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Cell Biology International (2008) 32, 384393 (Printed in Great Britain)
Mesenchymal stem cells from human bone marrow or adipose tissue differently modulate mitogen-stimulated B-cell immunoglobulin production in vitro
Ivan Bocheva, Gabriel Elmadjiana, Dobroslav Kyurkchievb, Liubomir Tzvetanovc, Iskra Altankovab, Peter Tivchevc and Stanimir Kyurkchieva*
aDepartment of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences,73 Tzarigradsko shosse, 1113 Sofia, Bulgaria
bLaboratory of Clinical Immunology, University Hospital “St. Ivan Rilski” Sofia, Bulgaria
cDepartment of Orthopedics and Traumatology, University Hospital “Tzaritza Ioana”, Sofia, Bulgaria
Mesenchymal stem cells (MSC) have been characterized as multipotent cells which are able to differentiate into several mesodermal and nonmesodermal lineage cells and this feature along with their extensive growth and comprehensive immunomodulatory properties establish them as a promising tool for therapeutic applications, including cell-based tissue engineering and treatment of immune-mediated disorders. Although bone marrow (BM) is the most common MSC source, cells with similar characteristics have been shown to be present in several other adult tissues. Adipose tissue (AT), large quantities of which can be easily obtained, represents an attractive alternative to BM in isolating adipose tissue-derived MSC (AT-MSC). BM-MSCs and AT-MSCs share some immunomodulatory properties as they are both not inherently immunogenic and suppress the proliferation of alloantigen- or mitogen-stimulated T-cells. Our purpose was to comparatively examine under appropriate in vitro conditions, phenotypes, morphology and some functional properties of BM-MSCs and AT-MSCs, such as differentiation potential and especially the ability to suppress the immunoglobulin production by mitogen-stimulated B-cells. While the morphological, immunophenotypical, colony-forming and adipogenic characteristics of both types of cells were almost identical, AT-MSCs showed less potential for osteogenic differentiation than BM-MSCs. We found that AT-MSCs not only inhibited the Ig-production but also suppressed this B-cell function to a much greater extent compared to BM-MSC. This finding supports the potential role of AT-MSCs as an alternative to BM-MSCs for clinical purposes.
Keywords: Mesenchymal stem cells, Immunosuppression, Bone marrow, Adipose tissue, Osteogenesis, Adipogenesis, Immunoglobulin production.
*Corresponding author. Tel.: +359 2 8723890.
Mesenchymal stem cells (MSC) represent a population of multipotent somatic stem cells identified in various tissues including bone marrow, skeletal muscle, brain, adipose tissue, umbilical cord blood, peripheral blood, connective tissues of the dermis, etc. (da Silva Meirelles et al., 2006; Javazon et al., 2004). These cells, regardless of their source, share some characteristic properties such as the capacity for clonal expansion and prolonged self-renewal under controlled conditions, spindle-shaped fibroblast-like morphology and potential for mesodermal or even nonmesodermal lineage differentiation. Expanded MSCs could be guided, either in vitro or in vivo, to terminally differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes, tenocytes, cells of visceral mesoderm, neurons, hepatocytes and endothelium. Phenotypically MSCs have been defined as positive for CD29, CD44, CD73, CD90, CD105, CD106 and CD166 and negative for hematopoietic lineage markers and HLA-DR (Javazon et al., 2004; Short et al., 2003; Croft and Przyborski, 2004).
