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Donor age negatively affects the immunoregulatory properties of both adipose and bone marrow derived mesenchymal stem cells
Transplant Immunology 30 (2014) 122–127
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Brief communication
Donor age negatively affects the immunoregulatory properties of both adipose and bone marrow derived mesenchymal stem cells Lehao W. Wu a,b, Yen-Ling Wang a,c, Joani M. Christensen a, Saami Khalifian a, Stefan Schneeberger a,d, Giorgio Raimondi a, Damon S. Cooney a, W.P. Andrew Lee a, Gerald Brandacher a,⁎ a
Vascularized Composite Allotransplantation (VCA) Research Laboratory, Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA Division of Plastic and Reconstructive Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, PR China Center for Vascularized Composite Allotransplantation, Department of Plastic Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan d Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria b c
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 3 March 2014 Accepted 4 March 2014 Available online 12 March 2014 Keywords: Age Bone marrow Adipose tissue Mesenchymal stem cells Immunoregulation
a b s t r a c t Purpose: Age negatively impacts the biologic features of mesenchymal stem cells (MSCs), including decreased expansion kinetics and differentiation potential. Clinically, donor-age may be within a wide spectrum; therefore, investigation of the role of donor's age on immunoregulatory potential is of critical importance to translate stem cell therapies from bench to bedside. Methods: Adipose and bone marrow derived MSCs (ASCs and BMSCs) were isolated in parallel from Lewis and Brown Norway rats of young (less than 4-week old) and senior groups (older than 15-month). The presentation of cells and time required for growth to 90% confluence was recorded. FACS sorting based on the expression of CD90 and CD29 double positive and CD45 CD11 double negative quantified the proportions of MSCs. After expansion, ASCs and BMSCs from different age groups were co-cultured in mixed lymphocyte reaction (MLR; Lewis vs. Brown Norway) assays. The suppression of CD3+CD4+ and CD3+CD8+ T cell populations by different sources of MSCs were compared. Results: The kinetics of cell growth was slower in old animals (17.3 ± 2 days) compared with young animals (8.8 ± 3 days), and cell morphology was irregular and enlarged in the senior groups. The yield of MSCs by FACS sorting was significantly higher in young groups compared to senior groups (p b 0.02). With regard to immunoregulatory potential, senior ASCs failed to induce any CD3+CD4+ T cell suppression (p N 0.05). In addition, young BMSCs-induced suppression was more prominent than seniors (p b 0.05). Conclusions: Donor age should be taken into consideration when using recipient MSC of either bone marrow or adipose origin in clinical applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The utilization of stem cell-based therapies, mesenchymal stem cells (MSCs)-based therapy in particular, has been proven to be a promising strategy to achieve transplant tolerance [1]. Despite their extensive proliferative and regenerative capacity, studies suggest that physical and biological properties of stem cells usually decline with advanced donor age [2]. This is primarily due to years of exposure to environmental damage and stress, which eventually leads to deterioration of tight control of multiple aspects of physiology, cell proliferation, and/or signal transduction [3].
Abbreviations: MSCs, mesenchymal stem cells; ASCs, adipose derived stem cells; BMSCs, bone marrow derived stem cells; MLR, mixed lymphocyte reaction. ⁎ Corresponding author at: Ross Research Building 749D, 720 Rutland Avenue Baltimore, MD 21205, USA. Tel.: +1 443 287 6679 (office); fax: +1 410 614 1296. E-mail address: [email protected] (G. Brandacher).
http://dx.doi.org/10.1016/j.trim.2014.03.001 0966-3274/© 2014 Elsevier B.V. All rights reserved.
Bone marrow derived mesenchymal stem cells (BMSCs) have shown great potential in regulating both alloreactive and non-specific immune responses when co-cultured in vitro through suppression of alloreactive T-cells [4,5]. However, accumulating evidence has shown that BMSCs exhibit both diminished differentiation and growth factor secretion with increasing donor age [6]. Yet, both transplant recipients and potential organ donors may be of any age. Thus, understanding the behavior and kinetics of these alterations in the quality and function of MSCs due to donor age is of great importance, as there is currently limited knowledge on how these potentially ‘functionally compromised’ MSCs would modulate transplantation outcomes. Adipose-derived mesenchymal stem cells (ASCs) are robust, multipotent cells, with a protein expression phenotype comparable to that of BMSCs [7]. Several in vitro studies have established that ASCs share immunological qualities with BMSCs including low immunogenicity, immunoregulatory effects, and similar cytokine expression profile [8–10]. Furthermore, ASCs are comparable to BMSCs in treating severe graft-versus-host disease [11] and alleviating arthritis and colitis [12].
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Fig. 1. The calculation of percentage of proliferated cell populations in a MLR. The percentage of all CD3+/CD4+ or CD3+/CD8+ cells was taken that no longer expressed CFSE as CD4 or CD8 T cells that had proliferated. Suppression was defined a lower percentage of cells proliferating. If the percentage of cells was the same with or without added MSC, this was defined as no suppression.
Clinically, ASCs are more widely available, easier to harvest and expand in cell culture, and cause less donor-site morbidity than MSCs from other sources. Therefore, ASCs present a promising alternative to BMSCs in many clinical applications. To our knowledge, MSCs derived from either bone marrow or adipose tissue have not been directly compared between young and aged donors for immunoregulatory capacities. Many different groups and investigators are now advocating ASCs as a potential alternative to the traditional BMSCs. This leads to the second questions we aimed to address in this study: does donor's age affect in the same way both BMSCs and ASCs? 2. Materials & methods 2.1. Animals Lewis and Brown Norway rats (Harlan, Indianapolis, IN) were divided into young (age under 4-week old) and senior (older than 15-month) groups, with n = 3 in both age groups from each strain. All animal procedures were approved by and performed in accordance with the Johns Hopkins University Animal Care and Use Committee. 2.2. Isolation of Rat BMSCs and ASCs Animals were euthanized by carbon dioxide suffocation and sterilized by iodine and ethanol solution. Soft tissue was stripped from femur & tibia, and the femur was disarticulated from the tibia. Long bone ends are cut and bone marrow flushed from the bones into a petri dish. The suspension was centrifuged at 1380 rpm for 5 min. Supernatant was aspirated, the pellet dislodged, and 20 cc of complete media consisting of DMEM/F12 with 10% heat-inactivated fetal bovine
serum, 1% penicillin/streptomycin and 1% Fungizone added, then suspensions of 50,000 cells were plated into T-50 flasks. All reagents were purchased from Invitrogen, CA unless otherwise noted. Inguinal fat pads were removed and minced with scissors in collagenase solution consisting of Hanks' balanced salt solution, bovine serum albumin and 1% type II collagenase (3.0 mg/g of fat) (Worthington Biochemical Corporation, Lakewood, NJ). Centrifuge tubes were then shaken at 100 rpm for 50 min at 37 °C. Following digestion, the content of each tube was filtered through double-layered sterile gauze. The filtrates were then centrifuged at 1000 rpm for 10 min at 37 °C. The pellets were suspended in the aforementioned plating medium and then preparations of 50,000 cells were added in 50 cm2 flasks and placed in 20% carbon dioxide incubator. Adherent ASCs were expanded for a period of 3 days at 37 °C, and the medium changed every 2 days until the cells achieved confluence.
