不同类型的人诱导多能干细胞异种嵌合能力的比较研究

Comparative study on interspecies chimera potential for different types of human induced pluripotent stem cells

目的比较不同培养方法建立不同类型的人诱导多能干细胞(hiPSC)的嵌合情况,筛选高异种嵌合能力的hiPSC。方法2022年9月至2023年10月,中山大学附属第一医院显微创伤手外科根据文献报道的方法,使用100只雌性C57小鼠、20只雄性C57小鼠和10只多重免疫缺陷鼠(NOD/SCID),通过不同的化学小分子诱导培养方法建立8细胞期胚胎样细胞(8CLC)、潜能扩展多能干细胞(EPS)、原始态多能干细胞(Naïve iPSC)、形成态多能干细胞(Formative iPSC)和始发态多能干细胞(Primed iPSC)。利用形态学观察、细胞免疫荧光、实时荧光定量PCR和畸胎瘤形成进行细胞系鉴定。将各种不同的细胞系使用CRISRR/CAS 9基因编辑技术标记GFP,筛选并扩增阳性细胞后,进行小鼠囊胚期注射,每次注射10个细胞形成嵌合胚胎,并将嵌合胚胎进行体外胚胎培养,观察评估嵌合情况。所有计量资料以均值±标准差(Mean±SD)表示,各亚组间的比较均先采用ANOVA单因素方差分析,再通过Bonferroni法进行两两比较,P<0.05表示差异有统计学意义。结果依据形态学、细胞免疫荧光、实时荧光定量PCR、畸胎瘤形成实验的鉴定与验证,成功培养出了8CLC、EPS、Naïve、Formative、Primed这5种代表胚胎发育不同阶段的iPSC细胞系。小鼠囊胚期注射后进行体外培养,发现8CLC和Naïve iPSC相比其他细胞系,第3天(两细胞系分别为74.28%和56.00%)和第5天(分别为62.85%和40.00%)的GFP阳性率更高,差异有统计学意义(P<0.05)。结论8CLC和Naïve iPSC作为供体细胞更有利于人-鼠异种嵌合胚胎的体外培养,有益于提高人-鼠异种嵌合后胚胎的嵌合率。

Objective To improve interspecies chimeric potential of human induced pluripotent stem cell(hiPSC)by establishing cell lines that represented distinct developmental stages through specific culture methods.Methods From September 2022 to October 2023,the study was performed by the Department of Microsurgery,Trauma and Hand Surgery,the First Affiliated Hospital of Sun Yat-sen University.A total of 100 female C57 mices,20 male C57 mices,and 10 multiple immunodeficient mices/severe combined immune deficiency(NOD/SCID)were used in the study.Five hiPSC lines were established through distinct culture methods for small molecules induction:eight-cell-like cells(8CLC),extended pluripotent stem cells(EPS),Naïve iPSC,Formative iPSC,and Primed iPSC.Cell line identities were confirmed by morphological observation and assays of cell immunofluorescence,real-time quantitative PCR,and teratoma formation.Using CRISPR/CAS9 gene editing,the five cell lines were labeled with GFP fluorescence.Following positive cell selection and expansion,10 iPSC from each line were microinjected into mouse blastocysts for generation of chimeric embryos.These embryos were then cultured in vitro for chimeric status assessment.All measurement data were represented by Mean±SD.ANOVA one-way analysis of variance was first used for comparison among subgroups,and then Bonferroni method was used for pound-wise comparison.P<0.05 indicated statistically significant differences.Results Employing morphological observation and assays of cell immunofluorescence,real-time quantitative PCR,and teratoma formation,5 iPSC cell lines,which represent different stages of embryonic development,such as 8CLC,EPS,Naïve iPSC,Formative iPSC,and Primed iPSC,were successfully established.Notably,8CLC and Naïve iPSC at 3 days post-injection into mouse blastocysts were 74.28%and 56.00%,respectively.And that at 5 days were 62.85%and 40.00%,respectively.They exhibited significantly higher GFP positivity in comparison with other cell lines in vitro.The difference was statistically significant(P<0.05).Conclusion The 8CLC and Naïve iPSCs hold greater potential as donor cells for the in vitro culture of human-mouse interspecies chimeric embryos.This has improved interspecies chimerism and paves the way for enhanced chimeric efficiency of hiPSCs.