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TISSUE-SPECIFIC STEM CELLS |
a Cognate Therapeutics, Inc., Baltimore, Maryland, USA;
b Stem Cell Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA;
c Artecel Sciences, Durham, North Carolina, USA;
d CuraGen Corporation, Branford, Connecticut, USA;
e Lineberger Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA;
f Duke University Medical Center, Durham, North Carolina, USA
Key Words. Human • Mixed lymphocyte reaction • Immunosuppressive • Immunogenic • Multipotent • Tissue engineering • Regenerative medicine • Adipose-derived stem cells
Correspondence: Jeffrey M. Gimble, M.D., Ph.D., Stem Cell Laboratory, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808, USA. Telephone: 225-763-3171; Fax: 225-763-0273; e-mail: gimblejm{at}pbrc.edu
Received May 24, 2005;
accepted for publication December 28, 2005.
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ABSTRACT |
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INTRODUCTION |
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We and others have demonstrated that adipose tissue is a potential source of adult stem cells capable of differentiation along multiple lineage pathways, including adipocytes, chondrocytes, endothelial cells, epithelial cells, hematopoietic supporting cells, hepatocytes, myocytes, neuronal-like cells, myocytes, and osteoblasts [1–15]. These cells are isolated by collagenase digestion of the adipose tissue, yielding an initial stromal vascular fraction (SVF) that is further processed by plating and adherence to plastic [1]. Clonal analyses demonstrate that a single adherent human adipose tissue cell can give rise to a daughter cell population capable of multilineage differentiation potential, consistent with the definition of a stem cell [16]. We have quantified the frequency of adherent colony-forming unit cells and found that they represent approximately 3% of the SVF population [17]. These values exceed those of human bone marrow aspirates by at least three orders of magnitude [18]. In accordance with a consensus reached by investigators attending the Second Annual International Fat Applied Technology Society meeting (October 3–5, 2004, Pittsburgh, PA), we will refer to this adherent cell population as adipose-derived stem cells (ASCs). A single milliliter of liposuction tissue aspirate can generate one-quarter million ASCs within a single passage, and these can expand an additional 64-fold within a 26-day period [17]. Thus, a single liposuction procedure, which routinely generates liters of waste tissue, could potentially yield several billion cells with minimal time in culture.
A number of studies from our laboratory and others have determined that allogeneic bone marrow-derived mesenchymal stem cells or stromal cells (BMSCs) fail to stimulate an immune response based on in vitro assays of T cell function [19–23]. The immunophenotype of BMSCs changes as a function of time in culture [24], and this may be contributory to their distinct immunogenic properties. We have found that the immunophenotype of human adipose-derived cells changes significantly in a similar manner as a function of adherence to cultureware and expansion [17]. Based on this and other parallels with BMSCs, we set out to determine whether the immunophenotype changes of the human adipose-derived cells correlated with alterations in their immunogenic properties based on in vitro mixed lymphocyte reaction. The results suggest that allogeneic transplantation of human ASCs may prove to be possible.
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MATERIALS AND METHODS |
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ASC Cell Isolation and Expansion
All protocols were reviewed and approved by the Pennington Biomedical Research Center Institutional Research Board prior to the study. Liposuction aspirates from subcutaneous adipose tissue sites were obtained from male and female subjects undergoing elective procedures in local plastic surgical offices. Tissues were washed three or four times with phosphate-buffered saline (PBS) and suspended in an equal volume of PBS supplemented with 1% bovine serum and 0.1% collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ, http://www.worthington-biochem.com) prewarmed to 37°C. The tissue was placed in a shaking water bath at 37°C with continuous agitation for 60 minutes and centrifuged for 5 minutes at 300–500g at room temperature. The supernatant, containing mature adipocytes, was aspirated. The pellet was identified as the SVF. Portions of the SVF were resuspended in cryopreservation medium (10% dimethylsulfoxide, 10% Dulbecco’s modified Eagle’s medium [DMEM]/Ham’s F-12, 80% fetal bovine serum), frozen at –80°C in an ethanol-jacketed closed container, and subsequently stored in liquid nitrogen. Portions of the SVF were used in colony-forming unit assays (see below). The remaining cells of the SVF were suspended and plated immediately in T225 flasks in stromal medium (DMEM/Ham’s F-12, 10% fetal bovine serum [HyClone, Logan, UT, http://www.hyclone.com], 100 U of penicillin/100 µg of streptomycin/0.25 µg of Fungizone) at a density of 0.156 ml of tissue digest per cm2 of surface area for expansion and culture. This initial passage of the primary cell culture was referred to as passage 0 (P0). Following the first 48 hours of incubation at 37°C at 5% CO2, the cultures were washed with PBS and maintained in stromal media until they achieved 75%–90% confluence (approximately 6 days in culture). The cells were passaged by trypsin (0.05%) digestion and plated at a density of 5,000 cells per cm2 (passage 1). Cell viability and numbers at the time of passage were determined by trypan blue exclusion and hemacytometer cell counts. Cells were passaged repeatedly after achieving a density of 75%–90% (approximately 6 days in culture) until passage 4.
