Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method

Abstract

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro, and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>10¹³ particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro, showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.

Keywords: cardiac progenitor cells; exosomes; good manufacturing practices; large-scale production; quality control.

FIGURE 1

Isolation and culture of CPC. (A) Representative images of CPC outgrowth from cardiac fragments (a–c) and CPC expansion (d–f), under the GMP grade methods I and II (CPC-I and -II) and the research grade method (CPC-R). The cumulative population doublings (CPD) over time is also shown (g) for the same representative experiment. (B) Doubling times (mean + SD, n = 5) of CPC cultured in the different conditions. NS, not significant; ^∗p < 0.05, one-way ANOVA with Student’s t-test with Bonferroni correction. (C) CPC number at harvest from the outgrowth plates, by culture conditions. CPC-I and -II: n = 5; CPC-R: n = 3.

FIGURE 2

FACS analysis of CPC cultured in different conditions. (A) Comparison of surface markers expression on CPC cultured in GMP conditions (CPC-I and -II, n = 5) or research conditions (CPC-R, n = 3), before (PRE) and after (POST) 7-day in vitro starvation for Exo production. Bars: mean + SD. (B) Histogram plots of surface markers expression of CPC-I, before (PRE) and after (POST) starvation for Exo production. Gray peaks: staining with isotype control antibodies; black peaks: staining with antibodies specific for the reported markers.

FIGURE 3

RT-PCR analysis of CPC cultured in different conditions. Mesodermal markers: TBX5 and TBX18; cardiac markers: GATA4 and MESP1. CPC samples were taken before (PRE) and after (POST) starvation for Exo production. Positive control: cardiac tissue. Amplification control: GAPDH. Negative controls (run in duplicate): reverse transcription negative sample with GAPDH primers. Gel images were cut above the primer dimers bands.

FIGURE 4

NTA profiles of Exo released by CPC cultured in different conditions. Size distribution graphs of the Exo from CPC expanded in GMP and research conditions. Red error bars indicate ±1 standard error of the mean. The mean ± standard error (n = 5) of the distribution modes from each replicate are shown.

FIGURE 5

In vitro anti-apoptotic activity of Exo released by CPC cultured in different conditions. Apoptotic agent: 1 mM staurosporine. CRL + = 10% FBS; black dotted bars = Exo-CPC-I and control (vehicle = Plasma-Lyte A^®); white dotted bars = Exo-CPC-R and control (Exo-F). Exo were added at 3 × 10⁷ particles/ml (100 μl/well), while the vehicle was added at the same volume as Exo-CPC-I. (A) Results from calcein staining. The treatment with complete medium + staurosporine is the 100% survival reference. (B) Results from PI staining. The treatment with the basal medium + staurosporine is the 100% cell death reference. Bars: mean + SD (n = 5). NS, not significant, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way ANOVA with Student’s t-test with Bonferroni correction.

FIGURE 6

Optimization and scale-up of Exo-CPC production. (A) TSG101 content and total protein content in CM from CPC-I and -II. Production scale: 15 ml; cells starved for 7 days before medium collection. Each dot represents the analyte concentration in CM from a single culture. (B) TSG101 content and total protein content in CM from CPC-I on different culture volumes; cells starved for 7 days before medium collection. Each dot represents the analyte concentration in CM from a single culture. (C) Time course of TSG101 and total protein content in CM collected from CPC-I at 1 week (7 ± 1 days) and 2 weeks (14 ± 1 days) during starvation. Production scale: 0.5 and 8 l. Paired Student’s t-test at 2 weeks versus 1 week starvation: p < 0.05 for TSG101 content, not significant for total proteins content.

FIGURE 7

Optimization and scale-up of Exo-CPC isolation. (A) Quantitative analysis of TSG101 and total proteins before and after concentration (a–d) and diafiltration (c,d) by different techniques. Mean + SD with n = 3 (a–c; CM collected after 1 week starvation) or n = 2 (d, CM collected after 2 weeks starvation). (B) TSG101 and total protein recovery as a percentage of the quantity measured in the initial CM (pre-concentration). Each dot is the result of an independent experiment. Pre-c, before concentration; post-c, after concentration; post-d, after diafiltration.

FIGURE 8

Large-scale GMP-Exo-CPC production chart. The upstream and downstream processes are grouped by the dotted boxes. See Section “Materials and Methods” for details.

FIGURE 9

Characterization of GMP-Exo-CPC. (A) Flow cytometry analysis. Two lots of GMP-Exo-CPC were analyzed with the MACSPlex Exosome Kit. Data are represented as APC mean fluorescence intensity normalized to the mean signal intensity obtained with the anti-CD9, anti-CD63, and anti-CD81 beads. (B) Representative TEM images.

FIGURE 10

In vitro anti-apoptotic activity of GMP-Exo-CPC. Apoptotic agent: 1 mM staurosporine. CRL + = 10% FBS; GMP-Exo-CPC doses: 0.5 and 5 ng of TSG101/ml (100 μl/well); vehicle = Plasma-Lyte A^® added at the same volume as GMP-Exo-CPC. (A) Representative images at 10x magnification of the cells after the indicated treatments. (B) Results from calcein staining. The treatment with the complete medium + staurosporine is the 100% survival reference. (C) Results from PI staining. The treatment with the basal medium + staurosporine is the 100% cell death reference. Bars: mean + SD (n = 5). NS, not significant, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way ANOVA with Student’s t-test with Bonferroni correction.

FIGURE 11

In vitro pro-angiogenic activity of GMP-Exo-CPC. GMP-Exo-CPC doses: 3 and 15 ng of TSG101/ml (0.5 ml/well). CRL + = VEGF; CRL – = suramin; vehicle = Plasma-Lyte A. (A) Representative images at 4x magnification of cells after the indicated treatments. CD31-stained tubules are visible in black. (B) Total tubule length detected by the AngioSys 2.0 Image Analysis Software. The mean length in VEGF treated cells was set to 100%. (C) CD31 expression detected by ELISA. The expression in VEGF treated cells was set to 100%.

FIGURE 12

In vivo therapeutic effect of GMP-Exo-CPC. (A) Representative echocardiographic M-mode images of hearts injected with vehicle and GMP-Exo-CPC, taken at day 28 after MI. (B) LVEF in sham-operated rats at day 28 (SHAM, n = 5), in GMP-Exo-CPC injected rats (black bars, n = 10), and vehicle injected rats (gray bars, n = 5) 7 and 28 days after MI. Bars: mean + SD. Measurements of LVESV (C) and LVEDV (D) in GMP-Exo-CPC injected rats (black line) and vehicle injected rats (gray line) 24 h, 7 and 28 days after MI. Mean ± SD is shown. Vehicle = PlasmaLyte A^®. ^∗p < 0.05, Student’s t-test.