2.3. Cell sorting, re-expansion and certification After the MSCs reached confluence, they were detached by a treatment with 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) and then approximately 105 cells for each of the senior groups, and 2.5 × 105 cells for the young groups were stained with Sytox-Blue (Invitrogen, Life Science, CA), CD29-FITC, CD90-PE, CD45-PerCP, and CD11b/c-AlexaFluor647 (BioLegend, US). Labeled (single) cells were sorted by gating on the CD29-FITC and CD90-PE double positive population, and excluding the Sytox-Blue, CD45-PerCP, and CD11-APC positive populations. We set 2 × 104 cells as target number of sorted cells to collect (when possible). Post-sorting cells were then expanded in culture for an additional 5 or 12 days and then used for purity analysis by flow cytometry, staining with anti-MHC class II-Alexa 647 (BD Biosciences, San Jose, CA), anti-CD31-eFluor 660 (eBioscience, San Diego,
Table 1 The biological difference in isolation of ASC. Brown Norway Senior (n = 3) Body weight (g) Adipose tissue harvested (g) Cell yield from adipose tissue(×104) Cell/g Body weight Cell/g Adipose tissue(×103) Days to reach confluence
497 4.63 13.58 273 29.3 15.0
± ± ± ± ± ±
Lewis Young (n = 3)
7.81 0.54 1.23 29.26 4.3 0
41.67 0.37 5.42 1303 146.5 7.3
± ± ± ± ± ±
Senior (n = 3) 1.27 0.04 0.52 161.98 21.2 0.58
497.33 5.24 12.00 241 22.9 17.6
± ± ± ± ± ±
Young (n = 3) 7.77 0.69 1.15 20.08 3.7 1.15
44.33 0.48 5.67 1279 118.1 8.0
± ± ± ± ± ±
1.37 0.06 0.29 79.54 16 0
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Fig. 2. Representative morphology of BMSCs and ASCs, 24-h after isolation from Lewis rat. (10× magnification.)
CA) and anti-CD105 (Antibodies-online, Atlanta, GA), or for the following cell function experiments. 2.4. CFSE-based mixed lymphocyte reaction (MLR) assay The detailed protocol has been published previously (J Robinson et al 1998.)[13] Briefly, spleens were harvested from both Lewis and Brown Norway rats then strained into cell suspension and separated by Ficoll-Paque (GE Health, TX). The mononucleocytes were collected and washed in DPBS at 400 g three times for 5 min. CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, CA, US) with a working concentration of 1 μM was added for every 5 million cells in 1 ml PBS. Cells were incubated at 37 °C for 10 min; staining was then quenched by adding 20 ml culture media consisting of RPMI, 10% FBS, 1% Penicillin/Streptomycin solution, and 15 mM Mercaptoethanol (Sigma, MO) for 5 min on ice. Cells were pelleted by centrifugation and washed three times. CFSEstained Lewis mononucleocytes were used as responders (105 cells) and irradiated Brown Norway splenocytes (5 × 104 cells irradiated with 2500 rads) were used as stimulators in a 96-well round bottom plate. MSCs were isolated from their respective rat strains as aforementioned and co-cultured in triplicates with a ratio of 1:1000 regulators vs. responders. On day 5, cells were harvested and stained with CD4-PE, CD8-PerCP and CD3-AlexaFlour 647 (all from Biolegend, CA, US) for detection of T cell subsets proliferation. 2.5. Comparison of CFSE-MLR assays and Statistical Analysis MLR analysis was conducted by flow cytometry by first gating on the lymphoid population on forward scatter/side scatter, followed by gating on CD3+CD4+ and CD3+CD8+ and then assessing proliferation to determine the effect of regulation by MSCs. Proliferation was calculated via quantification of the proportion of cells with CFSE staining lower than those that maintained the same level as the initial staining (nonproliferating cells; Fig. 1). Each experiment was conducted with three replicates and group means determined. Suppression was determined
Table 2 The percentage of MSCs (CD29+CD90+CD11−CD45−) in the entire cell preparation as assessed during FACS sorting (n = 3).
