Human Cardiac Progenitor Cells Enhance Exosome Release and Promote Angiogenesis Under Physoxia

Abstract

Studies on cardiac progenitor cells (CPCs) and their derived exosomes therapeutic potential have demonstrated only modest improvements in cardiac function. Therefore, there is an unmet need to improve the therapeutic efficacy of CPCs and their exosomes to attain clinically relevant improvement in cardiac function. The hypothesis of this project is to assess the therapeutic potential of exosomes derived from human CPCs (hCPCs) cultured under normoxia (21% O₂), physoxia (5% O₂) and hypoxia (1% O₂) conditions. hCPCs were characterized by immunostaining of CPC-specific markers (NKX-2.5, GATA-4, and c-kit). Cell proliferation and cell death assay was not altered under physoxia. A gene expression qPCR array (84 genes) was performed to assess the modulation of hypoxic genes under three different oxygen conditions as mentioned above. Our results demonstrated that very few hypoxia-related genes were modulated under physoxia (5 genes upregulated, 4 genes down regulated). However, several genes were modulated under hypoxia (23 genes upregulated, 9 genes downregulated). Furthermore, nanoparticle tracking analysis of the exosomes isolated from hCPCs under physoxia had a 1.6-fold increase in exosome yield when compared to normoxia and hypoxia conditions. Furthermore, tube formation assay for angiogenesis indicated that exosomes derived from hCPCs cultured under physoxia significantly increased tube formation as compared to no-exosome control, 21% O₂, and 1% O₂ groups. Overall, our study demonstrated the therapeutic potential of physoxic oxygen microenvironment cultured hCPCs and their derived exosomes for myocardial repair.

Keywords: angiogenesis; cardiac progenitor cells; cardiac repair; extracellular vesicles; hypoxia; stem cells.

FIGURE 1

hCPCs morphology and cardiac gene expression under normoxic and hypoxic microenvironments. Human CPCs were cultured under 21, 5, and 1% O₂ for 48 h. DIC imaging shows the typical morphology of cells, which is unchanged under hypoxia. Immunofluorescent staining for cardiac lineage markers NKX-2.5 (green, nuclear), GATA-4 (green, nuclear), and c-kit (green) showed their expression was maintained under all oxygen conditions. Nuclei are stained blue and F-actin is stained red.

FIGURE 2

hCPCs maintain normal function under hypoxia. hCPCs were cultured under 21, 5, and 1% O₂. (A) XTT assay for cell proliferation shows that cells maintained a similar level of proliferation over 24 h. Data represents mean ± SD, n = 4. (B) Flow cytometry for Annexin V/PI show that cells are equally healthy under the various oxygen conditions as the number of non-apoptotic/non-necrotic cells remains similar after 48 h of culture.

FIGURE 3

Culturing hCPCs under hypoxia modulates hypoxic gene expression. hCPCs were cultured under 21, 5, and 1% O₂ for 48 h then total RNA was harvested and gene expression analyzed via real-time PCR. Gene expression was normalized to five housekeeping genes and calculated as a fold change relative to levels at 21% O₂. (A) 5% O₂ culturing of hCPCs significantly increased expression of five genes, and significantly decreased expression of four genes. Data represented as mean ± SD, n = 3, all have p < 0.05 as compared to 21% O₂. (B) 1% O₂ culture altered expression of numerous hypoxia-related genes. 23 genes were significantly increased in expression and 8 genes were significantly decreased in expression, as compared to 21% O₂. Data represented as mean ± SD, n = 3, all have p < 0.05 as compared to 21% O₂. (C) Venn diagram illustrating similarly and differentially regulated genes under 5 and 1% O_2, as compared to 21% O₂.

FIGURE 4

Characterization of EVs derived from hCPCs under varying oxygen microenvironment. hCPCs were cultured under three different oxygen condition (21, 5, and 1% O₂) to generate EVs for analysis. (A) Cryo-TEM of isolated EVs shows their characteristic morphology of a round shape with a lipid bilayer (yellow arrows), which was identified under all three conditions. (B) EVs derived from 21, 5, and 1% O₂ hCPCs were analyzed with an exosome antibody array for known exosome markers and a negative exosome marker. The isolated EVs from all three O₂ conditions met the ISEV minimum requirements for identification. (C) NTA of triplicate samples shows the size and concentration distribution of EVs. (D) Mean and mode sizes of isolated EVs were similar and within the accepted range for extracellular vesicles, data is mean ± SD, n = 3. (E) The concentration of EVs isolated from hCPCs cultured under 5% O₂ microenvironment was higher than 21 and 1% O₂ groups, data is mean ± SD, n = 3. **p < 0.01, ***p < 0.001.

FIGURE 5

Effect of hCPC-derived EVs on endothelial cell migration. BAECs were grown to confluence, wounded with a pipet tip, and treated with 100 μg/ml of total EV protein from each condition. (A) Representative images from 0 and 8 h show the extent of cell migration, with dotted lines approximating the cell front. (B) Wound healing assay demonstrates that treating ECs with hCPC-derived EVs increased cell migration significantly when compared to No-EVs group. However, no significant differences were observed between 5 and 1% O₂ derived EVs. Data are shown as mean ± SD, n = 4, *p < 0.05 vs. No-EVs control.

FIGURE 6

EVs derived from hCPCs under physoxia enhances angiogenesis in vitro. BAECs were seeded onto Matrigel-coated wells with 100 μg/ml of total EV protein from each condition and incubated for 16 h. (A) Representative images illustrate the degree of tube formation for each group. (B) 1% O₂-derived EVs significantly increased tube formation in terms of the number of master junctions as compared to No-EVs control. However, 5% O₂-derived EVs significantly increased tube formation as compared to all groups in terms of the number of master junctions, the total master segment length, and the total mesh area. Data shown represents mean ± SD, n = 3, *p < 0.05 for comparison.