Edited by: Ulrike Koehl, Hannover Medical School, Germany
Reviewed by: Susanne Petri, Hannover Medical School, Germany; Laurent Garderet, Assistance Publique Hopitaux De Paris, France
This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology
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Mesenchymal stem cells (MSCs) are well-known for their beneficial potential in a variety of conditions since discovery of their multi-facetted ability to proliferate, differentiate, heal, regenerate and modulate (
Inherent advantages of ASCs over their bone-marrow counterpart have shifted the focus accordingly (
Not only the anatomical cell origin is of relevance, but also the species may have impact on the functionality of MSCs: while porcine and human MSCs seem to possess similar characteristics suggesting that results can be extrapolated from preclinical studies and be applied to clinical protocols (
Cell-based therapies for tolerance induction in transplantation are usually applied in conjunction with immunosuppressants employed as conditioning or maintenance medication, and these may indeed have an influence on viability and function of the applied cells. It has been shown previously that drugs such as mycophenolic acid (MPA) and rapamycin (Rapa) can disturb viability and proliferation of MSCs (
Here, we aimed at directly comparing porcine MSCs harvested from three distinct anatomical locations head-to-head to their counterpart of human origin
The cells were isolated from domestic Yorkshire pigs post-mortem (
Isolation data.
3.8 ± 1.0 | 49.43 ± 11.3 | 112.9 ± 36.4 | 44.71 ± 7.3 | 4 ± 0.0 | 8.9 ± 8.8 | 19.4 ± 17.0 | 13.8 ± 1.6 |
45.1 ± 13.8 | 88.5 ± 18.6 | 115 ± 45 | 110 ± 43.6 | 35.5 ± 15 | 8.9 ± 4.4 | 30.7 ± 15 | 12.4 ± 5.2 |
Deceased tissue donors were referred after informed consent by the Center for Organ Recovery and Education (CORE). All tissue donors (
For isolation of SC-ASC and O-ASC, the tissues were minced with sterile scissors and handled with sterile forceps under a laminar flow hood until a relatively homogenous fat mass was obtained. The tissues were distributed into 50 mL conical tubes at 5 mL aliquots and 35 mL of sterile enzymatic solution added. The enzymatic solution was composed of type II collagenase (Worthington Biochemical Corp, Lakewood, NJ, USA), Proteinase K (Sigma-Aldrich) and Hanks' balanced saline solution (HBSS; Fisher Scientific) (for 100 mL of harvested fat: 1.4 g collagenase and 175 mg proteinase in 700 mL HBSS). The tubes were placed in a shaking water bath at 37°C for 90 min. Next, the digestate was filtered through 12-ply sterile gauze that had been unfolded twice (final gauze filter was 3-ply). The tubes were centrifuged at 1,000 rpm for 10 min. at room temperature (RT) and supernatant discarded. 10 mL red blood cell (RBC) lysis buffer (NH4Cl, A-0171; KHCO3; P-7682; EDTA, E-5134, all from Sigma; pH adjusted to 7.4, solution with de-ionized water) was added to each tube and cell pellets were disrupted by pipetting up and down gently. Lysates from 4 tubes were combined into 50 mL tube yielded approximately 40 mL buffer per tube. Additional gauze filtration was used as needed. Tubes were centrifuged again at 1,000 rpm for 10 min. at RT and supernatant discarded. Cells were resuspended in endothelial growth factor 2 medium (EGM-2MV, Lonza, CC-3156 & CC-4147) supplemented with penicillin/streptomycin 1% (Invitrogen), gentamycin 1% (Invitrogen) and amphotericin B 1% (initial seeding only; Invitrogen), counted and either plated or immediately cryopreserved in freezing medium (DMSO10%/FBS90%). The cells were initially seeded at 10,000/cm2. After overnight incubation for cell attachment, non-adherent cells were removed using a PBS (Sigma-Aldrich) wash (culture incubator at 37°C, 5% CO2, 95–98% humidity). For BM-MSC isolation, the solution with the flushed bone marrow was added to an equal part of enzymatic digestion solution and placed in a shaking water bath at 37°C for 30 min. After that, the isolation process was identical to the above-mentioned ASC isolation. The cells were initially seeded at 100,000/cm2 and washed with PBS the day after. The cells were allowed to get to 80–90% confluence and then harvested by means of 0.25% trypsin (Corning). MSCs were always counted by an automated, calibrated counting machine (Countess machine and Countess® Cell Counting Chamber Slides, Invitrogen) and re-plated at 5,000 cells/cm2 in T175 or T75 flasks (Corning), 20 or 10 mL were added, respectively, to the flasks and changed thrice weekly. Amphotericin B was added during the first plating to avoid any fungal infection and then withdrawn at the first passaging.
