Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells

Introduction Mesenchymal progenitor cells (MPCs) are multipotent progenitor cells in adult tissues, for example, bone marrow (BM). Current challenges of clinical application of BM-derived MPCs include donor site morbidity and pain as well as low cell yields associated with an age-related decrease in cell number and differentiation potential, underscoring the need to identify alternative sources of MPCs. Recently, MPC sources have diversified; examples include adipose, placenta, umbilicus, trabecular bone, cartilage, and synovial tissue. In the present work, we report the presence of MPCs in human tonsillar tissue. Methods We performed comparative and quantitative analyses of BM-MPCs with a subpopulation of adherent cells isolated from this lymphoid tissue, termed tonsil-derived MPCs (T-MPCs). The expression of surface markers was assessed by fluorescent-activated cell sorting analysis. Differentiation potential of T-MPCs was analyzed histochemically and by reverse transcription-polymerase chain reaction for the expression of lineage-related marker genes. The immunosuppressive properties of MPCs were determined in vitro in mixed lymphocyte reactions. Results Surface epitope analysis revealed that T-MPCs were negative for CD14, CD31, CD34, and CD45 expression and positive for CD29, CD44, CD90, and CD105 expression, a characteristic phenotype of BM-MPCs. Similar to BM-MPCs, T-MPCs could be induced to undergo adipogenic differentiation and, to a lesser extent, osteogenic and chondrogenic differentiation. T-MPCs did not express class II major histocompatibility (MHC) antigens, and in a similar but less pronounced manner compared with BM-MPCs, T-MPCs were immunosuppressive, inhibiting the proliferation of T cells stimulated by allogeneic T cells or by non-specific mitogenic stimuli via an indoleamine 2,3-dioxygenase-dependent mechanism. Conclusion Human palatine T-MPCs represent a new source of progenitor cells, potentially applicable for cell-based therapies.

(page number not for citation purposes) osteoblasts, chondrocytes, adipocytes, and myocytes [1][2][3]. Due to their differentiation capacities, MPCs have emerged as a promising tool for therapeutic applications in tissue engineering and cell and gene therapy. Animal studies have shown that MPC implantation can repair critical bone fracture in a rat model of femoral segmental defect [4] and that, after systemic injection, MPCs localize to the site of experimentally induced fractures [5]. Pilot clinical studies have demonstrated the feasibility of allogeneic BM transplantation in the treatment of osteogenesis imperfecta. BM-derived MPCs (BM-MPCs) engrafted and generated donor-derived osteoblasts that improved the clinical signs associated with the disease and enhanced total body weight [6]. Besides their multilineage potential, MPCs display immunoregulatory properties that have prompted consideration of their use in BM transplantation. Indeed, a recent study reports the successful use of MPCs to treat severe grade IV acute graft-versus-host disease in one patient after allogeneic hematopoietic stem cell transplantation [7]. Although the exact immunosuppressive mechanisms are unknown, the capacity of MPCs to suppress T-cell proliferation stimulated by allogeneic lymphocytes, dendritic cells, and phytohemaglutinin (PHA) is well documented [8]. Mechanisms involving cell contact. [9] as well as soluble factors [10,11] have been proposed, particularly the involvement of interferon-gamma (IFN-γ) via its induction of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the catabolism of tryptophan, an essential amino acid required for protein synthesis and T-cell proliferation [12,13].
At present, BM is considered the most accessible source of adult MPCs. However, BM-MPC derivation has complications, including pain, donor site morbidity, and low cell yields upon harvest. Furthermore, the number of BM-MPCs and their proliferation rate and differentiating potential have been shown to decrease with donor age. [14]. Given that MPCs undergo a decline in their differentiation and expansion capacity with physiological aging, identification of potential sources of MPCs easily accessible from young donors is currently of main interest for cell-based therapy. [15]. The search for alternative sources of MPCs is thus of significant value. To date, MPCs have been isolated from a number of adult tissues, including trabecular bone [16], fat [17,18], synovium. [19,20], skin [21], thymus [22], periodontal ligament. [23] as well as prenatal and perinatal tissues such as umbilical cord blood. [24], umbilical cord [25], and placenta. [26].
This study explores the possibility of identifying and isolating MPCs from human palatine tonsils. Tonsillar epithelium is derived from the second pharyngeal pouch (of endodermal origin) and during fetal development is invaded by lymphoid tissue (of mesodermal origin). Therefore, embryologically, tonsils could be a source of MPCs. Because of the prevalence of tonsillectomy procedure, tonsils are easily accessible, particularly from young donors and, if necessary, tonsillar biopsy can be easily obtained without major complications under local anesthesia. Our results show that MPCs exist in the stroma of palatine tonsils and can be isolated and expanded in culture. These tonsil-derived MPCs (T-MPCs) show multipotent differentiation properties and share similar immunosuppressive characteristics as BM-MPCs in mixed lymphocyte reaction (MLR). The immunosuppressive activity is significant and dose-dependent, though at a lower level than that of BM-MPCs. The difference in immuosuppressive activity correlates with the level of cell surface IFN-γ receptor (IFN-γR) as well as the differential ability of IFN-γ to stimulate IDO activity by T-MPCs compared with BM-MPCs.

