||Defined MicroRNAs Induces Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell Derived Cardiomyocytes
||Institute of Clinical Medicine
||Desy S Lee
stem cell-derived cardiomyocyte
至今，心衰竭仍是一種無法徹底治癒的嚴重病症且預後不佳。近期的研究發現幹細胞療法(stem-cells therapy)為心衰竭的治療開啟新的契機。胚胎幹細胞(embryonic stem cells)和誘導式多潛能幹細胞(induced pluripotent stem cells, iPS cells)具有潛力可無限量的提供心肌細胞作為幹細胞療法的細胞來源。然而，從這些細胞誘導分化的心肌細胞屬於未成熟的；它們不具有成熟的肌節構造(sarcomeric structure)和間隙接合蛋白(gap junction protein)、 無法共同跳動，提高了心律不整(arrhythmia)的危險性若使用它們作為治療的細胞來源。
Advances in our ability to differentiate pluripotent stem cells into somatic cells of many lineages provide us with a platform for drug screening, disease modeling and regenerative medicine. Due to the high prevalence of cardiovascular disease, cardiac cells are of particular interest. Several outstanding groups have developed reproducible and efficient systems to differentiate pluripotent cells into cardiac cells. However, these pluripotent stem cell-derived cardiomyocytes often display the structural and functional attributes of fetal cardiomyocytes rather than adult cardiomyocytes. This significantly limits the use of these cells for applications such as drug screening or regenerative medicine. Thus, a method for increasing the maturity of pluripotent stem cell-derived cardiomyocytes is highly desirable.
Studies of anatomy and developmental biology suggest that the function of heart is considerably dependent on endothelial cells. We have shown that endothelial cells play a role in enhancing the maturation of murine ES-derived cardiomyocytes. We have identified a cluster of miRNAs (miR-125b-5p, miR-199a-5p, miR-221, and miR-222) that are upregulated in cardiomyocytes upon their coculture with endothelial cells. Exogenous addition of this combination of miRNAs was able to significantly increase the molecular, structural and functional maturation of ES-derived CMs. Bioinformatics revealed ErbB4 as the target of these four miRNAs, and a luciferase reporter assay confirmed that these four miRNAs together targeted ErbB4. This finding was further confirmed by siRNA-induced downregulation of ErbB4, resulting in the enhanced maturation of ES-CMs.
To apply these findings to human cardiomyocyte maturation, we tested the same miR combination in human ES-derived cardiomyocytes. The enforced expression of this miR-combo was able to increase maturation of ES-derived cardiomyocytes into a more adult-like state, evidenced by an increased binucleation ratio, lower ANF expression, improved respiratory capacity, more negative resting membrane potential and polarized Connexin-43. In conclusion, we present a novel approach for improving the maturity of cardiomyocytes differentiated from pluripotent stem cells. This is a significant step towards realizing the full potential of iPS and ES-derived cardiomyocytes as platforms for drug screening and disease modeling.
Table of Contents vi
List of Table/Figures x
Chapter 1 1
1.1 Stem Cell Therapy 2
1.1.1 Pluripotent Stem Cells as Cardiomyocytes Sources 2
A. Embryonic Stem Cells 2
B. Induced Pluripotent Stem Cells 3
1.1.2 Challenges in Isolation of Cardiomyocytes 4
A. Differentiation Efficiency 4
B. Purification of ES-CMs 5
C. Maturity Status of ES-CMs 8
1.2 MicroRNA 11
1.2.2 MicroRNA: Biogenesis and Target Recognition 11
1.2.2 MicroRNA in Heart Development 12
1.3 Thesis Aim 14
Chapter 2 16
Materials and Methods 16
2.1 Cardiac Differentiation and Culture of Mouse ESCs 16
2.2 Cardiac Differentiation and Culture of Human ESCs 17
2.3 Real-time Quantitative Polymerase Chain Reaction 17
2.4 Immunostaining and Fluorescence Microscopy 18
2.5 Transmission Electron Microscopy 18
2.6 Measurement of Ca2+ Transients 19
2.7 Action Potential Measurements 19
2.8 Exiqon miRNA Microarray 20
2.9 Transient Transfection 21
2.10 Western blotting 21
2.11 Preparation of Conditioned Medium, Endothelial Cell Lysate and Extracellular Matrix 21
2.12 Dual-Luciferase Reporter Assay 21
2.13 siErbB4 Microarray 22
2.14 Data Analysis 23
Chapter 3 24
3.1 Differentiation and purification of mouse ES-CMs 24
3.2 Coculture with endothelial cells improves the alignment of ES-CMs 24
3.3 Culture along with endothelial cells improves the maturation of ES-CMs 25
3.4 Microarray-based identification of microRNA modulated upon coculture 27
3.5 A combination of miRNAs is more effective than individual miRNA in promoting the maturation of ES-CMs 28
3.6 The introduction of miR-combo promotes the morphological maturation of ES-CMs 29
3.7 The structural maturation of ES-CMs is accompanied by enhanced functional maturation after miR-combo delivery 29
3.8 ErbB4 as a target of miR-combo 30
3.9 siRNA knockdown of ErbB4 enhanced several maturation-associated phenotypes in mES-CMs 31
3.10 The establishment of human cardiac differentiation platform 32
3.11 miR-combo transfection leads to hES-CMs structural changes in accordance to enhanced maturation 33
3.12 Effects on miR-combo on human ES-CM electrophysiological properties 34
3.13 The effects of miR-combo delivery after a longer culture period 35
Chapter 4 37
4.1 Differentiation and purification of ES-CMs 37
4.2 Endothelial cells promote structural and functional maturation of ES-CMs 38
4.3 Multiple origins of endothelial cells can improve ES-CM maturity 39
4.4 Four defined microRNAs, elevated during EC coculture, sufficiently promote the maturation of mouse ES-CMs 40
4.5 ES-CM microRNA elevation appears to depend on the presence of endothelial cells 41
4.6 ErbB4 is the predicted target gene of these four microRNAs 43
4.7 ErbB4 is a known player in cardiac development and implicated in maturation 43
4.8 An extensive range of assays reveal improved maturation of human ES-CMs following transfection of miR-combo 46
4.9 Fully adult-like ES-CMs still remain elusive 47
4.10 Summary 48
4.11 Conclusion 50
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