Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates.

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.

Hyperglycemia decreases the expression of ATP synthase ß subunit and enolase 2 in glomerular epithelial cells.

Glomerular epithelial cells (GECs) are known to play a key role in maintaining the structure and function of the glomerulus. GEC injury induced by hyperglycemia is present in early-stage diabetic nephropathy (DN), which is the most common cause of renal failure. In an attempt to identify target proteins involved in the pathogenesis of GEC injury at early DN, we performed the proteomic analysis using primary cultures of GECs, prepared from the dissected rat glomeruli. The protein expression profiles in the two-dimensional electrophoresis gels were compared between GECs treated for three days with normal glucose (5 mM) and those with high glucose (30 mM) concentrations. These concentrations correspond to blood glucose concentrations under normoglycemia and hyperglycemia, respectively. Proteins with differential expression levels were identified using ESI-Q-TOF tandem mass spectrometry. The primary GECs cultured in hyperglycemic conditions showed cellular hypertrophy and increased production of reactive oxygen species, both of which reflect the GEC injury. Our proteomic analysis identified eight proteins with differential expression profiles, depending on glucose concentrations. Among them, we selected ATP synthase ß subunit and enolase 2 that are related to energy metabolism and are down-regulated under hyperglycemia, and confirmed that hyperglycemia decreased the expression levels of ATP synthase ß subunit and enolase 2 proteins by western blotting analysis. Hyperglycemia may impair mitochondrial function and alter glycolysis in GECs by down-regulating the expression of ATP synthase ß subunit and enolase 2. The present study may provide a better understanding of the pathogenic mechanisms of GEC injury in early DN.

Combinations of growth factors enhance the potency of islets in vitro.

OBJECTIVES: Recent studies have demonstrated the impressive expansion of beta cells in vitro. But unfortunately, expanded beta cells do not function in the same way as fully differentiated beta cells. Therefore, we developed a condition that would allow islet cells to proliferate while maintaining their endocrine function. METHODS: We tested the different use of growth factors in a different culture period. And we tested the possibility of adult islets, which expanded during a short period, as a clinical source of islet cells by comparing the efficiency of transplantation of cultured islets with that of fresh islets. RESULTS: The islets showed a time-dependent increase in proliferative activity, reaching 32.2% on day 5. After 5 days of culture, the efficiency of transplantation of cultured islets was increased (2-fold) in comparison to that of noncultured islets. Moreover, islet transplantation immediately induced normoglycemia at a level equal to native islets. CONCLUSIONS: These findings suggest that adult beta cells have the potential to proliferate while maintaining their endocrine function, which can be improved through careful regulation of proliferation and differentiation.

A fibrin gel carrier system for islet transplantation into kidney subcapsule.

Islet transplantation is a promising therapeutic option for type 1 diabetes, and actively performed in the clinic as well as in the animal experiments. For the rodent experiments, islet transplantation into kidney subcapsule is widely used to assess islet quality, however, it is often difficult to do using a polyethylene tubing and fine needle because of inherent dead volume of needle and stickiness of the tubing to islets. This problem makes it difficult to interpret the physiological response to different islet doses. Here, we developed a simple fibrin gel carrier system for islet transplantation into kidney subcapsule and utilized it to determine the marginal islet mass sufficient for correction of hyperglycemia in diabetic nude mice.