Abstract Background The coordinated development of the mesenchymal and epithelial primordia of the murine ureter requires intense tissue communication. Previous studies have revealed the crucial role of the FGFR2-SHH-FOXF1-BMP4 signaling axis in the proliferation and differentiation programs of both the epithelium and the mesenchyme. Canonical (β-Catenin/CTNNB1-dependent) WNT signaling in the mesenchymal primordium has been reported to influence mesenchymal development by enhancing proliferation and favoring the smooth muscle cell fate over adventitial fibrocytes. Methods To investigate the role of mesenchymal WNT signaling in early ureter development further, we analyzed the ureters of Ctnnb1-deficient mouse mutants for cellular and molecular changes during embryogenesis. Using pharmacological inhibition and activation approaches in explant cultures of embryonic ureters, we assessed the contribution of altered signaling activities to differentiation changes in Ctnnb1-deficient ureters. Transcriptional profiling of embryonic ureters with short-term WNT signaling inhibition allowed us to identify the primary targets of this signaling pathway in the ureteric mesenchyme. Results We demonstrate that both mesenchymal and epithelial differentiation are impaired in ureters lacking mesenchymal Ctnnb1. This defect is linked to an inability to activate pro-differentiation transcription factors in either tissue primordia. Epithelial changes can be attributed, at least in part, to the loss of BMP4 signaling and the gain of canonical WNT signaling in the epithelial primordium. Mesenchymal WNT signaling directly activates transcription of multiple components of the SHH-FOXF1-BMP4 signaling axis, thereby enhancing this module in the inner region of the ureteric mesenchyme. Conclusions Our work improves the understanding of the signaling network that coordinates cytodifferentiation in the early ureter. Mesenchymal WNT signaling plays a central role in mesenchymal fate decisions but also in promoting epithelial differentiation through enhancement of the SHH-FOXF1-BMP4 axis and inhibition of epithelial WNT-signaling.

The patterned array of basal, intermediate and superficial cells in the urothelium of the mature ureter arises from uncommitted epithelial progenitors of the distal ureteric bud. Urothelial development requires signaling input from surrounding mesenchymal cells, which, in turn, depend on cues from the epithelial primordium to form a layered fibro-muscular wall. Here, we have identified FGFR2 as a crucial component in this reciprocal signaling crosstalk in the murine ureter. Loss of Fgfr2 in the ureteric epithelium led to reduced proliferation, stratification, intermediate and basal cell differentiation in this tissue, and affected cell survival and smooth muscle cell differentiation in the surrounding mesenchyme. Loss of Fgfr2 impacted negatively on epithelial expression of Shh and its mesenchymal effector gene Bmp4. Activation of SHH or BMP4 signaling largely rescued the cellular defects of mutant ureters in explant cultures. Conversely, inhibition of SHH or BMP signaling in wild-type ureters recapitulated the mutant phenotype in a dose-dependent manner. Our study suggests that FGF signals from the mesenchyme enhance, via epithelial FGFR2, the SHH-BMP4 signaling axis to drive urothelial and mesenchymal development in the early ureter.

Cell-based therapies have become a popular approach in the field of regenerative medicine. Human fibroblast cells, one of the cell types widely used in clinical applications, have been used for skin regeneration and wound healing procedures. Furthermore, they are utilized for aesthetic purposes since fibroblasts lose their abilities such as collagen synthesis with age. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of fibroblasts derived from adult human skin as a final product under good manufacturing practice-compliant conditions.

Mesenchymal stem cells have gained popularity in cell-based therapies due to their regenerative capabilities, immunomodulation properties, and paracrine activity through trophic factors. It is of utmost importance to establish clinical-grade procedures for the preparation of the mesenchymal stem cells for clinical applications. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of mesenchymal stem cells derived from umbilical cord as a final product under good manufacturing practices-compliant conditions.

