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Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

ABSTRACT: Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized that the administration of VEGF-expressing human MSCs would stimulate neurite outgrowth and proliferation of cell-types involved in neural repair. When cultured with dorsal root ganglion (DRG) explants in vitro, control and VEGF-expressing MSCs (VEGF-MSCs) increased neurite extension and proliferation of Schwann cells (SCs) and endothelial cells, while VEGF-MSCs stimulated significantly greater proliferation of endothelial cells. When embedded within a 3D fibrin matrix, VEGF-MSCs maintained overexpression and expressed detectable levels over 21 days. After transplantation into a murine sciatic nerve injury model, VEGF-MSCs maintained high VEGF levels for 2 weeks. This study provides new insight into the role of VEGF on peripheral nerve injury and the viability of transplanted genetically engineered MSCs. The study aims to provide a framework for future studies with the ultimate goal of developing an improved therapy for nerve repair.

SUBMITTER: Man AJ

PROVIDER: S-EPMC4830493 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

Man Alan J AJ Kujawski Gregory G Burns Travis S TS Miller Elaine N EN Fierro Fernando A FA Leach J Kent JK Bannerman Peter P

Cellular and molecular bioengineering 20160113 1

Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized t ...[more]

PMID: 27087859

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Project description:The possibility of generating neural cells from human bone marrow-derived mesenchymal stem cells by simple in vitro treatments is alluring both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of the cells’ transcriptome. Video time-lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of focal adhesion contacts. Analysis of markers revealed that mesenchymal stem cells constitutively expressed multi-lineage traits, including several pertaining to the neural one. However, the applied “neural induction” protocol neither significantly modulated the expression of such markers, nor induced de novo translation of other neural specific proteins. Similarly, global expression profiling of over 21,000 genes demonstrated that gene transcription was poorly affected. Most strikingly, we found that the set of genes whose expression was altered by the inductive treatment did not match the set of genes found to be differentially expressed when comparing untreated mesenchymal stem cells and immature neural tissues. Conversely, by comparing these gene expression profiles with that obtained from comparisons between the same cells and an unrelated non-neural organ, such as liver, we found that the adopted “neural induction” protocol was no more effective in redirecting human mesenchymal stem cells toward a neural phenotype than toward an endodermal hepatic pathway. Keywords: Differentiation analysis

Project description:Background/objectives:Accelerating the process of bone regeneration is of great interest for surgeons and basic scientists alike. Recently, umbilical cord mesenchymal stem cells (UCMSCs) are considered clinically applicable for tissue regeneration due to their noninvasive harvesting and better viability. Nonetheless, the bone regenerative ability of human UCMSCs (HUCMSCs) is largely unknown. This study aimed to investigate whether Wnt10b-overexpressing HUCMSCs have enhanced bone regeneration ability in a rat model. Method:A rat calvarial defect was performed on 8-week old male Sprague Dawley rats. Commercially purchased HUCMSCsEmp in hydrogel, HUCMSCsWnt10b in hydrogel and HUCMSCsWnt10b with IWR-1 were placed in the calvarial bone defect right after surgery on rats (N = 8 rats for each group). Calvaria were harvested for micro-CT analysis and histology four weeks after surgery. CFU-F and multi-differentiation assay by oil red staining, alizarin red staining and RT-PCR (real-time polymerase chain reaction) were performed on HUCMSCsEmp and HUCMSCsWnt10b in vitro. Conditioned media from HUCMSCsEmp and HUCMSCsWnt10b were collected and used to treat human umbilical cord vein endothelial cells in Matrigel to access vessel formation capacity by tube formation assay. Results:Alizarin red staining, oil red staining and RT-PCR results showed robust osteogenic differentiation but poor adipogenic differentiation ability of HUCMSCsWnt10b. Furthermore, HUCMSCsWnt10b could accelerate bone defect healing, which was likely due to enhanced angiogenesis after the HUCMSCsWnt10b treatment, because more CD31+ vessels and increased vascular endothelial growth factor-A (VEGF-A) expression were observed, compared with the HUCMSCsEmp treatment. Conditioned media from HUCMSCsWnt10b also induced endothelial cells to form vessel tubes in a tube formation assay, which could be abolished by SU5416, an angiogenesis inhibitor. Conclusion:To our knowledge, this is the first study providing empirical evidence that HUCMSCsWnt10b can enhance their ability to heal calvarial bone defects via VEGF-mediated angiogenesis. The translational potential of this article:HUCMSCsWnt10b can accelerate critical size calvaria and are a new promising therapeutic cell source for fracture nonunion healing.

Project description:BackgroundsAllergic airway inflammation is prevalent worldwide and imposes a considerable burden on both society and affected individuals. This study aimed to investigate the therapeutic advantages of mesenchymal stem cells (MSCs) overexpressed interleukin-10 (IL-10) for the treatment of allergic airway inflammation, as both IL-10 and MSCs possess immunosuppressive properties.MethodsInduced pluripotent stem cell (iPSC)-derived MSCs were engineered to overexpress IL-10 via lentiviral transfection (designated as IL-10-MSCs). MSCs and IL-10-MSCs were administered intravenously to mice with allergic inflammation induced by ovalbumin (OVA), and the features of allergic inflammation including inflammatory cell infiltration, Th cells in the lungs, and T helper 2 cell (Th2) cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. MSCs and IL-10-MSCs were co-cultured with CD4+ T cells from patients with allergic rhinitis (AR), and the levels of Th2 cells and corresponding type 2 cytokines were studied. RNA-sequence was performed to further investigate the potential effects of MSCs and IL-10-MSCs on CD4+ T cells.ResultsStable IL-10-MSCs were established and characterised by high IL-10 expression. IL-10-MSCs significantly reduced inflammatory cell infiltration and epithelial goblet cell numbers in the lung tissues of mice with allergic airway inflammation. Inflammatory cell and cytokine levels in BALF also decreased after the administration of IL-10-MSCs. Moreover, IL-10-MSCs showed a stronger capacity to inhibit the levels of Th2 after co-cultured with CD4+ T cells from patients with AR. Furthermore, we elucidated lower levels of IL-5 and IL-13 in IL-10-MSCs treated CD4+ T cells, and blockade of IL-10 significantly reversed the inhibitory effects of IL-10-MSCs. We also reported the mRNA profiles of CD4+ T cells treated with IL-10-MSCs and MSCs, in which IL-10 played an important role.ConclusionIL-10-MSCs showed positive effects in the treatment of allergic airway inflammation, providing solid support for the use of genetically engineered MSCs as a potential novel therapy for allergic airway inflammation.

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Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

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Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

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