Due to their multipotentiality, accessibility and expandability, MSCs could be regarded as a promising source of starting material for tissue engineering, cellular and somatic gene therapy applications (Quarto et al., 2001; Fuchs et al., 2003; Horwitz et al., 1999; Chen et al., 2004; Koc et al., 2000; Pereira et al., 1995; Schwartz et al., 2002; Dai et al., 2005). Although bone marrow represents the main source for the isolation of multipotent MSC there are some features that could restrict or make the use of bone marrow-derived MSCs unacceptable, such as the highly invasive harvesting procedure and the significant decline in the differentiation potential of the cells with increasing age. Adipose tissue, which can be obtained in a less traumatic manner and in larger quantities than bone marrow, could serve as an alternative MSC source. Within human lipoaspirates, an adherent stem cell population known as adipose-derived stem cells (AT-MSCs) (Zuk et al., 2001, 2002) has been previously identified and it was shown to exhibit stable growth and proliferation kinetics under standard tissue culture conditions and potential for multilineage mesodermal differentiation when treated with lineage-specific factors in vitro (Zuk et al., 2001, 2002). Thus, adipose-derived MSCs (AT-MSCs), like bone marrow MSCs (BM-MSCs), appear to be a very encouraging resource for regenerative cell therapy.
Recently, it has become clear that BM-MSCs, together with AT-MSCs, could escape the immune system in vitro and also possess immunoregulatory properties. Both cell populations have very similar immunophenotypical features, in that they either do not express, or they express negligibly low levels of HLA class II antigens and co-stimulatory molecules, such as CD80 and CD86 (Tse et al., 2003; McIntosh and Bartholomew, 2000; McIntosh et al., 2006). Therefore, they should not activate alloreactive T-cells (McIntosh et al., 2006; Rasmusson et al., 2003; Le Blanc et al., 2003a), which indicates that these cells are not inherently immunogenic. Moreover, both cell types could inhibit T-cell activation and proliferation in mixed lymphocyte cultures (MLCs) (McIntosh et al., 2006; Di Nicola et al., 2002; Le Blanc et al., 2003b) in a dose dependent manner, thus suggesting that they exert an active immunosuppressive effect. In addition, BM-MSCs have been shown to suppress T-cell proliferation in response to nonspecific mitogens or recall antigens (Di Nicola et al., 2002; Krampera et al., 2003; Potian et al., 2003) and to inhibit the development and function of nearly all immunocompetent cells, including NK cells (Sotiropoulou et al., 2006) and monocyte-derived dendritic cells (Zhang et al., 2004; Jiang et al., 2005). Some of these properties have already been successfully clinically exploited for the treatment of disorders such as osteogenesis imperfecta (Horwitz et al., 1999) and acute graft-versus-host disease in humans (Le Blanc et al., 2004) and for the prolonged survival of MHC-mismatched skin grafts after administration in baboons (Bartholomew et al., 2002).
While there is some data about the effects of BM-MSCs on B-cell differentiation and activities, no information is currently available about the influences of AT-MSCs on B-cell functions, which may be significant for their potential therapeutical applications. The aim of this study was to compare the differentiation capacity and immunosuppressive effect on B-cell immunoglobulin production of mesenchymal stem cells from bone marrow or adipose tissue.
2 Materials and methods
Samples of human bone marrow aspirates and adipose tissue were obtained from the same patients (aged 37–81
2.1 Isolation and expansion of human bone marrow-derived and adipose tissue-derived mesenchymal stem cells 2.1.1 Mesenchymal stem cells from bone marrow MSCs were generated from human bone marrow (BM) aspirates (5–10
2.1.1 Mesenchymal stem cells from bone marrow
MSCs were generated from human bone marrow (BM) aspirates (5–10
2.1.2 Mesenchymal stem cells from adipose tissue
AT-MSCs were isolated as described previously (Kern et al., 2006). The fresh samples of adipose tissue (AT) (2–3
2.2 Colony-forming unit fibroblast assay