BMSC Cell Isolation and Expansion
Human bone marrow was purchased from Cambrex (Walkersville, MD, http://www.cambrex.com) or AllCells, LLC (Berkeley, CA). Bone marrow aspirates were collected with heparin and fractionated over a 1.073 g/ml density gradient (Lymphocyte Separation Medium [LSM], Cambrex), and mononuclear cells collected at the interface were plated in HyQ DMEM-Low Glucose (HyClone) containing 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, http://www.jrhbio.com) that was selected based on its ability to support BMSC expansion. Nucleated cells were plated at a density of 30 x 107 cells per T185-cm2 flask. Cells were grown in primary cultures (P0) for 12–17 days with medium changes every 3 or 4 days. When the cells became confluent, the culture was passaged using 0.05% trypsin (Gibco, a subsidiary of Invitrogen, Grand Island, NY, http://www.invitrogen.com) to remove adherent cells and re-plated as P1 cells at 1 x 106 cells per T185-cm2 flask. From this point on, the BMSCs were passaged every 7 days, with one medium change every 3–4 days. At final harvest, BMSC were cryopreserved using a freeze solution containing 10% DMSO (Edwards Life Sciences, Irvine, CA, http://www.edwards.com) and 5% human serum albumin (JRH Biosciences) in plasmalyte (Baxter Health Care, Deerfield, IL, http://www.baxter.com). Expanded BMSCs (P2–P4) represented a homogenous population that was fibroblastic in appearance and negative for hematopoietic markers (CD45, CD14, CD3, major histocompatability class II antigens) and positive for stromal markers (CD13, CD29, CD44, CD90, CD105). BMSCs were multipotent at P2 and P4 as shown by their ability to differentiate along the osteogenic and adipogenic lineages (data not shown).
Flow Cytometry
Flow cytometry was performed as described [17]. Antibodies directed against the following antigens (catalog #) were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen) unless otherwise indicated and used at the vendor-recommended quantities: CD11a antigen presenting cell (APC) (550852), CD14 APC (555394), CD40 APC (555591), CD45 FITC (555482), CD54 APC (559771), CD80 FITC (Caltag MHCD8001), CD86 PE (Caltag MHCD8601), HLA-ABC APC (555555), histocompatible locus antigen-DR (HLA-DR) APC (559868). Isotype-matched negative controls were used to define background staining. Analysis gating included ~1.5% of the negative control fluorescence; thus, populations containing ~1.5% positive events should be considered negative.
Mixed Lymphocyte Reaction
Human Lymphocyte Populations. Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation of leuko-pheresed peripheral blood cells (AllCells; LLC, Emeryville, CA, http://www.allcells.com) over an LSM density gradient. T cells were purified from a portion of the PBMCs by negative selection using magnetic beads. Briefly, PBMCs were treated with a cocktail of monoclonal antibodies (mAbs) (all from Serotec, Inc., Raleigh, NC, http://www.serotec.com) chosen to bind to monocytes (anti-CD14; clone UCHM1), B cells (anti-CD19; clone LT19), natural killer cells (anti-CD56; clone ERIC-1), and cells expressing MHC class II antigens (anti-MHC class II DR; clone HL-39). PBMCs were mixed with magnetic beads coated with anti-mouse IgG antibody (Dynal Corp., Lake Success, NY). Bead-bound cells were removed using a magnet, leaving a population of purified T cells (>90% T cells by flow cytometry using anti-CD3 mAb). Both PBMCs and purified T cells were aliquoted and cryopreserved in liquid nitrogen.
