Abstract 15113: A Novel Purification Method for Cardiomyocytes and Endothelial Cells Derived From Human Pluripotent Stem Cells Using Microrna-responsive Messenger Rna

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kenji Miki ◽  
Seiya Takahashi ◽  
Shunsuke Funakoshi ◽  
Shinya Yamanaka ◽  
Hirohide Saito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Messenger Rna ◽  
Pluripotent Stem Cell ◽  
Troponin T ◽  
Target Cells ◽  
Mouse Heart ◽  
Effective Manner ◽  
High Purification

MicroRNAs (miRNAs) are small non-coding RNA that controls gene expression through translational regulation or mRNA cleavage. Recent progress in synthetic biology demonstrated that synthetic “RNA switches” enable to regulate gene expression depending on the intracellular environment. Here we show that synthetic mRNA technologies that modulate reporter protein expression by sensing miRNA activities enable purification of target cells in a safe and effective manner. To select cardiac-specific miRNA candidates, we performed miRNA microarray analyses using human induced pluripotent stem cell (hiPSC) line integrated MYH6 promoter driven EGFP cassette. We found 14 candidates, and synthesized individually these 14 miRNA-responsive OFF switch mRNAs (miR-switches) containing the sequence of blue fluorescent protein (TagBFP). We individually transfected them with EGFP mRNA into differentiated cells including cardiomyocytes derived from human pluripotent stem cell (hPSCs). We found that several cells transfected with miR-1- or miR-208-switches diminished TagBFP intensity, and sorting of these cells yielded high populations of troponin T-positive cardiomyocytes reproducibly and significantly (e.g., up to >98% in five hPSC lines). In addition, we developed a non-sorting system for purification of cardiomyocytes using miR-switch that code apoptosis-inducible protein (up to >97%). These cardiomyocytes purified by miR-switches were successfully engrafted in mouse heart and did not form tumor even when injected into testes of SCID mice. We next examined whether the system can be applied to purify endothelial cells, and confirmed that several miR-switches enabled high purification of CD31+ cells from differentiated derivatives of hPSCs (up to >98%), which have the function of tube formation like HUVEC. Moreover, the use of multiple miRNA-responsive mRNAs enabled simultaneous and high purification of cardiomyocytes and endothelial cells from heterogeneous population derived from hPSCs induced by single differentiation method (up to >98%). Our results suggest that miR-switch technology can be used for a variety of purposes including purification in any type of cells.

Abstract 498: Combinatorial Extracellular Matrix Microarrays for Probing Endothelial Differentiation of Human Induced Pluripotent Stem Cells

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Luqia Hou ◽  
John Coller ◽  
Vanita Natu ◽  
Ngan Huang
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Endothelial Differentiation ◽  
Induced Pluripotent

Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are a promising cell source for vascular regeneration in patients with peripheral arterial disease. However, a critical bottleneck to their clinical translation is the ability to differentiate the cells reproducibly at high yields. Since endothelial cells interact with the basement membrane extracellular matrix (ECM), we sought to examine the role of ECMs on endothelial differentiation using combinatorial ECM microenvironments. ECM microarrays were developed by covalent conjugation of ECMs (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, matrigel) and the multi-component combinations thereof. The pluripotent stem cells attached to the ECMs and subsequently differentiated over the course of 5 days. Endothelial differentiation was semi-quantitatively scored based on the degree of CD31 staining. Our results demonstrated greater levels of CD31staining when cultured on gelatin + matrigel + laminin (G+M+L) or fibronectin + laminin + heparan sulfate (F+L+H), compared to other combinations across three human pluripotent stem cell lines (iPSC-Huf5, iPSC-CON1, and ESC-H1). This enhancement in endothelial differentiation on the microscale was confirmed at larger cell culture platforms in which a marked increase in CD31+ cells was observed in G+M+L modified-dishes (> 5 fold), and F+L+H combination (> 10 fold), compared to gelatin-modified dishes. RT-PCR further confirmed the transcriptional upregulation in endothelial markers for CD31 (> 2 fold) and VE-cadherin (> 4 fold) on G+M+L, compared to gelatin-modified dishes. To elucidate the role of cell-ECM interactions on endothelial differentiation, gene expression of integrin subunits were examined. Gene expression was markedly upregulated in integrins α1 (>10 fold); α4, α5, and αV (>5 fold); and β1, β3 (>50 fold), and β4, when comparing differentiated cells on day14 to undifferentiated cells. The upregulation of integrin subunits was concomitant with upregulation in endothelial genes. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.

scholarly journals Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell-Derived Endothelial Cells

Stem Cells ◽  
2012 ◽  
Vol 31 (1) ◽  
pp. 92-103 ◽  
Author(s):  
Mark P. White ◽  
Abdul J. Rufaihah ◽  
Lei Liu ◽  
Yohannes T. Ghebremariam ◽  
Kathryn N. Ivey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Gene Expression Variation ◽  
Expression Variation ◽  
Induced Pluripotent

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kimimasa Tobita ◽  
Jason S Tchao ◽  
Jong Kim ◽  
Bo Lin ◽  
Johnny Huard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Immature Myocardium

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

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