The Transformation Of Human Umbilical Cord Mesenchymal Stem Cells Into Hematopoietic Cells

After40years of development, Hematopoietic stem cell transplantation has becomean effective method for the treatment of various types of hematologic malignancies.Transplant cases worldwide each year are on the rise. Transplant patients without thedisease-free survival up to over30years of successful implantation of hematopoietic stemcells play a therapeutic role in HSCT host by HLA consistency in the clinical treatment.Infusion of hematopoietic stem cells is the insufficient number of high-dose chemotherapybefore transplantation caused by bone marrow stromal damage and the occurrence ofgraft-versus-host disease will result in hematopoietic stem cell implant failure orimplantation delay, so that the transplantation of an increase in mortality, makes thetechnology is facing great difficulties in practical applications.Especially in the treatment of some hematologic malignancies, due to the long-term,multiple sources of allogeneic HSC can easily lead to fatal lung injury and infection aftertransplantation.Therefore, to resolve the source of HSCT in the HSC and its securityproblems, stability, and efficiency of hematopoietic stem cells or cells with hematopoieticpotential to become the key to further promote the HSCT, which not only has importantmedical implications, and will have huge social and economic benefits.Past the point ofview, the undifferentiated cells differentiate to form specific types of terminal cell of thisprocess is not reversible. But in recent years, development of biological and stem celltechnology can be successfully transformed into a type of tissue-specific cells to formanother type of cell. In HSCT patients, also supports these arguments. Few months afterHSCT, found in the patient's bone marrow or hematopoietic tissue derived from the hostepidermal cells, liver cells, and even appeared in the digestive tract epithelial cells. Theseresults indicate that human stem cells in vivo environment, there is a certain degree ofuncertainty in the differentiation process.Epigenetic studies may also provide for the mutual conversion between the terminalcell.Experiments that can be said that the epidermal cells or other cells inducedhematopoietic cells. Mesenchymal cells are easy to obtain the more naive stem cells, anddifferentiation potential, and are easy to proliferate in vitro. If the mesenchymal cells canbe successfully induced the formation of hematopoietic cells, proved the mechanism oftransformation, will have broad application prospects.This topic to be from humanumbilical cord mesenchymal stem cell precursor cells, passage purification after the single cell cloning cultivation, sub-elected with multilineage differentiation potential of Asiatotipotent cell populations more homogeneous, Explore the feasibility of differentiationtoward the hematopoietic cells, further study of cell differentiation Cheng Zhongqiepigenetic changes. With adult sources, such as bone marrow mesenchymal stem cellscompared to hUMSCs naive lower degree of HLA antigen expression weaker,amplification and multi-differentiation ability is also strong; Compared with the HSC, bothof which are derived from extraembryonic mesoderm, mesenchymal tissue, so in theory thepossibility of transformation.HSCT in the MSC has been used as an auxiliary cells, but if a stable MSC inducedthe formation of the HSC will HSCT in the existence of an unstable and allogeneictransplantation and play an invaluable role in clinical practice.Part1. Isolation and Identification of hUMSCsMethods:(1) attached method and enzyme digestion of primary cultured hUMSCspassaged purified single cell cloning cultivation, sorting the hUMSCs cell populations.(2)light microscopy and electron microscopy to observe the hUMSCs primary and passagedmorphological features.(3) take the4th generation (P4) hUMSCs immunofluorescencestaining and flow cytometry identification compare two specific cell surface molecules:Vimentin, of α-SMA, Oct-4, Sox,2, and HLA-1, CD29, CD34and of CD133and CD44and CD45expression.(4) Take the P4hUMSCs flow cytometry cell cycle DNA content.(5) For the cloning hUMSCs in vitro induced differentiation, identified the potential ofadipose differentiation of osteoblasts direction.Results: The primary cultured after7days, visible culture flasks clone-like growth ofcell mass, the subculture of single cell cloning:(1) spindle or spindle morphology,abundant cytoplasm; Transmission electron microscopy revealed P4hUMSCsfusiformnucleus different chromatin less prominent nucleoli.(2) surface signs, immune fluorescencecell chemical detection display, the P4hUMSCs are the expression of stromal cell skeletonprotein "Vimentin", early smooth muscle cell surface marker alpha SMA, the pluripotencytranscription factors Oct4and Sox2, between the charge and quality of stem cell surfaceantigen CD29and CD44, of which23%of the cells express CD133,5%cells expressingHLA-1,).5%of the cells express CD34and CD44.(3) cell cycle analysis, hUMSCs morethan80%of cells in the G0/G1phase cells of the quiescent cells in S+G2+M's anaverage of about17%, in line with the characteristics of stem cells in the cell cycle.(4) a single cell sources of P4hUMSCs has the potential of terminal cell differentiation to bonecells and fat cells in vitro.Conclusion:(1) we have obtained hUMSCs with most of the mesenchymal stem cellmarker, and CD133-positive rate is higher that relatively naive cell numbers moreHLA-positive rate was low showed that stem cells derived from umbilical cord tissueweakimmune markers, and more suitable for the process of stem cell transplantation.