First-Step In Vitro Human Umbilical Cord Blood Mesenchymal Stem Cells Differentiated Into Hepatocyte-Like Cells

Objective: Obtain and culture mesenchymal stem cells(MSCs) from human umbilical cord blood(HUCB) in vitro. Investigate a better one by several different culture environment of HUCB-MSCs. We also study the condition in which MCSs differentiated into hepatocyte-like cells.Methods:1.UCB was collected after the baby was delivered .Separated mononuclear cells(MNCs) from UCB by gradient centrifugation .2.Using adherent culture, MSCs was obtained. And compare the effect of the density of MNCs, serum concentrations for MSCs.3.Collected forth- to eighth-passage(P4-P8) cells, CD29, CD44, CD34, CD45 was detected by flow cytometry.4.Using conventional methods, cryopreservation and recovery mesenchymal stem cells.Observe the growth of MSCs after the recovery.5.Collected forth- to eighth-passage cells, induction is divided into five groups,which respectively cultured with LO2 commonly ,or cultured with several cytokines at the same time,or cultured with several cytokines step by step. All of them had been observed for 6w.6.By RT-PCR, albumin (ALB),alpha-fetoprotein (AFP),cytokeration (CK19) mRNA was detected. Results:1 .It was best that the density of 10~8/ml and the first time of changing culture medium after 7d for the growth and proliferation of MSCs in vitro.2.Adherence was difficult to be observed in 5% serum.The growth and proliferation of MSCs was best in 15%-20% serum.But MSCs can keep primary characteristic more easily in 15% surem than in 20% surem.3.The result of flow cytometry: CD29 (+), CD44 (+), CD34 (-), CD45 (-). So the adherent cells were proved to be MSCs. The result of RT-PCR: ALBmRNA(-),AFPmRNA(-)及CK19mRNA(-) in MSCs。4. In this study, proliferation of MSCs was significantly weakened after passage 8(P8). Size of cells was larger and shape of cells was irregular. MSCs of passage 10(P10) were unable to survive .After recovery, MSCs undergo a platform period for 1w ,then gradually restored the pre-frozen state.5.First group(LO2 common culture) and second group(A medium) and third group(common culture LO2 +A group) were observed no change and ALBmRNA(-), AFPmRNA (-), CK19mRNA (-) after 6w.6.Forth group(B medium) also was observed no change and ALBmRNA(-), AFPmRNA (-), CK19mRNA (-) after 6w.7.Fifth group(C medium) was observed changing of cell morphology and AFPmRNA (+),ALBmRNA(-),CK19mRNA (-) in 4w.In 6w, AFPmRNA, ALBmRNA, CK19mRNA were detected.But cell density decreased significantly.Conclusion:1 .There are mesenchymal stem cells in human Umbilical cord blood . But the amount is very small. Biological differences also exist between different individuals. It was later that the adherence time of MSCs from HUCB than that from HBM.And HUCB-MSCs showed high concentration and high-FCS(fetal culf serum) depended.10-15%FCS is best.But high-FCS(>20%) made MSCs differentiate more easily.2. In vitro, capacity of passage is limited for MSCs. Vitality of MSCs (within P4-P8) is good. But the cells(within P10) can not survive.So it is impossible to achieve unlimited proliferation in vitro now. After recovery, MSCs undergo a platform period for 1w ,then gradually restored the pre-frozen state.3.It was difficult that MSCs differentiate into hepatocyte. In five-induced group of this study, hepatocyte-like cells was obtained only in fifth group. RT-PCR detected AFPmRNA(+), ALBmRNA(+), CK19mRNA(+).4. In the process of induction, cell adherent is poor, the weak vitality, amplified difficulties. In order to achieve clinical application ,we must solve the problem of amplification of induced cells in vitro.