- Email: [email protected]
Suppression of human peripheral blood lymphocyte proliferation by immortalized mesenchymal stem cells derived from bone marrow of Banna Minipig inbred-line
Suppression of Human Peripheral Blood Lymphocyte Proliferation by Immortalized Mesenchymal Stem Cells Derived From Bone Marrow of Banna Minipig Inbred-Line. J. Liu, X.F. Lu, L. Wan, Y.P. Li, S.F. Li, L.Y. Zeng, Y.Z. Zeng, L.H. Cheng, Y.R. Lu, and J.Q. Cheng ABSTRACT This study sought to investigate whether mesenchymal stem cells (MSC) derived from Banna Minipig Inbred-line (BMI-MSC) suppressed human peripheral blood lymphocyte (hPBLs) proliferation in a one-way mixed lymphocyte reaction system. BMI-MSC failed to stimulate proliferative responses by hPBLs, which were activated by allogenic endothelial cells, BMI-PBLs and non-specific mitogenic stimuli. Furthermore, BMI-MSC also suppressed proliferation of hPBLs stimulated by mismatched allogenic, as well as xenogenic PBLs, and the mitogenic stimulus ConA. The suppression occurred in dose-dependent fashion when the ratio of hPBLs to BMI-MSC varied from 1 to 5 fold; fewer, BMI-MSC (0.001 to 0.01 times) showed no obvious suppression. When BMI-MSC were added to hPBLs stimulated for 72 hours, the proliferative suppression was still evident. Addition of anti-FasL or anti-TGF-1 antibody attenuated the proliferative suppression, while antibody against IL-10 had no effect on it. Further immunofluorescence analysis demonstrated that FasL and TGF-1 constitutively expressed BMI-MSC. These findings suggest that BMI-MSC suppress hPBLs proliferation relying on FasL and TGF-1 mediated pathways.
A
LLOGENIC mesenchymal bone marrow stem cells (MSC) could survive in human and animal transplantation models.1,2 Cotransplantation with autologous or allogeneic MSC prolongs skin graft survival3 and promotes hematopoietic stem cell (HSC) engraftment.4 Namely, allogenic MSC were believed to show low immunogenicity as well as immunomodulation properties, possibly related to their ability to inhibit T cell proliferation.5–7 The Banna Minipig Inbred Line (BMI), highly inbred porcines, has been suggested to be a hopeful candidate for xenotransplantation. Bone marrow MSC derived from this highly inbred pig have been useful for basic and clinical research of cell therapy. Therefore, this study investigated whether BMI-MSC suppressed human peripheral blood lymphocyte (hPBL) proliferation. Explore the underlying mechanism. MATERIALS AND METHODS BMI-MSC Mesenchymal stem cells isolated from bone marrow of 18th generation BMI were transfected with plasmids containing simian virus 40 large T antigen gene. Proliferation, expression of stem cell markers, potential of differentiation, senescence and tumorgenicity were investigated as previously described.8 BMI-MSC at PD20
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (GIBCO BRL) supplemented with 10% calf serum and incubated at 37°C in a humidified atmosphere supplemented with 5% CO2.
