Successful isolation, culturing, proliferation and transdifferentiation of stem cells obtained from Umbilical Cord blood and Wharton jelly

Abstract

ntroduction Mesenchymal stem cells (MSCs) and Haemopoietic stem cell (CD34) could be isolated from human umbilical cord blood. In addition to that MSCs like, could also be isolated from Wharton jelly which represent a rich source of primitive cells, with the same properties of cord blood and bone marrow MSCs. Mesenchymal stem cells and haemopoietic stem cells have the capacity of self-renewal, proliferation and in viva or ex vivo differentiation. This study was conducted to compare stem cells from umbilical cord blood and Wharton jelly, focusing on isolation techniques, identification and proliferation using various medium, (MesenCult and DMEM), and differentiation in vivo (albino rats) and ex viva (MethoCult medium), transdifferentiation into hepatocyte like cells and expression of CD34,CD45 and CD10. Materials and Methods Thirty female albino-rats age 5-6 week-old; weight 40-60 g, 20 were used for ‘human cells engraftment, whereas the remaining l0 served as negative controls. Twenty to fourty ml of 25 cord blood samples and length of 12 cm of 25 umbilical cords for Whaiton jelly samples were obtained from 25 newborns delivered after full tenn following ethical consent. The cord blood samples were grouped according to the time of collection after birth into three groups. Group one (G1) include samples processed after 5 - 7 hours, group (G2) after 3 - 9 hours and 9-13 hours for group three (G3), whereas Wharton jelly samples were grouped as G1 which include samples processed afier 56 - 93 hours, G2 afier 19 - 24 hours and 42 - 48 hours for G3 and time of trypsin digestion was 45 minutes for G1 and 30 minutes for G2 &G3.Ficoll Paque was used to obtain the mononuclear cells from cord blood samples while trypsin enzymatic treatment was used for Wharton jelly samples. Purification of CD34 was done using RosetteSep and EasyScp techniques. The expression and count of CD34, CD45 and CD10 was detected by desired monoclonal antibodies and analyzed using tlowcytometric. The ex viva proliferation of MSCs was performed in DMEM at day 7 for G2 and day 14 for G1, whereas for MesenCult media it was performed at day 14 for G3. The differentiation process was done by CFU-F fibroblast assay and hepatocyte growth factor protocol, and checked by monitored microscopic, immunocytochemistry and micro albumin assay. CD34 positive cells were cultured for 16 days in MethoCu]t media afier which viable cells count, colonies count assays, and calculation of fold expansion and plating efficiency were done. In vivo engrattment and cell proliferation was tested using CD34 positive cells in experimental irradiated albino rats (n=l0) and CD34 improved by human MSCs (n=l0) infused by intravenous and intraperitoneal modality. Statistical methods The data was analyzed using SPSS 13, p.value considered statistically significant below 0.05. Descriptive statistics (mean and standard deviation) obtained for numerical variables while frequency distribution and percent for categorical variables. Mean differences were tested by t-test or ANOVA. Results The results showed no significant differences in the count of mononuclear cells isolated from cord blood and Wharton jelly samples based on mother age, weeks of gestation, length of umbilical cord and time of processing. The results also showed that the MesenCult media gave the best MSCs viable cells count and fold expansion compared with DMEM. According to the incubation time, DMEM produced the best viable cells count and fold expansion in 7 days in contrast to the 14 days incubation. A statistical significant difference (p-value = 0.04) was observed when compared the MSCs viable cells count and fold expansion of cord blood group 1 and Wharton jelly group 2 in DMEM. The results also showed that the plating efficiency and colonies count of MSCs to form CFU-fibroblast from Wharton jelly samples was higher with no significant statistical difference (p-value % 0.2) than MSCs from cord blood samples. The activation of hepatocyte cell like obtained from the Wharton jelly samples was higher compared with MSCs obtained from umbilical cord blood. However, the difference turned to be statistically insignificant. Although CD10 was expressed by MSCs, we observed no significant difference between cord blood and Wharton jelly samples. Although EasySep technique gave the best purification count of CD34 and haemopoietic colonies count (CPU-E, BFU-E and CPU-GM) in ex vivo methoCult media, R0setteSep gave the best self renewal, proliferation into CD45 and engrafiment in albino rats. In addition the combination of CD34 and MSCs gave the best engraftment and the best site for administration was the intravenous one. Conclusions According to the results, Wharton jelly was the best source of MSCs when as compared to cord blood. Regarding CD10 expression, the cord blood was the best compared to Wharton jelly. The best media for stem cell research was the MesenCult obtained from Stem Cell Technologies Company. The best technique for CD34 isolation was the positive selection conceming EasySep technique, although the best in vivo engrafiment can be obtained by R0setteSep technique. MSCs look to have the same properties of bone marrow stroma, when it is combined with the haemopoietic stem cell (CD34) in case of in vivo engraftment.