SC- and O-ASCs were isolated according to our established protocol (
BM-MSCs were isolated according to a protocol previously published by Wolfe et al. (
Passage 2 to 3 MSCs were lifted by means of trypsin, counted and put in sterile Falcon tubes at aliquots of 1 × 106 cells/tube at least. The following antibody panel was used for characterization of porcine cells: CD14 (AbD Serotec), CD29 (BD Pharmingen), CD31 (BD Biosciences), CD34 (Abcam + Goat anti-rabbit IgG (PE-cy5.5), Life Technologies), CD44 (Biolegend), CD45 (Genway Biotech), CD73 (Biolegend), CD90 (BD Biosciences), CD105 (Bioss Antibodies), CD146 (Genetex). For human cells, following antibodies were used CD45, CD90, CD105, CD73, CD235a, CD34 (BD Biosciences), CD31 (BioLegend), CD33, CD14 (Beckman Coulter), and CD146 (Miltenyi Biotec) (antibodies summarized in
Porcine panel.
CD14 | FITC | AbD Serotec |
CD29 | Alexa Fluor® 647 | BD Bioscience |
CD31 | PE | BD Bioscience |
CD34 | PE-cy5.5 Ig-G | Abcam/Life Technologies (Dye) |
CD44 | Alexa Fluor 700 | Biolengend |
CD45 | DyLight®680 | Genway Biotech |
CD73 | Brilliant Violet 421 | Biolegend |
CD90 | DyLight®755 | BD Bioscience |
CD105 | PE-cy7 | Bioss Antibodies |
CD146 | FITC | Genetex |
Human panel.
CD14 | PC5 | Beckman coulter |
CD31 | PE-Cy7 | Biolegend |
CD33 | PC5 | Beckman coulter |
CD34 | AF700 | BD bioscience |
CD45 | APC-Cy7 | BD bioscience |
CD73 | PE | BD bioscience |
CD90 | APC | BD bioscience |
CD105 | FITC | BD bioscience |
CD146 | VioBlue | Miltenvi biotec |
CD235a | PE-Cy5 | BD bioscience |
For all the cell lines in culture, cell numbers were recorded at each passage and re-seeded at 5,000 cells/cm2. Proliferation was assessed until passage 7. The population doubling time (PDT) was calculated using following equation: time/log2(harvested cells/seeded cells) (
Mesenchymal stem cells derived from the same individual/animal were plated at passage 3 at a density of 40,000 cells/cm2 in 96-well plates using EGM. After 24 h, medium was replaced with adipogenic differentiation medium [STEMPRO® Adipogenesis Differentiation Kit (Invitrogen)] that was changed every 3–4 days over the course of 2 weeks. Control cells were cultured in regular EGM for 2 weeks that was changed every 3–4 days. After 2 weeks the cells were the washed with PBS and each well was filled with 200 μL PBS with addition of 5 μL Adipored® staining solution and incubated for 10 min. The fluorescence readout was performed using a microplate reader (Infinite® 200 PRO NanoQuant, Tecan). After readout, cells were imaged with bright-field microscopy.
Responder splenocytes were isolated from spleens of naïve pigs and human donors and were stimulated by phytohemagglutinin (PHA; Sigma-Aldrich). In the suppressor assay, 200,000 cells/well MSCs were added at different ratios to responder cells, namely at 1:4, 1:8, and 1:16, in 96-well plates with round bottom. Co-cultures were performed in triplicates until confluence (3–5 days) with RPMI-1640 medium supplemented with L-Glutamine. To assess splenocyte proliferation, the cells were pulsed with [3H]thymidine (1 mCi/well) for the final 8 h and [3H]thymidine incorporation was measured as counts per minute in a liquid scintillation counter (Perkin Elmer). A total of 6 and 3 experiments were performed for porcine and human MLRs, respectively. For MLR testing under influence of immunosuppressants, the MSCs were incubated with the different compounds for 36 h and then added to MLR as described above. Following, clinically relevant concentrations were used for these experiments: 10 ng/mL (Tac), rapamycin 10 ng/mL (Rapa), Cyclosporin A 250 ng/mL (CsA).