Immunogenicity Assay.
The one-way mixed lymphocyte reaction (MLR) assay was used to determine the immunogenicity of fat-derived cell populations. The MLR was performed in 96-well microtiter plates using Iscove’s modified Dulbecco’s medium supplemented with sodium pyruvate, nonessential amino acids, antibiotics/antimycotics, 2-mercaptoethanol (all reagents from Gibco, Grand Island, NY, http://www.invitrogen.com), and 5% human AB serum (Pel-Freez, Rogers, AK, http://www.pel-freez.com). Purified T cells derived from two different donors were plated at 2 x 105 cells per donor per well. Different donors were used to maximize the chance that at least one of the T cell populations was a major mismatch to the fat-derived test cells. Stimulator cells used in the assay included autologous PBMCs (baseline response), allogeneic PBMCs (positive-control response), and the test fat-derived cell populations. Stimulator cells were irradiated with 5,000 rads of 
radiation delivered by a cesium irradiator prior to being added to the culture wells at various numbers, typically ranging from 5,000 to 20,000 cells per well. Additional control cultures consisted of T cells plated in medium alone (no stimulator cells). Triplicate cultures were performed for each treatment. The cultures were incubated at 37°C in 5% CO2 for 6 days, pulsed with [3H]thymidine (1 µCi per well; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) for 16 hours, and the cells were harvested onto glass fiber filter mats using a Skatron 96-well cell harvester (Molecular Devices Corp., Sunnyvale, NY, http://www.moleculardevices.com). Radioactivity incorporated into the dividing T cells deposited on the filters was determined using a scintillation counter (Microbeta Trilux Scintillation and Luminescence Counter; Wallac Inc., Gaithersburg, MD, http://las.perkinelmer.com/).
Three criteria were used in assessing the immunogenicity of cell populations: 1) a statistically significant difference in the T cell-proliferative response (cpm) relative to that induced by autologous PBMCs (p < .05, Student’s t test); 2) a stimulation index (S.I.; cpm induced by the test population divided by cpm induced by autologous PBMCs) of at least 3.0; and 3) a difference of at least 750 cpm from the response induced to autologous PBMCs. Test populations that passed all three criteria were considered immunogenic. The rationale for selecting both an S.I. and a minimum 
cpm was to ensure that the proliferative response to the test population was sufficiently higher than the background response to autologous PBMCs, which can be very low (often <100 cpm). Based on our MLR database of hundreds of assays to over a dozen different nonhematopoietic stimulator cells, 750 cpm was chosen as a reasonable threshold since the majority of T cell-proliferative responses exhibiting an S.I. 
3 also exhibited a 
cpm of 750 or greater.
Suppression Assay.
The two-way MLR assay was used to evaluate suppression by adipose-derived cell populations. PBMCs from two different donors were used as the "responder cells" in the MLR. These were mixed in complete culture medium at 2 x 105 cells per donor per well in 96-well microtiter plates. Adipose tissue-derived cells, either SVF cells or ASCs at progressive passages, were added to the MLRs at cell concentrations of 5,000, 10,000, or 20,000 cells per well. Control MLR cultures had no adipose-derived cells added, or human splenic fibroblasts (CRL-7433; American Type Culture Collection, Manassas, VA, http://www.atcc.org) were added at concentrations of 5,000, 10,000, or 20,000 cells per well. Cultures were pulsed with [3H]thymidine on day 6 and harvested for scintillation counting as described above. Previous studies had shown that splenic fibroblasts displayed the least suppressive fibroblastic cell type when added to MLR assays (personal observations on six different fibroblast lines). Therefore, splenic fibroblasts were used in the present experiments to determine nonsuppressive dosing ranges in which to evaluate suppression by test cells. The percentage of suppression was calculated by the following formula: Percentage suppression = (1 – [Test cell + MLR cpm ÷ MLR cpm]) x 100. Statistical significance between control and test cultures was evaluated using Student’s t test.
