Evaluation of Porcine Versus Human Mesenchymal Stromal Cells From Three Distinct Donor Locations for Cytotherapy

Abstract

Background: Mesenchymal stromal cell (MSC)-based cytotherapies fuel the hope for reduction of chronic systemic immunosuppression in allotransplantation, and our group has previously shown this capability for both swine and human cells. MSCs harvested from distinct anatomical locations may have different behavior and lead to different outcomes in both preclinical research and human trials. To provide an effective reference for cell therapy studies, we compared human and porcine MSCs from omental fat (O-ASC), subcutaneous fat (SC-ASC) and bone marrow (BM-MSC) under rapid culture expansion with endothelial growth medium (EGM). Methods: MSCs isolated from pigs and deceased human organ donors were compared for yield, viability, cell size, population doubling times (PDT), surface marker expression and differentiation potential after rapid expansion with EGM. Immunosuppressant toxicity on MSCs was investigated in vitro for four different standard immunosuppressive drugs. Immunomodulatory function was compared in mixed lymphocyte reaction assays (MLR) with/without immunosuppressive drug influence. Results: Human and porcine omental fat yielded significantly higher cell numbers than subcutaneous fat. Initial PDT was significantly shorter in ASCs than BM-MSCs and similar thereafter. Viability was reduced in BM-MSCs. Porcine MSCs were positive for CD29, CD44, CD90, while human MSCs expressed CD73, CD90 and CD105. All demonstrated confirmed adipogenic differentiation capacity. Cell sizes were comparable between groups and were slightly larger in human cells. Rapamycin revealed slight, mycophenolic acid strong and significant dose-dependent toxicity on viability/proliferation of almost all MSCs at therapeutic concentrations. No relevant toxicity was found for Tacrolimus and Cyclosporin A. Immunomodulatory function was dose-dependent and similar between groups. Immunosuppressants had no significant adverse effect on MSC immunomodulatory function. Discussion: MSCs from different harvest locations and donor species differ in terms of isolation yields, viability, PDT, and size. We did not detect relevant differences in immunomodulatory function with or without the presence of immunosuppressants. Human and pig O-ASC, SC-ASC and BM-MSC share similar immunomodulatory function in vitro and warrant confirmation in large animal studies. These findings should be considered in preclinical and clinical MSC applications.

Keywords: adipose-derived stromal cells; bone marrow stromal cells; endothelial growth medium; immunomodulation; immunosupressants; mixed lymphocyte reaction; omentum majus; transplantation.

Figure 1

Cell isolation. (A) Cell yields after isolation following specific protocols for each cell type. The resulting cell population after isolation is the so called ≪stromal vascular fraction≫ (SVF). BM-MSC are not displayed since no precise tissue amount could be determined due to flushing. Expressed in number of cells per mL of harvested tissue. Cross = mean, whiskers = 5–95 percentile. (B) Cell yields after first passaging of plated cells at a density of 10,000 cells/cm², passaged at 80–90% confluency. Expressed in number of cells per cm². Cross = mean, whiskers = 5–95 percentile. (C) Viability of freshly isolated cells as determined by Tryptan blue exclusion. Expressed in viable cell % of total cells. Error bars = SD. Unpaired t-test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 2

Cell culture. (A) Population doubling times (PDT) are shown for the three cell lines (O-ASC, SC-ASC, and BM-MSC) over 7 passages. Median ± interquartile range, expressed in hours. (B) The cell viability is displayed up to passage 7 for the three porcine and human cell lines (O-ASC, SC-ASC and BM-MSC) during culture with endothelial growth medium (EGM-2MV, Lonza). Determined by Tryptan blue exclusion. Cells lifted at 80–90% confluency. Expressed in %. Error bars = SD. One-way ANOVA with Tukey post-test. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Cell surface marker phenotype and adipogenic differentiation. The three cell lines (O-ASC, SC-ASC, and BM-MSC) were analyzed for their surface marker phenotype by flow cytometry (A; Bencton FACS Aria). CD73 showed no cross-reaction. Porcine cells: O-ASC n = 9, SC-ASC n = 7, BM-MSC n = 4 runs. Human cells: O-ASC n = 4, SC-ASC n = 4, BM-MSC n = 3 runs. Expressed as %. Error bars = SD. Adipogenic differentiation assays were performed with passage 3 cells in triplicates. Differentiation was quantified by fluorescence after Adipored® staining. (B left panel) Representative microscopic pictures. (B right panel) Results expressed as x-fold increased intensity compared to controls. Error bars = SD. Two-way ANOVA with Tukey post-test. ***p < 0.001, ****p < 0.0001.

Figure 4

Cell size analysis. Diameters of lifted cells were measured in passage 2–3 cells for the three cell lines (O-ASC, SC-ASC, and BM-MSC). (A) Cell diameters expressed in μm. Error bars = SD. One-way ANOVA with Tukey post-test. *p < 0.05. (B) Representative microscopic image of one cell in the hemocytometer showing how diameters were manually measured with ImageJ (NIH). Bar = 20 μm.

Figure 5

Immunosuppressive function. Mixed lymphocyte reaction assays (MLR) were performed to assess the immunomodulatory potential of the three cell lines (O-ASC, SC-ASC, and BM-MSC) head-to-head of both porcine (A) and human (B) origin. Yorkshire splenocytes were stimulated with PHA and co-cultured with the different cell lines for 3–5 days. Error bars = SD. Expressed as percentage proliferation in relation to positive control. Two-way ANOVA with Tukey post-test. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control (splenocytes+PHA).

Figure 6

Susceptibility of MSCs to immunosuppressive agents. The three cell lines (O-ASC, SC-ASC, and BM-MSC) were exposed to 4 different immunosuppressive agents commonly used in transplantation for 36 h to assess the effect on cell proliferation using AlamarBlue. Expressed in % viable cells compared to control (no drug = vehicle+medium only). Error bars = SD. Red boxes indicate the approximate therapeutic ranges (Tacrolimus: 5–20 ng/mL, Rapamycin 16–24 ng/mL, Cyclosporin A 100–400 ng/mL, Mycophenolic Acid 1-3.5 ug/L). Two-way ANOVA with Tukey post-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control (PBS only).

Figure 7

Susceptibility of MSC immunosuppressive function to immunosuppressive agents. Mixed lymphocyte reaction assays (MLR) were performed to assess the immunomodulatory potential of the three cell lines (O-ASC, SC-ASC, and BM-MSC) head-to-head of both porcine (A) and human (B) origin after incubation with immunosuppressive agents at therapeutic dosage (Tacrolimus 10 ng/mL, Rapamycin 10 ng/mL, Cyclosporin A 250 ng/mL) for 36 h. Yorkshire splenocytes were stimulated with PHA and co-cultured with the different cell lines at a 4:1 ratio. Expressed as percentage proliferation in relation to positive control. Error bars = SD. Two-way ANOVA with Tukey post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control (splenocytes+PHA).