sBA yBA sBB yBB sLA yLA sLB yLB
Total event (×103)
Yield (×103)
Percentage
80.6 56.5 77.8 81.7 81.2 128.7 66.2 95.7
10.3 ± 1.3 20.0 8.1 ± 1.7 20.0 3.3 ± 0.8 20.0 5.7 ± 0.5 20.0
12.8 36.0 10.3 25.0 4.1 23.9 8.7 21.0
± ± ± ± ± ± ± ±
8.6 7.8 7.1 10.8 6.8 19.2 2.9 6.2
± ± ± ± ± ± ± ±
BA: BN-ASCs BB: BN-BMSCs LA: Lewis-ASCs LB: Lewis BMSCs; s: Senior y: Young Total event: number of cells passed through sorter Yield: number of positively-sorted cells
0.6% 4.6% 1.3% 3.6% 1.3% 3.7% 0.8% 1.4%
as a statistically significant lower value of group proliferation mean in comparison to control group. Student's t-test was used to determine statistical significance between group means. Significant results (p b 0.05) are denoted on figures by an asterisk. Continuous variables are presented as the mean (range ± standard deviation). 3. Results 3.1. Differences in primary isolation between young and senior donors Although the donors were 10 times smaller in size and tissue weight, the yield of cells on a per gram of body weight indicated a higher proportion of cells in adipose tissues of young versus old animals (Table 1). Additionally, as indicated by the time required to reach confluence during the first culture, stem cells isolated from young subjects were more vigorous with regard to expansion kinetics (Table 1). The morphology of MSCs isolated from young groups consisted of spindle-shaped adherent cells, as opposed to large, round, irregular shaped cells in the senior groups (Fig. 2). 3.2. Flow cytometry-based sorting The percent yield of the target population (CD29+CD90+ and CD11−CD45−) after sorting was significantly higher (p b 0.02) in young subjects regardless of source or strain. Additionally, the yield of MSCs (CD29+CD90+CD11−CD45−) isolated from sorted cells was also significantly higher in young groups. (Table 2) After re-expansion for 5 days, ASCs isolated from young or senior rats showed 87%–95% of CD105 expression, but no expression of MHC class II or CD31. Similarly, BMSCs expressed 93%–97% of CD105, but not MHC class II or CD31. These findings were also confirmed after expansion for 12 days. These data suggest that the isolated CD90+CD29+CD45−CD11− cells still maintain their MSC phenotype even after re-expansion for a prolonged period of time and do not exhibit any significant heterogeneity or contamination with endothelial cells (Fig. 3A and B). 3.3. ASC and BMSCs differentially regulate alloreactivity in a mixed lymphocyte reaction In the CFSE-based assay, as expected, BMSCs isolated from either Brown Norway and Lewis rats showed significant inhibitory activity irrespective of donor age (Fig. 4A and B). However, higher suppressive function of young BMSCs was evident as compared with senior BMSCs (p = 0.02 in Brown Norway; p = 0.003 in Lewis rats) A different scenario was observed with ASCs. While young ASCs maintained regulatory properties in both groups (p = 0.01 for Brown Norway; p = 0.004 for Lewis rats), ASCs isolated from senior rat groups failed to regulate CD3+CD4+ cell proliferation in an MLR, regardless of strain (p = 0.22 for Brown Norway; p = 0.07 for Lewis rats). (Tables 3A and 3B). Interestingly, this effect was restricted to CD4 T cells, as there was no difference between young and old ASCs in regulating CD3+CD8+ population in MLR assays (Fig. 4B).
4. Discussion The process of aging alters the immune system in a global and complex way, as evidenced by diminished primary and secondary immune responses, increased incidence of inflammatory pathologies, and weakened vaccine efficiency [14]. This concept of “immunosenescence” [15], can affect all immune compartments, including humoral and cellmediated immunity. Similarly, the effect of immunosenescence on stem cells may alter their function and phenotype, as many of their intrinsic beneficial functions deteriorate with age, and other misbalanced regulatory events lead to alterations in their immunoregulatory potential. In this study, the impact of age on the biological quality of MSCs from Lewis and Brown Norway was investigated, and we found that isolating
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Fig. 3. A) The CD90+CD29+CD45−CD11− ASCs from young (3 weeks of age) or senior (over 12 months of age) rats were isolated by cell sorting. Sorted cells were cultured for 5 days and further analyzed for additional surface markers (MHC class II, CD31 and CD105. B) BMSCs shared the same protocol of ASC isolation, and were re-expanded for 12 days. Each group contained three animals and data are presented as mean ± standard error of the mean (SEM).
ASCs from senior subjects using standard protocols led to inadequate cell yields and viability. Furthermore, in parallel experiment in which ASCs from all 3 senior ACI rats did not survive after the cell sorting process, the following experiment could not be performed. This further demonstrated the lower vitality of senior MSCs. These findings are in line with what has been reported by other groups in human donors, where the frequency and expansion capacity of MSC decreased with donor age [16]. Stenderup et al. [17] showed that donor age positively correlates with the rate of in vitro MSC senescence. Specifically, the maximal life span of MSCs between the young (18–25 years old) and old (N66 years old) group were found to be significantly different [17]. In general, donor age has a significant impact and is a well described factor in the harvest of bone marrow hematopoietic stem cells in particular when comparing pediatric versus elderly donors. Significantly higher mean total CD34 + cell counts are usually seen with young donor age as well as a higher ability in colony forming unit (CFU) assays.
Clinical implications of immunosenescence are broad and include increased risk of infections, malignancies, autoimmune disorders, and neuro-degenerative changes [18]. Furthermore, the consequences of immunosenescence also have significant ramifications in the field of organ transplantation, as increased donor or recipient age may require specific adaptations to immunosuppressive protocols [19]. In terms of immunoregulatory potential, assessed by in vitro MLR, we were able to demonstrate that senior ASCs not only failed to induce suppression in CD3+CD4+ proliferation, but also, allogeneic ASCs from Brown Norway rats actually appeared to stimulate proliferation, suggesting a possible increase of immunogenicity. Previous research has shown that ASCs inhibit proliferation of CD4+ and CD8+ cells in a non-selective and non-specific way [20] and several cytokines have been implicated in the regulatory process. Interferon-gamma and prostaglandin E2 (PGE2), amongst others, are immunosuppressive mediators selectively produced by MSCs that may convert pro-inflammatory environments into an
Fig. 4. A. MSCs regulating CD3+CD4+ proliferation assay (n = 3): In a MLR assay, CFSE-stained Lewis lymphocytes were stimulated by irradiated BN splenocytes (L + Bx) at a 2:1 ratio without or with ASCs (A) or BMSCs (B) from young (y) or senior (s) subjects of different strains. After 5 days of co-culture, the percentage of proliferated CD3+CD4+ and CD3+CD8+ were compared. ASCs and BMSCs from young groups have significantly better potential in regulating CD3+CD4+ responses (p b 0.05). Senior ASCs failed to induce CD3+CD4+ suppressions. All experiments were performed in triplicates. B. The percentage of proliferated CD3+CD8+. Young BMSCs were still more potent than senior BMSCs; however, such difference was not observed in ASCs groups. BA: BN-ASCs BB: BN-BMSCs LA: Lewis-ASCs LB: Lewis BMSCs; S: Senior Y: Young L: Lewis Splenocytes Bx: Irradiated BN Splenocytes Lx: Irradiated Lewis Splenocytes.
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Table 3A Percentage of proliferating T-cells regulated by young or senior Brown Norway ASCs/BMSCs, to demonstrate the efficacy of these cells in regulating allo-reactions.