PBLs PBLs were obtained by means of Lymphoprep (a mixture of 9.6% (w/v) sodium metrizoate and 5.6% (w/v) Ficoll; 1.077 g/mL) gradient centrifugation separation. In brief, 10 mL fresh heparinized peripherial blood from health volunteers or BMI were diluted with an equal volume of PBS. Five milliliters diluted blood was carefully layered on 3 mL Lymphoprep for centrifugation at (2000 rpm/20 min). The lymphocyte layer was separated and treated with 0.83% NH4Cl for From the Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University; and Laboratory of Banna Minipig Inbred Line (Y.Z.Z.), Yunnan Agriculture University, Kunming, China. Supported by the National Basic Research Program of China (Grant No. 2003CB515504) and the Natural Science Foundation of China (Grant No. 30200065 and 30270679). Address reprint requests to Youping Li and Jingqiu Cheng, Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Kunming, 610041 P. R. China. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.090
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3272
Transplantation Proceedings, 36, 3272–3275 (2004)
SUPPRESSION OF HPBL
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Fig 1. Proliferative responses of hPBLs to ConA, ECV-304, BMIPBLs and BMI-MSC. Statistically significant (P⬍.01) when compared with control (Student t test). (a) hPBLs. (b) hPBLs⫹ConA. (c) hPBLs⫹ECV304. (d) hPBLs⫹BMI-PBLs. (e) hPBLs⫹BMI-MSC. (f) BMI-MSC. 5 minutes to remove erythrocytes. After three times washing with five volumes of PBS, precipitated cells were resuspended in RPMI-1640 medium (GIBCO BRL) containing 100 U/mL penicillin, 100 g/mL streptomycin, 20 mmol/L HEPES and 20% heat-inactivated calf serum.
FasL (0.1 g/mL, 1 g/mL); TGF-beta1 (0.1 g/mL, 1 g/mL) and IL-10 (0.1 g/mL, 1 g/mL) and BMI-MSC were added to a ConA stimulated one-way MLR system. The proliferation of hPBLs was measured by an MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega).9
One-Way Mixed Lymphocyte Reaction
Statistical Analysis
BMI-MSC and other cellular stimuli were pretreated with 25 g/mL mitomycin (30 min/37°C) before being cultured with responder hPBLs. To determine whether BMI-MSC stimulated a proliferative response, hPBLs (7*104) were cultured with the same amount of BMI-MSC, BMI-PBLs, human endothelial cells (ECV304) or 10 g/mL ConA separately in 96-well microtitre plates for 6 days in 0.2 mL RPMI-1640 medium. To evaluate their suppressive effect on hPBL proliferation, different amounts of BMI-MSC were added to MLR systems containing hPBLs stimulated with 10 g/mL ConA, 0.8% PHA, mismatched hPBLs or BMI-PBLs. To identify possible factors responsible for the inhibitory effect, antibodies against
The data were presented as stimulation index (SI) values calculated by using the following formula proliferation of stimulated lymphocyte with or without MSC/proliferation of unstimulated lymphocyte alone. The data were expressed as mean values ⫾ standard deviations of 3 separate experiments. The Student t test was used to evaluate the significance of differences between the samples with P ⬍ .05 accepted as significant.
RESULTS
As shown by Fig 1, when 7 ⫻ 104 BMI-MSC were used to
Fig 2. Dose-dependent suppressive effect of immortalized BMIMSC on human T-lymphocyte proliferation induced by 10 g/mL ConA (stripe bars) or 0.8% PHA (white bars). Stimulated hPBLs were cultured with or without BMIMSC in the presence of increasing numbers of BMI-MSCs from 1:0.0001 to 1:5 ratios. Statistically significant (P⬍.01) when compared with control MLRs containing hPBLs and stimuli (Student t test).
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LIU, LU, WAN ET AL
Fig 3. Modulation of hPBLs proliferation to ConA by antibodies against FasL, TGF-1 and IL-10. Statistically significant (P⬍.05) when compared with control MLRs containing hPBLs, stimuli and BMI-MSC (Student t test). *Control: stimulated hPBLs⫹BMI-MSCs(1:3).
