Objectives: Mesenchymal stem cells are easy to obtain and expand, with characteristics of low immuno­genicity and strong tissue repair capacity. In this study, our aim was to investigate the role of mesenchymal stem cells in chronic immune rejection of heterotopic small intestine transplant in rats.

Materials and Methods: After successfully constructing a rat chronic immune rejection model of heterotopic small intestine transplant, we infused mesenchymal stem cells into the animal recipients. We observed mesenchymal stem cell location in the recipients, recipient survival, pathology changes, and the ex­pression of CD68, transforming growth factor β1, and platelet-derived growth factor C in the donor intestine.

Results: Mesenchymal stem cells inhibited the lym­phocyte proliferation caused by concanavalin A in vitro. After stem cells were infused into recipients, they were mainly located in the donor intestine, as well as in the spleen and thymus. Recovery after transplant and pathology changes of the donor intestine in rats with stem cell infusion were better than in the control group; however, we observed no differences in survival time, accompanied by downregulated ex­pression of CD68, transforming growth factor β1, and platelet-derived growth factor C.

Conclusions: Mesenchymal stem cells, to a certain extent, could inhibit the process of chronic rejection. The mechanisms may include the inhibited function of these cells on lymphocyte proliferation, reduced infiltration of macrophages, and reduced expression of transforming growth factor β1 and platelet-derived growth factor C.

Key words : Chronic rejection, CD68, Mesenchymal stem cells, Platelet-derived growth factor, Transforming growth factor β1

Introduction

Small intestine transplant has become the standard treatment for irreversible intestinal failure.¹ However, the immune response is more intense and complicated than with other organs, due to a large number of lymph nodes and bacteria in the small intestine.² Thus, many complications still need to be solved in the clinical application of small intestine transplant. With improvements in surgical techniques, enhanced management during the perioperative period, and the application of immunosuppressant, acute rejection is more efficiently controlled.³ However, chronic rejection is the main reason for loss of the long-term function of the transplanted intestine.⁴ Chronic rejection usually occurs in a few months or years after transplant, and graft function gradually deteriorates, with intimal proliferation and obvious fibrosis as the typical histologic findings. Patients usually present with refractory diarrhea and sustained weight loss.⁵ The whole vascular system of the transplants may be damaged, resulting in intimal thickening, calcification, and obstruction of aortic and pulmonary homografts. Other manifestations include swelling of the endothelial cells, accumulation of foam cells, and subcutaneous infiltration of T lymphocytes and macrophages, with vascular inflammation around the sustainable space.⁶

Macrophage infiltration is one of the characteristics of an allograft immune response; however, no immune factors, such as ischemia-reperfusion, could aggravate the infiltration of macrophages.⁷ Thus, the role of macrophages in chronic rejection have re­cently aroused interest. The accumulation of monocytes and macrophages around blood vessels observed at early stages of chronic rejection may facilitate the artery narrowing process.⁸ Many studies have suggested that extended macrophage infilt­ration during the early stage after transplant may be an important predictive indicator of chronic kidney disease in the later period.⁹ As shown in rat renal transplant models (F344 to Lewis), the application of macrophage inhibitors or transfection of anti­macrophage factors could prevent chronic kidney disease,¹⁰ which was further confirmed using immunofluorescence and immunohistochemical technologies. With the development of molecular biology technology and the further understanding of transplant immunity, many genes have been found to be associated with chronic rejection, such as trans­forming growth factor β1 (TGF-β1) and platelet-derived growth factor C (PDGFC). Studies have suggested that interstitial fibrosis is associated with the high expression of TGF-β1 in the transplanted organs.^11,12 Down-regulating the expression of TGF-β could significantly relieve the extent of interstitial fibrosis and cardiac hypertrophy, thus improving graft function.¹³ As shown in various studies, PDGFC could strongly promote the division of vascular smooth muscle cells and fibroblast interaction with PDGF-α receptors^14-17 and could play an important role in normal embryonic development, wound healing, and angiogenesis together with other growth factors.^18-20 Recent studies have shown that over-expression of PDGFC was related to fibrosis of the heart, liver, lung, and kidney.^21-25 Tuuminen and associates showed that PDGFC participated in the chronic rejection process, showing a significant elevation.²⁶

