ABSTRACT
Objective: Worldwide, diabetes mellitus [DM] is an ever-increasing metabolic disorder
A promising approach to the treatment of DM is the implantation of insulin producing cells [IPC] that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells [hUCMs] into IPCs and measured insulin production
Materials and Methods: In this experimental study, we exposed hUCMs cells to pancreatic medium and differentiated them into IPCs in monolayer and suspension cultures
Pancreatic medium consisted of serum-free Dulbecco's modified eagle's medium Nutrient mixture F12 [DMEM/F12] medium with 17.5 mM glucose supplemented by 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, and B-27 serum-free supplement. After differentiation, insulin content was analyzed by gene expression, immunocytochemistry [IHC] and the chemiluminesence immunoassay [CLIA]
Results: Reverse transcription-polymerase chain reaction [RT-PCR] showed efficient expressions of NKX2.2, PDX1 and INSULIN genes in both groups. IHC analysis showed higher expression of insulin protein in the hanging drop group, and CLIA revealed a significant higher insulin production in hanging drops compared with the monolayer group following the glucose challenge test
Conclusion: We showed by this novel, simple technique that the suspension culture played an important role in differentiation of hUCMs into IPC. This culture was more ef- ficient than the conventional culture method commonly used in IPC differentiation and cultivation
Subject(s)
Diabetes Mellitus , Umbilical Cord , Mesenchymal Stem Cells , Pancreas , Cell Culture TechniquesABSTRACT
Background: Fetal bovine serum [FBS] is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded
Objective: To find FBS substitute, we tested human umbilical cord blood serum [hUCS] for proliferation of human umbilical cord matrix derived mesenchymal stem cells [hUC-MSCs] and human bone marrow-derived mesenchymal cells [hBM-MSCs]
Materials and Methods: Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium [IMDM] by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining
Results: The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium
Conclusion: Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies
ABSTRACT
Retinoids have been suggested to play a role in oogenesis and oocyte survival. In the present study the effects of retinol palmitate were investigated on differential follicular counts in response to superovulation as well as follicle quality after vitrification of ovaries. Ten, 4 week old female BALB/c mice were randomly assigned to either paraffin [n=5] or retinol palmitate [n=5] administration. Vitamin A administered animals received [i.p.] 250 IU retinol palmitate, dissolved in 0.1 ml of paraffin oil on days one and ten followed by superovulation with 10 IU PMSG. Paraffin administered mice were only treated with 0.1 ml of paraffin oil. The collected left ovaries from both paraffin and vitamin A administered groups were considered as non-vitrified and the collected right ovaries from both treated groups underwent vitrification. Ovaries in the vitrified group were frozen sequentially by placing into two vitrification solutions [VS1: 10% ethylene glycol [EG], 10% DMSO in holding medium] TCM-199 + 20% FBS: HM] and VS2: 20% EG, 20% DMSO in HM]. After warming, recovered ovaries as well as nonvitrified ovaries were serially sectioned and examined histopathologically. The proportion of antral follicles in the non-vitrified ovaries from vitamin A administered mice was statistically higher than the non-vitrified ovaries from paraffin administered group [29.4% vs. 15.6%, respectively; p<0.001]. No difference due to retinol palmitate injection was observed for the rate of small follicles between the two non-vitrified groups. The percentage of damaged follicles did not show any significant differences between the two vitrified groups [76% vs. 79%]. Our results demonstrate that administration of retinol palmitate may improve the response to superovulation through the shift of follicular growth towards antral follicle development. However, no positive effect of retinol palmitate in the quality of follicles is probable when ovaries are vitrified