| The main sources of hematopoietic stem cells(HSCs)are mobilized adult peripheral blood and umbilical cord blood.However,due to the difficulty of large-scale expansion of hematopoietic stem cells in vitro,few sources for acquisition,low resuscitation activity,and low proportion of CD34+cells,the current widespread stored cord blood loses its value in treating malignant blood diseases.As a result,this precious biological resource is wasted.Mesenchymal stem cells(MSCs)have a wide range of sources,and have important functions such as tissue repair and immune regulation.It is one of the most widely used stem cells in clinical applications.However,due to different methods of obtaining and different donor conditions,mesenchymal stem cells are far from satisfying the constant Increasing clinical and scientific needs.Induced pluripotent cells(iPSCs)technology is to transfer stromal cells or other stem cells into Oct-4,Sox-2,Klf-4 and c-myc and other four transcription factors.To reprogram these cells into pluripotent stem cells with biological characteristics similar to embryonic stem cells.This article uses induced pluripotent stem cell technology to reprogram hematopoietic stem cells to turn them into induced pluripotent stem cells.We expanded the best induced pluripotent stem cell lines on a large scale.Multi-step medium replacement method is adopted to induce induced pluripotent stem cells to differentiate into mesenchymal stem cells.This can provide a sufficient number of mesenchymal stem cells for clinical applications and scientific research.The research contents of this article are as follows:1.We conduct a pathogen check on cord blood to confirm that it is not infected by bacteria and viruses,and use density gradient centrifugation to isolate monocytes in cord blood.We use flow cytometry to detect monocytes in order to distinguish each group of monocytes.Then use immunomagnetic beads to separate hematopoietic stem cells from monocytes.We chose a fully artificial medium to culture CD34+cells in vitro and used flow cytometry to detect their surface markers and cell purityThree mixed free circular gene vectors were used to reprogram hematopoietic stem cells.Then 6 human induced pluripotent stem cell lines were obtained.Expand and collect induced pluripotent stem cells.The cloning ability,growth level,surface marker expression rate,and three germ layer differentiation potential of each cell line were tested.The above factors were analyzed to obtain the best cell line for targeted differentiation.2.Induce the iPSCs from the best cell line with whole artificial medium to make them differentiate into iPSC-derived mesenchymal stem cells(iMSCs).The Wharton's Jelly from the same donor was collected and the umbilical cord mesenchymal stem cells(UMSCs)were isolated from it.The three differentiation capabilities of mesenchymal stem cells were verified.In order to obtain more accurate data,it is necessary to determine the amount of flow cytometry antibody and the corresponding number of cells to be tested through experiments.After the previous step is completed,the surface markers of mesenchymal stem cells were detected by flow cytometry.The microbial contamination and cell nuclear chromosomes of two kinds of mesenchymal stem cells were detected.3.In order to have enough test samples,a large amount of two iMSCs and UMSCs need to be amplified.STR analysis was performed on both cells to confirm that iMSCs and UMSCs were from the same donor.In the experiment,we completed the test of two cell growth levels.The cell surface markers,cell cycle,apoptosis level,exosome-specific antibody expression rate and the expression rates of various cytokines in the culture medium of the two cells of different passages were detected and analyzed.Result:1.The amount of CD34+cells isolated from pathogen-free umbilical cord blood is too low.The expansion is required.The total number of CD34+cells obtained from the expansion is 2.32*10~6.We use flow cytometry to detect these HSCs,and the result was the CD34 expression rate is 99.4%and the cell purity is high.The colonies of iPSCs were visible to the naked eye after 20 days after transfection.The 6 cell lines obtained at the same time all can strongly express the pluripotent stem cell surface markers SSEA4,Oct4,SOX2,and the cell growth curve is normal,the cells grow rapidly from the third day after adherence.From the analysis of the above data,the 6 cell lines are all iPSCs,of which the optimal cell line is cell line 1.and the mice can grow teratoma with three germ layers cells.The cells line 1 is expanded in large numbers and injected into immunodeficient mice.The mice can develop teratomas with triple germ cells.2.Using the two-step method described in this article can make iPSCs differentiate into mesenchymal stem cells.The obtained iMSCs and the same donor(UMSCs)are both long fusiform and grow in a vortex shape.The cells can differentiate into osteoblasts,fat cells,and chondrocytes.According to the gradient test,the best flow cytometry antibody dosage is 2.5?L,and the corresponding optimal cell number is 1*10~5.Identify primary cells using flow cytometry.Both MSCs have cell surface positive antigens CD73,CD90,CD105 with an expression rate of greater than 95%,and CD14,CD34,CD45,CD79a,HLA-DR antigens with an expression rate of less than 1%,It was confirmed that the cells of the two sources were mesenchymal stem cells.and the chromosome morphology of the two cells was normal.3.The STR analysis results of the two cells are identical,confirming that they have the same source and no foreign gene contamination.Cell surface markers of iMSCs can be expressed normally when cultured to 13 generations in vitro.Although the expression rate of the positive markers of P13 generation iMSCs decreased slightly,the expression rates of the positive markers CD73,CD90 and CD105 were all higher than 95%,which could be used in clinical treatment..The expression rates of CD73,CD90 and CD105 in the surface markers of P13generation UMSCs were all lower than 95%,and the negative markers were all less than 1%,which could not be used in clinical treatment.Cell cycle analysis and apoptosis level detection of the P3 and P13 generations of the two MSCs showed that the cells in the apoptotic phase increased after passage in vitro.The ratio of P3apoptotic cells of both cells was less than 1%.The apoptotic cell rate of iMSCs in P13 generation was 6.87%,and the apoptotic cell rate of UMSCs was 44.41%.The late apoptotic and dead cell rates of iMSCs were lower than those of the same generation UMSCs.The expression rate of exosome-specific protein of the two cells also decreased with the increase of the number of passages.The expression rates of exosome-specific surface antigens CD63,CD29,and CD73 of MSCs of the same generation were detected,and the results showed that iMSCs were significantly higher than UMSCs.The content of 4 factors including hepatocyte growth factor,insulin-like growth factor 1,interleukin-6,and transforming growth factor-?1 in the supernatants of both cell culture media were significantly higher than that of UMSCs in iMSCs.As the number of passages increased,the secretion of various exocrine factors of iMSCs decreased,while the amount of exocrine factors of UMSCs decreased significantly.Only the amount of interleukin-8 in the supernatant of p3 generation UMSCs medium was higher than that of iMSCs,but the content of this factor in the supernatant of P13 generation UMSCs cell medium was lower than that of iMSCs.The secretion of vascular endothelial growth factor in iMSCs was significantly higher than that of UMSCs,and there was no significant decrease with passage.Conclusion:In this study,a fully artificial induction protocol for umbilical cord blood-derived hematopoietic stem cells was constructed so that it could be reprogrammed into induced pluripotent stem cells.The quality of iPSCs cell lines were evaluated,and the optimal cell line was selected to differentiate into mesenchymal cells.The method of fully artificial directed differentiation of iPSCs into MSCs was determined so that they could be differentiated into iMSCs.And the biological characteristics of iMSCs obtained by this method in each generation in vitro are superior to the UMSCs from the same donor. |
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