UCB is a rich source of MSCs 20. The capability to obtain effective MSCs-derivedhepatocytes would improve celltherapy for liver diseases 24. Wesuccessfully isolated and characterized MSCs from 11 samples of 30 (36.67%) with high viability and proliferationcapacity. In support of our findings, Denner et al. 25 succeeded to isolate UCB-derived MSCs from 16 samples of 42(38.09%), and Kazemnejad et al. 26 separated MSCs from 4 of 11 cord blood samples (36.
4%). In this study, the cultured cells had specific characters of MSCs (they were spindle shaped cells attached to thesurface with high proliferation capacity). Thesecells were positive for MSCs markers, including CD90 (41.
3 ± 1.7 %) and CD105(73.5 ± 3.
8 %) but negative for hematopoietic markers, including CD34 and CD45. This was alsoreported by Semedo et al. 27. Effective trans-differentiationof MSCs towards hepatocyte-like cells in vitro was routinely achievedusing an array of recombinant growth factors HGF, fibroblast growth factor(FGF), epidermal growth factor (EGF), cytokines Oncostatin M (OSM) andchemical compounds (dexamethasone, nicotinamide, insulin etc.) either as acocktail or in a sequential manner 14.
However, hepatic inductionswith multiple recombinant growth factors are not optimal for clinicalapplications as it is expensive and time-consumingtechnique. HGF is the key factor for liver growth and function. In ourstudy, we successfully differentiated UCB-derived MSCs into hepatocyte-likecells by HGF and we tried to detect the best time for harvesting the maximumnumber of functional hepatocytes. In thepresent study, differentiation was evident by cytoplasmic contraction,accumulations of cytoplasmic granules and change in fibroblastic morphology ofMSC to cuboidal, polygonal morphology of hepatocytes. Themorphological change and confluence of induced cells were time dependent and by the end of the day 20 differentiatedcells were very similar to hepatocytes appearance with the highest confluence.
The viability of induced MSCs was high along the culture duration, with a narrowrange (87%-98%). So, it didn’t significantly correlate with any of the measuredvariables (culture duration, confluence, urea production and albuminsecretion). Our data demonstrated that the number andviability of induced cells increased significantly during culture till day 20 thatmay be due to anti-apoptotic and anti-necrotic effect of HGF. The viabilityof induced cells reached its maximum ratio after 20 days of induction and thenthe viable cells decreased significantly with longer culture duration, whichmay be due to decreased amount of nutrient and increased wastes formed by thecells in the crowded media.
It may also be that, the cells reached a maximumnumber of divisions and were unable to divide anymore. This goes with theresults reported by Kang et al. 28, who found that hepatocyte-likecells can be obtained from MSCs after 21 daysof culture in hepatogenic mediumwith high number and viability. On theother hand, Nonome etal. 17 cultured human MSCs for 28 daysin hepatogenic medium containing HGF and FGF and found that the viability ofcultured cells decreased with time. In the currentwork, The concentration of ureaincreased significantly on day 12 and reachedmaximum concentration on day 20 then significantly decreased with longerculture duration, which may be due to significant decrease in viability. Ureaproduction is significantly positively correlated with culture duration,confluence of induced MSCs and albumin secretion. Our results were supported bythose demonstrated by Yang et al.
29 andWang et al. 30, who found that during differentiation of MSCs, urea production was detected in low level on day 12 then ureaproduction gradually increased until day20 of induction. The immunocytochemicalanalysis, herein, showed that the percentage of AFP-producing cells andintensity of brown color increased significantly till reach maximum (45% ±2.887) after 20 days of induction with HGF.
Theseresults could be explained by the concomitant increase in the percentageof cells which showedmorphological transformation from bipolarfibroblast-like morphology to round or oval-shaped cells, which reached ? 45%on day 20. After day 20, the amount ofsecreted AFP was decreased gradually with longer culture duration, which may be attributed to the observed significantdecrease in viability of induced cells after day 20. Similar results were recorded by Tang et al. 31, whostudied the hepatic differentiation of human UCB stem cells by HGF and FGF andfound that the percentage of AFP producing cells increased with culture timetill day 21 at which about 50% of differentiated cells expressed AFP. In the present work, induced MSCsexpressed AFP and albumin after incubation with HGF in a time dependent manner.
The concentration of secreted albumin was significantly positively correlatedwith culture duration, confluence of cells, and the amount of secreted urea. Quantitative gene expression study, herein, showed a significantincrease in gene expression of albumin (8.1 fold) on day 20, then a decrease on day 24 (6.
3 fold), and a further decrease after 28days of culture (5.8 fold). Our results can be interpreted by the workof Bishi et al.
14, who reported that albumingene is a late hepatic gene which was produced in higher concentration inmature hepatocytes than immature hepatocyte in developing stage.Conclusion: HumanUCB-derived MSCs can differentiate into hepatocyte-like cells in vitro whencultured in nutrient media containing HGF. These hepatocyte-like cells showedhigh functional capacity as evidenced by albumin production, urea secretion andAFP expression. The process of differentiation was time dependent with its peakon day 20 of culture. Our study provides a simple and cheap strategy for in vitrodifferentiation of human UCB-derived MSCs into hepatocyte-like cells during 20day using HGF.