The following drugs were used (all Sigma-Aldrich, St. Louis, MO, USA): tacrolimus (T-049), rapamycin (S-015), cyclosporin A (30024), and mycophenolic acid (M3536; MPA). AlamarBlue (AbD Serotec) was quality-controlled as suggested by the manufacturer and optimal cell seeding density and incubation time determined. Positive and negative controls were performed. A stock solution was created for each drug and then serially diluted. 5000 cells/well were seeded in 96-well plates (Corning) and incubated with the drugs for 36 h. Experiments were performed in quadruplicates and passage 3 to 4 cells were used. Following serial dilutions were used: Tac and Rapa 0 – 2 - 10 – 50 – 250 - 1250 ng/ml, CsA 0 – 10 – 50 – 250 – 1250 - 6250 ng/ml, and MPA 0 – 0.2 – 1 – 5 – 25 – 125 ug/ml. Sole addition of PBS served as control. At the end of incubation, 20 μl AlamarBlue was added to each well and incubated for 5 h. After that, 200 μl of supernatant were transferred from each well to a new, black 96-well plate with clear flat bottom (Fisher Scientific) and directly assessed for fluorescence in a plate reader (Tecan Infinite M200 Pro). A total of 8 readings per well was performed and mean calculated.
Plates with detached cells were imaged and under an Axiovert 25 microscope (Carl Zeiss Microscopy GmbH) as described previously (
Prism 8.0 (GraphPad, San Francisco, CA, USA) was used for statistical analysis. Data are presented as means ± SD unless otherwise specified. Differences between the groups were assessed by unpaired
O-SVF of both porcine and human origin showed the highest yields per mL of tissue (pO-SVF 0.49 ± 0.4 and hO-SVF 0.894 ± 0.05 x106/mL tissue), significantly higher than the SC counterpart (pSC-SVF 0.076 ± 0.05 and hSC-SVF 0.154 ± 0.09 × 106/mL tissue;
Cell isolation.
Cell yields after passaging the cultured SVF showed a trend for higher numbers in pO-ASC and pSC-ASC compared to BM-MSC (37,390 ± 16,268 and 39,859 ± 21,606 vs. pBM-MSC 22,917 ± 9,814 cells/cm2; n.s.). For human cells, however, there was a statistically significant higher proliferation in O-ASC and SC-ASC compared to their BM counterpart (hO-ASC 33,973 ± 2,936, and hSC-ASC 29,695 ± 1,336;
While SVF viability upon isolation was comparable between O-SVF and SC-SVF from both porcine and human donors (pO-SVF 94.31 ± 5.15%, pSC-SVF 89.87 ± 11.89%, hO-SVF 96.25 ± 1.89%, hSC-SVF 92.5 ± 3.78%; p>0.05;
While first passage PDT was significantly longer in BM cells (pBM-MSC 96.80 ± 3.86 h, hBM-MSC 76.37 ± 5.98 h) compared to O- and SC-MSC (pO-ASC 55.1 ± 14.71 h,
Cell culture.