CD3+CD4+ CD3+CD8+
L + Bx
L + Lx
sBA
yBA
sBB
yBB
36.10% ± 2.39% 85.47% ± 1.52%
2.52% ± 0.27% 11.19% ± 1.64%
40.47% ± 7.38% 66.03% ± 8.69%
20.30% ± 1.43% 66.49% ± 1.43%
24.26% ± 1.86% 70.04% ± 1.86%
19.45% ± 4.85% 61.52% ± 4.85%
. BA: BN-ASCs BB: BN-BMSCs S: Senior Y: Young L: Lewis Splenocytes Bx: Irradiated BN Splenocytes Lx: Irradiated Lewis Splenocytes
Table 3B The percentage of proliferated T-cells as regulated by young and senior Lewis-ASCs/BMSCs, to demonstrate the efficacy of these cells in regulating allo-reactions. LA: Lewis-ASCs LB: Lewis BMSCs.
CD3+CD4+ CD3+CD8+
L + Bx
L + Lx
sLA
yLA
sLB
yLB
36.10% ± 2.39% 85.47% ± 1.52%
2.52% ± 0.27% 11.19% ± 1.64%
33.31% ± 4.35% 66.11% ± 12.67%
19.09% ± 4.20% 61.08% ± 12.34%
21.42% ± 2.05% 67.12% ± 5.99%
16.38% ± 12.34% 56.33% ± 14.56%
anti-inflammatory cytokine milieu, effectively altering the cytokine secretion profile of dendritic and T-cell subsets [21]. Clearly, age is an important determinant of the immunoregulatory potential of MSCs, and this effect is much more prominent in ASCs as compared to BMSCs. This may be explained by increased senescence of stem cell populations, which manifests in vitro as increasingly flawed indicators of genetic robustness, genomic integrity, and regulation of transcription as donors gradually age [22]. These effects ultimately lead to alteration of differentiation capacity, which can result in adverse effects such as the accumulation of fat deposits in bone and muscles, impaired healing and fibrosis after severe injury, or altered hematopoiesis and autoimmunity [18]. Furthermore, aged stem cells become prone to release different set of molecules, including metalloproteinases, inflammatory cytokines, and growth factors [23]. For MSCs, specifically, several key cytokines may be involved. TGF-beta was found to be decreased in somatic cells [24] and in MSCs from aged [25] and SAMP (Senescence Accelerated Prone Strain) mice [26]. Similarly, expression of interleukin-6 (IL-6), a cytokine capable of regulating proliferation, has been shown to be increased by human MSCs as donors age [27]. Furthermore, interleukin-11 (IL-11) activity and mRNA expression are reduced in MSCs from aged mice [28] and humans [29]. This process of distinct functional and phenotypic alterations in aging has been termed “inflamm-aging” in other literature [30] (Fig. 5). The concepts of in vitro culture-related senescence and cell isolation, however, from senior donors both warrant further exploration. This becomes particularly evident as despite some considerable amount of research and data, still no definite statement of age-related effects of MSCs can be made [30]. Although there is a notable tendency for CFU numbers to decrease during aging a review by Sethe et al. has shown that aged adipose tissue was routinely found to have increased CFU
capacity, not decreased CFU capacity [30]. These somewhat contradictory findings also raise the question how relevant and applicable in vitro data for stem cell based differentiation and immunoregulation studies generally might be. However, until better tools are available and given the fact that BMSCs are a very rare population (0.01% to 0.001%) [31], the ability to culture and expand MSCs in vitro is critical, as these cells have significant potential to make a profound impact on regenerative medicine and the field of transplantation. Since telomere length shortens after each cycle of cell division, the gradual progression towards senescence is inevitable, and this is also applicable to cells that have been expanded in vitro [32,33]. Thus, in this study we attempted to minimize the effect of culture-induced senescence by only using the first passage after sorting. Nevertheless, the time required for cell expansion before and after sorting may have increased the influence of culture-related senescence on the results. Notably, all groups of MSCs performed similarly in regulating CD3+CD8+proliferation, regardless of donor age or strain. These different outcomes between CD4+ and CD8+ populations suggest that MSCs might exert suppressive effect on these two populations through different mechanisms, and it also suggests that donor's age affects ASCs and BMSCs ability to inhibit CD4 versus CD8 T cells even more differentially. These results warrant further investigation for the clarification of these potential differential mechanisms of suppression. The knowledge gained regarding the senescence of MSCs is still expanding. Our data adds to the current understanding of how age affects the immunoregulatory potential of MSCs. This information may help guide future translational and clinical studies in which MSCs are utilized to harness their immunoregulatory properties in the fields of transplantation, or regenerative medicine.
Fig. 5. MSCs quality changes with age: (‘Inflammaging’).
L.W. Wu et al. / Transplant Immunology 30 (2014) 122–127
5. Conclusion MSCs have significant immunological potential that continues to be elucidated by multiple groups around the world. As these stem cells begin to enter the clinical arena, several nuances should be considered that may affect their immunoregulatory capacity. In particular, when considering the use of ASCs donor age should be taken into account, as increasing donor age has been shown to limit regulatory capacity, as well as proliferative and differentiation potential. In addition, the current armamentarium of phenotypic markers used to define young versus senior MSCs may be inadequate. Further characterization may reveal that subsets in both ASCs and BMSC that are not differentially affected by age. Therefore, continued research on the role of aging in MSCs is warranted, especially as it pertains to the clinical application of these stem cells in various immunological conditions. Acknowledgments We greatly appreciate Dr. Alan Hess and Mr. Christopher Thoburn for stimulating discussions; and the expert technical support from Lee Blosser and Ada Tam. References [1] Uccelli A, Moretta L, Pistoia V. Immunoregulatory function of mesenchymal stem cells. Eur J Immunol 2006;36:2566–73. [2] Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006;441:1080–6. [3] Zaim M, Karaman S, Cetin G, Isik S. Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells. Ann Hematol 2012;91:1175–86. [4] Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99:3838–43. [5] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815–22. [6] Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 2008;129:163–73. [7] Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–60. [8] Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol 2005;129:118–29. [9] McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, et al. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells 2006;24:1246–53. [10] Lombardo E, DelaRosa O, Mancheno-Corvo P, Menta R, Ramirez C, Buscher D. Toll-like receptor-mediated signaling in human adipose-derived stem cells: implications for immunogenicity and immunosuppressive potential. Tissue Eng Part A 2009;15:1579–89. [11] Yanez R, Lamana ML, Garcia-Castro J, Colmenero I, Ramirez M, Bueren JA. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells 2006;24:2582–91.