The colony-forming unit fibroblast assay was performed as described by DiGirolamo et al., (1999). BM-MSCs or AT-MSCs at the 4th passage were plated at 10 cells/cm2 on 34
2.3 Flow cytometric analysis
For immunophenotypic characterization BM-MSCs or AT-MSCs, both at 3rd passage, were trypsinized (0.05% trypsin/1.0
Immunofluorescent analysis was performed on cell-coated cover slips. Briefly, cells were fixed with 4% paraformaldehyde for 10
2.5 SDS-PAGE and Western blotting
BM-MSCs and AT-MSCs, at 3rd passage were harvested with 0.05% trypsin/1.0
2.6 Isolation of peripheral blood mononuclear cells (PBMCs)
The leukocyte concentrate (50
2.7 Osteogenic differentiation
To promote osteogenic differentiation, cells (4th passage) were replated at a density of 1
For mineralized deposits detection, von Kossa staining was performed as described by Cheng et al. (1994). Cells were washed in water, stained with 1% silver nitrate solution for 60
2.8 Adipogenic differentiation
Isolated cells at the 4th passage were seeded at 1
2.9 Immunoglobulin detection assay
PBMCs at a concentration 1.0
2.10 Statistical analysis
Quantitative data are expressed as means
3.1 Early morphological and cell expansion characteristics of the BM-MSCs and AT-MSCs
At the 2nd and 4th day after the initial cultivation of AT and BM cell fractions, respectively, the first single adherent fibroblast-like spindle-shaped cells could be observed between the numerous nonadherent nuclear cells (Fig. 1A). One week later, numerous early colonies of 15–20 fibroblast-like cells appeared in the cultures (Fig. 1B), which expanded (Fig. 1C), and by the 15th day of AT cell culturing an almost homogeneous confluent layer of fibroblast-like cells occupied the whole plastic surface. The respective confluent state was achieved at day 20–22 by BM-MSC cells (Fig. 1D).
Fibroblast-like spindle-shaped cells in the primary culture of human bone marrow derived cells. The morphology and growth confluence of BM-MSCs after (A) 4, (B) 12, (C) 18, and (D) 22
The cultured cells, regardless of their origin, retained their fibroblast-like morphology and grew forming colonies for >8 consecutive passages.
3.2 Colony-forming unit fibroblast activity of the cells isolated from BM and AT
One of the characteristic in vitro features of MSC when seeded at low density is their capacity to generate single-cell derived colonies of adherent cells. The single precursor cells with colony-forming ability are also termed colony-forming unit-fibroblasts (CFU-F) and are usually used as an indicator for mesenchymal progenitor potential. Results from three CFU-F assays showed that when seeded for 2
CFU-F profile of BM-MSCs and AT-MSCs. Left panel: Size (0.5–2.5
3.3 Comparative immunophenotypic characterization of BM-MSCs and AT-MSCs
Cells isolated from both sources were analyzed by fluorescence-activated cell sorting for cell surface antigens that have been previously reported to be determinative for the MSC phenotypic profile. Both types of cells were negative for the hematopoietic markers such as CD34, CD45, CD3, CD19, CD14, CD16, CD56 and HLA-DR (Fig. 3A and B), while they displayed strong expression (>90%) typical of MSC markers like CD73, CD29 and CD90 (Fig. 3A and B) which is in agreement with other reports on BM-MSC and AT-MSC.
Comparative immunophenotypic characterization of BM-MSCs (A) and AT-MSCs (B). Flow cytometry analyses from a representative donor are shown. Both types of cells were negative for CD3, CD19, CD45, CD14, CD16, CD56, CD34 and HLA-DR, but were positive for CD90, CD29 and CD73. Horizontal bars indicate the positive region; the grey lines indicate the isotype-matched monoclonal antibody control staining.
For further characterization of the BM and AT obtained cells, the indirect immunofluorescence revealed positive staining for vimentin and endoglin (CD105) (Fig. 4A) and no reaction for cytokeratin and von Willebrand Factor (data not shown). The expression of vimentin and endoglin from both types of cells was confirmed by Western blot of cell lysates (Fig. 4B).
Expression of CD105 and vimentin in BM-MSCs and AT-MSCs. (A) Positive reaction for both markers was detected by indirect immunofluorescence (magnification, ×400). (B) Western blot analysis of whole-cell lysates confirmed the presence of CD105 and vimentin in BM-MSCs and AT-MSCs.