Cell viability of porcine cells was comparable throughout passaging up to passage 6, whereas at passage 7 there was a decline in cell viability percent in BM-MSC group (86.50 ± 19.09% vs. pO-ASC 98.00 ± 2.09% and pSC-ASC 98.66 ± 1.15%;
All cells of porcine origin showed high expression of CD29, CD44, and CD90, and negative for CD14, CD45, CD73, CD105, and CD146, while showing low expression for CD31 and CD34. Cells of human origin had a surface marker phenotype expressing high levels of CD73, CD90, and CD105 and negative, or low expression, for CD14, CD31, CD33, CD34, CD45, CD146, and CD235 (
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 (
Swine-derived cells showed no relevant difference in terms of adipogenic differentiation potential between the cell types. X-fold fluorescence increase compared to control was 8.53 ± 1.93 for pO-ASC, 7.56 ± 3.03 for pSC-ASC and 6.45 ± 1.28 for pBM-MSC, respectively (n.s.;
In contrast, in human cells there was a marked difference between the cell types: the highest adipogenic differentiation was in hO-ASC (20.27 ± 6.10 fold), followed by 14.95 ± 5.40 in hSC-ASC and 4.20 ± 1.20 in hBM-MSC (
Mean porcine cell sizes did not differ between the groups and were 13.93 ± 0.82 μm in pO-ASC, 13.45 ± 0.35 μm in pSC-ASC and 13.98 ± 0.38 μm in pBM-MSC. Compared to that, human cells showed slightly higher mean sizes, significant only for BM-MSCs (hO-ASC 17.70 ± 3.32 μm, hSC-ASC 16.26 ± 3.96 μm, hBM-MSC 19.17 ± 3.30 μm; hBM-MSC
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).
All three porcine MSCs demonstrated immunomodulatory function
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
Human cells, had a similar but stronger trend compared to porcine-derived counterparts, with all cell types being able to reduce allo-reactivity more consistently in a dose-dependent way; however, there was no significant difference between the groups (O-ASC 4:1 31.64% ± 13.69, 8:1 60.50% ± 31.11, 16:1 72.12% ± 24.79; SC-ASC 4:1 40.92% ± 6.39, 8:1 64.21% ± 23.44, 16:1 72.27% ± 23.91; BM-MSC 4:1 29.85% ± 7.06 8:1 44.43% ± 20.84 16:1 68.10% ± 33.27; all 4:1
All cell types from swine and human donors showed no significant differences in proliferation with Tac compared to controls at any concentration. A similar picture was found with CsA, but pO-ASC revealed a slight, yet significant higher proliferation starting from 50 ng/mL upwards (110.80 ± 2.3%,
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 *
In co-cultures with Rapa, however, there was a differential effect on cell proliferation: while porcine O-ASCs were not affected and human BM-MSCs only affected at high doses (87.55 ± 3.77 at 50 and 77.95 ± 4.95% at 250 ng/mL), pSC-ASC and pBM-MSC revealed diminished proliferation starting from Rapa doses of 10 ng/mL (83.74 ± 2.13%
ASCs' and BM-MSCs' immunomodulatory function was not significantly reduced in porcine cells when under the influence of Tac, Rapa or CsA compared to PBS control. There was, however, a minor reduction in suppression of allo-reaction with Tac (O-ASC 41.21 ± 2.86% vs. PBS 32.82 ± 3.18%; SC-ASC 40.86 ± 2.53% vs. 26.28 ± 4.15% PBS; BM-MSC 52.36 ± 9.48% vs. 37.65 ± 41.23% PBS) and Rapa (O-ASC 51.09 ± 6.37%, SC-ASC 53.81 ± 3.35%; BM-MSC 56.76 ± 3.59%) in all groups and with CsA only in O-ASC (56.96 ± 27.21%) and SC-ASC (47.22 ± 19.26%)(
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
For human cells there were similar results with no significant alteration of the immunomodulatory ability of ASCs and BM-MSCs when co-cultured with the immunosuppressants. In hO-ASC proliferation was 63.95 ± 13.56% with Tac, 43.77 ± 10.87% with Rapa and 53.48 ± 3.81% with CsA vs. 79.00 ± 18.10% with PBS (
This study is unique in that it evaluated the potential of MSCs for cytotherapies from three different anatomical donor sites from both swine und human origin after rapid culture-expansion with endothelial growth medium (EGM). As main finding, there was no significant difference between the three cell types in terms of immunomodulatory function or susceptibility to immunosuppressive agents, making them all ideal candidates for transplant-related cytotherapies in conjunction with immunosuppressive agents at usual therapeutic ranges. However, there are some advantages from an isolation, cultivation and proliferative point of view for the omental and, to a less extent, subcutaneous counterparts, which might favor their use, even more so when accounting for ease of harvest from these donor sites with low morbidity. The uniqueness of the present study is seen also in the direct comparison of the different cell lines between different species: the results suggest that immunomodulatory function and cell susceptibility to immunosuppressants found in swine are similar to that of human cells, enabling extrapolation of results from large animal studies to clinical application.
