Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

The role of epigenetic modifiers in the hepatic differentiation of human umbilical cord derived mesenchymal stem cells.

Human umbilical cord (hUC) derived mesenchymal stem cells (MSCs) can be progressively differentiated into multiple lineages including hepatic lineages, and thus provide an excellent in vitro model system for the study of hepatic differentiation. At present, hepatic differentiation protocols are based on the use of soluble chemicals in the culture medium and provide immature hepatic like cells. Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are two important epigenetic modifiers that regulate stem cell differentiation. Therefore, this study aimed to investigate the role of HDACi, valproic acid (VPA) and DNMTi,5-azacytidine (5-aza) along with a hepatic inducer in the hepatic differentiation of hUC-MSCs. hUC-MSCs were characterized via immunocytochemistry and flow cytometry. The final concentrations of VPA and 5-aza were optimized via MTT cytotoxicity assay. All treated groups were assessed for the presence of hepatic genes and proteins through qPCR and immunocytochemistry, respectively. The results showed that the pretreatment of epigenetic modifiers not only increased the hepatic genes but also increased the expression of the hepatic proteins. VPA induces hepatic differentiation in hUC-MSCs with significant gene expression of hepatic markers i.e., FOXA2 and CK8. Moreover, VPA pretreatment enhanced the expression of hepatic proteins AFP and TAT. The pretreatment of 5-aza shows significant gene expression of hepatic marker LDL-R. However, 5-aza treatment failed to induce hepatic protein expression. The results of the current study highlighted the effectiveness of epigenetic modifiers in the hepatic differentiation of hUC-MSCs. These differentiated cells can be employed in cell-based therapeutics for hepatic diseases in future.

Metformin for preventing gestational diabetes in women with polycystic ovarian syndrome.

OBJECTIVE: To assess the effect of metformin in controlling Gestational Diabetes Mellitus (GDM) in women with Polycystic Ovarian Syndrome (PCOS). STUDY DESIGN: Comparative cohort study. PLACE AND DURATION OF STUDY: Gynecology Clinics of Mamji Hospital, Karachi, from 2008 to 2010. METHODOLOGY: Patients who had been diagnosed Polycystic Ovarian Syndrome (PCOS) with hyperinsulinemia and conceived and continued pregnancy, were divided in two groups; 50 patients received metformin throughout pregnancy and 32 did not. Development of GDM was ascertained in both groups. The patients were followed throughout pregnancy and in puerperium with OGTT as per WHO criteria. Primary outcome measure was development of gestational diabetes mellitus. Comparison of continuous variables was done using student 't' test. For categorical variables, frequency and percentages are reported while, odds ratio is also estimated for GDM during pregnancy. RESULTS: A total of 82 women with PCOS were included in this study, out of whom, 50 patients received metformin treatment while 32 patients did not. Pregnant women with PCOS in both groups were comparable in age, weight, parity and BMI. Mean fasting insulin levels at beginning of study entry were 17.22 ± 2.3 mIU/L and 16.93 ± 2.28 mIU/L in metformin and no metformin group respectively (p=0.589). Mean fasting blood sugar levels were 94.54 mg/dl in metformin and 99.59 mg/dl in no metformin group p < 0.001. A total of 5 (10%) patients in metformin group developed GDM while 11 (34.37% OR 4.71, p = 0.01) developed GDM in no metformin group. Patients not receiving metformin were 4.7 times likely to have GDM (OR: 4.71) compared to those who received it. CONCLUSION: The frequency of gestational diabetes, was significantly higher in patients with PCOS who had not received metformin compared to those who did.