The Study Of Biological Characteristics Of Mesenchymal Stromal Cells Derived From Wharton's Jelly Cells And Their Cocultivation With Brain Tumor Stem Cells

Background and Objective:The doctrine of cancer stem cells, which reckon that a small minority of tumor cells with the ability of self-renewal and potentially differentiation in tumor tissue are responsible for proliferation, growth, metastasis and recurrence of tumor, has been put forward more than 30 years ago. In 1992, Reynolds put forward the concept of neural stem cells (NSCs) based on the research on mice.5 years later, the article, wrote by Mckay concerned about NSCs, published in Science has been recognized. In 2003, Singh cultured and identified brain tumor stem cells (BTSCs) from different pathological types of human brain tumor. BTSCs has the capability of proliferation, self-renewal, differentiation and expression of neural stem cell surface marker CD133, which makes it become a new target for treatment of brain tumors. In recent years, numerous studies confirm that BTSCs is the origin of the brain tumor cells, play a decisive role in the tumor occurrence and development, and is also the roots of radiation therapy and chemotherapy tolerance.The BTSCs-related molecular markers and tumorigenic mechanism is a hot research field, but also for the future of brain tumor research and clinical treatment.It is proven that once NSCs has been transplanted into the brain tumor, it could acquire the ability of directional migration, proliferation surrounding the brain tumor, thereby reducing the invasiveness of brain tumors.However, graft-versus-host disease and the potential tumorigenicity after transplantation and ethical issues of sources of tissue make people pay more attention to the mesenchymal stem cells (MSCs).Mesenchymal stem cells (MSCs) is derived from the number of embryos in stem cells, with a high degree of self-renewal capacity and more potential to divide, in appropriate in vivo or in vitro environment, MSCs not only differentiate into hematopoietic cells, Has divided hematopoiesis outside for a variety of organizations, especially in the cells of the embryo's ability, and even cross-layer differentiation of embryonic nerve cells of the tumor of origin potential, so they have a high degree of plasticity, is a new type of seed cells in cell transplantation and tissue engineering that have broad application prospects. Previous studies have focused on the study of bone marrow MSCs, but there are also other organizations that MSCs, including the mobilization of peripheral blood, umbilical cord blood, adipose tissue, placenta tissue and deciduous, however, these organizations have a limited number of cell restrictions on the research and development.The study found that in recent years people from the AMT, the umbilical cord, amniotic fluid can also be extracted from a good proliferation of MSCs, which for better access to MSCs to provide a new way, and with the in-depth study found that MSCs, MSCs Can also be induced to differentiate into cardiac cells, liver cells and neurons and other cells, MSCs a variety of differentiation potential of the research staff to be more and more attention. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. Whereas the source of umbilical cord is quite rich, the use of it is without ethics problem.This study is to be concentrated mainly on the research of the biological characteristics of WJCs, whether it can influence the biological characteristics of BTSCs after the two type cells co-cultured in vitro, and then provide a theoretical basis for cell therapy.Methods:The isolation and cultivation of WJCs:Taking about 10cm umbilical cord being removed the umbilical cord membrane, the two umbilical arteries and one umbilical vein were, and retained Wharton's jelly. Wharton's jelly is fully washed by PBS buffer and cut into pieces to the size of 2mm×2mm×2mm.WJCs immunophenotype identified:flow cytometry CD29, CD44, HLA-ABC, HLA-DR, CD34, CD45.WJCs immunofluorescence identified:GFAP and Nestin.Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:flow cytometry CD29, CD44, HLA-ABC.Comparison of Proliferation ability of WJCs came out from Wharton's jelly at different times by CCK-8:mapping the cell growth curve of 7 days.The isolation and cultivation of BTSCs:wash fresh tumor tissue with PBS buffer and then remove the necrotic tissue and capillary from it. Filter the tissue with 80 mesh and then digest in the 2.5 g/L trypsin and 1.0 g/L collagenaseⅣat 37℃for 30 minutes. After digestion, filter the digestive juice with 200 mesh then remained single-cell suspension. Counting of living cells in a final concentration of 2×105/ml, then inoculate it in serum-free medium (SFM) and placed in T75 culture flask added 10ml culture medium into.BTSCs immunophenotype identified:flow cytometry