stimulate an equal number of hPBLs, no proliferative response was detected. However, hPBLs showed significant proliferative responses to allogenic endothelial cells, to xenogenic PBLs or to a nonspecific mitogenic stimulus ConA (P ⬍ .01). Thus, BMI-MSC possess low immunogenicity. After BMI-MSC varied from 1 to 5 times the number of hPBLs added into a ConA or PHA stimulated MLR system, a significant and dose dependent reduction in lymphocyte proliferation was evident (P ⬍ .01). However, fewer BMI-MSC (.0001 to .01 times) did not suppress and maybe even enhanced hPBL proliferation to nonspecific mitogenic stimuli (Fig 2). Similarly, BMI-MSC also inhibited proliferative responses of hPBLs to mismatched allogeneic and xenogeneic PBLs (data not shown). This suggested that the suppression was dose-dependent, but antigen-independent. BMI-MSC induced proliferative suppression was evident when MSC were added after hPBLs had been stimulated for 72 h. Compared with control MLRs containing hPBLs plus ConA and mitomycin pretreated BMI-MSCs, addition of anti-FasL or anti-TGF-1 antibody
at 1 g/mL attenuated the inhibitory activity of BMI-MSC significantly (P ⬍ .05). The attenuation of both antibodies was also in a dose dependent fashion. In contrast, addition of antibody against IL-10 had no influence on the inhibitory effect (Fig 3). Expression of FasL and TGF-1 on BMIMSC was further proven by immunofluorescence analysis under a laser confocal scanning microscope (Fig 4). DISCUSSION
We have showed that (1) BMI-MSC fail to stimulate a strong proliferative response by hPBLs; (2) BMI-MSC suppressed proliferation of hPBLs to other stimuli in dose-dependent, but antigen-independent fashion; (3) FasL and TGF-1 expression on BMI-MSC were closely related to this suppressive ability. These results on immunogenicity and immunomodulatory properties of xenogenic MSC are consistent with the descriptions on allogenic MSC by Nicola6 and Krampera.7 We suggested that similar to the effects allogenic MSC, xenogenic MSC might also be trans-
Fig 4. Immunofluorescence of FasL (Left, ⫻200) and TGF-1 (Right, ⫻400) on BMI-MSC under Laser confocal scanning microscope.
SUPPRESSION OF HPBL
plantable, due to their low immunogenicity as cell negative immunomodulators for organ or hematopoietic stem cell transplantation. REFERENCES 1. Horwitz EM, Gordon PL, Koo WK, et al: Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. PNAS 99:8932, 2002 2. Satoshi, Noriko, Yukiji, et al: In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Experimental Cell Research 288: 51, 2003 3. Bartholomew A, Surgeon C, Siatkas M, et al: Mesenchymal stem cells can prolong skin graft survival in the baboon. Blood 94:135, 1999 4. Koc ON, Gerson SL, Cooper BW, et al: Rapid hematopoietic recovery after coinfusion of autologous blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced
3275 breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307, 2000 5. Tse WT, Pendleton JD, Beyer W, et al: Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75:389, 2003 6. Di Nicola M, Carlo-Stella C, Magni M, et al: Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99:3838, 2002 7. Krampera M, Glennie S, Dyson, J, et al: Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 101(9): 3722–3729, 2003 8. Liu J, Lu X.F, Wan L, et al: Immortalization of bone marrow mesenchymal stem cells from inbred pig for regenerative medicine. Key Eng Mater 288:43, 2005 9. Malich G, Markovic B, Winder C: The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 124:179, 1997
A
LLOGENIC mesenchymal bone marrow stem cells (MSC) could survive in human and animal transplantation models.1,2 Cotransplantation with autologous or allogeneic MSC prolongs skin graft survival3 and promotes hematopoietic stem cell (HSC) engraftment.4 Namely, allogenic MSC were believed to show low immunogenicity as well as immunomodulation properties, possibly related to their ability to inhibit T cell proliferation.5–7 The Banna Minipig Inbred Line (BMI), highly inbred porcines, has been suggested to be a hopeful candidate for xenotransplantation. Bone marrow MSC derived from this highly inbred pig have been useful for basic and clinical research of cell therapy. Therefore, this study investigated whether BMI-MSC suppressed human peripheral blood lymphocyte (hPBL) proliferation. Explore the underlying mechanism. MATERIALS AND METHODS BMI-MSC Mesenchymal stem cells isolated from bone marrow of 18th generation BMI were transfected with plasmids containing simian virus 40 large T antigen gene. Proliferation, expression of stem cell markers, potential of differentiation, senescence and tumorgenicity were investigated as previously described.8 BMI-MSC at PD20
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (GIBCO BRL) supplemented with 10% calf serum and incubated at 37°C in a humidified atmosphere supplemented with 5% CO2.