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Brief communication
Donor age negatively affects the immunoregulatory properties of both adipose and bone marrow derived mesenchymal stem cells Lehao W. Wu a,b, Yen-Ling Wang a,c, Joani M. Christensen a, Saami Khalifian a, Stefan Schneeberger a,d, Giorgio Raimondi a, Damon S. Cooney a, W.P. Andrew Lee a, Gerald Brandacher a,⁎ a
Vascularized Composite Allotransplantation (VCA) Research Laboratory, Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA Division of Plastic and Reconstructive Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, PR China Center for Vascularized Composite Allotransplantation, Department of Plastic Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan d Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria b c
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 3 March 2014 Accepted 4 March 2014 Available online 12 March 2014 Keywords: Age Bone marrow Adipose tissue Mesenchymal stem cells Immunoregulation
a b s t r a c t Purpose: Age negatively impacts the biologic features of mesenchymal stem cells (MSCs), including decreased expansion kinetics and differentiation potential. Clinically, donor-age may be within a wide spectrum; therefore, investigation of the role of donor's age on immunoregulatory potential is of critical importance to translate stem cell therapies from bench to bedside. Methods: Adipose and bone marrow derived MSCs (ASCs and BMSCs) were isolated in parallel from Lewis and Brown Norway rats of young (less than 4-week old) and senior groups (older than 15-month). The presentation of cells and time required for growth to 90% confluence was recorded. FACS sorting based on the expression of CD90 and CD29 double positive and CD45 CD11 double negative quantified the proportions of MSCs. After expansion, ASCs and BMSCs from different age groups were co-cultured in mixed lymphocyte reaction (MLR; Lewis vs. Brown Norway) assays. The suppression of CD3+CD4+ and CD3+CD8+ T cell populations by different sources of MSCs were compared. Results: The kinetics of cell growth was slower in old animals (17.3 ± 2 days) compared with young animals (8.8 ± 3 days), and cell morphology was irregular and enlarged in the senior groups. The yield of MSCs by FACS sorting was significantly higher in young groups compared to senior groups (p b 0.02). With regard to immunoregulatory potential, senior ASCs failed to induce any CD3+CD4+ T cell suppression (p N 0.05). In addition, young BMSCs-induced suppression was more prominent than seniors (p b 0.05). Conclusions: Donor age should be taken into consideration when using recipient MSC of either bone marrow or adipose origin in clinical applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The utilization of stem cell-based therapies, mesenchymal stem cells (MSCs)-based therapy in particular, has been proven to be a promising strategy to achieve transplant tolerance [1]. Despite their extensive proliferative and regenerative capacity, studies suggest that physical and biological properties of stem cells usually decline with advanced donor age [2]. This is primarily due to years of exposure to environmental damage and stress, which eventually leads to deterioration of tight control of multiple aspects of physiology, cell proliferation, and/or signal transduction [3].
Abbreviations: MSCs, mesenchymal stem cells; ASCs, adipose derived stem cells; BMSCs, bone marrow derived stem cells; MLR, mixed lymphocyte reaction. ⁎ Corresponding author at: Ross Research Building 749D, 720 Rutland Avenue Baltimore, MD 21205, USA. Tel.: +1 443 287 6679 (office); fax: +1 410 614 1296. E-mail address: [email protected] (G. Brandacher).
http://dx.doi.org/10.1016/j.trim.2014.03.001 0966-3274/© 2014 Elsevier B.V. All rights reserved.
Bone marrow derived mesenchymal stem cells (BMSCs) have shown great potential in regulating both alloreactive and non-specific immune responses when co-cultured in vitro through suppression of alloreactive T-cells [4,5]. However, accumulating evidence has shown that BMSCs exhibit both diminished differentiation and growth factor secretion with increasing donor age [6]. Yet, both transplant recipients and potential organ donors may be of any age. Thus, understanding the behavior and kinetics of these alterations in the quality and function of MSCs due to donor age is of great importance, as there is currently limited knowledge on how these potentially ‘functionally compromised’ MSCs would modulate transplantation outcomes. Adipose-derived mesenchymal stem cells (ASCs) are robust, multipotent cells, with a protein expression phenotype comparable to that of BMSCs [7]. Several in vitro studies have established that ASCs share immunological qualities with BMSCs including low immunogenicity, immunoregulatory effects, and similar cytokine expression profile [8–10]. Furthermore, ASCs are comparable to BMSCs in treating severe graft-versus-host disease [11] and alleviating arthritis and colitis [12].
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Fig. 1. The calculation of percentage of proliferated cell populations in a MLR. The percentage of all CD3+/CD4+ or CD3+/CD8+ cells was taken that no longer expressed CFSE as CD4 or CD8 T cells that had proliferated. Suppression was defined a lower percentage of cells proliferating. If the percentage of cells was the same with or without added MSC, this was defined as no suppression.
Clinically, ASCs are more widely available, easier to harvest and expand in cell culture, and cause less donor-site morbidity than MSCs from other sources. Therefore, ASCs present a promising alternative to BMSCs in many clinical applications. To our knowledge, MSCs derived from either bone marrow or adipose tissue have not been directly compared between young and aged donors for immunoregulatory capacities. Many different groups and investigators are now advocating ASCs as a potential alternative to the traditional BMSCs. This leads to the second questions we aimed to address in this study: does donor's age affect in the same way both BMSCs and ASCs? 2. Materials & methods 2.1. Animals Lewis and Brown Norway rats (Harlan, Indianapolis, IN) were divided into young (age under 4-week old) and senior (older than 15-month) groups, with n = 3 in both age groups from each strain. All animal procedures were approved by and performed in accordance with the Johns Hopkins University Animal Care and Use Committee. 2.2. Isolation of Rat BMSCs and ASCs Animals were euthanized by carbon dioxide suffocation and sterilized by iodine and ethanol solution. Soft tissue was stripped from femur & tibia, and the femur was disarticulated from the tibia. Long bone ends are cut and bone marrow flushed from the bones into a petri dish. The suspension was centrifuged at 1380 rpm for 5 min. Supernatant was aspirated, the pellet dislodged, and 20 cc of complete media consisting of DMEM/F12 with 10% heat-inactivated fetal bovine
serum, 1% penicillin/streptomycin and 1% Fungizone added, then suspensions of 50,000 cells were plated into T-50 flasks. All reagents were purchased from Invitrogen, CA unless otherwise noted. Inguinal fat pads were removed and minced with scissors in collagenase solution consisting of Hanks' balanced salt solution, bovine serum albumin and 1% type II collagenase (3.0 mg/g of fat) (Worthington Biochemical Corporation, Lakewood, NJ). Centrifuge tubes were then shaken at 100 rpm for 50 min at 37 °C. Following digestion, the content of each tube was filtered through double-layered sterile gauze. The filtrates were then centrifuged at 1000 rpm for 10 min at 37 °C. The pellets were suspended in the aforementioned plating medium and then preparations of 50,000 cells were added in 50 cm2 flasks and placed in 20% carbon dioxide incubator. Adherent ASCs were expanded for a period of 3 days at 37 °C, and the medium changed every 2 days until the cells achieved confluence.