CD133.BTSCs immunofluorescence identified:GFAP and Nestin.CCK-8 assay of proliferation of BTSCs:mapping the cell growth curve of 7 days.WJCs co-cultured experiments with BTSCs:Co-culture of two kinds of cells in 24-well plates in serum-free medium without any growth factor. Collect the centrifuged suspension cells, and then analysis expression of CD133 by flow cytometry and at day 3 and 7 respectively. Analysis adherent cells expression of Nestin and GFAP by immunofluorescence assay at day 3 and 7 respectively. Co-culture supernatant (CCS) acquired at day 3 resuspend BTSCs. Compare the differences of cell growth curve between cultured in CCS and in normal suspension by Microplate Reader.Results:The isolation and cultivation of WJCs:It can be seen that a single spindle or triangular adherent cells come out from tissue being adherent after 3 to 5 days. After 1 week, adherent cells are swimming out of tissue. Cells gradually become from the multi-layer to single-layer, loose to intensive, and small to big. Cell morphology is mostly full of long spindle-shaped or flat fibroblast-like cells with prominent nucleoli. Two weeks later, it turns out to be uniform spindle cell morphology similar to fibroblasts, showing parallel growth or vortex growth.WJCs immunophenotype identified:high expression of CD29 (91.50%), low expression of CD44 (36.92%), HLA-ABC (28.10%), not express CD34 (0.72%), CD45 (0.11%) and HLA-DR (0.80%)WJCs immunofluorescence identified:None-induced WJCs does not express Nestin and GFAP.Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:no difference in expression of CD29, CD44, HLA-ABC.Comparison of Proliferation ability of MSCs came out from WJCs at different times by CCK-8:no difference in cell growth curve.The isolation and cultivation of BTSCs:1 days after inoculation, it is observed under the inverted microscope that a large number of suspended growth and diversity of tumor cell clusters and some single-cell suspension with not obvious refraction.7 days later it can be seen suspension bulk of cells are composed by 2 to 5 the size of cells. After 10 days bulk of cell volume significantly increased with the regular shape and strong refraction. Disperse then subculture the cells, the majority of cells formed news bulk of cells 1 day later.BTSCs immunofluorescence identified:Without induced BTSCs express Nestin but GFAP.WJCs co-cultured experiments with BTSCs:After 24h separately incubation of WJCs in the 24-well plate, then wash plate with PBS to remove non adherent cells. It can be seen most of the spindle, homogeneous scattered growth of the typical WJCs in the bottom of the plate. After 1 day co-cultivation, it is shown two phenomena. One is that BTSCs can form a typical sphere of cells, it can be viewed 3 to 5 clone and connected with adherent WJCs, while the number and form of WJCs is almost no difference with negative control; another is that BTSCs can hardly form a typical sphere of cells, they are more dispersed as single cells, floating in the culture medium covering WJCs. Compared with negative control, WJCs rapidly proliferate and form many cell colony with the elongation of cell body connected with each other. Negative control of BTSCs began to form typical sphere of cells floating and rolling in the medium.After 3 day co-cultivation, it is shown two phenomena. One phenomenon is that sphere of BTSCs connected with WJCs begin adherent to the plate. Tumor cells are climbing out from Sphere of BTSCs with the astral, spindle morphological, and format the radial synapse and connection with proliferation of WJCs around. Adherent BTSCs is much smaller than the WJCs. Another is that BTSCs is still scattered floating in the medium without morphology changing compared to the first day. But WJCs is proliferating obviously, becoming wider and bigger and occupying the range of 70%of the vision.After 7 day co-cultivation, there is shown two phenomena. One phenomenon is that most of sphere of BTSCs connected with WJCs are adherent to the plate. Another is that BTSCs is scattered floating in the medium or adherent to the plate. Negative control of BTSCs and WJCs has normal morphology.We can see the results that the higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was and CD133+in BTSCs declined when co-cultured with WJCs.Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously.Conclusions:1. WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. The original WJCs came out from Wharton's jelly at different times express stable positive immunophenotype and have stable growth curve. That indicates more WJCs can be gained and used.2. With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated.3. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+in BTSCs declined when co-cultured with WJCs.4. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro.