| Hematopoietic stem/progenitor cell(HSPC)has the ability of self-renewal and multidirectional differentiation,which has a broad application prospect in the treatment of various blood diseases and immune deficiency diseases.Umbilical cord blood is the main source of hematopoietic stem/progenitor cells due to its advantages of convenient collection,a wide range of sources and strong cell expansion ability.Due to the limited volume of umbilical cord blood,the absolute number of hematopoietic stem/progenitor cells in a single portion of umbilical cord blood is not enough to meet the needs of clinical application,which has become a major bottleneck restricting its clinical application.Ex vivo expansion is the main method to solve the shortage of HSPCs in clinical application.Hematopoietic stem/progenitor cells have asymmetric division,differentiated symmetric division and self-renewal symmetric division.Among them,the number of HSPC cells could be increased by self-renewing symmetric division mode.HSPCs inevitably differentiate into CD34~-cells during ex vivo expansion.CD34~-cells may also influence the ex vivo expansion of HSPCs.In order to explore and solve this problem,this paper first investigated the effect of CD34’ cells on ex vivo expansion and division mode selection of HSPC.On this basis,the strategy of reducing the concentration of TGF-β1 in the culture supernatant through periodically enriching CD34~+cells and all medium refreshing was adopted to increase the proportion of self-renewal symmetric division of CD34~+cells in the culture process,promote the expansion of total cells and CD34~+cells and maintain of physiological functions.The changes in expression levels of TGF-β signaling pathway related genes were analyzed to explore the possible mechanism of CD34~-cells affecting the ex vivo expansion of CD34~+cells.Starting from strengthening the inhibition of AhR signaling pathway,the combination of AhR and TGF-β signaling pathway further increased the proportion of HSPC choosing self-renewal symmetric division in the process of ex vivo expansion and improved the ex vivo expansion fold of HSPC on the basis of maintaining the physiological function of HSPC.These provide technical support for the establishment of optimal ex vivo expansion process of HSPC.In this paper,umbilical cord blood CD34~+cells were taken as the research object in serumfree culture system.Three groups of six ex vivo expansion conditions were designed to explore the effect of CD34~-cells in culture system on the expansion and division mode selection of HSPC.The results showed that the presence of CD34~-cells inhibited the ex vivo expansion of HSPC.The alignment relationship was found between the proportion of CD34~-cells and the expansion of CD34~+ cells,the expansion of CD34~+CD38-cells and the proportion of HSPC selecting self-renewal symmetric division mode.The fitting curve equations were Y=-1.132x+2.204,Y=-1.474x+2.695 and Y=-0.3431x+1.361.In addition,the proportion of HSPC in the culture during ex vivo expansion was also aligned with the proportion of HSPC selecting self-renewal symmetric division mode.The linear equation is Y=1.894x-0.9724.In order to reduce the influence of CD34~-cells in culture on HSPC ex vivo expansion,a periodically enriching CD34~+cells strategy was adopted to remove CD34’ cells during ex vivo expansion.In control group,half of fresh medium was updated on day 4 and day 7 in culture,while in enriched CD34~+cell group,CD34~+cells were periodically enriched,and all medium was updated in culture.The results showed that with 10 day-culture,the expansion fold of CD34~+cells was 10.75±2.75 folds,which was significantly higher than 7.15±1.10 folds of in control group(p<0.05).The proportion and expansion of CD34~+CD38-cell and primitive hematopoietic stem/progenitor cells in enriched CD34~+cell group were significantly higher than those in control group(p<0.05).The proportion of HSPC selecting self-renewal symmetrical division in enriched CD34~+cell group was 38.85±4.49%,which was significantly higher than 29.77±0.85%in control group(p<0.05).By analysing the colony-forming ability,secondary expansion ability and the expression of HSPC self-renewal and function-related genes,it was found that periodical enrichment of CD34~+cells promoted the maintenance of physiological function of expanded HSPC.It was found that with 10-day culture,the concentration of total TGF-β1 and activated TGF-β1 in enriched CD34~+cell group was 69.15± 4.81 pg/mL and 4.33 ±1.09 pg/mL,which were significantly lower than 625.54 ± 58.76 pg/mL and 313.07± 12.81 pg/mL in control group(p<0.05),TGF-β signaling pathway related genes expression was also significantly down-regulated.In conclusion,it is speculated that the strategy of periodical enrichment of CD34~+ cells in the culture process can increase the proportion of HSPC selecting self-renewal symmetric division mode in the culture process and promote the expansion of total cells and CD34~+ cells and the maintenance of physiological functions by reducing the concentration of TGF-β1 in the culture supernatant