3.4 Potential for osteogenic/adipogenic differentiation
Osteogenic differentiation of BM-MSCs and AT-MSCs was induced by treatment with osteo-inductive factors as described in Section
In vitro differentiation potential of BM-MSCs (E–H; I) and AT-MSCs (A–D; J). (C, G) Osteogenesis was indicated by calcium deposition visualized by von Kossa reaction (D and H are non-induced controls; magnification, ×100) and (I, J) quantitatively measured by pNPP assay (black bars, induced cells; white bars, non-induced cells; results are shown as mean
Adipogenic differentiation of BM- and AT-derived cells was demonstrated by the formation of triglyceride-containing vacuoles in the cell cytoplasm, visualized by Oil Red O staining (Fig. 5A and E). In contrast, the control cells maintained only in LG-DMEM/10% FCS did not display an adipogenic phenotype (Fig. 5B and F).
3.5 Comparison of the BM-MSC/AT-MSC effects on immunoglobulin production
The immunomodulatory effect of both types of mesenchymal stem cells on B-cell immunoglobulin production was comparatively studied by sandwich-ELISA. PBMCs were incubated without or with isolated BM-MSCs and AT-MSCs at 10:1 ratio in the presence or absence of PWM, and Ig amounts were assessed. As measured by ELISA, in the presence of mesenchymal stem cells, regardless of their source, Ig levels in culture medium were significantly lower with respect to the control of PBMCs treated only with PWM (P
MSC-induced suppression of B-cell immunoglobulin production. PBMCs were incubated for 7
The major results from the comparative analysis of BM-MSC and AT-MSC are schematically summarized in Table 1.
Schematic presentation of the main results from the characterization of BM-MSCs and AT-MSCs
In order to compare the differentiation and immunomodulatory properties of AT-MSCs with those of their well-characterized bone-marrow counterparts (Croft and Przyborski, 2004; Di Nicola et al., 2002; Le Blanc et al., 2003b; Krampera et al., 2003; Corcione et al., 2006), we have isolated adherent cell populations from human bone marrow and adipose tissue and have demonstrated that these two cell types exert similar characteristics according to morphological and immunophenotypical parameters but differ by some functional aspects when assessed in vitro. The immunophenotypical patterns of both cell types determined by flow cytometry, immunofluorescence and Western blot, together with their fibroblast-like morphology and colony-forming capacity, are very similar and correspond to previous studies (Mitchell et al., 2006; Pittenger et al., 1999). Neither AT-MSCs nor BM-MSCs express the hematopoietic markers CD34, CD45, CD3, CD19, CD14, CD16, CD56 or HLA-DR, but they are positive for classical MSC marker proteins such as CD29, CD73, CD90 and CD105. In addition, the cells are also positive for vimentin, a cytoskeleton filament protein that is widely expressed in mesenchymal cells, and negative for cytokeratin (a marker of epithelial differentiation) and von Willebrand Factor (endothelial cell marker). The immunofluorescent microscopy revealed a qualitatively comparable pattern of expression of CD105 and vimentin between BM-MSCs and AT-MSCs.
The main functional difference between the isolated BM-MSCs and AT-MSCs at the very early stages of their in vitro cultivation under the specified culture conditions concerns their growth rate and especially the finding that newly isolated AT-MSCs expanded faster and reached full confluence earlier than do BM-MSCs. Similar observations were made by Kern et al. (2006), who reported higher expansion potential of AT-MSCs compared to BM-MSCs through 4–6th passage.
We have demonstrated a bipotential differentiation capacity for BM-MSCs and AT-MSCs and successfully directed their development toward adipo- and osteo-genic lineages. In response to appropriate stimulation under the same conditions, both types of cells exhibited identical potential for adipogenic differentiation revealed by Oil Red O staining and there was no difference between BM-MSCs and AT-MSCs with regard to the amount of neutral lipid-vacuoles.
