have primarily been explored with bulk sequencing methods. In this work, we hypothesized that differences in CSC populations and subsequent cell therapy outcomes may arise from differing cell subtypes within donor CSC samples. We performed single-cell RNA sequencing on four neonatal CSC (nCSC) and five child CSC (cCSC) samples. Subcluster analysis revealed cCSC-enriched clusters upregulated in several fibrosis- and immune response-related genes. Module-based analysis identified upregulation of chemotaxis and ribosomal activity-related genes in nCSCs and upregulation of immune response and fiber synthesis genes in cCSCs. Further, we identified versican and integrin alpha 2 as potential markers for a fibrotic cell subtype. By investigating differences in patient-derived CSC populations at the single-cell level, this research aims to identify and characterize CSC subtypes to better optimize CSC-based therapy and improve patient outcomes.
| S-EPMC9693599 | biostudies-literature
Project description:Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies.
| S-EPMC7069099 | biostudies-literature
Project description:Background Salivary glands produce saliva that play essential roles in digestion and oral health. Derivation of salivary gland organoids from pluripotent stem cells (PSCs) provides a powerful platform to model the organogenesis processes during development. A few studies attempted to differentiate PSCs into salivary gland organoids. However, none of them could recapitulate the morphogenesis of the embryonic salivary glands, and most of the protocols involved complicated manufacturing processes. Methods To generate PSC-derived salivary gland placodes, the mouse embryonic stem cells were first differentiated into oral ectoderm by treatment with BMP4 on day 3. Retinoic acid and bFGF were then applied to the cultures from day 4 to day 6, followed by a 4-day treatment of FGF10. The PSC-derived salivary gland placodes on day 10 were transplanted to kidney capsules to determine the regenerative potential. Quantitative reverse transcriptase–polymerase chain reaction, immunofluorescence, and RNA-sequencing were performed to identify the PSC-derived SG placodes. Results We showed that step-wise treatment of retinoic acid and FGF10 promoted the differentiation of PSCs into salivary gland placodes, which can recapitulate the early morphogenetic events of their fetal counterparts, including the thickening, invagination, and then formed initial buds. The PSC-derived salivary gland placodes also differentiated into developing duct structures and could develop to striated and excretory ducts when transplanted in vivo. Conclusions The present study provided an easy and safe method to generate salivary gland placodes from PSCs, which offered possibilities for studying salivary gland development in vitro and developing new cell therapies. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-03033-5.
| S-EPMC9330698 | biostudies-literature
Project description:There is a need for an artificial salivary gland as a long-term remedy for patients suffering from salivary hypofunction, a leading cause of chronic xerostomia (dry mouth). Current salivary gland tissue engineering approaches are limited in that they either lack sufficient physical cues and surface area needed to facilitate epithelial cell differentiation, or they fail to provide a mechanism for assembling an interconnected branched network of cells. We have developed highly-ordered arrays of curved hemispherical "craters" in polydimethylsiloxane (PDMS) using wafer-level integrated circuit (IC) fabrication processes, and lined them with electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers, designed to mimic the three-dimensional (3-D) in vivo architecture of the basement membrane surrounding spherical acini of salivary gland epithelial cells. These micropatterned scaffolds provide a method for engineering increased surface area and were additionally investigated for their ability to promote cell polarization. Two immortalized salivary gland cell lines (SIMS, ductal and Par-C10, acinar) were cultured on fibrous crater arrays of various radii and compared with those grown on flat PLGA nanofiber substrates, and in 3-D Matrigel. It was found that by increasing crater curvature, the average height of the cell monolayer of SIMS cells and to a lesser extent, Par-C10 cells, increased to a maximum similar to that seen in cells grown in 3-D Matrigel. Increasing curvature resulted in higher expression levels of tight junction protein occludin in both cell lines, but did not induce a change in expression of adherens junction protein E-cadherin. Additionally, increasing curvature promoted polarity of both cell lines, as a greater apical localization of occludin was seen in cells on substrates of higher curvature. Lastly, substrate curvature increased expression of the water channel protein aquaporin-5 (Aqp-5) in Par-C10 cells, suggesting that curved nanofiber substrates are more suitable for promoting differentiation of salivary gland cells.
| S-EPMC3755621 | biostudies-literature
Project description:ObjectiveHyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro.Materials and methodsSingle cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response.ResultsSingle cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland.ConclusionsMurine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.
| S-EPMC7818507 | biostudies-literature
Project description:New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.
| S-EPMC7903845 | biostudies-literature
Project description:Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.
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Project description:Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.
| S-EPMC6181987 | biostudies-literature
Project description:Embryonic stem cell (ESC) self-renewal and pluripotency is maintained by an external signaling pathways and intrinsic regulatory networks involving ESC-specific transcriptional complexes (mainly formed by OCT3/4, Sox2 and Nanog proteins), the Polycomb repressive complex 2 (PRC2) and DNA methylation [1-8]. Among these, Nanog represents the more ESC specific factor and its repression correlates with the loss of pluripotency and ESC differentiation [9-11]. During ESC early differentiation, many development-associated genes become upregulated and although, in general, much is known about the pluripotency self-renewal circuitry, the molecular events that lead ESCs to exit from pluripotency and begin differentiation are largely unknown. Snai1 is one the most early induced genes during ESC differentiation in vitro and in vivo [12,13]. Here we show that Snai1 is able to directly repress several stemness-associated genes including Nanog. We use a ESC stable-line expressing a inducible Snai1 protein. We here show microarray analysis of embryonic stem cells (ESC) expressing Snail-ER at various time points of induction with 4-OH. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference GSE57854 and here: http://epigenetics.hugef-research.org/data.php.
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