Purpose

The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks.

Methods

hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus.

Results

hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC₄(5), an electrically responsive dye.

Conclusion

Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

中文翻译：

---

使用人类成脂间充质干细胞衍生的Purkinje细胞对心脏Purkinje系统进行3D生物打印。

目的

这项研究的目的是对人类脂肪形成的间充质干细胞（hADMSC）进行重编程，以形成Purkinje细胞，并使用经过重编程的Purkinje细胞对Purkinje网络进行生物打印。

方法

通过使用转录因子ETS2和MESP1的多步骤过程，将hADMSCs重新编程以形成Purkinje细胞，首先形成心脏祖细胞，然后由SHOX2和TBX3形成Purkinje细胞。以Pluronic酸为牺牲材料，以I型胶原为结构材料，开发了一种新颖的生物打印方法。重新编程的浦肯野细胞与新型生物印染方法结合使用，可以对浦肯野网络进行生物印染。评价印刷的构建体的功能蛋白连接蛋白40（Cx40）的保留以及响应生理刺激而发生膜电位变化的能力。

结果

根据IRX3，IR​​X5，SEMA和SCN10的表达模式，成功地将hADMSC重新编程以形成Purkinje细胞。将重新编程的浦肯野细胞掺入1型胶原生物墨水中，并使用牛浦肯野系统的解剖图像作为参考来打印左心室浦肯野网络。优化研究表明，1.8 mg / mL的I型胶原蛋白的播种密度为每200 µ L 300,000个细胞，可产生最具功能的生物打印浦肯野网络。此外，生物打印的浦肯野网络形成了连续的合胞体，保留了重要的功能性间隙连接蛋白Cx40的印后表达，并通过DiBAC ₄评估了响应电刺激和乙酰胆碱的膜电位变化。（5），电响应染料。

结论

基于这项研究的结果，成功地将hADMSCs重新编程以形成Purkinje细胞，并进行生物印刷以形成Purkinje网络。