In contrast, the osteogenic differentiation of AT-MSCs, as demonstrated by measuring the ALP activity, was statistically less than that of the BM-MSCs. Moreover, the qualitative determination of matrix mineralization by von Kossa staining confirmed the lower effectiveness of AT-MSC osteogenesis under the appropriate conditions when compared with BM-MSCs.
Our results are in agreement with previous studies (Im et al., 2005) concerning BM-MSC versus AT-MSC osteogenesis which also reported less sensitivity of AT-MSCs towards the osteogenic differentiation. Nevertheless, there are authors who demonstrated no statistically significant differences in ALP activity between BM-MSCs and AT-MSCs when undergoing osteogenic differentiation (De Ugarte et al., 2003). All these contradictory data could be due to the different cultivation conditions, experimental approaches or isolation and stimulation protocols used by various researchers. It could be possible that, under optimized conditions, AT-MSCs could enhance their osteogenic differentiation capacity, which is the reason why we could not definitively assume that AT-MSCs are less potent osteoblast precursors than BM-MSCs.
Despite the notable disparity in the effectiveness of osteogenic stimulation between the BM- and AT-derived cells, it is essential that we clearly confirmed their bidirectional differentiation potential. All these findings concerning some phenotypical and functional features of the cultivated BM and AT fibroblast-like cells, together with their high growth potential for 7–8 consecutive passages, strongly suggest their mesenchymal stem cells identity.
Recent studies have shown that BM-MSCs and AT-MSCs share very similar in vitro immunomodulatory activities as they are both unable to induce the response of allogenic T-lymphocytes and both suppress the proliferation and the production of inflammatory cytokines (TNF-α, IFN-γ, and IL-12) of T-cells activated either by mitogens or allogeneic PBMCs (McIntosh et al., 2006; Di Nicola et al., 2002; Le Blanc et al., 2003b; Bartholomew et al., 2002; Puissant et al., 2005). Moreover, these adult stem cell types are both successfully exploited for the in vivo amelioration of GVHD in human (Le Blanc et al., 2004), or in animal models (Yañez et al., 2006). According to these immunomodulating similarities, we aimed to investigate to what extent the similarities between BM-MSCs and AT-MSCs are valid regarding their in vitro impact on B-cell function and, in particular, the immunoglobulin production after mitogen stimulation. We have demonstrated here that after cultivation with BM-MSCs or AT-MSCs, B-cell differentiation to immunoglobulin-producing cells was significantly inhibited at PBMC/BM-MSC (AT-MSC) ratio of 10:1. In this respect, it is highly significant to point out that higher levels of immunosuppression produced by AT-MSCs on PWM activated PBMCs than with their BM counterparts were found. Although these data are the first demonstration of the more powerful AT-MSC versus BM-MSC inhibitory impact on B-cell function, there is another study which previously reported that in some in vitro circumstances AT-MSCs could possess more effective immunosuppressive properties than BM-MSCs. Puissant et al. (2005) observed that the lymphocyte PWM-induced proliferation was more regularly sensitive to inhibition by AT-MSCs than to the inhibition by BM-MSCs.
Our findings, in addition to all other observations that AT-MSCs exhibit in vitro and in vivo immunomodulatory properties similar to or even exceeding those of the BM-MSCs, open new perspectives on the therapeutic use of adipose-tissue derived mesenchymal stem cells to lessen transplant rejection and immune-mediated disorders. Moreover, adipose tissue provides some advantages such as a simple surgical procedure under local anesthesia, easy and repeatable access to large quantities of subcutaneous adipose tissue and uncomplicated isolation procedures that make it the most attractive alternative to bone marrow as a source of autologous mesenchymal stem cells.
This work was supported by Grant no. G-4-01/2005 and Grant no. VU-L-201/06 from the National Science Fund of the Ministry of Education and Science, Sofia, Bulgaria.
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Received 18 June 2007/23 July 2007; accepted 22 December 2007doi:10.1016/j.cellbi.2007.12.007