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RESULTS |
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Further analyses examined the expression of hematopoietic-associated surface antigens (Table 1
, Fig. 1). The initial SVF cells freshly isolated from collagenase-digested adipose tissue and the initial adherent P0 ASCs contained a subset of cells that were positive for a panel of hematopoietic markers, including the common leukocyte antigen CD45, the monocyte/macrophage markers CD11a and CD14, the MHC class II DR histocompatibility antigen, and the co-stimulatory molecule CD86 (Table 1). This population disappeared by P1 according to decreased expression for most of the aforementioned markers (Table 1). The presence of cells bearing these surface markers in the freshly isolated adipose tissue-derived cell population is significant, as these cells are potentially immunogenic, could induce an allogeneic-rejection response, and may be a source of proinflammatory cytokines.
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). The range of change varied between 1.3%–66% for CD40 and 67%–92% for HLA-ABC. The high level of class I antigen expression coupled with intermediate to high levels of molecules associated with costimulatory activity (CD40, CD54, CD80) would suggest that these cells could function as antigen presenting cells in the mixed lymphocyte reaction. This was investigated as described below.
Immunogenicity
One-way MLR assays were performed to assess the immunogenicity of human adipose-derived cells. The proliferation of T cells was measured based on [3H]thymidine incorporation in the presence of increasing doses of irradiated stimulator cells. Autologous and allogeneic PBMCs served as negative and positive stimulator cell controls, respectively. Human SVF cells elicited a dose-dependent MLR response comparable to that of allogeneic PBMCs (Fig. 2). With progressive passage, the human adipose-derived cells elicited a decreased response that fell to undetectable levels by P1. Immunogenicity of adipose-derived cell populations from multiple donors is shown in Table 2. Positive and negative designations for immunogenicity are based on criteria described in Materials and Methods and are shown for the highest cell dose in each experiment, which ranged from 20,000 cells per well (donors 902–917) to 30,000 cells per well (donors 407–611). Based on positive responses for either or both T cell populations, the following populations were immunogenic: SVF cells (four of seven donors), P0 cells (seven of nine donors), and P1 cells (four of seven donors). The remaining passaged cell populations (P2–P4) did not induce T cell proliferation in MLR assays, with the exception of P2 cells from one donor.
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, splenic fibroblasts suppressed the MLR cultures only at the highest dose (20,000 cells per well). Using the lower two doses as being valid (no artifactual suppression), significant suppression was mediated by all ASC passages except the SVF population. Percentage suppression of the control MLR response (no cells added) mediated by P0–P4 ASCs ranged from 33%–63%. The results from four donors are summarized in Table 3. Percentage suppression was determined at the lowest dose of cells (5,000 cells per well) since there was no suppression of the MLR at this dose of splenic fibroblasts in any of these experiments. Mean suppression by the SVF population was minimal (10%), whereas suppression by P0–P4 cells averaged 32.0% ± 3.2%. This degree of suppression is significant in view of the low percentage of ASCs in these cultures (1.3%).
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). The magnitude of suppression by ASCs exceeded that of BMSCs by up to 13%, but the difference was not statistically significant.
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DISCUSSION |
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There are several explanations to account for these observations. First, heterogeneity within the SVF and P0 populations may include the presence of a significant percentage of hematopoietic-derived APCs. The abundance of these hematopoietic-derived cells decline with progressive passage and expansion of the adherent ASCs. Alternatively, the adherent ASCs may themselves express the APC-related surface proteins initially during culture and thereby serve as immunogenic stimuli in the MLR. Certainly, the ASCs express a number of cell surface molecules through passage P4, which can exhibit costimulatory activity, including CD54 [26], CD40 [27], CD80, and CD86 [28]. However, in the absence of CD11, CD14, HLA-DR, and other APC-related proteins, these surface antigens may not be sufficient to endow ASCs with APC function. In addition, other mechanisms, such as the ASC’s development of active immunosuppression, may override any inherent immunogenicity of the ASCs in culture.
