Abstract
Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3−7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.
Highlights
When we analyzed the microarray data by normalizing (1) the undifferentiated induced pluripotent stem cells (iPSCs) as standard, we found 110 genes that were upregulated in cell stages (2), (3), and (4) on day 4
Since we were interested in identifying new surface markers, we looked for G-protein-coupled receptor (GPCR) genes among the 110 upregulated genes, and discovered four genes: lysophosphatidic acid receptor 4 (LPAR4), Latrophilin-2 (LPHN2), chemokine (C-X-C motif), receptor 4 (CXCR4), and the regulator of G-protein signaling 5 (RGS5; Figure 1D)
LPHN2 was published as a novel cardiac lineage marker that is expressed when pluripotent stem cells (PSCs) differentiate into cardiac progenitor cells (CPCs) and maintain the expression until CMC differentiation.[16]
Summary
The precise manipulation of embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) and the understanding of the characteristics of adult cardiac progenitor cells (CPCs) are essential for clinical applications.[1,2,3] Cell-based therapy shows great potential for several clinical applications, for tissue repair, including heart repair.[4,5,6] its application to the regeneration of the injured cardiac tissue is limited by two major issues, i.e., the requirement to induce efficient lineage-specific stem cell differentiation[7,8,9,10] and the need to deliver CPCs or immature cardiomyocytes (CMCs) efficiently to the damaged heart.[5,11,12] The identification of lineagespecific markers for CPCs could help the development of methods to drive CMC differentiation. Even if cardiac differentiation is achieved, the effective delivery of CPCs or CMCs to the injured heart remains a substantial challenge. To solve these issues, we searched for novel markers that specify cardiac lineage, using microarray analysis of four cell populations that differ in terms of the degree of enrichment of cardiac progenitors during differentiation of ESCs/iPSCs toward CMCs. We found that the G-protein-coupled receptor (GPCR)[13] lysophosphatidic acid receptor 4 (LPAR4) is a strong candidate. The effectiveness of LPAR4 as a cardiac progenitor-specific marker and its functions were further evaluated based on its spatiotemporal expression patterns in the mouse heart during development and cardiac differentiation. We used a mouse myocardial infarction (MI) model to highlight the concept of cell-free regeneration therapy with the optimal protocol to modulate the signaling of LPAR4
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