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Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro.

ABSTRACT: Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff's reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.

SUBMITTER: Luengen AE

PROVIDER: S-EPMC9247357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe development of the human placenta is tightly coordinated by a multitude of placental cell types, including human chorionic villi mesenchymal stromal cells (hCV-MSCs). Defective hCV-MSCs have been reported in preeclampsia (PE), a gestational hypertensive disease characterized by maternal endothelial dysfunction and systemic inflammation. Our goal was to determine whether hCV-MSCs are ciliated and whether altered ciliation is responsible for defective hCV-MSCs in preeclamptic placentas, as the primary cilium is a hub for signal transduction, which is important for various cellular activities.MethodsIn the present work, we collected placental tissues from different gestational stages and we isolated hCV-MSCs from 1st trimester, term control, and preeclamptic placentas. We studied their ciliation, functionality, and impact on trophoblastic cell lines and organoids formed from human trophoblast stem cells (hTSCs) and from the trophoblastic cell line JEG-3 with various cellular and molecular methods, including immunofluorescence staining, gene analysis, spheroid/organoid formation, motility, and cellular network formation assay. The statistical evaluation was performed using a Student's t test (two-tailed and paired or homoscedastic) or an unpaired Mann-Whitney U test (two-tailed).ResultsThe results show that primary cilia appeared abundantly in normal hCV-MSCs, especially in the early development of the placenta. Compared to control hCV-MSCs, the primary cilia were truncated, and there were fewer ciliated hCV-MSCs derived from preeclamptic placentas with impaired hedgehog signaling. Primary cilia are necessary for hCV-MSCs' proper signal transduction, motility, homing, and differentiation, which are impaired in preeclamptic hCV-MSCs. Moreover, hCV-MSCs derived from preeclamptic placentas are significantly less capable of promoting growth and differentiation of placental organoids, as well as cellular network formation.ConclusionsThese data suggest that the primary cilium is required for the functionality of hCV-MSCs and primary cilia are impaired in hCV-MSCs from preeclamptic placentas.

Project description:BACKGROUND:Radiotherapy remains one of the cornerstones to improve the outcome of colorectal cancer (CRC) patients. Radiotherapy of the CRC not only help to destroy cancer cells but also remodel the tumour microenvironment by enhancing tumour-specific tropism of bone marrow-derived mesenchymal stromal cell (BM-MSC) from the peripheral circulation. However, the role of local MSCs and recruited BM-MSC under radiation were not well defined. Indeed, the functions of BM-MSC without irradiation intervention remained controversial in tumour progression: BM-MSC was previously shown to modulate the immune function of major immune cells, resulting in an impaired immunological sensitivity and to induce an increased risk of tumour recurrence. In contrast, it could also secrete various cytokines and possess anticancer effect. METHODS:Three co-cultivation modules, 3D culture modules, and cancer organoids were established. The induction of cytokines secretion in hBM-MSCs after irradiation was analysed by ELISA array and flow cytometry. AutoMac separator was used to separate hBM-MSC and CRC automatically. Cells from the co-cultured group and the control group were then irradiated by UV-C lamp and X-ray. Proliferation assay and viability assay were performed. RESULTS:In this study, we show that BM-MSCs can induce the EMT progression of CRC cells in vitro. When irradiated with low doses of ultraviolet radiation and X-rays, BM-MSCs show an anti-tumour effect by secreting certain cytokine (TNF-?, IFN-?) that lead to the inhibition of proliferation and induction of apoptosis of CRC cells. This was further verified in a 3D culture model of a CRC cell in vitro. Furthermore, irradiation on the co-culture system induced the cleavage of caspase3, and attenuated the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase in cancer cells. The signal pathways above might contribute to the cancer cell death. CONCLUSIONS:Taken together, we show that BM-MSC can potentially promote the effect of radiotherapy in CRC.

Project description:Human Mesenchymal Stromal Cells (hMSCs) are a promising source for cell-based therapies. Yet, transition to phase III and IV clinical trials is remarkably slow. To mitigate donor variabilities and to obtain robust and valid clinical data, we aimed first to develop a manufacturing concept balancing large-scale production of pooled hMSCs in a minimal expansion period, and second to test them for key manufacture and efficacy indicators in the clinically highly relevant indication wound healing. Our novel clinical-scale manufacturing concept is comprised of six single donor hMSCs master cell banks that are pooled to a working cell bank from which an extrapolated number of 70,000 clinical doses of 1x106 hMSCs/cm2 wound size can be manufactured within only three passages. The pooled hMSC batches showed high stability of key manufacture indicators such as morphology, immune phenotype, proliferation, scratch wound healing, chemotactic migration and angiogenic support. Repeated topical hMSCs administration significantly accelerated the wound healing in a diabetic rat model by delivering a defined growth factor cargo (specifically BDNF, EGF, G-CSF, HGF, IL-1α, IL-6, LIF, osteopontin, VEGF-A, FGF-2, TGF-β, PGE-2 and IDO after priming) at the specific stages of wound repair, namely inflammation, proliferation and remodeling. Specifically, the hMSCs mediated epidermal and dermal maturation and collagen formation, improved vascularization, and promoted cell infiltration. Kinetic analyses revealed transient presence of hMSCs until day (d)4, and the dynamic recruitment of macrophages infiltrating from the wound edges (d3) and basis (d9), eventually progressing to the apical wound on d11. In the wounds, the hMSCs mediated M2-like macrophage polarization starting at d4, peaking at d9 and then decreasing to d11. Our study establishes a standardized, scalable and pooled hMSC therapeutic, delivering a defined cargo of trophic factors, which is efficacious in diabetic wound healing by improving vascularization and dynamic recruitment of M2-like macrophages. This decision-making study now enables the validation of pooled hMSCs as treatment for impaired wound healing in large randomized clinical trials.

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Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro.

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