Identification of Latrophilin-2 as a Novel Cell-Surface Marker for the Cardiomyogenic Lineage and Its Functional Significance in Heart Development

Identification of a lineage-specific marker that enables monitoring of subsets would be valuable for establishing the conditions under which pluripotent stem cells (PSCs) differentiate into cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs). The demand for PSC-derived CMCs for use in studies on cardiovascular disease has increased in recent years.¹ Here, we report a new cardiac lineage marker and demonstrate its functional significance in heart development.

We established a protocol for directed PSC differentiation into CMCs after exposing cells to various combinations of cytokines for different times, based on the biology of embryonic development.² When mouse PSCs were stimulated with multiple cytokines, they differentiated into cardiac cells (Figure [A]). To screen surface markers of CPC during PSC differentiation, we performed microarray analysis. We aimed to discover molecules whose expression increased sequentially during cardiac lineage differentiation and exclusively in the CPC-enriched population expressing both Flk-1 (fetal liver kinase 1) and PdgfR-α (platelet-derived growth factor receptor-α).² Therefore, we compared gene expression profiles in 4 different cell populations: (1) undifferentiated PSCs (group 1; Flk-1⁺ PdgfR-α⁺ [F⁺P⁺], 4.9±1.1%), (2) spontaneously differentiated cells at day 4 (group 2; F⁺P⁺, 11.8±5.2%), (3) cells that underwent optimized cardiac differentiation at day 4 (group 3; F⁺P⁺, 33.0±5.8%), and (4) cells enriched for CPCs after fluorescence-activated cell sorting (group 4; F⁺P⁺, 90.4±4.3%; Figure [B]). In groups 1 to 4, we identified 7 genes whose expression increased continuously; among the groups, these genes showed the highest expression in group 4 (Figure [C]). Among these 7 candidate genes, we focused on the little-studied G protein–coupled receptor, latrophilin-2 (Lphn2), which is expressed on the cell surface and may have functional significance (Figure [D]).

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To confirm that Lphn2 is necessary for cardiac development, we established Lphn2-knockout (KO) embryonic stem cells. Reverse transcription–polymerase chain reaction and Western blot analyses confirmed that Lphn2 expression increased during cardiac differentiation in wild-type embryonic stem cells, whereas the expression did not appear in Lphn2-KO embryonic stem cells (Figure [E] and [F]). During cardiac differentiation, Lphn2-KO cells failed to express cardiac-related genes, such as Gata4 and Nkx2.5, which encode transcription factors central for heart development; Tbx5, a transcription factor of the primary heart field; Isl1, a transcription factor of the secondary heart field; and cTnT, a structural protein of CMCs (Figure [G]).

To verify the in vivo functional significance of Lphn2 during development, we generated Lphn2-KO mice (Figure [H] and [I]). All animal experiments were approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital. Although Lphn2 heterozygous (Lphn2^+/−) mice were alive and fertile, homozygous Lphn2 (Lphn2^−/−) mice showed embryonic lethality. Immunofluorescent staining of E11.5 embryos of Lphn2^+/− mice revealed no significant gross abnormality compared with wild-type mice embryos, whereas Lphn2^−/− embryos exhibited serious defects in the development of the right ventricle, right atrium, and outflow tract (Figure [J]). Additionally, the muscle mass of the left ventricle was much smaller than those of wild-type and heterozygous embryos (Figure [J]).

Next, we investigated the mRNA expressions of regulatory genes involved in heart development during embryogenesis. Because Lphn2-KO cells did not express cardiac-related genes in vitro, we assessed how Lphn2 deficiency functions in heart development. Notably, similar results were observed in the quantitative polymerase chain reaction analysis of embryonic tissues (Figure [K]). Lphn2^+/− embryos were not significantly different from wild-type embryos. In contrast, Lphn2^−/− embryos showed markedly reduced expression of genes encoding transcription factors that are regulators of heart development, including Gata4, Nkx2.5, Tbx5, and Isl1, as well as a cardiac structural gene, cTnT (Figure [K]). Also, we analyzed wild-type, Lphn2^+/−, and Lphn2^−/− embryos to further investigate the role of Lphn2 in the late stage of heart development. Interestingly, the hearts of Lphn2^−/− embryos at embryonic day 15.5, unlike those of wild-type and Lphn2^+/− embryos, had a small and single ventricle, revealing the defect of cardiomyogenic development (Figure [L]).

Extensive studies on preclinical and clinical cell therapies for heart disease have used several types of cells for cardiac repair. Although CPCs have been identified using multiple markers,³ it is very challenging to isolate PSC-derived CPCs and CMCs in vitro, because most cardiac-specific markers are intracellular molecules or transcription factors.^4,5 Thus, cell-surface markers are needed to purify CPCs and CMCs from heterogeneous cell populations during stem cell differentiation. In conclusion, we have demonstrated that Lphn2 is a unique cell-surface marker of cardiomyogenic cells. These findings provide a valuable tool for isolating CPCs from PSCs, as well as novel insights into heart development.

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