Stem Cell Rev. 2014 Feb;10(1):79-85. doi: 10.1007/s12015-013-9478-8.

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

Mohamed B. Ezzelarab1, Jiang Li1,2, David Ayares3, David K.C. Cooper1

1 The Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA. 2 Department of Transplantation Surgery, Tianjin First Central Hospital, Tianjin Medical University, Tianjin, China. 3 Revivicor Inc., Blacksburg, VA, USA.

 

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.

PMID: 24142483

 

Supplement

Mesenchymal stromal cells (MSCs) can be isolated from various tissues of mammals, where blood, bone marrow, and adipose tissue have to date formed the major sources. However, the number of cells that can be isolated from any tissue is minimal. For example, MSCs represent only 0.001% to 0.01% of the total nucleated cells in the bone marrow. Accordingly, MSCs have been grown in vitro and expanded in culture to adequate numbers for therapeutic purposes.

MSCs are known to have regenerative, anti-inflammatory, and immunomodulatory effects. They have attracted particular attention for their therapeutic potential in a range of acute and chronic diseases. After in vivo administration, MSCs migrate to sites of inflammation. Of particular interest to the field of transplantation, it is well-documented that MSCs can target several subsets of lymphocytes, including CD4+ and CD8+T cells, B cells and natural killer cells, and induce regulatory T cells. Furthermore, MSCs can regulate immune cells through cell-cell contact, as well as contact-independent mechanisms through the secretion of several immunoregulatory mediators.

 

Do mesenchymal stem cells function across species barriers?

For the purposes of xenotransplantation, it is important to know whether MSCs function across species barriers. By the end of 2011, there had been more than 90 reports of in vivo cross-species administration of MSCs, where the majority of these studies (more than 90%) indicated that MSCs engrafted and functioned across the species barrier, while there was evidence of failure to function only in six cases (less than 10%). Of interest, human-derived MSCs were demonstrated to function in no fewer than seven different recipient species, including mouse, rat, sheep, hamster, dog, rabbit, and pig. There are, therefore, extensive indications that pig MSCs would function satisfactorily across species barriers. However, to date there are no in vivo data from a pig-to-primate model.

 

Genetically-engineered pig mesenchymal stromal cells (pMSCs)

In our own laboratory we have had the opportunity of studying MSCs from various genetically-engineered pigs. We have successfully harvested and cultured MSCs from pig bone marrow and adipose tissue. In accordance with the 2006 International Society of Cellular Therapy defined criteria for human MSCs, we identified a fibroblast-like morphology of genetically-engineered pig (p)MSCs. We have demonstrated the differentiation of these pMSCs into adipocytes, osteoblasts, and chondroblasts. Additionally, we have documented that these pMSCs express CD29, CD44, CD73, CD105, and CD166, and are negative for CD31 and CD45. Of importance, pMSCs from α1,3-galactosyltransferase gene-knockout (GTKO) pigs do not express the galactose-α1,3-galactose (Gal) antigen (the major target of human natural antibodies), compared to pMSCs isolated from wild-type pigs. Additionally, we have shown that these pMSCs can express human proteins, for example the human complement-regulatory protein CD46.

 

Potential advantages of genetically-engineered pMSCs over human MSCs

In regard to xenotransplantation, there are several potential advantages of pMSCs over human MSCs:

  1. In the case of organ or cell transplantation, pMSCs can be obtained from the specific organ- or cell-source donor pig or from an identical (cloned) pig. This would enable the MSCs to be genetically identical to the organ or cells to be transplanted. There would be no need for third-party donors, as is the case with most human MSCs.
  2. Unlimited supply of pMSCs which are easy to obtain in large numbers. Therefore, no need for prolonged ex vivo expansion of pMSCs with its disadvantages, which include cell senescence and the risk of malignant changes.
  3. With advanced technology of genetic engineering, donor pigs can be genetically modified to produce pMSCs which express specific human proteins according to the therapeutic goals, which is clearly not possible for hMSCs

 

Immunoregulatory effects of genetically-engineered pMSCs compared to human MSCs

We initially investigated bone marrow-derived and adipose-tissue pMSCs from both wild-type and genetically-engineered pMSCs. Following activation with pig interferon-gamma (pIFN-γ), less than 1% of pMSCs upregulated swine leukocyte antigen (SLA) class II, similar to human MSCs. Also, activated pMSCs (particularly those isolated from genetically-engineered pigs) induced minimal human T cell proliferation. In addition, human and nonhuman primate antibodies binding to genetically-engineered pMSCs was significantly less than that to wild-type pMSCs.

Next, we evaluated the immunoregulatory potential of pMSCs obtained from genetically-engineered GTKO pigs expressing the human complement-regulatory protein CD46 (GTKO/CD46). In vitro studies indicate that GTKO/CD46 pMSCs significantly suppress both human CD4+ and CD8+ T cell proliferation in response to pig antigens. The suppressive capacity of GTKO/CD46 pMSC was comparable to or significantly more efficient than of human MSCs (Figure 1). Further studies indicate that human T cells upregulate CD69 following coculture with GTKO/CD46 pMSC, suggesting that genetically engineered pMSCs may regulate human T cell activation in a similar manner to as reported with human MSCs.

 

Conclusions

Recent reports suggest that genetically-engineered GTKO pMSCs can survive and differentiate in vivo in comparison to wild-type pMSCs. Furthermore, GTKO pMSCs were less immunogenic than wild-type pMSCs when transplanted into rodents. These studies indicate that genetically-engineered pMSCs (i) can be efficiently isolated from genetically-engineered pigs, where these pMSCs exhibit most of the defined criteria required by International Society of Cellular Therapy, (ii) express human proteins with known regulatory function, and (iii) regulate human T cell responses to pig antigens as efficiently as human MSCs

In summary, therefore, we believe that genetically-modified pMSCs may have considerable potential in xenotransplantation. Not only are they available in large numbers, which reduces the necessity for prolonged ex vivo expansion, but they can be obtained from a herd of identical pigs, thus significantly reducing the variation seen when they are obtained from pooled human sources. This may be a significant advantage over hMSCs. Although not yet tested, their anti-inflammatory and regenerative capacities in humans are likely to be no different from allogeneic MSCs.

 

fig1

Figure 1: Genetically engineered pMSC downregulate human CD4+T cell proliferation in response to pig endothelial cells:

Human CD4+T cells were cocultured with GTKO pig aortic endothelial cells (GTKO pAEC; A), human MSCs (B), GTKO pMSCs (C) or GTKO/CD46 pMSCs (D) at 1 to 10 (CD4+T cells) ratio for 5 days. The proliferation of human CD4+T cells in response to both human MSCs and pMSCs was minimal and comparable, and significantly lower than that to GTKO pAEC (** p<0.001). Additionally, human CD4+T cells were cocultured with GTKO pAEC in the presence of human MSCs (E), GTKO pMSCs (F) or GTKO/CD46 pMSCs (G). MSCs to pAEC ratio was 1:1. Both human MSC and pMSC significantly reduced human CD4+T cell proliferation to pAEC (* p<0.01). Furthermore, GTKO/CD46 pMSCs reduced human CD4+T cell proliferation more efficiently than human MSCs (# p<0.05). Figure adapted from Ezzelarab et al, Xenotransplantation. 2011 May-Jun;18(3):183-95. (CPM = counts per minute)

 

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