(2)hUMSCs to the differentiation of fat cells and osteoblasts, suggesting that theirdifferentiation potential.Part2. Chemical inducting hUMSCs differentiation toward the hematopoietic cellsMethods:(1) hUMSCs direction in vitro differentiation of hematopoietic cellsinduced by the model: the establishment of the5-Aza and hematopoietic growth factor GFcommon induction for the experimental group, the GF induction and for the inductiongroup as a control group. Experiment with the basic medium for the methyl cellulose ofmedium induced concentration set at2.5μg/ml5-Aza,50ng/ml GM-CSF and SCF, the setinduced1d,3d,5d,7d and10d of the different observation time, as far as possible toincrease the proportion of positive cells for CD34.(2) after the induction of cell surfacemarkers detected: the microscope after the induction of cell morphology and ultrastructureof the change; flow cytometry detection of surface markers, including CD34, CD45, ofCD133, CD11b and CD44and for CD29; immune fluorescence detection of morphologicalchanges when the cell expression HOXB4and Vimintin,; PCR reaction related signalingmolecules within the hematopoietic cells were detected Hoxb4, Wnt3a and Gata2, Scl-1.(3)detection of the induced cells of hematopoietic function: the use of experimental classicalspleen colony will the induced hUMSCs injected into the receiving lethally irradiated nudemice, nude blood, spleen and bone marrow biopsies detected after15days.Results:(1)5-Aza/GF induced by the combined group of cells suspended in culturemedium as the induction time, showing a set of drop-like growth form. TEM observation,the decrease in the number of organelles within cells, cell morphology nucleoplasm furtherincrease, nuclear chromatin distribution similar to the hematopoietic cells.(2) cell surfacemarkers: CD34, CD45and CD133positive cell proportion as the induction time andgradually increased for CD29and CD44was gradually decreased. Immunofluorescenceassay on cells induced5d stretched, some of which form the rounded nucleus has highexpression of Hoxb4, but still the spindle cells still expressed in the cytoplasm Vimintin; PCR detection of hematopoietic signal expression of Hoxb4can be seen-Scl-1, Wnt3a andgata2expression as induced increase in the number of days increase.(3) of hematopoieticfunction tests: the spleen set off the experiment, collecting peripheral blood of miceinduced by blood analysis inoculation5-Aza/GF granulocytes increased in the cell group ismore obvious, statistically significant; mouse spleen section staining and bone marrowanalysis shows that a large number of cells in the tissue expression of HLA-1.Conclusion: After induced by5-Aza/GF hUMSCs in morphology do not have themorphological characteristics of mesenchymal cells, similar to the gradual and adult HSC,on the surface of molecular markers, a large proportion of cells begin toexpresshematopoietic stem cell-specific molecule CD34and CD45, and a more primitivecell marker CD133also increased, cell shape change, intracellular signaling moleculesinvolved in the hematopoietic process Hoxb4expression was significantly increased, butalso by the spleen colony experiment can provethe induced hUMSCs in the body with thefunction of hematopoietic reconstitution. Be seen in the5-Aza/GF induced by thecombined effect, hUMSCs toward the direction of differentiation of hematopoietic cells,hematopoietic cells with the morphological characteristics and features.Part3. Changes of MiRNA in the differentiation of hUMSCs to hematopoietic cellsMethods:(1) according to the of MiRNA Library forecasts and reported in theliterature, selected miR-126miR-451, miR-181a, the miR-150miR-218is the main objectof study, the use of more than five primer6.0biology software designArticle by reversetranscription of RNA and RealtimePCR primers.(2) to collect5-Aza/GF induced by thecombined3d,7d and10d hUMSCs separate GF induced experimental control, HSC andhUMSCs for positive and negative controls, the cell extract RNA, Trizol RNA extractionreagent lysis collected. The role of MMLV reverse transcriptase downstream reversetranscription reaction to detect the differences of the different time points MiRNA,syberGREEN system realtimePCR reaction.(3) large selection of differential expression ofMiRNA vitro synthesis MiRNA fragment, liposomes and transfected into culturedhUMSCs in24hours after the Realtime PCR, to detect the expression changes, the use ofmethyl cellulose of medium the cells, collected1w after the transfected cells by Westernblot detection of hematopoietic factor Hoxb4expression to verify that the MiRNA in theprocess of hematopoietic differentiation. Results:(1) RealtimePCR reaction detection of the miR-126miR-451, miR-181a, themiR-150and miR-218and other MiRNA expressed in the hUMSCs induced increases inboth, and further increase in the expression levels increased with timewhere the miR-218and miR-451increased more significantly.(2) Select the miR-218to verify its downstreamfunctions, miR-218transfected into the hUMSCs cells, cells after24h miR-218expressionwas elevated prove transfected effective. After1w cells within the hematopoieticassociated factor HoxB4expression is further increased.Conclusion: hUMSCs to hematopoietic cell differentiation process of the miR-126miR-451, miR-181a, the miR-150miR-218expression were increased, the miR-218hematopoietic function starts with a certain sense.Research findings and significance1. The application of chemically induced means of reprogramming to specificallystem cells. Induced by the application of chemical, set the number of concentration andreference conditions, the more likely conditions for the stability of hematopoietic cells.2. Proven mesenchymal type of cells to hematopoietic cells in the direction from theperspective of epigenetic mechanisms. As a result of changes in cell epigenetic induction,so we can direct attention to MiRNA changes, and provides a theoretical basis for thedeterminants of the cell transformation process.3Provide a new concept for clinical HSCT. The study tried to change the epigeneticexpression on a more primitive between mesenchymal cells to transform provides a newmethod for HSCT.