Objective: To explore the role of over-expression of TBX3 and TBX18 in inducing human induced pluripotent stem cells (HiPS) to enrich and differentiate into sinoatrial node-like cells.
Methods: The expression of stemness markers OCT3/4, SOX2, and NANOG in HiPS was detected by real-time fluorescence quantitative PCR (qRT- PCR), and compared with human embryonic stem cells (hESCs). Immunofluorescence staining was used to observe the expression of HiPS stemness markers OCT3/4, NANOG, SSEA4, and TRA-1-60. The HiPS were directional differentiated into cardiomyocytes, the expressions of ISL1, NK2 homeobox 5 (NKX2-5), ACTN1, and TNNT2 were detected by qRT-PCR, and human adult cardiomyocytes (hACM) were used as positive control. Immunofluorescence staining was used to observe the expressions of NKX2-5, cardiac troponin (cTnT), α-actinin, atria myosin light chain 2A (MLC-2A), and ventricular myosin light chain 2V (MLC-2V). The positive rate of α-actinin was detected by flow cytometry. On the 3rd day after HiPS were differentiated into cardiomyocytes (mesodermal stage), lentiviral over-expressions of sinoatrial node-related genes TBX3 and TBX18 were carried out for 21 days. The relative expressions of specific markers TBX3, TBX18, SHOX2, NKX2-5, HCN4, and HCN1 in sinoatrial node cells were detected by qRT-PCR, and compared with enhanced green fluorescent protein blank virus.
Results: OCT3/4, SOX2, and NANOG were highly expressed in HiPS and ESCs, and there was no significant difference in the relative expression of each gene ( P>0.05); OCT3/4 and NANOG were specifically distributed in the nucleus of HiPS, while SSEA4 and TRA-1-60 were distributed in the cell membrane. The relative expressions of ISL1 gene at 5, 7, 21, and 28 days and NKX2-5 gene at 7, 21, and 28 days of HiPS differentiation into cardiomyocytes were significantly higher than those of hACM ( P<0.05), and the relative expressions of ACTN1 and TNNT2 genes at 3, 5, 7, and 21 days of HiPS differentiation into cardiomyocytes were significantly lower than those of hACM ( P<0.05). NKX2-5 was expressed in most of the nuclei, cTnT and α-actinin, MLC-2A and MLC-2V signals were localized in the cytoplasm, presenting a texture-like structure of muscle nodules. Flow cytometry results showed that HiPS was successfully induced to differentiate into cardiomyocytes. The expressions of TBX18, SHOX2, HCN4, and HCN1 in the over-expression TBX3 group were up-regulated when compared with the control group, and difference in the relative expression of SHOX2 gene was significant ( P<0.05); the relative expression of NKX2-5 gene was lower than that in the control group, but there was no significant difference ( P>0.05). There was no significant difference in the relative expression of each gene between the over-expressed TBX18 group and the control group ( P>0.05).
Conclusion: HiPS and hESCs have similar pluripotency, and we have established a stable method for maintaining and culturing the stemness of HiPS. A technological platform for the efficient differentiation of HiPS into cardiomyocytes has been successfully established. Although TBX3 and TBX18 do not play a significant role in promoting the enrichment and differentiation of HiPS into sinoatrial node-like cells, TBX3 shows a certain promoting trend, which can be further explored in the future.
目的: 探索过表达 TBX3、TBX18 在人源诱导多能干细胞（human induced pluripotent stem cells，HiPS）向窦房结样细胞富集分化的作用。.
方法: 取 HiPS，采用实时荧光定量 PCR（real-time fluorescence quantitative PCR，qRT-PCR）检测其干性标记物 OCT3/4、SOX2、NANOG 表达，并与人胚胎干细胞（human embryonic stem cells，hESCs）比较；免疫荧光染色观察 HiPS 干性标记物 OCT3/4、NANOG、SSEA4 和 TRA-1-60 表达。将 HiPS 向心肌细胞定向分化，qRT-PCR 检测心肌前体细胞特异性基因 ISL1、NKX2-5（NK2 homeobox 5）以及心肌细胞特异标记物 ACTN1、TNNT2 表达，以成人心肌细胞（human adult cardiomyocytes，hACM）作为阳性对照；免疫荧光染色观察心肌前体细胞特异核转录因子 NKX2-5，心肌细胞特异性收缩蛋白肌球蛋白（cardiac troponin，cTnT）、α-辅肌动蛋白（α-actinin），以及心房肌球蛋白轻链（myosin light chain 2A，MLC-2A）和心室肌球蛋白轻链（myosin light chain 2V，MLC-2V）表达；流式细胞术检测 α-actinin 阳性率。在 HiPS 向心肌细胞定向分化第 3 天（中胚层阶段），以慢病毒方式过表达窦房结相关基因 TBX3、TBX18，继续培养至 21 d，采用 qRT-PCR 检测窦房结细胞特异性标记物 TBX3、TBX18、SHOX2、NKX2-5、HCN4、HCN1 相对表达量，以增强绿色荧光蛋白空白病毒为对照。.
结果: OCT3/4、SOX2、NANOG 在 HiPS 和 ESCs 中均高表达，各基因相对表达量比较差异无统计学意义（ P>0.05）；OCT3/4 和 NANOG 在 HiPS 中特异分布于细胞核中，SSEA4 和 TRA-1-60 分布于细胞膜上。HiPS 向心肌细胞分化 5、7、21、28 d 时 ISL1 基因以及分化 7、21、28 d 时 NKX2-5 基因相对表达量均显著高于 hACM（ P<0.05），分化 3、5、7、21 d ACTN1 和 TNNT2 基因相对表达量均显著低于 hACM（ P<0.05）。NKX2-5 在绝大部分细胞核中表达；cTnT 和 α-actinin，MLC-2A 和 MLC-2V 信号定位于细胞质中，并初步呈现出肌小结纹理样结构。流式细胞术检测示 HiPS 向心肌细胞诱导分化成功。过表达 TBX3 组中，TBX18、SHOX2、HCN4、HCN1 较对照组表达均有上调，且 SHOX2 基因相对表达量差异有统计学意义（ P<0.05）；NKX2-5 基因相对表达量虽低于对照组，但差异无统计学意义（ P>0.05）。过表达 TBX18 组中，各基因相对表达量与对照组比较差异均无统计学意义（ P>0.05）。.
结论: HiPS 和 hESCs 的多能性相近，建立了稳定的 HiPS 干性维持及培养方法；成功建立 HiPS 向心肌细胞高效分化技术平台；尽管 TBX3、TBX18 在 HiPS 向窦房结样细胞富集分化中的促进作用不显著，但 TBX3 呈现出一定促进趋势，未来可进一步探索。.
Keywords: Human derived induced pluripotent stem cells; TBX18; TBX3; sinoatrial node-like cells.