| IntroductionRelated studies have shown that human placenta is a potent hematopoietic niche containing hematopoietic stem cells(HSCs)as early as week 6 in gestation,throughout fetal development.There are a large number of HSCs in the placenta with its characters of convenient to fetch and not prone to ethical problems.At present,ex vivo expansion of Umbilical Cord Blood(UCB)Hematopoietic Stem and Progenitor Cells(HSPC)for clinical transplantation has been successful.Using expanded UCB HSPC for transplantation can effectively solve the problem of delayed hematopoietic recovery which cause by the insufficient number of hematopoietic stem cells.The HSC implantation rate unchanged before and after ex vivo expansion.Placentas derived HSC are more primitive than UCB derived HSC and they were all perinatal HSCs.Now there is no research literature in the world about ex vivo expansion of placenta derived HSPC.ObjectiveThe purpose of this research is to compare the efficiency of methods of HSC extraction from placenta by mechanical treatment combined with enzymatic digestion and recirculating AMD3100 perfusion.We hope to construct a new method of HSC extraction from placenta for clinical application with actual origin and fixed composition.To investingatethe effect of umbilical cord MSCs in combination withUM171 on amplification of cord blood CD34+cells based on the theory of mimic hematopoietic microenvironment.Isolation and extraction of HSC derived from placenta according to the optimized method.Evaluation of amplification effect of the new method based on the theory of mimic hematopoietic microenvironment and HSPC function after amplification.Try to expand the clinical HSC source.Methods1.Ten healthy full-term placentas were divided into two groups accordance with different treatment methods: Group A(mechanical treatment combined with enzyme digestion group),Group B(AMD3100 perfusion group).The cell number and phenotype were compared among the two groups.In group A,the placenta was dissected and divided into groups.The tissue groups were washed with phosphate buffered saline(PBS),and then collect thecleaning solution.The tissue was digested by collagenase,hyaluronidase and DNA enzyme.The digested solution was filtered through 100 ?m nylon mesh,and then collects the filtrate.Peristaltic pump connected the umbilical artery and vein.Placental vascular system was circulating by normal saline(NS)and NS containing AMD3100 perfusion.The irrigating solution was collected.The cell numbers were compared between the two groups.The number of cells and the phenotype of flow cytometry were measured at different stages of the two methods.2.CD34+ HSCs were isolated from using CD34+ progenitor cell isolated kit by a high-gradient magnetic cells sorting system.Umbilical cord was cultured with DMEM/F12 medium containing 0.2% type II collagenase after cutting into small tissues.MSCs were cultured,proliferated and purified by passage culture.The UCB CD34+ cells and umbilical cord MSCs were divided into four groups: Group A(control group),Group B(UM171 culture group),Group C(MSCs co-culture group)and Group D(UM171 andMSCs co-culture group).Human CD34+ cells were cultured in HSC expansion media consisting of StemSpan supplemented with human 100 ng/ml stem cell factor(SCF),100 ng/ml FMS-like trysine kinase 3 ligand(FLT3),50 ng/ml thrombopoietin(TPO).1x105CD34+ UCB cells were injected into 6-well.Group B need to add superfluity 100 ng/ml UM171.Group C use the third generation MSCs as the feeder.The MSCs were irradiated at the doses of 10 Gy by 60 Co ?-ray until 80% confluence of cells.Group D need to add superfluity 100 ng/ml UM171 compared with group C.The cell number,phenotype and colony formation ability were compared among different groups after amplification in vitro for 10 days.3.CD34+ cells in the NS and NS containing AMD3100 irrigating solution were separated and purified by magnetic-activated cell separation(MACS)purification system.CD34+ cells were cultured by the UM171 andMSCs co-culture method.To evaluate the effect of amplification,flow cytometry and colony culture.ResultsTNC collected in group A was(45.19±9.41)×107 and(42.69±11.19)×107 in group B without significant difference.CD34+ cells collected in group A