| The differentiation of hematopoietic stem cells is a hotspot in the field of stem cells.The study of hematopoietic differentiation of stem cells from different sources and the effects of related factors on hematopoietic differentiation are important foundations for clinical transformation in the field of stem cells.It has been found that EZH2 plays an important role in early embryo development and mesoderm development,suggesting that this gene may be involved in the process of hematopoietic differentiation.OBJECTIVE:To observe the expression of EZH2 in different types of thalassemia hiPS cell lines,and to explore the role of EZH2 in hematopoietic differentiation of hESC/hiPSC.METHODS:The laboratory established a technology platform for differentiation of human ES cells and human iPS cells.On the basis of this,combined with EZH2 in the study of hematopoietic differentiation function,combined with thalassemia cell lines for further exploration.H1 is a normal human ES cell,which is isolated from the blastocyst inner cell mass.AF888 is a normal human iPS cell derived from amniotic fluid cells.At the same time,it has established four different types of thalassemia for Hainan regional genetic disease thalassemia.The iPS cell line includes the mild beta thalassemia hiPS cell line AF038,AF806,and the severe beta thalassemia hiPS cell line AF077,AF884.To study the role of EHZ2 in hematopoietic differentiation,EHZ2 knockout cell line was established by using Crispr/Cas9 technology in the above hESC/hiPSC,EHZ2 gene overexpressing cell line was established by lentivirus infection,and by q RT-PCR and Western Blotting Methods The EZH2 gene knockout and overexpression were verified.The hematopoietic differentiation of hESCs/hiPSCs was induced by the embryoid body pathway,and the embryoid bodies of d0,d4,d8,d12 and d16 were collected,and the pluripotency genes OCT4,SOX2,NANOG and internal,middle and middle were detected by q RT-PCR.The changes of the outer trisomy marker genes GATA4,MSX1,and PAX6 were detected by flow cytometry to detect the ratio of CD34~+-CD43~+,CD34~+,and CD43~+in the above EBs.RESULT:The expression of EZH2 in wild-type cell lines of normal hESC/hiPSC and6 kinds of hiPSC cells with different thalassemia types was detected by qRT-PCR.The normal ES cells were used as control and the results showed severe thalassemia.The expression of EZH2 was decreased in hiPSC;and cell lines stably knocking out and overexpressing EZH2 gene were established in the above 6 cell lines.The hESC/hiPSCs were induced to hematopoietic differentiation by the embryoid body pathway,and the embryoid bodies(EBs)of d0-d8 were cultured in suspension.EBS spontaneously aggregated to form globular shape after 24 hours of preparation.The EBs was cultured in the d8,and the EBs were inoculated into the petri dish coated with Matrigel.The EBs adhered to the EBs and grew into clones.With the prolongation of the culture time,the EBs after adherence were differentiated. The pluripotency and expression of the three germ layer marker genes in normal ESC/iPSC during EBs induction.Compared with AF888,in terms of dry maintenance,knocking out the EZH2 gene in H1 can enhance the maintenance of dryness,while knocking out EHZ2 in AF888 inhibits the maintenance of dryness;overexpression of EZH2 gene in H1 can still be expressed.To promote dry maintenance,overexpression of the EHZ2 gene in AF888 showed an inhibitory effect on dryness.In the differentiation of three germ layers,knocking out the EZH2 gene in H1 can promote the differentiation into the endoderm and ectoderm,and inhibit the differentiation of mesoderm,while the differentiation of the inward,middle and outer three germ layers after knocking out EHZ2 in AF888 It is a promoting effect;overexpression of EZH2gene in H1 is to inhibit the differentiation of the three germ layers inward,middle and outer.In the AF888 line,the differentiation of the EHZ2 gene into the inner and outer three donor layers is also inhibited.The pluripotency of the mildly poor iPSCs during EBs induction and the expression of the three germ layer marker genes.Compared with AF038,AF806 knocked out the EZH2 gene in AF806 to enhance the maintenance of dryness,while the EHZ2knockout in AF038 promoted the maintenance of dryness in the early stage of differentiation with the prolongation of differentiation time.The effect is weakened;overexpression of the EZH2 gene in AF806 may still be shown to promote