Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions

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Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to increased incidence of osteoporosis. culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease CCT239065 state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts in high glucose levels. In contrast, MSCs had no reduction of viability or clonogenicity when cultured with adipocytes in CCT239065 high glucose conditions and adipogenic gene expression indicated that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis. CCT239065 Introduction Diabetes is associated with insulin deficiency (Type I) or resistance (Type II) and consequential dysregulation CCT239065 in adipose tissue and energy metabolism.1 Notably, both type I and II diabetes are associated with increased risk of osteoporosis, a skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone.2 Among other cell types, adipocytes and osteoblasts are dysregulated during the progression of diabetes and resulting secondary osteoporosis.3 As both cell types are differentiated from mesenchymal stem cells (MSCs) and are components of the bone marrow microenvironment,1,3,4 it is possible that the progression of these diseases involves altered MSC behavior.3 The stem cell microenvironment, where stem cells derive signals from the extracellular matrix (ECM), cellular contacts, and both short and long range soluble factors,5,6 has been seen to change in disease states and has recently gained interest as a potential new target for disease therapies.5,6 Within the bone marrow compartment, MSCs are directed to differentiate to osteoblasts or adipocytes, a process that is tightly regulated, partially by cellular communication between MSCs and the osteoblasts and adipocytes in the immediate microenvironment.3 Irregular MSC behavior has been observed in abnormal environments, such as the tumor microenvironment, where MSCs home and potentially participate in tumor pathogenesis.7 Similarly, in an model of Gaucher disease, MSCs were seen to have reduced proliferative capacity and may contribute to increased bone resorption.8 As it has been hypothesized that alterations in the MSC microenvironment both contribute to and result from interactions Rabbit Polyclonal to HS1 with bone and adipose tissues,3 understanding how environmental changes inherent to diabetes impact these interactions may provide insight into the role MSCs play in the progression of diabetes and concomitant osteoporosis. Clinically, diabetes is often associated with hyperglycemic conditions due to the bodys inability to properly regulate the amounts of glucose in the blood.4 Studies have shown that elevated glucose levels have negative effects on MSCs, adipocytes and osteoblasts, all of which are cell types that influence the MSC microenvironment. Data suggest that at high glucose levels, MSCs undergo increased apoptosis and senescence as well as lose colony forming capacity and osteogenic potential.9C12 Adipocytes have demonstrated decreased insulin sensitivity, unregulated triglyceride storage, increased production of reactive oxygen species and pro-inflammatory cytokines, and decreased adiponectin secretion when cultured in high glucose conditions.13C15 Finally, osteoblasts cultured in high glucose have shown reduced proliferative capacity, mineralization capabilities, collagen I synthesis, and expression of differentiation markers.16C19 However, how these individual consequences impact cellular cross-talk between all three cell types remains to be fully understood, though previous work has shown that intercellular communication is affected in the context of diabetes. For instance, murine osteoblasts in co-culture with bone marrow cells from diabetic mice undergo increased cell death as compared to those co-cultured with bone marrow cells from normal mice.20 This indicates that MSCs derived from diabetic tissues may have an altered secretome, but how these changes influence interactions between MSCs and neighboring cell types in the bone marrow niche remains largely unexplored. Understanding how hyperglycemic conditions influence MSCs both directly and indirectly (through soluble signaling from neighboring osteoblasts and adipocytes) may provide insight into how the altered stem cell microenvironment contributes to tissue dysregulation, particularly in the development of diabetes-related osteoporosis. To gain such biological insight, it is necessary to use an culture system that permits the co-culture of multiple cell types but still allows specific cell population analyses. As opposed to experiments, systems can be advantageous by eliminating the confounding factors present in animal models and by permitting use of human cells. In past studies, both 2D techniques, such as transwell-based systems and cell patterning,21,22 and 3D techniques, involving various biomaterial-based scaffolds,23,24 have provided useful information about cellular interactions. However, in many of these systems, both separation of unique cell populations for analysis and retrieval of live cells for further.