Abstract MP215: Endothelial Colony Forming Cell Derived Exosomes Promote Angiogenesis And Cardiac Repair Post Myocardial Infarction

Background: Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance. Methods and Results: Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of MI prevented cardiac remodeling and heart failure. The acute MI resulted in severely decreased left ventricle ejection fraction (Sham 71.2% ± 5 .87, MI+Saline 32.9% ± 2.32) and fractional shortening (Sham 29.5% ± 3.20, MI+Saline 13.6% ± 2.87), and the administration of ECFC-exosomes prevented MI-induced cardiac dysfunction (ejection fraction: MI+ECFC-Exo 64.3% ± 8.74; fractional shortening: MI+ECFC-Exo: 26.4% ± 3.13). Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. Increased nuclear HIF-1α levels in response to ECFC-exosome administration, which may aid in the transcriptional function of HIF-1α, corroborated the role of exosomal miRNA in myocardial angiogenesis. We also found decreased levels of PTEN in response to ECFC-exosome treatment, which is a key negative regulator of PI3K/Akt pathways, survival pathways of heart. We also identified the relative angiogenesis expression profile of the peri-infarcted area in response to ECFC-exosome treatment. The ECFC-exosome administration upregulated the levels of VEGF, IGFBP-1 and PDGF, among others proangiogenic factors, and downregulated the levels of angiostatic factors as IP-10 and Thrombospondin-2. Conclusion: Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair after MI.

CD62L Expression Level Dictates the Cell Fate of Myeloid Progenitors in Mice and Humans

Hematopoietic cells are hierarchically differentiated from hematopoietic stem cells through several progenitors. Recent studies showed that each progenitor population has significant heterogeneity and some of the subsets have skewed differentiation potential. However, it has not been elucidated how and when common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) acquire different fates. We hypothesized that progenitor cells with skewed differentiation potential had acquired a part of gene expression profiles of more differentiated cells. By analyzing publicly available single-cell RNA sequencing data of human and murine CMPs and GMPs, we thoroughly explored surface markers with heterogeneous expression patterns and identified CD62L as a candidate to refine the differentiation potential of CMPs and GMPs. First, at CMP level, we clarified that CD62L-low CMPs had genuine CMP potential, whereas CD62L-high CMPs were mostly restricted to GMP potentials in both mice and humans. CD62L expression on CMPs was widely distributed and when divided into low, middle, and high fraction, colony forming-cell assay showed that murine CD62L-high CMPs mostly differentiated into CD11b-positive granulocytes and macrophages (97.4 ± 1.7%), whereas CD62L-low CMPs produced many erythroid and megakaryocytic colonies (38.7 ± 3.3%), and human CMPs had similar tendency. Also, liquid culture assay showed that murine CD62L-low CMPs differentiated into both GMPs (43.3 ± 4.1%) and megakaryocyte-erythrocyte progenitors (MEPs) (20.6 ± 3.7%), while CD62L-high CMPs mainly produced GMPs (82.8 ± 1.7%) and the proportion of MEPs was only 1.3 ± 0.1%. As for in vivo kinetics, we transplanted CD62L-low and high CMPs from GFP-expressing mice into irradiated wild-type mice, thus donor-derived platelets can be detected as GFP-positive. On day 7 after transplantation of each 15,000 cells, GFP-positive platelets from CD62L-low CMPs accounted for 9.8 ± 2.7% of all platelets, meanwhile CD62L-high CMPs accounted for only 0.20 ± 0.14%, which reinforced our hypothesis. Moreover, we performed transcriptome analysis using data of murine and human CMPs and focused on several transcription factors. Gata1, Klf1, Tal1 and Gfi1b are important transcription factors for erythroid and megakaryocytic differentiation, and these expressions were exclusively high in CD62L-low CMPs. On the other hand, Spi1 and Irf8 are important for differentiation into granulocytes and monocytes, and these expressions were higher in CD62L-high CMPs, which further corroborated the role of CD62L as a marker for refining the differentiation fate of CMPs. Second, at GMP level, we found that part of CD62L-neg GMPs possess remaining CMP potential and CD62L-low GMPs were skewed to granulocyte differentiation. CD62L expression on GMPs was mostly positive, but when we defined lowest 10% of GMPs as CD62L-neg GMPs, colony-forming cell assay in mice showed that part of CD62L-neg GMPs produced erythroid and megakaryocytic colonies (3.9 ± 2.6%), which suggested that this subset still possesses CMP potential. In vivo transplantation experiment using GFP-positive mice showed that CD62L-neg GMPs produced GFP-positive platelets slightly (0.02 ± 0.01%), but no platelets from CD62L-positive GMPs. Also, single-cell RNA-seq data revealed that part of CD62L-neg GMPs had CMP-specific gene expression pattern, suggesting that the bona fide GMPs were restricted to CD62L-positive GMPs. Next, we divided GMPs into CD62L-low and high GMPs, and performed colony-forming cell assay. CD62L-low GMPs produced granulocyte colony (CFU-G) 73.2 ± 6.1% and macrophage colony (CFU-M) 17.4 ± 3.3%, whereas CD62L-high GMPs produced CFU-G 46.6 ± 1.7% and CFU-M 42.9 ± 2.7%, which suggested that CD62L-low GMPs were granulocyte-skewed population. Also, we performed in vivo transplantation assay using Ly5.1 and Ly5.2 mice. On day 5 after transplantation, murine CD62L-low GMPs produced more neutrophils (87.6 ± 3.1%) in spleen than bulk GMPs (78.9 ± 1.9 %), which further confirmed this hypothesis. In summary, our in vitro and in vivo experiment and transcriptome analysis revealed that CMPs and GMPs had high heterogeneity. CD62L expression level refines the definition of CMPs and GMPs in both mice and humans, and elucidates the differentiation mechanism of myeloid cells in more detail. Disclosures Nakahara: Bristol-Myers Squibb Company: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria. Kagoya:NIPPON SHINYAKU CO.,LTD.: Research Funding; Bristol-Myers Squibb Company: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding. Kurokawa:Eisai: Research Funding, Speakers Bureau; Teijin: Research Funding; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Shire Plc: Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Sanwa-Kagaku: Consultancy; Pfizer: Research Funding; Otsuka: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application

Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process. The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+). Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. Disclosures No relevant conflicts of interest to declare.