Department of Cytology, Institute of Developmental Biology and Biomedical Sciences, Faculty of Biology, University of Warsaw, Warsaw, Poland.
Laboratory of Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Stem Cell Res Ther. 2020 Jun 17;11(1):238. doi: 10.1186/s13287-020-01742-3.
Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population.
In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers.
We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired.
Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.
多能干细胞具有自我更新和分化为构建生物体的任何细胞类型的能力。重要的是，大量关于胚胎干细胞 (ESC) 分化的证据来自体外研究。然而，体外培养的 ESC 不一定表现为体内分化的细胞。出于这个原因，我们使用畸胎瘤来研究体内 ESC 成肌分化的早期和晚期阶段以及 Pax7 在这个过程中的作用。Pax7 转录因子在胚胎发育过程中骨骼肌肉前体细胞的形成和分化中起着至关重要的作用。它控制着其他成肌调节因子的表达，也作为一种抗细胞凋亡因子。它还参与卫星细胞群体的形成和维持。
我们使用体内方法涉及多能干细胞衍生的畸胎瘤的生成和分析。这种模型允许分析组织分化的早期和晚期阶段，例如成肌作用的晚期阶段，包括有神经支配和血管化的成熟肌纤维的形成。
我们确定了缺乏 Pax7 功能如何影响不同肌纤维类型的产生。与 Pax7+/+ 相比，Pax7-/- 畸胎瘤中的骨骼肌组织面积明显更小。在 Pax7+/+ 和 Pax7-/- 畸胎瘤中，表达 Myh3 和 Myh2b 的肌纤维比例没有差异。然而，Pax7-/- 中的 Myh7 和 Myh2a 肌纤维面积明显较低。骨骼肌的分子特征表明，Pax7-/- 畸胎瘤中编码 Myh 同工型的 mRNA 水平明显降低。Pax3 的 mRNA 水平显著升高，而 Pax7-/- 畸胎瘤中 Eno3、Mck、Mef2a 和 Itga7 的表达水平明显低于 Pax7+/+ 畸胎瘤。我们证明 Pax7-/- 畸胎瘤中的卫星细胞数量明显减少。最后，使用 iDISCO 方法制备的样本中神经肌肉接头定位的分析证实，Pax7-/- 畸胎瘤中神经肌肉接头的组织受损。
与 Pax7+/+ 细胞相比，Pax7-/- ESC 在体内更易分化为胚胎成肌细胞。在缺乏功能性 Pax7 的情况下，成肌分化的启动得到促进，结果是中胚层胚胎成肌细胞标记物的表达上调。然而，在缺乏功能性 Pax7 的情况下，神经肌肉接头的形成异常，导致 Pax7-/- ESC 在成肌作用的晚期阶段分化潜力降低。