Mesenchymal stem cells (MSC) have become an important topic of investigations. They have the ability to differentiate into a variety of tissue cells, such as osteoblasts, endothelial cells, and nerve cells under appropriate conditions. These kinds of cells have good application prospects in the field of organ transplant, since they are easy to obtain and expand in vitro, have low immunogenicity, and have strong capability for tissue repair.²⁷

Materials and Methods

Isolation, culture, and identification of mesenchymal stem cells
Mesenchymal stem cells were obtained from bone marrow cells of Lewis rats using density gradient centrifugation. Cells were maintained in L-Dulbecco’s modified eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% glutamine-penicillin-streptomycin. All cell culture experiments were performed in an incubator at 37°C in a 5% CO₂ humidified atmosphere. Cells were obtained after 3 to 5 passages when the purity was more than 90%. Flow cytometry analyses were performed according to standard protocols using the following antibodies (clones): CD29-fluorescein isothiocyanate (FITC), CD34-FITC, CD45-FITC/­CD90-phycoerythrin, or relevant isotype controls (all Biolegend, San Diego, CA, USA).

To identify the characteristics of MSCs, we induced MSC differentiation into osteoblasts using osteoblast-inducing culture (L-Dulbecco’s modified eagle’s medium, 10% fetal bovine serum, 100 U/ML penicillin, 100 μg/mL streptomycin, l0 mmol/L P-sodium glycerophosphate, 10^-8 mol/L hexa­decadrol, 50 μg/mL ascorbic acid), using alkaline phosphatase and alizarin red staining for detection. In addition, we also induced chondrogenic dif­ferentiation using dexamethasone (5 ng/mL) and TGF-β1 (5 μg/mL) to observe the morphologic changes of the cells.

Mixed lymphocyte culture
Mononuclear cells, separated from spleens of F344 rats and stained with carboxy fluorescein succinimidyl ester (CFSE), were cocultured in 96-well plates with MSCs separated from the bone marrow of Lewis rats. The experimental culture groups were as follows: mononuclear cell group, mononuclear cells plus concanavalin A (conA) group, mono­nuclear cells plus MSC group, and mononuclear cells plus MSCs plus conA group. The number of mononuclear cells in each well was 5 × 10⁵ and the number of MSCs was 10⁵. The final concentration of conA was 10 μg/mL. After 3 days of coculture, cells were collected for flow cytometry analyses (CD3-FITC, CD4-PE, CD8-peridinin chlorophyll protein, CFSE).

Heterotopic small intestine transplant and tissue analyses
All procedures pertaining to animal experiments were approved by our Animal Care and Use Committee. Our heterotopic small intestine trans­plant models were prepared using male F344 rats (weighing 250 g) as small intestine donors and male Lewis rats (weighing about 250 g) as recipients. All animals were bred and maintained under specific pathogen-free conditions. The surgical procedures are shown in Figure 1.

To determine the status of chronic rejection, we divided animals into 3 groups: isogeneic control group, allogeneic control group, and allogeneic treatment group. In the allogeneic treatment group, rats were given a subcutaneous injection of cyclosporine (5 mg/kg) from days 1 to 14 after transplantation. The rats in the isogeneic control group and allogeneic control group were given the same doses but of saline. All rats were observed daily for general status and weight. On days 7, 14, 30, 60, and 90, we cut tissues of anastomosis to perform biopsies. Biopsy tissues were fixed with 10% formalin and paraffin embedded for Verhoeff and hematoxylin-eosin staining. Results of pathologic histology examinations allowed us to determine the occurrence of chronic rejection.

