Single Cell Level Resolution Of The Effect Of Ex Vivo Expansion On Cord Blood HSC Self-Renewal And Differentiation

?Objective?To explore the optimum system of inducing HSC ex vivo expansion,cord blood?CB?derived hematopoietic stem cells?HSCs?expanded by cytokines combined with different small molecular compounds.And the engraftment and self-renewal potential of expanded cells was evaluated by serial transplantation in NOD-Prkdc^scid Il2rg^null?NPG?mice.At last,the key molecules regulating HSC self-renewal were screened by single cell RNA sequencing?sc RNA-seq?,and signal pathway were elucidated.The effect of ex vivo expansion system on HSC self-renewal and differentiation was revealed from the single cell level.?Methods??1?CB-derived CD34⁺cells were cultured in the presence of four cytokines?SCF,FLT3-L,TPO,IL-6?alone or in combination with UM171,SR1 and K1 for 10 days.Colony forming units?CFU?assay was used to evaluate the ability of the expanded cells to differentiate into progenitors.?2?A quantity of 500 to 10,000 uncultured CD34⁺CB-derived cells or the a fraction of the final culture equivalent to 500 to 10,000 starting cells were transplanted into primary sub-lethally irradiated NPG recipients.The proportion of h CD45⁺cells in NPG mice peripheral blood?PB?at 5,8,12,16 weeks post-transplantation and bone marrow?BM?at 16 weeks post-transplantation was detected by flow cytometry.?3?2×10⁶,5×10⁶ and 1×10⁷ BM cells from primary recipient mouse of 10,000 starting cells group transplanted into secondary sub-lethally irradiated NPG recipients.The proportion of h CD45⁺cells in NPG mice BM at 16 weeks post-transplantation detected by flow cytometry.?4?The severe-combined immunodefcient mouse repopulating cells?SRC?frequency quantified by Limit dilution analysis?LDA?.?5?Single cell RNA sequencing?sc RNA-seq?was used to screen the key molecules and signal pathway regulating HSC self-renewal,and reveal the effect of ex vivo expansion system on HSC proliferation and differentiation.?Results??1?Proportion of CD34⁺cells in UM171,SR1,K1 or USK group was higher than that in vehicle group?P<0.001?.Proportion of CD34⁺CD38^-cells in UM171 and USK group was significantly higher than that in vehicle group?P<0.001?,but there was no significant difference between SR1,K1 group and vehicle group?P>0.05?.Percentage in vehicle group?P<0.001?,and there was no significant difference between other groups and vehicle group?P>0.05?.There was no significant difference in the proportion of CD34⁺cells between the USK group and the Unculture group?P>0.05?.group was higher than that in Unculture group.?2?Fold of CD34⁺cell expansion in SR1 group and USK group increased significantly compared with vehicle group?P<0.05?.Fold of CD34⁺CD38^-cell expansion in UM171group increased significantly compared with vehicle group?P<0.05?.Vehicle,UM171,SR1,K1,USK treatment led to a 61.58-,248.56-,210.05-,133.08-,and 543.33-fold significantly higher than that in vehicle group?P<0.001?.?3?Proportion of CD3^-CD56⁺cells in the USK and SR1 groups was higher than that in the vehicle group?P<0.01?.Proportion of CD33⁺cells in the UM171 group was higher than that in the vehicle group?P<0.01?.Proportion of CD41⁺cells in the UM171 group was lower than that in the vehicle group?P<0.05?.Proportion of CD235a⁺,CD19⁺and CD3⁺cells was no significant difference in all groups?P>0.05?.Number of CFU-granulocyte?CFU-G?,macrophage?M?,granulocyte/macrophage?GM?,erythrocyte?E?and granulocyte/erythrocyte/macrophage/megakaryocyte?GEMM?in USK,K1,and UM171 groups was no significant difference when compared to vehicle group and Unculture group?P>0.05?.Number of CFU-G and GEMM in SR1 group was lower than that in Unculture group?P<0.05?.Number of GEMM in Vehicle group was also lower than that in Unculture group?P<0.05?.?4?When 500 initial cells were transplanted,proportion of h CD45⁺cells in the primary NPG recipients PB of USK group was significantly higher than that of Unculture and vehicle groups at 5 weeks post-transplantation?P<0.05?.There was no significant difference in the proportion of h CD45⁺cells in PB and BM between USK group and Unculture group at 16 weeks after transplantation?P>0.05?.When 2500 initial cells were transplanted,proportion of h CD45⁺cells in the primary NPG recipients PB and BM of USK and Vehicle groups was significantly lower than that of Unculture group at5,8,12 and 16 weeks post-transplantation?P<0.05?.When 10000 initial cells were transplanted,the proportion of h CD45⁺cells in the primary NPG recipients PB of USK group was significantly lower than that of Unculture group?P<0.05?.?5?When 2×10⁶,5×10⁶ and 1×10⁷ BM cells from primary recipient mouse transplanted into secondary NPG recipients,proportion of h CD45⁺cells in NPG