dry maintenance,whereas in AF038,the expression of the EHZ2 gene is shown to inhibit dry performance.In terms of trigeminal differentiation,knockout of EZH2 gene in AF806 promotes differentiation into mesoderm and ectoderm,while inhibiting endoderm differentiation,while knocking out EHZ2 in AF038 promotes differentiation into endoderm,while inhibiting metastasis Germ and ectoderm differentiation.Overexpression of EZH2 gene in AF806 can still inhibit the differentiation into endoderm and promote the differentiation into mesoderm and ectoderm.In AF038,the expression of EHZ2 gene promotes differentiation into endoderm and ectoderm,and inhibits the middleward.Germ layer differentiation.The pluripotency of the severely depleted iPSCs during EBs induction and the expression of the three germ layer marker genes.Compared with AF884,AF077enhanced the maintenance of dryness after knocking out the EZH2 gene in AF077,but also enhanced the maintenance of dryness after knocking out EHZ2 in AF884;overexpressing EZH2 gene in 077 In the early stage of differentiation,it still appears to promote dry maintenance.As the differentiation progresses,this promotion disappears,and in AF884,the EHZ2 gene is expressed to promote dry maintenance.In terms of three germ layer differentiation,knocking out the EZH2 gene in AF077promotes differentiation into the inner,middle,and outer ectoderm,and knocking out EHZ2 in AF884 also promotes differentiation into the inner,middle,and outer three germ layers.Overexpression of EZH2 gene in AF077 still promoted the differentiation of the inner,middle and outer germ layers,but the promotion was weaker than AF077-EZH2-KO.In AF884,the EHZ2 gene was expressed inward and inward.The differentiation also showed a promoting effect,but the promotion was weaker than AF884-EZH2-KO.The ratio of CD34~+-CD43~+,CD34~+,CD43~+in EBs on the above different days was detected by flow cytometry.In H1,the presence of CD34~+-CD43~+,CD34~+,and CD43~+cells was detected in d4 induced by H1-WT,H1-EZH2-KO,and H1-EZH2-OE,and the changes were first increased and then decreased.Compared with the EBs induced by H1-WT in the corresponding days,the ratio of CD34~+-CD43~+and CD43~+ells(no hematopoiesis at d0)was 0.28 and 0.52,respectively,and the relative change with the differentiation time.The trend of increasing gradually,the relative differentiation ratio of d16 was 2.03,2.81,the relative change trend of CD34~+cell ratio increased first,and the peak of d12 appeared,the relative ratio was 34.29,and then decreased.Compared with the EBs induced by H1-WT,the ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells was 0.17,0.40,and 0.27,respectively.The relative change trend of slow reduction,the peak appeared at d12,and the relative change ratios were 1.66,3.59,and 1.96,respectively.(See Figure3.10,Table 3.7)In AF888 lines,AF888-WT,AF888-EZH2-KO,and AF888-EZH2-OE induced EBs,CD34~+-CD43~+,CD34~+,and CD43~+cells were detected in d4,and the changes were first increased and then decreased.Compared with the EBs induced by AF888-WT,AF888-EZH2-KO increased the ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells,and the relative ratios of d4 were 1.16,1.45 and 1.31,respectively.The relative increase in the trend of d16 is 2.07,2.03,and 2.5,respectively.Compared with the EBs induced by AF888-WT,AF888-EZH2-OE increased the ratio of C CD34~+-CD43~+,CD34~+,and CD43~+cells,and the relative ratios of d4 were 3.04,7.07,and 1.63,respectively.The trend of reduction,d16 first comparison is 1.31,1.26,1.45.(See Figure 3.10,Table 3.8) Both AF806 and AF038 are iPS cell lines induced by amniotic fluid-derived cells in mildly thalassemia patients.In AF806 line,AF806-WT,AF806-EZH2-KO,AF806-EZH2-OE induced EBs,CD34~+-CD43~+,CD34~+,CD43~+cells were detected in d4,and the changes were first increased and then decreased.Both appear at d12.Compared with the EBs induced by AF806-WT,the ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells decreased,and the relative changes showed a decreasing trend.The relative ratios of d4 were 0.41 and 0.71,respectively.0.35,with the prolongation of differentiation time,the relative change gradually decreased,and the relative ratio of d16 was 0.17,0.17,and 0.18,respectively.The ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells was decreased in AF806-EZH2-OE compared with the corresponding days AF806-WT.The relative ratio of CD34~+-CD43~+and CD34~+cells to d4 was 0.76and 