To determine the role of MSCs on chronic rejection, we divided the experimental groups into a control group (n = 8) and an MSC group (n = 8). Animal recipients in the MSC group were injected with MSCs cells (about 10⁷, stained with CFSE) through the penis vein. The donor's small intestine was excised on day 7 to investigate the distribution of MSCs in the recipients. Results of pathologic histology examinations (isogeneic control group, allogeneic control group, and allogeneic treatment group) suggested that chronic rejection occurred on day 60; therefore, small intestines from donors were retrieved and analyzed using hematoxylin-eosin and Verhoeff staining on day 60. The expression of CD68, TGF-β1, and PDGFC were detected by real-time polymerase chain reaction and immuno­histochemical staining. Primers used were as follows: for TGF-β1, 5’-AGCAACAATTCCTGGCGTTAC-3’(forward) and 5’-GGACTGATCCCATTGATT TCC-3’(reverse); for PDGFC, 5’-AAAGCAAAGCGGT GAATCT-3’(forward) and 5’-AATTATGGAGGCA ACAGGC-3’(reverse); for β-actin, 5’-GTCGTACCA CTGGC ATTGTG-3’(forward) and 5’-CTCTCAGC TGTGGTGGTGAA -3’ (reverse).

Statistical analyses
Data are presented as means ± standard deviation (SD). For multiple group comparisons, one-way analysis of variance was performed, followed by the 2-sided, unpaired t test. The unpaired 2-tailed t test was used to compare 2 independent groups. For all statistical analysis, SPSS statistical software (version 15.0) was used (SPSS: An IBM Company, IBM Corporation, Armonk, NY, USA). P < .05 was considered statistically significant.

Results

Isolation, culture, and identification of mesenchymal stem cells
After centrifugation, the MSCs were located in the white cloud layer according to the density gradient of different cellular components in bone marrow cell suspension (Figure 2A). There was no direct way to identify MSCs; we mainly used the following characteristics to confirm MSCs: good proliferation characteristics with adherent culture, could be induced to differentiate into different kinds of cells (such as osteoblasts and chondrocytes), and showing specific expression of surface antigens, such as CD29+, CD90+, CD34-, and CD45-. After 3 days of culturing, cells were mainly circular with adherent culture (Figure 2A). Adherent cells then began to split and became fusiform or spindly fiber cells in a colony or spiral formation (Figure 2A). Passage 1 of MSCs was performed after 3 weeks of culturing, with time intervals then extended according to the growth situation. We allowed continuous passaging for about 2 or 3 generations, at which time cell morphology became more uniform, mostly like fiber sample cells. After 6 generations, cells had no obvious changes (Figure 2A). For our study, we used the third generation for analyses and counted the numbers for the next 9 days. Cell growth is illustrated in Figure 2B. In the differentiation experiment, after we added inducing factors, the differentiated MSCs grew slowly, with brown color stain (alkaline phosphatase) showing changes at 7 days and red color stain (alizarin red) showing changes at 14 days (Figure 2C). After we added dexamethasone (5 ng/mL) and TGF-β1 (5 μg/mL) to the culture medium for about 3 days, cells increased in size, with shapes turning round or oval. The nucleus also became asymmetric and kidney shaped. After 15 days of culturing, the cells could secrete an extracellular matrix, manifesting the characteristics of chondrogenic differentiation (Figure 2D). As shown in Figure 2E, the positive expression rates of CD29 and CD90 were above 96%, whereas expres­sion rates of CD34 and CD45 were below 3%.

Effect of mesenchymal stem cells on lymphocyte proliferation in vitro
As shown in Figure 3A, the number of cells in group B (mononuclear cells + conA group) was significantly increased compared with group A (only mono­nuclear cells). Interestingly, after MSCs were added to group B, cell numbers were not significantly different from group A and had the same result as group C (mononuclear cells + MSCs). The expression of CFSE in each group also showed the same trend (Figure 3B). In addition, we also analyzed the T-cell subsets in the coculturing systems. As shown in Figure 3C, flow cytometry results suggested that the percentage of CD4+ and CD8+ T cells in group D significantly decreased compared with the other groups (P < .01).