was(12.98±3.62)×107 and(1.87±0.87)×107 in group B with significant diffence,p=0.00.CD133+ cells collected in group A was(1.16±0.61)×107 and(1.15±0.63)×107 in group B without significant difference.TNC collected in cell suspension A1 of group A was(6.76±2.13)×107,and percentage of CD34+CD38-cells was(1.14±0.56)%.TNC collected in cell suspension A1 of group A was(6.76±2.13)×107,and percentage of CD34+CD38-cells was(1.14±0.56)%.TNC collected in cell suspension A2 of group A was(38.44±7.33)×107,and percentage of CD34+ CD38-cells was(30.31±10.75)%.TNC collected in cell suspension B1 of group B was(15.11±6.03)×107,and percentage of CD34+CD38-cells was(0.72±0.34)%.TNC collected in cell suspension B2 of group B was(27.58±11.14)×107,and percentage of CD34+CD38-cells was(5.56±1.78)%.2.Obtain the umbilical cord MSCs with good growth condition by cell subculture.The umbilical cord MSCs expressed CD105,CD73 andCD90,while didn't express CD14,CD34,CD19,CD45 and HLA-DR.After induction,the cells could differentiate into osteoblasts,adipocytes and chondrocytes.Afterthe CD34+ cells were cultured in vitro for 10 days,the total nuclear cells in the control group were amplified by3.7times,and CD34+ cells by3.5 times;the total nuclear cells in the UM171 culture group were amplified by 14 times,and CD34+ cells by13.5 times;the total nuclear cells in the MSCs co-culture group were amplified by 11 times,and CD34+ cells by 10 times;the total nuclear cellsin the UM171 and MSCs co-culture group were amplified by 22 times,andCD34+ cellsby 21 times.The ratio of CD34+CD38— cell in the UM171 and MSCs co-culture group was(91.49±2.67)% There was significant difference compared with the UM171 culture group((91.49±2.68)%)and the MSCs co-culture group((78.11±2.35)%).After amplification,the cells were cultured for 14 days,and the colony formation were observed in all groups.Besides,cells in the UM171 group formed more erythroid cellsand granulocytes than cells in the MSCs co-culture group.3.After the CD34+ cells from the placenta lavage with NS were cultured in vitro for 10 days,the total nuclear cells in the control group were amplified by 17 times,and CD34+ cells by 13 times;Almost all of the CD34+ cells from the placenta lavage with AMD3100 died after 10 days cultured.There was significant difference compared with the UCB control group and the placenta lavage with NS group on the TNC?CD34+ cells and CD133+ cells.The ratio of CD34+CD38—cell in the placenta lavage with NS group was(91.49±2.67)%.After amplification,the cells were cultured for 14 days,and the colony formation were observed in all groups.There was significant difference compared with the UCB control group and the placenta lavage with NS group on the ability of granulocyte reconstruction.ConclusionCD34+ cells can both effectively collected from placenta with the method of mechanical treatment combined with enzyme digestion and AMD3100 perfusion.The method of AMD3100 circulation perfusion can effectively reduce the risk of bacterial contamination while ensuring enough cells can be collected.The method of mechanical treatment combined with enzyme digestion can effectively improve separation efficiency.It can also reduce the adverse effect of maternal tissue on the placenta HSC.It has a greater advantage in collecting HSC from placenta compared with the method of mechanical treatment combined with enzymatic digestion.2.As feeder cells,umbilical cord MSCs can improve in vitro amplificationof CD34+ cells.UM171 can favorably maintain stemness of the cells in the amplification process.The optimalamplification can be achieved by combining UCB MSCs and UM171.3.CD34+ cells in the NS irrigating solution were amplified by 22 times in the UM171 and MSCs co-culture system and the ratio of CD34+CD38—cell was 82%.The results of colony forming experiment showed that the cells amplified in the culture system had the same lineage reconstruction ability as the UCB CD34+ cells.CD34+ cells in the NS containing AMD3100 irrigating solution were all died after being cultured in vitro for 10 days in the UM171 and MSCs co-culture system.The source and function of the CD34+ cells in the NS containing AMD3100 irrigating solution need to be further studied. |
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