In this study, we have shown that ASCs significantly suppressed T cell proliferation in the MLR. This property was pronounced in P0–P4 cells (mean suppression 32%), but not in the SVF population (mean suppression 10%). There are two alternative explanations for these findings. First, the SVF may contain a subpopulation of cells that possess immunosuppressive function but are not present in sufficient numbers to mediate their effect in an MLR. Alternatively, the ASCs may acquire their immunosuppressive properties only upon adherence and expansion. Theoretically, these two alternatives could be resolved by assessing the immunosuppressive properties in cells purified directly from the SVF population based on their immunophenotype. Although this experiment would be potentially challenging to perform, the results could be important in sorting out issues related to the immunogenicity of the SVF population as described previously (e.g., the presence of contaminating APCs or lack of suppression).
To avoid artifactual interpretation of results (i.e., suppression due to cell crowding), we performed suppression experiments at very high ratios of responding cells in the MLR to the test cells (80:1). Control splenic fibroblasts were not suppressive at this ratio. We compared suppression by ASCs to BMSCs since both cell types have similar phenotypic and functional characteristics and BMSCs have been shown to be immunosuppressive by their ability to inhibit T cell proliferation in MLR assays as well as to mitogenic stimulation [20–23]. Indeed, we found that ASCs and BMSCs exhibited similar magnitude of suppression. Our results confirm and extend those reported recently by Puissant et al. [29]. These investigators examined cells equivalent to our P0 population and found that ASCs stimulated a proliferative response that was significantly less than that elicited by allogeneic PBMCs, similar to the current findings. Likewise, their ASCs displayed a cell concentration-dependent immunosuppressive effect when added to mixed lymphocyte reactions.
Although there is little information on the mechanism of suppression by ASCs, Puissant et al. determined that the immunosuppressive effect of the ASCs was not entirely dependent on direct contact with lymphocytes, consistent with the release of a soluble factor [29]. BMSCs have been reported to elaborate suppressive molecules, including hepatocyte growth factor and transforming growth factor-ß [20], prostaglandins [30, 31], and indoleamine 2,3-dioxygenase [32]. Several different mechanisms have been proposed to account for BMSC-mediated suppression of lymphocyte proliferation. These include division arrest of activated T cells and B cells by inhibition of cyclin D2 expression [33], induction of regulatory T cells [30] or APCs [34], and interference with dendritic cell [30] and cytotoxic T cell maturation [35]. Further studies are needed to determine whether ASCs mediate suppression by similar mechanisms.
Our immunological data demonstrating that culture-expanded adipose-derived cells do not stimulate but actively suppress alloreactive T cell proliferation suggests that these cells can be transplanted across classical histocompatibility barriers. BMSCs have been reported to survive in immunocompetent allogeneic and xenogeneic recipients for longer than expected periods of time [30]. Whether this is due to their immunosuppressive properties remains to be determined, although there is evidence to suggest that BMSCs can mediate suppression in vivo [19, 34, 35]. Due to the immunogenic nature of the SVF population, it is likely that transplantation of SVF cells will be limited solely to autologous applications, although manipulation of the graft to remove monocytes may diminish immunogenicity of this population. The potential use of allogeneic ASCs as a source of cells for tissue repair or replacement has important implications with respect to the ready availability of adult stem cells for clinical practice and to the practical and commercial aspects of their manufacture and quality assurance. Additional in vivo studies using ASCs from a preclinical animal model will be necessary to determine the feasibility of such an approach.
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SUMMARY |
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ACKNOWLEDGMENTS |
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DISCLOSURES
J.M.G. and X.W. have financial interests in Artecel Sciences. S.G., J.M.G., L.H., K.M., J.B.M., and X.W. have financial interests in Cognate Therapeutics.
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REFERENCES |
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