Rat chronic rejection models of heterotopic small intestine transplant
The rats in the allogeneic control group had persistent diarrhea, weight loss, and postoperative intestinal secretions, which increased and also included hemorrhagic secretions. All rats died within 15 days; therefore, this group was excluded from the follow-up study. Rats in the other 2 groups survived more than 90 days, with no significant differences between the groups (Figure 4A). Rats in the isogeneic group had no obvious postoperative diarrhea. As shown in Figure 4B, rat weight started to increase on day 3 and was restored to preoperative levels by day 12. Weight increased gradually and was maintained at high levels. However, rats in the allogeneic treatment group had diarrhea (ranging from 1-8 days), and body weight significantly decreased. On day 9, a formed stool appeared, and weight began to increase, ultimately reaching to levels shown before surgery.

We observed histology changes in grafts using hematoxylin-eosin (Figure 4C) and Verhoeff staining (Figure 4D) at different times. In the isogeneic group, structure of the small intestinal mucosa had no obvious changes over our observation time. The intestinal wall structure was stable, with no obvious inflammatory cell infiltration. The villus structure was integrated, with no loss of goblet cells and no obvious intestinal gland atrophy or proliferation.

In the allogeneic treatment group, hematoxylin-eosin staining results showed that the intestinal mucosa underwent progressive failure. On day 7, the small intestinal mucosa structure was integrated, with roughly normal morphology and a small amount of inflammatory cell infiltration. On day 14, the small intestinal mucosa structure was still integrated with roughly normal morphology but obvious inflam­matory cell infiltration. On day 30, the villus structure was blunt, with some missing. The numbers of goblet cells decreased, with intestinal gland proliferation shown. The inflammatory cells infiltrated the mucosa lamina propria and muscle layer. The pathologic results suggested no significant differences between samples at day 60 versus day 90, with both times showing chaotic intestinal wall, missing villi structure, some residual adenomas, and diffuse inflammatory cell infiltration. Inflammatory cell infiltration to the perivascular region also showed obvious proliferated arterial intima and narrowing lumen. Verhoeff staining results sug­gested a significant difference between the 2 groups. In the isogeneic control group, grafts showed no obvious fibrosis in the intestinal mucosa, muscularis, and mesangial layers at days 30, 60, and 90. However, in the allogeneic group, biopsy spec­imens suggested a progressive fibrosis process over time. Pathologic investigation also showed no significant difference between day 60 and day 90, with obvious fibrosis and over-hyperplasia of endarterium, leading to 75% to 80% luminal narrowing.

Effect of mesenchymal stem cells on rat chronic rejection models of heterotopic small intestine transplant
Our results led us to conclude that, in the immune environment of mixed lymphocyte culture, MSCs could significantly inhibit the proliferation of lymphocytes in vitro. On day 7, the donor intestine and autologous small intestine, liver, spleen, heart, lung, and thymus were excised, with CFSE used to investigate the distribution of MSCs in the recipients. As shown in Figure 5A, the MSCs were mainly located in the small intestine allografts, with small amounts in the spleen and thymus. However, none were distributed in recipient’s heart, liver, or lung. The postoperative recovery of recipients that received MSCs was better than shown in the control group (Figure 5B), although no significant differences in survival time (all recipients could survival more than 60 days) were shown.

Normal hematoxylin-eosin staining of allografts in the control group showed mucosal degeneration, with diffuse monocyte infiltration, residual ade­nomas, and gland expansion, displaying an irregular shape (Figure 5C). With Verhoeff staining, we observed interstitial fibrosis, proliferation of artery intimal, and luminal narrowing. In the MSC group, the mucosa was still complete, with glands showing mild hyperplasia. However, there was less monocyte infiltration, interstitial fibrosis, and proliferation of the artery intimal versus the control group (Figure 5C). Results of real-time polymerase chain reaction and immunohistochemical staining (Figure 6A and 6B) showed that, on day 60 after transplantation, CD68, TGF-β1, and PDGFC expression levels in the MSC group were significantly less than shown in the control group (P < .01).