2.3. Cell sorting, re-expansion and certification After the MSCs reached confluence, they were detached by a treatment with 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) and then approximately 105 cells for each of the senior groups, and 2.5 × 105 cells for the young groups were stained with Sytox-Blue (Invitrogen, Life Science, CA), CD29-FITC, CD90-PE, CD45-PerCP, and CD11b/c-AlexaFluor647 (BioLegend, US). Labeled (single) cells were sorted by gating on the CD29-FITC and CD90-PE double positive population, and excluding the Sytox-Blue, CD45-PerCP, and CD11-APC positive populations. We set 2 × 104 cells as target number of sorted cells to collect (when possible). Post-sorting cells were then expanded in culture for an additional 5 or 12 days and then used for purity analysis by flow cytometry, staining with anti-MHC class II-Alexa 647 (BD Biosciences, San Jose, CA), anti-CD31-eFluor 660 (eBioscience, San Diego,
Table 1 The biological difference in isolation of ASC. Brown Norway Senior (n = 3) Body weight (g) Adipose tissue harvested (g) Cell yield from adipose tissue(×104) Cell/g Body weight Cell/g Adipose tissue(×103) Days to reach confluence
497 4.63 13.58 273 29.3 15.0
± ± ± ± ± ±
Lewis Young (n = 3)
7.81 0.54 1.23 29.26 4.3 0
41.67 0.37 5.42 1303 146.5 7.3
± ± ± ± ± ±
Senior (n = 3) 1.27 0.04 0.52 161.98 21.2 0.58
497.33 5.24 12.00 241 22.9 17.6
± ± ± ± ± ±
Young (n = 3) 7.77 0.69 1.15 20.08 3.7 1.15
44.33 0.48 5.67 1279 118.1 8.0
± ± ± ± ± ±
1.37 0.06 0.29 79.54 16 0
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Fig. 2. Representative morphology of BMSCs and ASCs, 24-h after isolation from Lewis rat. (10× magnification.)
CA) and anti-CD105 (Antibodies-online, Atlanta, GA), or for the following cell function experiments. 2.4. CFSE-based mixed lymphocyte reaction (MLR) assay The detailed protocol has been published previously (J Robinson et al 1998.)[13] Briefly, spleens were harvested from both Lewis and Brown Norway rats then strained into cell suspension and separated by Ficoll-Paque (GE Health, TX). The mononucleocytes were collected and washed in DPBS at 400 g three times for 5 min. CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, CA, US) with a working concentration of 1 μM was added for every 5 million cells in 1 ml PBS. Cells were incubated at 37 °C for 10 min; staining was then quenched by adding 20 ml culture media consisting of RPMI, 10% FBS, 1% Penicillin/Streptomycin solution, and 15 mM Mercaptoethanol (Sigma, MO) for 5 min on ice. Cells were pelleted by centrifugation and washed three times. CFSEstained Lewis mononucleocytes were used as responders (105 cells) and irradiated Brown Norway splenocytes (5 × 104 cells irradiated with 2500 rads) were used as stimulators in a 96-well round bottom plate. MSCs were isolated from their respective rat strains as aforementioned and co-cultured in triplicates with a ratio of 1:1000 regulators vs. responders. On day 5, cells were harvested and stained with CD4-PE, CD8-PerCP and CD3-AlexaFlour 647 (all from Biolegend, CA, US) for detection of T cell subsets proliferation. 2.5. Comparison of CFSE-MLR assays and Statistical Analysis MLR analysis was conducted by flow cytometry by first gating on the lymphoid population on forward scatter/side scatter, followed by gating on CD3+CD4+ and CD3+CD8+ and then assessing proliferation to determine the effect of regulation by MSCs. Proliferation was calculated via quantification of the proportion of cells with CFSE staining lower than those that maintained the same level as the initial staining (nonproliferating cells; Fig. 1). Each experiment was conducted with three replicates and group means determined. Suppression was determined
Table 2 The percentage of MSCs (CD29+CD90+CD11−CD45−) in the entire cell preparation as assessed during FACS sorting (n = 3).
