Discovering cell-surface markers based on a comprehensive understanding of development is utilized to isolate a particular cell type with high purity for therapeutic purposes. Given that latrophilin-2 (Lphn2) substantially contributes to cardiac differentiation, we examined whether Lphn2 regulates functional significance in heart development and repair. We performed whole-mount immunostaining followed by clearing technique of embryo, RNA sequencing related to Lphn2-knockout (KO) embryo, and in vivo functional analyses of Lphn2+ cells using echocardiography. After immunostaining the cleared embryo sample, Lphn2 was exclusively observed in cardiac cells expressing α-sarcomeric actinin at embryonic days E9.5 and E10.5. Homozygous Lphn2-KO mice were embryonically lethal and showed underdevelopment of the ventricular myocardium. However, Lphn2 was not required to develop vessels, including endothelial cells and smooth muscle cells. For the purpose of cardiac regeneration, we transplanted pluripotent stem cell (PSC)-derived Lphn2+ cells into the infarcted heart. PSC-derived Lphn2+ cells differentiated into cardiomyocytes and regenerated the myocardium when transplanted into the infarcted heart, unlike Lphn2- cells. Transplanted Lphn2+ cells improved left-ventricle systolic function and reduced infarct size. We demonstrated that Lphn2 exhibits potential as a cardiomyogenic marker to facilitate targeted stem cell therapy for heart repair in clinical practice.

Duffy antigen receptor for chemokines (DARC)/CD234, also known as atypical chemokine receptor 1 (ACKR1), is a seven-transmembrane domain protein expressed on erythrocytes, vascular endothelium, and a subset of epithelial cells (Peiper et al., 1995). Previously, we reported that ACKR1 was expressed in bone marrow macrophages. ACKR1 interacts with CD82 on long-term repopulating hematopoietic stem cells (LT-HSCs) to maintain the dormancy of LT-HSCs during homeostasis (Hur et al., 2016). We also demonstrated that ACKR1 interacts with CD82 in HSCs from human umbilical cord blood (hUCB). These findings demonstrated that CD82 is a functional surface marker of LT-HSCs and this molecule maintains LT-HSC quiescence by interactions with ACKR1-expressing macrophages in mice and humans.

Previously, we found that the delivery of mouse ES (mES) cell-derived proteins to adult fibroblasts enables the full reprogramming of these cells, converting them to mouse pluripotent stem cells (protein-iPS cells) without transduction of defined factors. During reprogramming, global gene expression and epigenetic status such as DNA methylation and histone modifications convert from somatic to ES-equivalent status. mES cell extract-derived iPS cells are biologically and functionally indistinguishable from mES cells in its potential in differentiation both in vitro and in vivo. Furthermore, these cells show complete developmental potency. However, the efficiency of generating iPS by treatment with extract from mES cells is still low. In this report, we demonstrated that protein extracts of mouse iPS cells that were previously generated by mES cell extract treatment were able to reprogram somatic cells to become ES-like cells (secondary protein-iPS cells). We confirmed that fetal animals (E12.5) could be derived from these cells. Surprisingly, the efficiency of forming Oct4-positive colonies was remarkably improved by treatment of somatic cells with mouse iPS cell extract in comparison to treatment with mES cell extract. By screening the genes differentially expressed between mouse iPS and mES cells, Zscan4, which is known to enhance telomere elongation and stabilize genomic DNA, was identified as a strong candidate to promote efficiency of reprogramming. Interestingly, treatment with protein extracted from mES cells overexpressing Zscan4 enhanced formation of Oct4-positive colonies. Our results provide an efficient and safe strategy for reprogramming somatic cells by using mouse iPS cell extract. Zscan4 might be a key molecule involved in the demonstrated improvement of reprogramming efficiency.

The inhibitors of CD26 (dipeptidyl peptidase-4; DPP4) have been widely prescribed to control glucose level in diabetic patients. DPP4-inhibitors, however, accumulate stromal cell-derived factor-1α (SDF-1α), a well-known inducer of vascular leakage and angiogenesis both of which are fundamental pathophysiology of diabetic retinopathy. The aim of this study was to investigate the effects of DPP4-inhibitors on vascular permeability and diabetic retinopathy. DPP4-inhibitor (diprotin A or sitagliptin) increased the phosphorylation of Src and vascular endothelial-cadherin (VE-cadherin) in human endothelial cells and disrupted endothelial cell-to-cell junctions, which were attenuated by CXCR4 (receptor of SDF-1α)-blocker or Src-inhibitor. Disruption of endothelial cell-to-cell junctions in the immuno-fluorescence images correlated with the actual leakage of the endothelial monolayer in the transwell endothelial permeability assay. In the Miles assay, vascular leakage was observed in the ears into which SDF-1α was injected, and this effect was aggravated by DPP4-inhibitor. In the model of retinopathy of prematurity, DPP4-inhibitor increased not only retinal vascularity but also leakage. Additionally, in the murine diabetic retinopathy model, DPP4-inhibitor increased the phosphorylation of Src and VE-cadherin and aggravated vascular leakage in the retinas. Collectively, DPP4-inhibitor induced vascular leakage by augmenting the SDF-1α/CXCR4/Src/VE-cadherin signaling pathway. These data highlight safety issues associated with the use of DPP4-inhibitors.

The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming.

Poor homing efficiency is one of the major limitations of current stem cell therapy. Magnetic bionanoparticles (MPs) obtained from Magnetospirillum sp. AMB-1 have a lipid bilayer membrane and ferromagnetic properties. We evaluated a novel priming strategy using MPs to enhance the homing of transplanted progenitor cells to target tissue.

Lysophosphatidic acid receptor 4 (LPAR4) exhibits transient expression at the cardiac progenitor stage during pluripotent stem cell (PSC)-derived cardiac differentiation. Using RNA sequencing, promoter analyses, and a loss-of-function study in human PSCs, we discovered that SRY-box transcription factor 17 (SOX17) is an essential upstream factor of LPAR4 during cardiac differentiation. We conducted mouse embryo analyses to further verify our human PSC in vitro findings and confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development. In an adult bone marrow transplantation model using LPAR4 promoter-driven GFP cells, we observed two LPAR4+ cell types in the heart following myocardial infarction (MI). Cardiac differentiation potential was shown in heart-resident LPAR4+ cells, which are SOX17+, but not bone marrow-derived infiltrated LPAR4+ cells. Furthermore, we tested various strategies to enhance cardiac repair through the regulation of downstream signals of LPAR4. During the early stages following MI, the downstream inhibition of LPAR4 by a p38 mitogen-activated protein kinase (p38 MAPK) blocker improved cardiac function and reduced fibrotic scarring compared to that observed following LPAR4 stimulation. These findings improve our understanding of heart development and suggest novel therapeutic strategies that enhance repair and regeneration after injury by modulating LPAR4 signaling.

Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we investigated the functioning of a cardiomyogenic cell-surface marker, latrophilin-2 (LPHN2), an adhesion G-protein-coupled receptor, in cardiac differentiation. LPHN2 was selectively expressed in cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation; cell sorting with an anti-LPHN2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knockdown or knockout PSCs did not express cardiac genes. We used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways, to assess molecular mechanisms underlying LPHN2-induced cardiac differentiation. LPHN2-dependent phosphorylation was the strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of LPHN2 signaling. These findings provide a valuable strategy for isolating CPCs and CMCs from PSCs and insights into the still-unknown cardiac differentiation mechanisms.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.