The potential role of genetically-modified pig mesenchymal stromal cells in xenotransplantation

Abstract

Mesenchymal stromal cells (MSCs) are known to have regenerative, anti-inflammatory, and immunodulatory effects. There are extensive indications that pig MSCs function satisfactorily across species barriers. Pig MSCs might have considerable therapeutic potential, particularly in xenotransplantation, where they have several potential advantages. (i) pMSCs can be obtained from the specific organ- or cell-source donor pig or from an identical (cloned) pig. (ii) They are easy to obtain in large numbers, negating the need for prolonged ex vivo expansion. (iii) They can be obtained from genetically-engineered pigs, and the genetic modification can be related to the therapeutic goal of the MSCs. We have reviewed our own studies on MSCs from genetically-engineered pigs, and summarize them here. We have successfully harvested and cultured MSCs from wild-type and genetically-engineered pig bone marrow and adipose tissue. We have identified several pig (p)MSC surface markers (positive for CD29, CD44, CD73, CD105, CD166, and negative for CD31, CD45), have demonstrated their proliferation and differentiation (into adipocytes, osteoblasts, and chondroblasts), and evaluated their antigenicity and immune suppressive effects on human peripheral blood mononuclear cells and CD4(+)T cells. They have identical or very similar characteristics to MSCs from other mammals. Genetically-modified pMSCs are significantly less immunogenic than wild-type pMSCs, and downregulate the human T cell response to pig antigens as efficiently as do human MSCs. We hypothesized that pMSCs can immunomodulate human T cells through induction of apoptosis or anergy, or cause T cell phenotype switching with induction of regulatory T cells, but we could find no evidence for these mechanisms. However, pMSCs upregulated the expression of CD69 on human CD4(+) and CD8(+) T cells, the relevance of which is currently under investigation. We conclude that MSCs from genetically-engineered pigs should continue to be investigated for their immunomodulatory (and regenerative and anti-inflammatory) effects in pig-to-nonhuman primate organ and cell transplantation models.

Figure 1. Differentiation of pMSCs

(A)Fibroblast-like morphology of GTKO/hCD46 pAdMSC (light microscopy 10×) (B) Adipogenic differentiation of GTKO/hCD46 pAdMSC – Oil red stain (20×). (Insert –fat droplets stained with oil red-40×). (C) Osteogenic differentiation of GTKO/hCD46 pAdMSC – Von Kossa stain (20×), showing morphological changes in pAdMSC and linear calcium deposition (black). (D) Chondrogenic differentiation of GTKO/hCD46 pAdMSC – Alcian blue stain (10×), showing strongly acidic mucosubstance (blue) and goblet cells (red nuclei). (Reproduced from Kumar G, et al, Cytotherapy 2012; 14:494–504 [14], with permission).

Figure 2. Pig MSC surface markers

By flow cytometry, GTKO pMSC were positive for CD29, CD44, CD73, CD105, and CD166, and negative for CD31 and CD45 (black); isotype control (gray). (Reproduced from Ezzelarab M, et al. Xenotransplantation 2011; 18:183–195 [13] with permission).

Figure 3. Immunogenicity of pMSCs

(Left) Using serum from two healthy human volunteers (one with high [Human 1] and one with low [Human 2] levels of anti-pig antibodies), IgM/IgG antibody binding to WT and GTKO pMSC was measured by flow cytometry. IgM/IgG binding to GTKO pMSC was less than that to WT pMSC. Relative mean immunoflorescence intensity (MFI) was calculated by dividing the MFI of the tested serum sample by the MFI of the background (secondary antibody binding to target cells). Data are representative of three different experiments. (Right) Proliferative responses of PBMC from two different healthy humans to WT and GTKO pMSC in mixed lymphocyte reaction (responder:stimulator ratio 1:10). In both cases, the response to GTKO pMSC was weaker than that to WT pMSC (p<0.01 vs. WT pMSC). (Reproduced from Ezzelarab M, et al. Xenotransplantation 2011; 18:183–195 [13] with permission).

Figure 4. Downregulation of human PBMC responses to GTKO pig (p)AEC by GTKO pMSC, GTKO/CD46 pMSC, and human (h)MSC

In mixed lymphocyte reaction, human PBMC proliferative responses to GTKO pAEC alone (A), hMSC alone (B), GTKO pMSC alone (C), and GTKO/CD46 pMSC alone (D) were measured. The human PBMC response was significantly weaker to all three types of MSC (B, C, and D) than to GTKO pAEC (A). When GTKO pAEC were mixed with hMSC (E), GTKO pMSC (F), or GTKO/CD46 pMSC (G) at a 1:10 responder:stimulator ratio, each type of MSC significantly reduced the human PBMC proliferative response, with GTKO/CD46 pMSC reducing the response more significantly than hMSC. Data representative of three different experiments (*p<0.01 and **p<0.001 vs. GTKO pAEC [A]; #p<0.05 vs. GTKO pAEC + hMSC [E]). (Reproduced from Ezzelarab M, et al. Xenotransplantation 2011;18:183–195 [13] with permission).

Figure 5. Suppression of human CD4⁺ and CD8⁺ T cell proliferation by genetically-modified GTKO/CD46 pMSC

In MLR, human CD4⁺ (left) or CD8⁺ (right) T cell proliferation was measured after coculture with GTKO/CD46 pMSC, or with GTKO pAEC (at a responder:stimulator ratio of 1:10). Also, human T cells were cocultured with GTKO/CD46 pMSC mixed with GTKO pAEC at a 1:1 ratio. GTKO/CD46 pMSC significantly downregulated the responses of CD4⁺ or CD8⁺ T cells in response to GTKO pAEC. The proliferation was measured after 5 days of culture, and 16h after addition of ³H-thymidine (1µCi/well) using a beta-scintillation counter (PerkinElmer, Waltham, MA). The results are represented as counts per minute (CPM). Data represent 2 different experiments, and are presented as mean ± SD of triplicates. * p<0.01