sBA yBA sBB yBB sLA yLA sLB yLB
Total event (×103)
Yield (×103)
Percentage
80.6 56.5 77.8 81.7 81.2 128.7 66.2 95.7
10.3 ± 1.3 20.0 8.1 ± 1.7 20.0 3.3 ± 0.8 20.0 5.7 ± 0.5 20.0
12.8 36.0 10.3 25.0 4.1 23.9 8.7 21.0
± ± ± ± ± ± ± ±
8.6 7.8 7.1 10.8 6.8 19.2 2.9 6.2
± ± ± ± ± ± ± ±
BA: BN-ASCs BB: BN-BMSCs LA: Lewis-ASCs LB: Lewis BMSCs; s: Senior y: Young Total event: number of cells passed through sorter Yield: number of positively-sorted cells
0.6% 4.6% 1.3% 3.6% 1.3% 3.7% 0.8% 1.4%
as a statistically significant lower value of group proliferation mean in comparison to control group. Student's t-test was used to determine statistical significance between group means. Significant results (p b 0.05) are denoted on figures by an asterisk. Continuous variables are presented as the mean (range ± standard deviation). 3. Results 3.1. Differences in primary isolation between young and senior donors Although the donors were 10 times smaller in size and tissue weight, the yield of cells on a per gram of body weight indicated a higher proportion of cells in adipose tissues of young versus old animals (Table 1). Additionally, as indicated by the time required to reach confluence during the first culture, stem cells isolated from young subjects were more vigorous with regard to expansion kinetics (Table 1). The morphology of MSCs isolated from young groups consisted of spindle-shaped adherent cells, as opposed to large, round, irregular shaped cells in the senior groups (Fig. 2). 3.2. Flow cytometry-based sorting The percent yield of the target population (CD29+CD90+ and CD11−CD45−) after sorting was significantly higher (p b 0.02) in young subjects regardless of source or strain. Additionally, the yield of MSCs (CD29+CD90+CD11−CD45−) isolated from sorted cells was also significantly higher in young groups. (Table 2) After re-expansion for 5 days, ASCs isolated from young or senior rats showed 87%–95% of CD105 expression, but no expression of MHC class II or CD31. Similarly, BMSCs expressed 93%–97% of CD105, but not MHC class II or CD31. These findings were also confirmed after expansion for 12 days. These data suggest that the isolated CD90+CD29+CD45−CD11− cells still maintain their MSC phenotype even after re-expansion for a prolonged period of time and do not exhibit any significant heterogeneity or contamination with endothelial cells (Fig. 3A and B). 3.3. ASC and BMSCs differentially regulate alloreactivity in a mixed lymphocyte reaction In the CFSE-based assay, as expected, BMSCs isolated from either Brown Norway and Lewis rats showed significant inhibitory activity irrespective of donor age (Fig. 4A and B). However, higher suppressive function of young BMSCs was evident as compared with senior BMSCs (p = 0.02 in Brown Norway; p = 0.003 in Lewis rats) A different scenario was observed with ASCs. While young ASCs maintained regulatory properties in both groups (p = 0.01 for Brown Norway; p = 0.004 for Lewis rats), ASCs isolated from senior rat groups failed to regulate CD3+CD4+ cell proliferation in an MLR, regardless of strain (p = 0.22 for Brown Norway; p = 0.07 for Lewis rats). (Tables 3A and 3B). Interestingly, this effect was restricted to CD4 T cells, as there was no difference between young and old ASCs in regulating CD3+CD8+ population in MLR assays (Fig. 4B).
4. Discussion The process of aging alters the immune system in a global and complex way, as evidenced by diminished primary and secondary immune responses, increased incidence of inflammatory pathologies, and weakened vaccine efficiency [14]. This concept of “immunosenescence” [15], can affect all immune compartments, including humoral and cellmediated immunity. Similarly, the effect of immunosenescence on stem cells may alter their function and phenotype, as many of their intrinsic beneficial functions deteriorate with age, and other misbalanced regulatory events lead to alterations in their immunoregulatory potential. In this study, the impact of age on the biological quality of MSCs from Lewis and Brown Norway was investigated, and we found that isolating
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Fig. 3. A) The CD90+CD29+CD45−CD11− ASCs from young (3 weeks of age) or senior (over 12 months of age) rats were isolated by cell sorting. Sorted cells were cultured for 5 days and further analyzed for additional surface markers (MHC class II, CD31 and CD105. B) BMSCs shared the same protocol of ASC isolation, and were re-expanded for 12 days. Each group contained three animals and data are presented as mean ± standard error of the mean (SEM).
ASCs from senior subjects using standard protocols led to inadequate cell yields and viability. Furthermore, in parallel experiment in which ASCs from all 3 senior ACI rats did not survive after the cell sorting process, the following experiment could not be performed. This further demonstrated the lower vitality of senior MSCs. These findings are in line with what has been reported by other groups in human donors, where the frequency and expansion capacity of MSC decreased with donor age [16]. Stenderup et al. [17] showed that donor age positively correlates with the rate of in vitro MSC senescence. Specifically, the maximal life span of MSCs between the young (18–25 years old) and old (N66 years old) group were found to be significantly different [17]. In general, donor age has a significant impact and is a well described factor in the harvest of bone marrow hematopoietic stem cells in particular when comparing pediatric versus elderly donors. Significantly higher mean total CD34 + cell counts are usually seen with young donor age as well as a higher ability in colony forming unit (CFU) assays.
Clinical implications of immunosenescence are broad and include increased risk of infections, malignancies, autoimmune disorders, and neuro-degenerative changes [18]. Furthermore, the consequences of immunosenescence also have significant ramifications in the field of organ transplantation, as increased donor or recipient age may require specific adaptations to immunosuppressive protocols [19]. In terms of immunoregulatory potential, assessed by in vitro MLR, we were able to demonstrate that senior ASCs not only failed to induce suppression in CD3+CD4+ proliferation, but also, allogeneic ASCs from Brown Norway rats actually appeared to stimulate proliferation, suggesting a possible increase of immunogenicity. Previous research has shown that ASCs inhibit proliferation of CD4+ and CD8+ cells in a non-selective and non-specific way [20] and several cytokines have been implicated in the regulatory process. Interferon-gamma and prostaglandin E2 (PGE2), amongst others, are immunosuppressive mediators selectively produced by MSCs that may convert pro-inflammatory environments into an
Fig. 4. A. MSCs regulating CD3+CD4+ proliferation assay (n = 3): In a MLR assay, CFSE-stained Lewis lymphocytes were stimulated by irradiated BN splenocytes (L + Bx) at a 2:1 ratio without or with ASCs (A) or BMSCs (B) from young (y) or senior (s) subjects of different strains. After 5 days of co-culture, the percentage of proliferated CD3+CD4+ and CD3+CD8+ were compared. ASCs and BMSCs from young groups have significantly better potential in regulating CD3+CD4+ responses (p b 0.05). Senior ASCs failed to induce CD3+CD4+ suppressions. All experiments were performed in triplicates. B. The percentage of proliferated CD3+CD8+. Young BMSCs were still more potent than senior BMSCs; however, such difference was not observed in ASCs groups. BA: BN-ASCs BB: BN-BMSCs LA: Lewis-ASCs LB: Lewis BMSCs; S: Senior Y: Young L: Lewis Splenocytes Bx: Irradiated BN Splenocytes Lx: Irradiated Lewis Splenocytes.
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Table 3A Percentage of proliferating T-cells regulated by young or senior Brown Norway ASCs/BMSCs, to demonstrate the efficacy of these cells in regulating allo-reactions.
