An Experimental Study On Isolation And Cultivation Human Umbilical Cord Blood Mesenchymal Stem Cells And Induction Differentiation Into Hepatocyte-like Cells

BackgroundStem cells are defined as cells that have clonogenic and self-renewing capabilities and that differentiate into multiple cell lineages. Stem cells lineages maintain itself by cellular fission and have the ability to generate any terminally differentiated cell in the body to constitute all kinds of complicated organization. As seeds cells of biological medicine tissueengineering technique, stem cells bring the new hope to dream about human health and longevial, became the hot spot of biological medicine research. According to the stages of stem cells differentiation, stem cells were divided into embryo stem cells (ESCs) and adult stem cells (ASCs). Though ESCs have omnipotent differentiation potentiality, there are considerable ethical issues surrounding their study and clinical application. ASCs resided in adult tissue, had characteristics of abundance resource, powerful plasticity, recent to mature cell and multiple differentiation potentiality, could conduct individualize treatment, had little immunological rejection problem, so that it was the seeds cells for clinical application prevalently, could conduct individualize treatment, had little immunological rejection problem, so that it was the seeds cells for clinical application prevalently.Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were studied widely among all of ASCs. Bone marrow is most common source of HSCs and MSCs, so is umbilical cord blood (UCB). It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells, and transplantation of cord blood has been part of clinical practice for more than 10 years. However, controversy exists as to whether UCB contains mesenchymal stem cells (MSCs), which are capable of differentiating into cells of different tissues and are the best candidates for tissues engineering.So we attempted to isolation and cultivation Human Umbilical Cord Blood Mesenchymal Stem from UCB , and induce them differentiation to hepatocyte-like cell in vitro, expected to conduct basic research work in ASCs transdifferentiation. Our study attempted to provide new feasibilitypathway for hepatocyte resource of clinical liver diseases treatment. ObjectiveTo establish isolation and culture method for UCBMSC sane observe cytobiology characteristics and phenotypes of MSCs in order to provide MSCs resource. To induce UCBMSCs differentiation to hepatocyte-like cell in vitro by the supernatant fluid of human hepatocyte line cultivation, and identify cytobiology characteristics and phenotypes of the induced UCBMSCs.Methods1. To isolate UCBMSCs by combination density gradient centrifugation and directly adherence growth, then set them culture and passage in the medium consists of DMED(LG) and 20% calf serum; observe cytobiology characteristics and phenotypes; draw primary growth curve; identify the espression of hepatocyte markers such as ALB and CK18 was measured with immunohistochemistry.2. To induce UCBMSCs differentiation to hepatocyte-like cell in vitro by the supernatant fluid of human hepatocyte line L-02 cultivation;3. Contrast with uninduced UCBMSCs and L-02 cells, observe cytobiology characteristics and phenotypes of induced UBCMSCs; identify induced UBCMSCs the expression of hepatocyte markers such as ALB and CK18 was measured with immunohistochemistry.Results1. After the mononuclear cells (MNCs), which were acquired by density gradient centrifugation, were set in culture plates for 24 hours, onlya few cells attached to the plastic culture dishes and grew round. After 48 hours, the adherent cells increased and part of them began to display fibroblastoid cells. Through repeatedly exchanging medium, all adherent cells mostly displayed fibroblastoid cells. The adherent cell began logarithmic growth about at the sixth day. Once adherent cells reached approximately 80-90% confluence, they were passaged.2. Second filial generation began to attach and adherent , then displayed fibroblastoid cells in 12 hours. After 3 days cells displayed Fusiform shape or polygon, and grew in uniformity scatter. It is about at the 10th day that adherent cells reached approximately 80-90% confluence. As passsging, they transfigure became faster and the cell appearance was more uniformity.3. UCBMSCs were cultured at different densities, 1×106 cells/ml and 1×107cell/ml. The densities of 1×107cell/ml was more suitable for cell growth. Among 6-12 days cell grew in period of proliferation, and grew in period of platform after the 12th day.4. Immune phenotypic analyses by flow cytometry showed that 16.8% of MNCs expressed CD44 and 29.6% expressed CD34. After 14-days culture, 16.8% of UCBMSCs expressed CD44 and 29.6% expressed CD34. 54.4% of UCBMSCs expressed CD44 and 6.0% expressed CD34 at passage 3, and 97.9% of UCBMSCs expressed CD44 and 0.5% expressed CD34 at passage 5.5. All cells including at primary, at passage 3 and passage 5 didn't express ALB and CK18 by immunohistochemistry.6. Contrast induced cells by the supernatant fluid of humanhepatocyte line cultivation, and uninduced UCBMSCs, cells' appearance didn't change in predominance.After UCBMSCs were induce and differentiation to hepatocyte-like cell in vitro by the supernatant fluid of human hepatocyte line L-02 cultivation in 14 days, the induces cells express ALB and CK18 by immunohistochemistry.7. Immune phenotypic analyses by flow cytometry showed that L-02 cells didn't express CD44 or CD34. But 96.5% of the induced UCBMSCs expressed CD44 and 93.1% expressed CD34.Conclusion1. UCBMSCs are isolated easily by combination density gradient centrifugation and directly adherence growth, and are uniformity by repeatedly changing medium. UCBMSCs proliferate prosperity in vivo, and there is not ethical controversy. So it is feasibility as seeds cells of tissue engineering reconstruction.2. Induced UCBMSCs express hepatocyte markers such as ALB and CK18. During UCBMSCs transdifferentiation into hepatocyte-like cell, UCBMSCs differentiate firstly to the precursor of hepatocyte, then differentiate to mature hepatocyte, hepatocy-like cell with positive hepatocyte marker are obtained under this experimental induction condition.