0.83,respectively.The relative change is gradually decreasing,and the relative ratio of d16 is 0.33 and 0.33 respectively.The relative change of CD43~+is fluctuating,and the peak appears at d8 with a relative ratio of 0.55. In AF038 cell line,AF038-EZH2-KO,AF038-EZH2-OE induced EBs,the presence of CD34~+-CD43~+,CD34~+,and CD43~+cells was detected in d4,among which AF038-WT and AF038-EZH2-OE were induced.The changes of EBs increased first and then decreased,and the peaks appeared in d8.The ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells in EBs induced by AF038-EZH2-KO increased with the differentiation time.Compared with the EBs induced by the corresponding days AF038-WT,the ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells decreased in d4-d12as a whole,and d12-d16 increased overall.The relative ratios at d4 were 0.42,0.49,and 0.53,respectively.With the extension of differentiation time,the relative change gradually increased.The relative ratios of d16 were 4.64,3.77,and 4.78,respectively.The ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells was decreased in AF038-EZH2-OE compared with the corresponding days AF038-WT-induced EBs.The relative ratios at d4 were 0.07,0.07,and 0.1,respectively.With the extension of differentiation time,the relative change showed a gradually increasing trend.The d16first comparison was 0.88,0.71,and 0.95,respectively. Both AF077 and AF884 were heavily depleted in iPS cell lines.In AF077 cell line,AF077-WT,AF077-EZH2-KO,AF077-EZH2-OE-induced EBs,the presence of CD34~+-CD43~+,CD34~+,CD43~+cells was detected in d4,which gradually increased during d4-d16.There are no obvious peaks.Compared with the EBs induced by AF077-WT,AF077-EZH2-KO showed a relative decrease in the ratio of CD34~+-CD43~+,CD34~+,and CD43~+cells.The relative ratios of d4 were 1.47,1.51,1.09,and troughs.At d12,they are:0.75,0.42,and 0.45.The ratio of CD34~+-CD43~+,CD34~+and CD43~+cells in AF077-EZH2-OE compared with the corresponding number of AF077-WT-induced EBs decreased in d4-d12 as a whole,and d4-d12increased in all after d12.The relative ratios at d4 were 0.21,0.17,and 0.55,respectively.The relative changes gradually increased with the prolongation of differentiation time.The relative ratios of d16 were 1.11,1.14,and 1.11,respectively.In AF884 cell line,AF884-WT,AF884-EZH2-KO,AF884-EZH2-OE induced EBs,CD34~+-CD43~+,CD34~+,CD43~+cells were detected in d4,and AF884-WT showed a trend of increasing,AF884-EZH2-KO,AF884-EZH2-OE showed a trend of increasing first and then decreasing.Compared with the EBs induced by AF884-WT,the ratio of CD34~+,CD43~+,CD34~+-CD43~+cells increased first and then decreased.The peak of CD34+-CD43+appeared at d12,the relative ratio was 2.36,the peaks of CD34+and CD43+appear at d8,and the relative ratios are:2.09 and 2.24,respectively.The ratios of CD34~+-CD43~+,CD34~+and CD43~+cells in the AF884-EZH2-OE compared with the corresponding days of AF884-WT increased firstly and then decreased.The peaks appeared in the d8 relative ratios of 1.52,1.49and 1.42,respectively.CONCLUSION:1.It was found that the expression of EZH2 gene was different in different types of thalassemia hiPS cell lines,which was significantly decreased in severe thalassemia;2.EZH2 gene promoted the expression of stem factor in stem cell lines;3.EZH2 gene knockout has Helps the expression of the relevant factors of germ layer;4,EZH2 has a dose effect on the regulation of stem cell dry maintenance and differentiation,and needs to be further verified by experimental results;5,embryonic stem cells and adult induced pluripotent stem cells in the expression of dry factors The dose changes of EZH2 showed different trends in the expression and hematopoietic differentiation potential of each related germ layer,suggesting that they differed in dry maintenance and differentiation induction.6.Induction of thalassemia with different mutation types The pluripotent stem cells showed different trends in the dose changes of EZH2 in the expression of dry factors and related germ layers and the hematopoietic differentiation potential,and the phenotypes of different mutant cell types were different.The specific mechanism needs further Analysis;7,establish different types of stem cell lines,especially carrying different disease mutation types Stem cell lines have significance,it can be used as model for studying the disease,clinical transformation and application value. |
PDF Full Text Request