Discussion

Mesenchymal stem cells derived from the mesoderm during early embryonic development have the ability of multidirectional differentiation. Under certain in vitro conditions, MSCs can differentiate into a variety of tissue cells, thus making them ideal for cell therapy. These cells exist in a variety of tissues and are most easily isolated from the bone marrow, although these are few in number.^28,29 Therefore, the separation method is important in cell therapy application. Current isolation methods are mainly density gradient centrifugation, adherent method, flow cytometric sorting, and magnetic bead separation. All of these methods have their own advantages and disadvantages. For our experiments, we used density gradient centrifugation combined with the adherent method. With these methods, our cells were uniform and displayed fast proliferation and stable growth traits. However, the centrifugal purification destroyed the bone marrow micro­environment; this was a chief drawback with extended generation. Original generation of bone marrow required about 3 weeks to achieve 80% to 90% fusion. After passaging, we found the growth rate to be relatively more constant and fast, only needing about 6 to 7 days. Cryopreservation and resuscitation did not affect MSC growth, and proli­feration and differentiation were also unaffected, consistent with that previous shown.³⁰

Currently, there are mainly 3 methods to identify MSCs³¹: proliferation characteristics with good appearance in the adherent culture, multiple dif­ferentiation in vitro, and specificity of the expression of surface antigens, such as CD29+, CD90+, CD3-, and CD45-. In our experiment, after the cultivation of MSCs to the third generation, obtained by density gradient centrifugation, the positive expression rates of CD29 and CD90 were above 99%, with CD34 and CD45 expression rates lower than 3%, similar to the literature.³² The results of osteoblast and chon­drogenic differentiation experiments further proved that the cells that we obtained were MSCs, not hematopoietic stem cells or fibroblasts.

In recent years, a variety of studies found that MSCs could obviously inhibit the proliferation of the immune cells in vitro, such as T cells, B cells, dendritic cells, and natural killer cells, as well as reduce the production of inflammatory cytokines,^33-35 which showed its prospect for the treatment of autoimmune diseases and transplant rejection. Mesenchymal stem cells have been shown to evade recognition by immune cells, suggesting that MSCs have low immunogenicity.³⁶ In addition, MSCs did not express the major histocompatibility complex I molecules (CD40, CD40L) or II molecules (B7-1, B7-2).³⁷ Coculture of MSCs and allogeneic peripheral blood mononuclear cells or allogeneic T cells showed that MSCs could not cause the proliferation of T cells or the release of interferon-gamma.³⁸ In our present experiments, coculture of MSCs obtained from Lewis rat bone marrow and spleen cells obtained from F344 rats also showed that MSCs could not stimulate the proliferation of lymphocytes in vitro. Further analyses of the cultivation system, through flow cytometry, showed that MSCs could inhibit CD4+ and CD8+ T-cell expression. In addition, MSCs could also inhibit T-cell proliferation caused by anti-CD3 and anti-CD28 antibodies.³⁹

Researchers found that manifestations of chronic rejection of small intestine transplant include refractory diarrhea and sustained weight loss.⁴⁰ In our models, all rats in the allogeneic group survived more than 200 days. Weights stayed at a high level, and no obvious diarrhea was observed. Therefore, we could not judge the appearance of chronic rejection according to the performance of weight and diarrhea. Biopsies of grafted intestine fistula allowed us to judge chronic rejection according to the histopathologic analyses.

According to the diagnostic criteria of the trans­plant center of Pittsburgh,⁴⁰ when the sub­mucosa, subserous layer, and mesenterium show visible occlusive arterial changes, chronic rejection can be determined. In our experiments, we analyzed pathology changes from 4 aspects: small intestinal mucosa structure, degree of inflammatory cell infiltration, degree of fibrosis, and the degree of artery intimal proliferation. Results showed that damage of the structure of the small intestinal mucosa began to appear on day 30; due to persistent chronic rejection, the injury of mucous membrane structure existed since then. The degree of inflammatory cell infiltration also showed the same tendency. However, Verhoeff staining results sug­gested that obvious fibrosis and artery intimal proliferation began to appear at day 60 versus day 30. However, there were no significant differences between day 60 and day 90. This showed that the degree of fibrosis and artery intimal hyperplasia on day 60 had reached a higher level after transplant. In conclusion, in our experiments, we assumed that the performance of typical chronic rejection appeared on day 60 after surgery.

