Synthetic mRNA switches for detection and purification of cardiomyocytes and endothelial cells derived from human pluripotent stem cells [mRNA]. Homo sapiens

We designed microRNA-responsive, modified mRNA switches (miR-switches) that quantitatively control their own translation level by determining the miRNA activities. We found that the three miR-switches (i.e., miR-1-, miR-208-, and miR-499-switches) enable to purify cardiomyocytes differentiated from hPSCs with high efficiency, accuracy, and safety. The purified cardiomyocytes were engrafted in mouse heart and did not form tumor. Simultaneous purification of two different cell types, cardiomyocytes and endothelial cells, was also performed by transfecting the mRNA responding to the both miR-208 and miR-126, into heterogeneous cell population derived from hPSCs. In addition, selective induction of apoptosis in noncardiomyocytes triggered by miR-1/208-Bim switch enriched cardiomyocytes without cell sorting. The mRNA switch could purify desired cell types and control cell death pathways by monitoring the miRNA expression dynamics during cell fate conversion. Overall design: MYH6-EIP4 iPSCs for mRNA, N=3 MYH6-EIP4 day18 cardiomyocytes (before transfection) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (miR-1-switch day1) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (miR-1-switch day7) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=3 201B7 d22 miR-208a-BFP sorted cells for mRNA, N=1 201B7 d22 miR-208a-BFP negative fraction sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- negative fraction sorted cells for mRNA, N=1 201B7 d22 VCAM1+ sorted cells for mRNA, N=1 201B7 d22 VCAM1+negative fraction sorted cells for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (miR-208a-Bim-switch day1) for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=1

本研究设计了响应微小RNA（microRNA, miRNA）的修饰型mRNA开关（miR-switches），该类开关可通过感知miRNA的活性水平，定量调控自身的翻译效率。研究结果显示，三款miR开关（分别为miR-1开关、miR-208开关与miR-499开关）能够以高效率、高精准度与高安全性，从人多能干细胞（human pluripotent stem cells, hPSCs）分化得到的细胞群中纯化心肌细胞。纯化后的心肌细胞可成功移植入小鼠心脏组织，且未形成肿瘤。通过将同时响应miR-208与miR-126的mRNA转染至源自hPSCs的异质性细胞群中，还可实现心肌细胞与内皮细胞这两种不同细胞类型的同步纯化。此外，由miR-1/208-Bim开关触发的非心肌细胞选择性凋亡，可在无需细胞分选的情况下实现心肌细胞的富集。综上，该mRNA开关可通过监测细胞命运转化过程中的miRNA表达动态，实现目标细胞类型的纯化，并精准调控细胞死亡通路。

整体实验设计：
1. 用于mRNA测序的MYH6-EIP4诱导多能干细胞，N=3
2. 用于mRNA测序的MYH6-EIP4第18天心肌细胞（转染前），N=3
3. 用于mRNA测序的MYH6-EIP4第19天心肌细胞（miR-1开关转染第1天），N=3
4. 用于mRNA测序的MYH6-EIP4第19天心肌细胞（未转染），N=3
5. 用于mRNA测序的MYH6-EIP4第25天心肌细胞（miR-1开关转染第7天），N=3
6. 用于mRNA测序的MYH6-EIP4第25天心肌细胞（未转染），N=3
7. 用于mRNA测序的201B7细胞系第22天miR-208a-蓝色荧光蛋白（Blue Fluorescent Protein, BFP）阳性分选细胞，N=1
8. 用于mRNA测序的201B7细胞系第22天miR-208a-BFP阴性分选细胞，N=1
9. 用于mRNA测序的201B7细胞系第22天信号调节蛋白α（Signal Regulatory Protein α, SIRPA）阳性、谱系阴性（LIN-）分选细胞，N=1
10. 用于mRNA测序的201B7细胞系第22天SIRPA+LIN-阴性分选细胞，N=1
11. 用于mRNA测序的201B7细胞系第22天血管细胞黏附分子1（Vascular Cell Adhesion Molecule 1, VCAM1）阳性分选细胞，N=1
12. 用于mRNA测序的201B7细胞系第22天VCAM1+阴性分选细胞，N=1
13. 用于mRNA测序的MYH6-EIP4第19天心肌细胞（miR-208a-Bim开关转染第1天），N=1
14. 用于mRNA测序的MYH6-EIP4第19天心肌细胞（未转染），N=1
15. 用于mRNA测序的MYH6-EIP4第21天心肌细胞（miR-208a-Bim开关转染第7天），N=1
16. 用于mRNA测序的MYH6-EIP4第21天心肌细胞（未转染），N=1
17. 用于mRNA测序的MYH6-EIP4第25天心肌细胞（miR-208a-Bim开关转染第7天），N=1
18. 用于mRNA测序的MYH6-EIP4第25天心肌细胞（未转染），N=1