We used a rapid culture-expansion protocol to achieve high cell yields in short culture time as proposed earlier by Suga et al. (
The use of omentum-derived ASCs in conjunction with subcutaneous ASCs for transplant related purposes has been proposed and investigated by our group previously (
Isolation yields in the O-ASC group were significantly higher from porcine and human tissues compared to SC-ASC, even though the amount of tissue harvested was usually less: this is probably due to the loose nature of the omentum majus, which makes disruption by the enzymatic solution more effective. In general, there was high variability in the number of isolated cells per volume from the omentum. Yields and viability at the first passage were higher in O-ASC and SC-ASC, suggesting that for BM-MSCs the actual isolation protocol is not able to eliminate non-MSC cells adequately during isolation and plating. Cell viability throughout cultivation to P7 was lower in BM-MSC, significantly in human cells, under rapid expansion with EGM.
In a study by Toyoda et al., isolation of human ASCs from omentum majus and subcutis yielded fewer and more cells, respectively, compared to our study, probably due to the differing isolation protocol (
Presence of FGF-2, as in the EGM, has been shown to promote ASC proliferation (
Although some authors reported similar adipogenic differentiation potential in bone marrow and adipose tissue derived MSCs (
There is a paucity of reports on the cell size for MSCs, especially comparing different cell types. Our analysis revealed slightly larger cell diameters in human MSCs compared to porcine ones, especially for BM-MSCs. We could not identify differences between the anatomical locations. Previously our group analyzed detached rodent ASCs and MSCs and found similar sizes for ASCs but larger diameters for BM-MSCs, which might be due to inter-species differences or different culture medium (
We assessed the immunomodulatory function of MSCs from the different anatomical locations
The influence of immunosuppressive medications on MSCs has been investigated previously: Hoogduijn et al. found that MPA and Rapa inhibit MSC proliferation at therapeutic doses. We were able to confirm these results for pig and human MSCs. While pre-incubation of MSCs with Tac revealed an increased immunosuppressive function according to Hoogduijn et al. (
To investigate whether MSCs are influenced by the immunosuppressants in their immunomodulatory functionality, we incubated the cells with Tac, Rapa and CsA at a clinically relevant dose in MLRs (MPA was excluded according to toxicity assay results). We found no significant impact of the different drugs on MSCs, but there was a non-significant trend showing slight worse suppressive action of porcine MSCs under immunosuppressant influence, while for human cells this was not the case.
While the cell isolation method applied in this study was not performed under a specific Food and Drug Administration approved protocol, it is analogous to the process employed in commercially available closed systems for cell isolation and has been shown by our group to result in a similar cell product as have been used in our own FDA sanctioned clinical trials (
In summary, MSCs from omentum, subcutaneous tissues, and bone marrow of both human and porcine origin share similar behavior in terms of surface marker phenotype, immunomodulatory function and susceptibility to immunosuppressants. However, the data show superiority of ASCs in isolation yields, viability and potential for rapid culture-expansion. EGM culture expansion seems to retain the functionality of both porcine and human MSCs, making is a useful tool for cell therapy studies in which the necessary cell dose exceeds the quantity of cells harvested.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
Deceased tissue donors were referred after informed consent by the Center for Organ Recovery and Education (CORE).
RS: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing. MW: provision of study material, collection and/or assembly of data, data analysis and interpretation. SO and CK: provision of study material, collection and/or assembly of data. WZ: collection and/or assembly of data, data analysis and interpretation. JP: data analysis and interpretation, manuscript writing. VG: conception and design, provision of study material, data analysis and interpretation. MS: conception and design, data analysis, and interpretation. LK: collection and/or assembly of data, data analysis and interpretation, and manuscript writing. KM: conception and design, provision of study material, and data analysis and interpretation. JR: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing, and final approval of manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Isaac James, MD, Debra Bourne, MD, and Ryan Schroth for occasional help during cell isolations.
adipose-derived mesenchymal stem cell
bone marrow MSC
cyclosporin A
systemic immunosuppression
mixed lymphocyte reaction assay
mycophenolic acid
mesenchymal stem (stromal) cell
omental ASC
population doubling time
rapamycin
room temperature
subcutaneous ASC
Stromal vascular fraction
tacrolimus
white blood cells.