To investigate the role of MSCs in chronic rejection, we observed the effects of MSCs on chronic rejection by infusing cells into the recipients. After infusion, these cells mainly distributed to the transplanted intestine in addition to immune organs such as the spleen and thymus, meaning that MSCs had the characteristics of directional movement to injury and then repaired damaged tissue by directional differentiation. During our construction of chronic rejection models of heterotopic small intestine transplant in rats, we found typical pathology changes at day 60 after transplantation and chose day 60 as our endpoint of observation. Normal hematoxylin-eosin and Verhoeff staining of allografts showed that the infused MSCs were efficient to control chronic rejection versus that shown in the control group. Molecular biology results showed that CD68, TGF-β1, and PDGFC expression levels in the MSC group were decreased, meaning that MSCs could probably restrain the chronic rejection by regulating the expression of CD68, TGF-β1, and PDGFC, but the specific regulation mechanism needs further study.

References:

1. Fryer JP. Intestinal transplantation: an update. Curr Opin Gastroenterol. 2005;21(2):162-168.
   CrossRef - PubMed
   
2. Suzuki J, Isobe M, Morishita R, Nagai R. Characteristics of chronic rejection in heart transplantation: important elements of pathogenesis and future treatments. Circ J. 2010;74(2):233-239.
   CrossRef - PubMed
   
3. Mazariegos GV, Squires RH, Sindhi RK. Current perspectives on pediatric intestinal transplantation. Curr Gastroenterol Rep. 2009;11(3):226-233.
   CrossRef - PubMed
   
4. Fryer JP. The current status of intestinal transplantation. Curr Opin Organ Transplant. 2008;13(3):266-272.
   CrossRef - PubMed
   
5. Sibley RK. Pathology and immunopathology of solid organ graft rejection. Transplant Proc. 1989;21(1 Pt 1):14-17.
   PubMed
   
6. Hruban RH, Beschorner WE, Baumgartner WA, et al. Accelerated arteriosclerosis in heart transplant recipients is associated with a T-lymphocyte-mediated endothelialitis. Am J Pathol. 1990;137(4):871-882.
   PubMed
   
7. Herrero-Fresneda I, Torras J, Cruzado JM, et al. Do alloreactivity and prolonged cold ischemia cause different elementary lesions in chronic allograft nephropathy? Am J Pathol. 2003;162(1):127-137.
   CrossRef - PubMed
   
8. Kitchens WH, Chase CM, Uehara S, et al. Macrophage depletion suppresses cardiac allograft vasculopathy in mice. Am J Transplant. 2007;7(12):2675-2682.
   CrossRef - PubMed
   
9. Pilmore HL, Painter DM, Bishop GA, McCaughan GW, Eris JM. Early up-regulation of macrophages and myofibroblasts: a new marker for development of chronic renal allograft rejection. Transplantation. 2000;69(12):2658-2662.
   CrossRef - PubMed
   
10. Yang J, Reutzel-Selke A, Steier C, et al. Targeting of macrophage activity by adenovirus-mediated intragraft overexpression of TNFRp55-Ig, IL-12p40, and vIL-10 ameliorates adenovirus-mediated chronic graft injury, whereas stimulation of macrophages by overexpression of IFN-gamma accelerates chronic graft injury in a rat renal allograft model. J Am Soc Nephrol. 2003;14(1):214-225.
    CrossRef - PubMed
    
11. Sharma VK, Bologa RM, Xu GP, et al. Intragraft TGF-beta 1 mRNA: a correlate of interstitial fibrosis and chronic allograft nephropathy. Kidney Int. 1996;49(5):1297-1303.
    CrossRef - PubMed
    
12. Morris-Stiff G. TGFbeta-1 and the development of chronic graft nephropathy: relative roles of gene, mRNA and protein. Ann R Coll Surg Engl. 2005;87(5):326-330.
    CrossRef - PubMed
    
13. Faust SM, Lu G, Wood SC, Bishop DK. TGFbeta neutralization within cardiac allografts by decorin gene transfer attenuates chronic rejection. J Immunol. 2009;183(11):7307-7313.
    CrossRef - PubMed
    
14. Uutela M, Lauren J, Bergsten E, et al. Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFD genes. Circulation. 2001;103(18):2242-2247.
    CrossRef - PubMed
    