PBLs PBLs were obtained by means of Lymphoprep (a mixture of 9.6% (w/v) sodium metrizoate and 5.6% (w/v) Ficoll; 1.077 g/mL) gradient centrifugation separation. In brief, 10 mL fresh heparinized peripherial blood from health volunteers or BMI were diluted with an equal volume of PBS. Five milliliters diluted blood was carefully layered on 3 mL Lymphoprep for centrifugation at (2000 rpm/20 min). The lymphocyte layer was separated and treated with 0.83% NH4Cl for From the Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University; and Laboratory of Banna Minipig Inbred Line (Y.Z.Z.), Yunnan Agriculture University, Kunming, China. Supported by the National Basic Research Program of China (Grant No. 2003CB515504) and the Natural Science Foundation of China (Grant No. 30200065 and 30270679). Address reprint requests to Youping Li and Jingqiu Cheng, Lab of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Kunming, 610041 P. R. China. E-mail: [email protected]
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.090
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3272
Transplantation Proceedings, 36, 3272–3275 (2004)
SUPPRESSION OF HPBL
3273
Fig 1. Proliferative responses of hPBLs to ConA, ECV-304, BMIPBLs and BMI-MSC. Statistically significant (P⬍.01) when compared with control (Student t test). (a) hPBLs. (b) hPBLs⫹ConA. (c) hPBLs⫹ECV304. (d) hPBLs⫹BMI-PBLs. (e) hPBLs⫹BMI-MSC. (f) BMI-MSC. 5 minutes to remove erythrocytes. After three times washing with five volumes of PBS, precipitated cells were resuspended in RPMI-1640 medium (GIBCO BRL) containing 100 U/mL penicillin, 100 g/mL streptomycin, 20 mmol/L HEPES and 20% heat-inactivated calf serum.
FasL (0.1 g/mL, 1 g/mL); TGF-beta1 (0.1 g/mL, 1 g/mL) and IL-10 (0.1 g/mL, 1 g/mL) and BMI-MSC were added to a ConA stimulated one-way MLR system. The proliferation of hPBLs was measured by an MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega).9
One-Way Mixed Lymphocyte Reaction
Statistical Analysis
BMI-MSC and other cellular stimuli were pretreated with 25 g/mL mitomycin (30 min/37°C) before being cultured with responder hPBLs. To determine whether BMI-MSC stimulated a proliferative response, hPBLs (7*104) were cultured with the same amount of BMI-MSC, BMI-PBLs, human endothelial cells (ECV304) or 10 g/mL ConA separately in 96-well microtitre plates for 6 days in 0.2 mL RPMI-1640 medium. To evaluate their suppressive effect on hPBL proliferation, different amounts of BMI-MSC were added to MLR systems containing hPBLs stimulated with 10 g/mL ConA, 0.8% PHA, mismatched hPBLs or BMI-PBLs. To identify possible factors responsible for the inhibitory effect, antibodies against
The data were presented as stimulation index (SI) values calculated by using the following formula proliferation of stimulated lymphocyte with or without MSC/proliferation of unstimulated lymphocyte alone. The data were expressed as mean values ⫾ standard deviations of 3 separate experiments. The Student t test was used to evaluate the significance of differences between the samples with P ⬍ .05 accepted as significant.
RESULTS
As shown by Fig 1, when 7 ⫻ 104 BMI-MSC were used to
Fig 2. Dose-dependent suppressive effect of immortalized BMIMSC on human T-lymphocyte proliferation induced by 10 g/mL ConA (stripe bars) or 0.8% PHA (white bars). Stimulated hPBLs were cultured with or without BMIMSC in the presence of increasing numbers of BMI-MSCs from 1:0.0001 to 1:5 ratios. Statistically significant (P⬍.01) when compared with control MLRs containing hPBLs and stimuli (Student t test).
