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Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

ABSTRACT: Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.

SUBMITTER: Boldyreva MA

PROVIDER: S-EPMC6627773 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Boldyreva Maria A MA Shevchenko Evgeny K EK Molokotina Yuliya D YD Makarevich Pavel I PI Beloglazova Irina B IB Zubkova Ekaterina S ES Dergilev Konstantin V KV Tsokolaeva Zoya I ZI Penkov Dmitry D Hsu Mu-Nung MN Hu Yu-Chen YC Parfyonova Yelena V YV

International journal of molecular sciences 20190624 12

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC tra ...[more]

PMID: 31238604

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Project description:ObjectiveRecent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes.Materials and methodsIn this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William's) medium supplemented with 4% N-CM or H-CM. Untreated William's and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells.ResultsWe observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063).ConclusionThe enrichment of William's basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.

Project description:Diabetes mellitus (DM) is one of the most prevalent feline endocrinopathies, affecting up to 1% of pet cats. De novo generation of functional insulin producing cell (IPC) clusters via transdifferentiation of feline adipose-derived multipotent stromal cells (ASCs) may not only provide a viable, functional cell therapy for feline DM, but may also serve as a platform for developing a comparable human treatment given feline and human DM similarities. Cells were induced to form IPCs with a novel, three-stage culture process with stromal or differentiation medium under static and dynamic conditions. Clusters were evaluated for intracellular zinc, viability, intracellular insulin, glucagon, and somatostatin, ultrastructure, glucose stimulated insulin secretion in the presence or absence of theophylline, and protein and gene expression. Isolated cells were multipotent, and cell clusters cultured in both media had robust cell viability. Those cultured in differentiation medium contained zinc and mono- or polyhormonal α-, β-, and δ-like cells based on immunohistochemical labeling and Mallory-Heidenhan Azan-Gomori's staining. Ultrastructurally, cell clusters cultured in differentiation medium contained insulin granules within vesicles, and clusters had a concentration-dependent insulin response to glucose in the presence and absence of theophylline which increased both insulin secretion and intracellular content. Expression of NK6.1, Pax6, Isl1, Glut2, RAB3A, glucagon, insulin, and somatostatin increased with differentiation stage for both sexes, and expression of nestin at stages 1 and 2 and Neurod1 at stage 2 was higher in cells from female donors. The cluster insulin secretion responses and endocrine and oncogene gene expression profiles were inconsistent with insulinoma characteristics. A total of 180 proteins were upregulated in differentiated clusters, and the majority were associated with biological regulation, metabolic processes, or stimulus response. Dynamic culture of IPC clusters resulted in clusters composed of cells primarily expressing insulin that released higher insulin with glucose stimulation than those in static culture. Collectively, the results of this study support generation of functional IPC clusters using feline ASCs isolated from tissues removed during routine sterilization. Further, cluster functionality is enhanced with dynamic, motion-driven shear stress. This work establishes a foundation for development of strategies for IPC therapy for short or long-term diabetes treatment and may represent an option to study prevention and treatment of diabetes across species.

Dataset Information

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Publications

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

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