Isolation And Identification Of Umbilical Cord Mesenchymal Stem Cells

In recent years, due to cell replacement therapy for many diseasesbrought hope, related research has become a focus of research in the lifesciences and medical field. So far, the umbilical cord mesenchymal stemcells have been in many ways is proved to be ideal cell autologous andallogeneic cell transplantation, treatment. Compared with bone marrowmesenchymal stem cells, umbilical cord mesenchymal stem cells aresimilar in biology and immunological properties, but in the proliferation,differentiation, than bone marrow mesenchymal stem cells have morepotential. More importantly, bone marrow mesenchymal stem cells havegreat limitations, based on in age, disease, injury of donor has affected itsfurther application. Umbilical cord mesenchymal stem cell source is wide,convenient, no adverse effects on the donor, at the same time, there hasno ethical restrictions. Therefore, two kinds of cells in contrast, expectmesenchymal stem cells has a broader application prospect.This experiment is through tissue culture method and enzymaticdigestion method to obtain the umbilical cord mesenchymal stem cellsout of the umbilical cord of fetal which in waste. The source of materialsis from the Department of Obstetrics and gynecology hospital after thebirth of informed consent. In enzyme digestion of umbilical cordWharton’s jelly process, we use three different digestion methods whichdigestion with collagenase type II only, trypsin only and collagenase typeⅡ mixed with pancreatin. Through the observation we found that thethird one is the best method which can get the most cells in the shortesttime. At the same time in the experiment, we testing the optimumconcentration of serum, the experiment found in primary culture in theprocess, the15%serum concentration is the most suitable for cell togrowth. To identify this cell, we use the flow cytometry for examine. The cell has expressed CD29, CD44, CD73and CD166which is the typical ofthe umbilical cord mesenchymal stem cells that we can establish cell linesthrough culture. In order to test the umbilical cord mesenchymal stemcells can grow stably, we testing different generations with random.When we get stability human umbilical cord mesenchymal stemcells, the third passage is induced the cell to differentiation direction.Because mesenchymal stem cells is pluripotent stem cells, it candifferentiate into three germ layers, Therefore, this experiment aboutmesenchymal stem cells had differentiate it into adipocytes, osteoblastsand neurocyte.When the third generation of hUC-MSCs growth about60%in petri dish, we discard basic culture medium and added intoadipogenic induction medium, osteoblasts induction medium andneurocyte induction medium. In adipogenic induction process, seven dayslater cells have change obviously, lipid droplets has created, fourteen dayslater, after oil red O staining confirmed, we successfully to get adipocytes;During osteogenic induction, about seven days the cells began to changeinto the polygonal cells, we found the calcium nodules in fourteenth day,with alizarin red staining confirmed the induction was successful toinduce mesenchymal stem cells into osteoblasts; In the process of inducedhUC-MSCs into neurocyte, the cells began to change after we added theinduction medium when6hours later, they began to out of protrusions,with the extension of time, the protrusion length increasingly, and formthe similar morphological with neurocyte after3days, though theimmunocytochemical staining, it shows strong positive expression ofNestin, confirmed to neurocyte induced was successful.Proved by experiments, the umbilical cord mesenchymal stem cellsare widely distributed in the umbilical cord tissues. Through theexperiments, we established a mature and stable method for isolation and amplification of umbilical cord mesenchymal stem cells in vitro, whichcan help a lot for its application in clinical treatment.