Over the past few years, a large number of clinical studies for advanced therapy medicinal products have been registered and/or conducted for treating various diseases around the world and many have generated very exciting outcomes. Media fill, the validation of the aseptic manufacturing process, is the simulation of medicinal product manufacturing using nutrient media. The purpose of this study is to explain the media fill procedure stepwise in the context of cellular therapy medicinal products. The aseptic preparation of patient individual cellular product is simulated by using tryptic soy broth as the growth medium, and sterile vials as primary packaging materials.

The mesothelial lining of the lung, the visceral pleura, and of the heart, the epicardium, derive from a common multipotent precursor tissue, the mesothelium of the embryonic thoracic cavity that also contributes to organ-specific mesenchymal cell types. Insight into mesothelial mobilization and differentiation has prevailedin the developing heart while the mesenchymal transition and fate of the visceral pleura are poorly understood. Here, we use the fact that the early mesothelium of both the lung and the heart expresses the transcription factor gene Wt1, to comparatively analyze mesothelial mobilization in the two organs by a genetic cre-loxP-based conditional approach. We show that epicardial cells are mobilized in a large number between E12.5 and E14.5, whereas pleural mobilization occurs only sporadically and variably in few regions of the lung in a temporally highly confined manner shortly after E12.5. Mesothelium-specific inactivation of unique pathway components using a Wt1creERT2 line excluded a requirement for canonical WNT, NOTCH, HH, TGFB, PDGFRA, and FGFR1/FGFR2 signaling in the mesenchymal transition of the visceral pleura but indicated a deleterious effect of activated WNT, NOTCH, and HH signaling on lung development. Epicardial mobilization was negatively impacted on by loss of HH, PDGFRA, FGFR1/2 signaling. Epicardial overactivation of WNT, NOTCH, and HH disturbed epicardial and myocardial integrity. We conclude that mesothelial mobilization in the developing lung and heart differs in timing, quantity and pathway dependency, indicating the organ specificity of the program.

The organized array of smooth muscle cells (SMCs) and fibroblasts in the walls of visceral tubular organs arises by patterning and differentiation of mesenchymal progenitors surrounding the epithelial lumen. Here, we show that the TBX2 and TBX3 transcription factors have novel and required roles in regulating these processes in the murine ureter. Co-expression of TBX2 and TBX3 in the inner mesenchymal region of the developing ureter requires canonical WNT signaling. Loss of TBX2/TBX3 in this region disrupts activity of two crucial drivers of the SMC program, Foxf1 and BMP4 signaling, resulting in decreased SMC differentiation and increased extracellular matrix. Transcriptional profiling and chromatin immunoprecipitation experiments revealed that TBX2/TBX3 directly repress expression of the WNT antagonists Dkk2 and Shisa2, the BMP antagonist Bmper and the chemokine Cxcl12 These findings suggest that TBX2/TBX3 are effectors of canonical WNT signaling in the ureteric mesenchyme that promote SMC differentiation by maintaining BMP4 and WNT signaling in the inner region, while restricting CXCL12 signaling to the outer layer of fibroblast-fated mesenchyme.

Stem cell-based applications have become a popular and promising approach for therapy for a number of disorders including neurodegenerative diseases, degenerative muscle diseases, and osteoporosis, as well as trauma, inflammations, burns, and injuries. Human tooth germ stem cells are an adult stem cell source; they have mesenchymal stem cell properties and show high proliferative and differentiation capacity. Melatonin has been demonstrated to regulate differentiation of human and mouse mesenchymal stem cells into various cell lineages in addition to its other functions in the body. In the current study, the effects of melatonin on osteogenic, neurogenic, adipogenic, chondrogenic, myogenic, and odontogenic differentiation of human tooth germ stem cells were investigated. The results showed that melatonin increases the viability of cells. It significantly augments osteogenic, neurogenic, chondrogenic, myogenic, and odontogenic differentiation of the cells, whereas it reduces adipogenic differentiation capability. These results suggest that melatonin has a great potential to increase differentiation capacity of human tooth germ stem cells and might be useful in regenerative therapy applications involving stem cell differentiations in addition to defining potential treatments for obesity because of its suppressor effects on adipogenesis.