15. Chen J, Han Y, Lin C, et al. PDGF-D contributes to neointimal hyperplasia in rat model of vessel injury. Biochem Biophys Res Commun. 2005;329(3):976-983.
    CrossRef - PubMed
    
16. Pontén A, Li X, Thorén P, et al. Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy. Am J Pathol. 2003;163(2):673-682.
    CrossRef - PubMed
    
17. Ponten A, Folestad EB, Pietras K, Eriksson U. Platelet-derived growth factor D induces cardiac fibrosis and proliferation of vascular smooth muscle cells in heart-specific transgenic mice. Circ Res. 2005;97(10):1036-1045.
    CrossRef - PubMed
    
18. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277(5323):242-245.
    CrossRef - PubMed
    
19. Ding H, Wu X, Bostrom H, et al. A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. Nat Genet. 2004;36(10):1111-1116.
    CrossRef - PubMed
    
20. Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol. 2000;2(5):302-309.
    CrossRef - PubMed
    
21. Campbell JS, Hughes SD, Gilbertson DG, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2005;102(9):3389-3394.
    CrossRef - PubMed
    
22. Zhuo Y, Hoyle GW, Shan B, Levy DR, Lasky JA. Over-expression of PDGF-C using a lung specific promoter results in abnormal lung development. Transgenic Res. 2006;15(5):543-555.
    CrossRef - PubMed
    
23. Grun K, Markova B, Bohmer FD, Berndt A, Kosmehl H, Leipner C. Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis. Eur Heart J. 2005;26(7):728-739.
    CrossRef - PubMed
    
24. Zhuo Y, Zhang J, Laboy M, Lasky JA. Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 2004;286(1):L182-188.
    CrossRef - PubMed
    
25. Eitner F, Bucher E, van Roeyen C, et al. PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis. J Am Soc Nephrol. 2008;19(2):281-289.
    CrossRef - PubMed
    
26. Tuuminen R, Nykanen AI, Krebs R, et al. PDGF-A, -C, and -D but not PDGF-B increase TGF-beta1 and chronic rejection in rat cardiac allografts. Arterioscler Thromb Vasc Biol. 2009;29(5):691-698.
    CrossRef - PubMed
    
27. Zou Z, Zhang Y, Hao L, et al. More insight into mesenchymal stem cells and their effects inside the body. Expert Opin Biol Ther. 2010;10(2):215-230.
    CrossRef - PubMed
    
28. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone. 1992;13(1):81-88.
    CrossRef - PubMed
    
29. Wekerle T, Sykes M. Mixed chimerism and transplantation tolerance. Annu Rev Med. 2001;52:353-370.
    CrossRef - PubMed
    
30. Doucet C, Ernou I, Zhang Y, et al. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol. 2005;205(2):228-236.
    CrossRef - PubMed
    
31. Ringe J, Kaps C, Schmitt B, et al. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res. 2002;307(3):321-327.
    CrossRef - PubMed
    
32. Lodie TA, Blickarz CE, Devarakonda TJ, et al. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue Eng. 2002;8(5):739-751.
    CrossRef - PubMed
    
33. Jiang XX, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood. 2005;105(10):4120-4126.
    CrossRef - PubMed
    
34. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood. 2006;107(4):1484-1490.
    CrossRef - PubMed
    
35. Oh JY, Kim MK, Shin MS, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells. 2008;26(4):1047-1055.
    CrossRef - PubMed
    
36. Maitra B, Szekely E, Gjini K, et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant. 2004;33(6):597-604.
    CrossRef - PubMed
    
37. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol. 2000;28(8):875-884.
    CrossRef - PubMed
    
38. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation. 2003;75(3):389-397.
    CrossRef - PubMed
    
39. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol. 2003;57(1):11-20.
    CrossRef - PubMed
    
40. Parizhskaya M, Redondo C, Demetris A, et al. Chronic rejection of small bowel grafts: pediatric and adult study of risk factors and morphologic progression. Pediatr Dev Pathol. 2003;6(3):240-250.
    CrossRef - PubMed