and downregulating the TGF-β signaling pathway transduction.Since the enrichment of CD34~+cells in the culture process lengthened the cell processing time,the all-medium refreshing method to reduce the concentration of TGF-β1 in the culture supernatant was adopted to reduce the cell processing time and verify that CD34~-cells inhibited the ex vivo expansion of HSPCs by secreting TGF-β1.By comparing HSPC ex vivo expansion and changes of TGF-β1 concentration in supernatant between half(control)and all medium refreshing strategies,it was found that the total concentration and activated concentration of TGF-β1 in supernatant in all medium refreshing group were significantly lower than those in control group(p<0.05).The total cell expansion fold,the percentage and expansion fold of CD34~+cell,CD34~+CD38-cell and the primitive hematopoietic stem/progenitor cell,and the percentage of HSPC choosing self-renewal symmetrical division in all medium refreshing group were significantly higher than those in control group(p<0.05).It was found that all medium refreshing also promoted the maintenance of physiological function of expanded HSPC.By further comparing the expansion effect of HSPC between the enriched CD34~+cells strategy and all medium refreshing strategy,it was found that the two strategies had the same ex vivo expansion effect of HSPC.In conclusion,TGF-β1 secreted by CD34~-cells is the key factor to inhibit the ex vivo expansion of HSPC.Previous studies have shown that inhibition of AhR signaling pathway can significantly improve the ex vivo expansion of HSPC.The activation of TGF-β signaling pathway can also inhibit the transcription of AhR signaling pathway,and the strategy of periodically enriching CD34~+cells or all medium refreshing to reduce the concentration of TGF-β1 in the culture process can inhibit the transduction of TGF-β signaling pathway,which may weaken the inhibitory effect of TGF-β signaling pathway on AhR signaling pathway.Therefore,the AhR signaling pathway and TGF-β signaling pathway were combined from the perspective of strengthening AhR signaling pathway.On the basis of determining the addition strategy of AhR signaling pathway inhibitor SR1 as 0.5 μM/12h,the ex vivo expansion of HSPC were compared between SR1 addition strategy and SR1 addition combined with all medium refreshing strategy.The results showed that with 10 day-culture,the expansion fold of CD34~+cells in the SR1+all medium refreshing group were 9.84±2.70 folds,which was significantly higher than 7.27±2.24 folds in SR1 group(p<0.05).The proportion of HSPC choosing self-renewal symmetric division was also significantly higher than that in SR1 group(p<0.05).In addition,by analyzing the colony formation ability,secondary expansion ability,it was found that SR1 addition combined with all medium refreshing strategy significantly promoted the maintenance of physiological function of expanded HSPC.In conclusion,on the basis of all medium refreshing,the addition of SR1 at 0.5 μM/12h increased the proportion of HSPC selecting self-renewal symmetric division mode and promoted the expansion of total cells and CD34~+cells and the maintenance of physiological functions.The ex vivo expansion effect of hematopoietic stem/progenitor cells was further verified by SR1 addition combined with periodically enriching CD34~+cells strategy.By comparing the in vitro amplification of HSPC between SR1 addition strategy and SR1 addition combined with periodically enriched CD34~+cells strategy,it was found that on day 10,the expansion fold of CD34~+cells were 28.91 ±5.98 folds,significantly higher than 11.53±1.51 folds in SR1 group(p<0.05).The percentage of HSPC choosing self-renewal symmetric division in the SR1+periodically enriched CD34~+cells group was significantly promoted(p<0.05).It was found that SR1+periodically enriched CD34~+cells group further enhanced the inhibition of SR1 on AhR signaling pathway.The expansion effect of SR1+ periodically enriched CD34~+cells group and SR1+all medium refreshing was further compared.It was found that these two strategies had the same ex vivo expansion effect of HSPC.Therefore,this study further increased the proportion of HSPC selecting self-renewal symmetric division mode in culture and promoted the expansion of total cells and CD34~+cells as well as the maintenance of physiological functions by enhancing the inhibitory effect of AhR signaling pathway and jointly inhibiting AhR signaling pathway and TGF-β signaling pathway.In this paper,the effect of CD34’ cells on the ex vivo expansion of HSPC was studied,and the inhibition of CD34’ cells on the ex vivo expansion of HSPC was eliminated by the strategy of periodically enriching CD34~+cells or all medium refreshing to reduce the concentration of TGF-β1 in culture supernatant.Combined inhibition of AhR and TGF-β signaling pathway improved the ex vivo expansion of HSPC effect of HSPC.These provide technical support for further establishment and optimization of HSPC ex vivo culture process and development of serum-free medium. |
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