recipients BM in the USK and vehicle groups was no significant difference when compared to Unculture group at 16 weeks post-transplantation?P>0.05?.?6?For primary transplants,USK treatment led to a five-fold increase in SRC frequency relative to vehicle group?P<0.05?,and led to a two-fold increase relative to unculture group?P>0.05?.For secondary transplants,there was no significant difference in SRC frequency in all groups?P>0.05?.?7?Data from sc RNA-seq visualized by UMAP and divided into 17 clusters.Comparing the changes of clusters between uncultured CB CD34⁺cells and cultured them with USK,the results showed that the cell number of Cluster 4?HSC?,Cluster 16?My SC?and Cluster 10?MESC?was significantly reduced or disappeared when CB-derived CD34⁺cells were cultured with USK for 10 days in vitro.While the cell number of Cluster 1?My RP?,Cluster 2?GMP?,Cluster 3?Granulocyte?,Cluster 5?EMP?,Cluster6?Ma/Ba/Eo?,Cluster 7?Monocyte/m DC?,Cluster 8?Platelet?,Cluster 15?Erythroid?was obviously increased when cultured with USK for 10 days.Comparing the changes of clusters between CB CD34⁺cells cultured with USK and cultured with Vehicle,the results showed that the cell number of Cluster 0?Multi RP?,Cluster 1,Cluster 10,Cluster 5,and Cluster 6 increased in USK cultured 10 days compared with vehicle culture.While the cell number of cluster 3,Cluster 7,Cluster 8,Cluster 13 and Cluster14?B/p DC?was significantly decreased when compared with Vehicle culture.?8?The gene expression analysis from sc RNA-seq showed that in addition to the reported HSC specific marker AVP and the transcription factors KLF2,HES1,MLLT3related to HSC"stemness",HOPX and ID1 also expressed on Cluster 4?HSC?and Cluster 16.?9?The results of differentially expressed genes?DEG?between USK treatment and vehicle treatment showed that USK treatment up-regulated gene expression of NRIP1,PRDX1 and SELENOW,and downregulated CYP1B gene expression.The results of DEG between USK treatment and Unculture showed that USK treatment up-regulated expression of cell cycle and proliferation related genes?MKI67,TOP2A,HISTH4C and TUBA1B?,myeloid genes?IGLL1 and MPO?,Mitochondrial related genes?MT-ND1and MT-CO3?,and down-regulated transcription factors JUN,JUND,FOS,FOSB,IRF1,REL and NFKBIA expression.?10?The results of DEG between Cluster 0,Cluster 1,Cluster 2,Cluster 10 and Cluster4 showed that the enriched up-regulated genes mainly included PCLAF,TYMS,CENPF,MPO,IGLL1,HIST1H4C,TESC and PGAM1,and the enriched down regulated genes included AVP,JUN,JUND,FOS,FOSB,KLF2,IRF1,NFKBIA,HES1,DNAJB1,HSPH1,HSPA1A and PNRC1.GO analysis showed that Cluster 0,Cluster 1,Cluster 2,Cluster 10 promoted the oxidative phosphorylation energy metabolism of mitochondria,and inhibited the negative regulation of cell differentiation and cell adhesion compared with Cluster 4.The pathway analysis showed that Cluster 0,Cluster 1,Cluster 2,and Cluster 10 positively regulated oxidative phosphorylation and energy metabolism pathway,and negatively regulated MAPK signaling pathway,FOXO pathway and REG/GR pathway compared with Cluster 4.?11?The results of SCENIC analysis from sc RNA-seq data showed that the transcription factors FOS,FOSB,JUN,JUND,IRF1 and REL were highly regulated in Cluster 4?HSC?and Cluster 16?My SC?.These transcription factors mainly interact with HSC-related genes,nuclear receptor genes,FOXO family,hypoxia-related genes,Hox family,NF-?B signal,TGF-?signal,Notch signal,m TOR signal and MAPK signal-related genes.?Conclusion??1?The combination of cytokines?SCF+FLT3-L+TPO+IL-6?and small molecules?UM171+SR1+K1?expand approximately 543-fold of cells with this study.?2?The cells with the phenotype of CD34+CD38-CD45RA-CD90+ expanded by USK can only short-term engraftment in NPG mice,but there was no advantage in long-term of functional HSC.?3?HSC,My SC and MESC decreased or disappeared significantly,and the number of regenerating progenitor cells increased when CB CD34⁺cells cultured with USK or Vehicle.Compared with cytokine culture system,USK treatment increased the number of MESC,multi RP,My RP,EMP,and reduced the number of granulocyte/monocyte,megakaryocyte/platelet and B lymphocyte.These findings suggest that CB CD34⁺cells cultured with USK and Vehicle have different degrees of differentiation,but the differentiation degree of USK group is lower than that of Vehicle group.?4?HOPX and ID1 may be phenotypic markers of human HSC.?5?FOS,FOSB,JUN,JUND,IRF1 and REL are the key molecules regulating HSC self-renewal.Promoting the expression of transcription factors FOS,FOSB,JUN,JUND,IRF1,REL and regulating the metabolic pathway,mitochondrial function and ROS level of HSC may contribute to supporting the self-renewal and inhibit the differentiation of HSC.