CD3+CD4+ CD3+CD8+
L + Bx
L + Lx
sBA
yBA
sBB
yBB
36.10% ± 2.39% 85.47% ± 1.52%
2.52% ± 0.27% 11.19% ± 1.64%
40.47% ± 7.38% 66.03% ± 8.69%
20.30% ± 1.43% 66.49% ± 1.43%
24.26% ± 1.86% 70.04% ± 1.86%
19.45% ± 4.85% 61.52% ± 4.85%
. BA: BN-ASCs BB: BN-BMSCs S: Senior Y: Young L: Lewis Splenocytes Bx: Irradiated BN Splenocytes Lx: Irradiated Lewis Splenocytes
Table 3B The percentage of proliferated T-cells as regulated by young and senior Lewis-ASCs/BMSCs, to demonstrate the efficacy of these cells in regulating allo-reactions. LA: Lewis-ASCs LB: Lewis BMSCs.
CD3+CD4+ CD3+CD8+
L + Bx
L + Lx
sLA
yLA
sLB
yLB
36.10% ± 2.39% 85.47% ± 1.52%
2.52% ± 0.27% 11.19% ± 1.64%
33.31% ± 4.35% 66.11% ± 12.67%
19.09% ± 4.20% 61.08% ± 12.34%
21.42% ± 2.05% 67.12% ± 5.99%
16.38% ± 12.34% 56.33% ± 14.56%
anti-inflammatory cytokine milieu, effectively altering the cytokine secretion profile of dendritic and T-cell subsets [21]. Clearly, age is an important determinant of the immunoregulatory potential of MSCs, and this effect is much more prominent in ASCs as compared to BMSCs. This may be explained by increased senescence of stem cell populations, which manifests in vitro as increasingly flawed indicators of genetic robustness, genomic integrity, and regulation of transcription as donors gradually age [22]. These effects ultimately lead to alteration of differentiation capacity, which can result in adverse effects such as the accumulation of fat deposits in bone and muscles, impaired healing and fibrosis after severe injury, or altered hematopoiesis and autoimmunity [18]. Furthermore, aged stem cells become prone to release different set of molecules, including metalloproteinases, inflammatory cytokines, and growth factors [23]. For MSCs, specifically, several key cytokines may be involved. TGF-beta was found to be decreased in somatic cells [24] and in MSCs from aged [25] and SAMP (Senescence Accelerated Prone Strain) mice [26]. Similarly, expression of interleukin-6 (IL-6), a cytokine capable of regulating proliferation, has been shown to be increased by human MSCs as donors age [27]. Furthermore, interleukin-11 (IL-11) activity and mRNA expression are reduced in MSCs from aged mice [28] and humans [29]. This process of distinct functional and phenotypic alterations in aging has been termed “inflamm-aging” in other literature [30] (Fig. 5). The concepts of in vitro culture-related senescence and cell isolation, however, from senior donors both warrant further exploration. This becomes particularly evident as despite some considerable amount of research and data, still no definite statement of age-related effects of MSCs can be made [30]. Although there is a notable tendency for CFU numbers to decrease during aging a review by Sethe et al. has shown that aged adipose tissue was routinely found to have increased CFU
capacity, not decreased CFU capacity [30]. These somewhat contradictory findings also raise the question how relevant and applicable in vitro data for stem cell based differentiation and immunoregulation studies generally might be. However, until better tools are available and given the fact that BMSCs are a very rare population (0.01% to 0.001%) [31], the ability to culture and expand MSCs in vitro is critical, as these cells have significant potential to make a profound impact on regenerative medicine and the field of transplantation. Since telomere length shortens after each cycle of cell division, the gradual progression towards senescence is inevitable, and this is also applicable to cells that have been expanded in vitro [32,33]. Thus, in this study we attempted to minimize the effect of culture-induced senescence by only using the first passage after sorting. Nevertheless, the time required for cell expansion before and after sorting may have increased the influence of culture-related senescence on the results. Notably, all groups of MSCs performed similarly in regulating CD3+CD8+proliferation, regardless of donor age or strain. These different outcomes between CD4+ and CD8+ populations suggest that MSCs might exert suppressive effect on these two populations through different mechanisms, and it also suggests that donor's age affects ASCs and BMSCs ability to inhibit CD4 versus CD8 T cells even more differentially. These results warrant further investigation for the clarification of these potential differential mechanisms of suppression. The knowledge gained regarding the senescence of MSCs is still expanding. Our data adds to the current understanding of how age affects the immunoregulatory potential of MSCs. This information may help guide future translational and clinical studies in which MSCs are utilized to harness their immunoregulatory properties in the fields of transplantation, or regenerative medicine.
Fig. 5. MSCs quality changes with age: (‘Inflammaging’).
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5. Conclusion MSCs have significant immunological potential that continues to be elucidated by multiple groups around the world. As these stem cells begin to enter the clinical arena, several nuances should be considered that may affect their immunoregulatory capacity. In particular, when considering the use of ASCs donor age should be taken into account, as increasing donor age has been shown to limit regulatory capacity, as well as proliferative and differentiation potential. In addition, the current armamentarium of phenotypic markers used to define young versus senior MSCs may be inadequate. Further characterization may reveal that subsets in both ASCs and BMSC that are not differentially affected by age. Therefore, continued research on the role of aging in MSCs is warranted, especially as it pertains to the clinical application of these stem cells in various immunological conditions. Acknowledgments We greatly appreciate Dr. Alan Hess and Mr. Christopher Thoburn for stimulating discussions; and the expert technical support from Lee Blosser and Ada Tam. References [1] Uccelli A, Moretta L, Pistoia V. Immunoregulatory function of mesenchymal stem cells. Eur J Immunol 2006;36:2566–73. [2] Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006;441:1080–6. [3] Zaim M, Karaman S, Cetin G, Isik S. Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells. Ann Hematol 2012;91:1175–86. [4] Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99:3838–43. [5] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815–22. [6] Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 2008;129:163–73. [7] Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–60. [8] Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol 2005;129:118–29. [9] McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, et al. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells 2006;24:1246–53. [10] Lombardo E, DelaRosa O, Mancheno-Corvo P, Menta R, Ramirez C, Buscher D. Toll-like receptor-mediated signaling in human adipose-derived stem cells: implications for immunogenicity and immunosuppressive potential. Tissue Eng Part A 2009;15:1579–89. [11] Yanez R, Lamana ML, Garcia-Castro J, Colmenero I, Ramirez M, Bueren JA. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells 2006;24:2582–91.
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