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LIU, LU, WAN ET AL
Fig 3. Modulation of hPBLs proliferation to ConA by antibodies against FasL, TGF-1 and IL-10. Statistically significant (P⬍.05) when compared with control MLRs containing hPBLs, stimuli and BMI-MSC (Student t test). *Control: stimulated hPBLs⫹BMI-MSCs(1:3).
stimulate an equal number of hPBLs, no proliferative response was detected. However, hPBLs showed significant proliferative responses to allogenic endothelial cells, to xenogenic PBLs or to a nonspecific mitogenic stimulus ConA (P ⬍ .01). Thus, BMI-MSC possess low immunogenicity. After BMI-MSC varied from 1 to 5 times the number of hPBLs added into a ConA or PHA stimulated MLR system, a significant and dose dependent reduction in lymphocyte proliferation was evident (P ⬍ .01). However, fewer BMI-MSC (.0001 to .01 times) did not suppress and maybe even enhanced hPBL proliferation to nonspecific mitogenic stimuli (Fig 2). Similarly, BMI-MSC also inhibited proliferative responses of hPBLs to mismatched allogeneic and xenogeneic PBLs (data not shown). This suggested that the suppression was dose-dependent, but antigen-independent. BMI-MSC induced proliferative suppression was evident when MSC were added after hPBLs had been stimulated for 72 h. Compared with control MLRs containing hPBLs plus ConA and mitomycin pretreated BMI-MSCs, addition of anti-FasL or anti-TGF-1 antibody
at 1 g/mL attenuated the inhibitory activity of BMI-MSC significantly (P ⬍ .05). The attenuation of both antibodies was also in a dose dependent fashion. In contrast, addition of antibody against IL-10 had no influence on the inhibitory effect (Fig 3). Expression of FasL and TGF-1 on BMIMSC was further proven by immunofluorescence analysis under a laser confocal scanning microscope (Fig 4). DISCUSSION
We have showed that (1) BMI-MSC fail to stimulate a strong proliferative response by hPBLs; (2) BMI-MSC suppressed proliferation of hPBLs to other stimuli in dose-dependent, but antigen-independent fashion; (3) FasL and TGF-1 expression on BMI-MSC were closely related to this suppressive ability. These results on immunogenicity and immunomodulatory properties of xenogenic MSC are consistent with the descriptions on allogenic MSC by Nicola6 and Krampera.7 We suggested that similar to the effects allogenic MSC, xenogenic MSC might also be trans-
Fig 4. Immunofluorescence of FasL (Left, ⫻200) and TGF-1 (Right, ⫻400) on BMI-MSC under Laser confocal scanning microscope.
SUPPRESSION OF HPBL
plantable, due to their low immunogenicity as cell negative immunomodulators for organ or hematopoietic stem cell transplantation. REFERENCES 1. Horwitz EM, Gordon PL, Koo WK, et al: Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. PNAS 99:8932, 2002 2. Satoshi, Noriko, Yukiji, et al: In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Experimental Cell Research 288: 51, 2003 3. Bartholomew A, Surgeon C, Siatkas M, et al: Mesenchymal stem cells can prolong skin graft survival in the baboon. Blood 94:135, 1999 4. Koc ON, Gerson SL, Cooper BW, et al: Rapid hematopoietic recovery after coinfusion of autologous blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced
3275 breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307, 2000 5. Tse WT, Pendleton JD, Beyer W, et al: Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75:389, 2003 6. Di Nicola M, Carlo-Stella C, Magni M, et al: Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99:3838, 2002 7. Krampera M, Glennie S, Dyson, J, et al: Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 101(9): 3722–3729, 2003 8. Liu J, Lu X.F, Wan L, et al: Immortalization of bone marrow mesenchymal stem cells from inbred pig for regenerative medicine. Key Eng Mater 288:43, 2005 9. Malich G, Markovic B, Winder C: The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 124:179, 1997