T-MPC and BM-MPC isolation and culture
With institutional review board approval (George Washington University, Washington, DC, USA), tonsils were obtained after informed consent from patients (4 to 15 years old) undergoing tonsillectomy as a result of recurrent episodes of acute tonsillitis. The tissue was minced and digested in RPMI medium (Gibco-BRL, now part of Invitrogen Corporation, Carlsbad, CA, USA) containing 210 U/mL collagenase type I (Invitrogen Corporation) and 90 KU/mL DNase (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C. Following filtration through a wire mesh, the cells were washed twice in 20% normal human serum (NHS)-RPMI and once with 10% NHS-RPMI. Mononuclear cells were obtained by Ficoll-Paque (Amersham, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK) density gradient centrifugation of digested tonsil tissue. Cells were plated after 24 to 48 hours in T-150 cm 2 tissue culture flasks (Corning Incorporated, Corning, NY, USA), and non-adherent cells were washed away with expansion medium consisting of Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corporation) with 10% fetal bovine serum (FBS) from selected lots (HyClone, Logan, UT, USA) and antibiotics (50 μg/mL streptomycin and 50 IU/mL penicillin; Invitrogen Corporation).
For BM-MPCs, BM was obtained after informed consent from patients (39 to 58 years old) undergoing lower extremity reconstructive surgery with institutional review board approval (University of Washington, Seattle, WA, USA, and George Washington University) and was processed by direct plating as described previously. [27]. BM aspirates were plated overnight in T-150 cm 2 culture flasks in the same expansion medium as T-MPCs, and adherent cells were obtained similarly. Both T-MPCs and BM-MPCs were culture-expanded in basal medium at 37°C and 5% CO 2 using T-150 Triple Flask (Nunc, Roskilde, Denmark), and medium changes were done twice weekly.
Cell proliferation, limiting dilution assays, and colonyforming unit-fibroblast assays To estimate cell proliferation, cultures of T-MPCs and BM-MPCs plated at 1 × 10 4 cells per square centimeter in 12-well plates in basal medium were analyzed on days 3, 4, 7, 10, 12, and 14, using MTS (methanethiosulfonate) assay according to the protocol of the manufacturer (Promega Corporation, Madison, WI, USA). T-MPCs plated into six-well plates in serial dilutions (1 × 10 7 , 1 × 10 6 , 1 × 10 5 , and 1 × 10 4 cells per well, in triplicate) were cultured in expansion medium for 2 weeks, fixed with 10% formalin, and stained with Giemsa (Sigma-Aldrich), and colonies of fibroblast-like cells were identified and counted based on the methods described by Castro-Malaspina and colleagues. [28]. Colony-forming unit-fibroblast (CFU-F) potential, the average number of cells required to produce one colony, was determined by plating aliquots (1,000 cells per square centimeter) of T-MPCs in expansion medium for 14 days and was analyzed as described previously [29].

Histology and histochemistry
Oil red O staining Three-week adipogenic cultures of MPCs were rinsed twice with PBS, fixed in 4% buffered paraformaldehyde, stained with Oil red O (Sigma-Aldrich) for 5 minutes at room temperature, and counterstained with Harris-hematoxylin solution (Sigma-Aldrich) to visualize lipid droplets.

Alizarin red S staining
MPCs cultured for 3 weeks in osteogenic medium were fixed with 60% isopropyl alcohol and stained for 3 minutes with 2% (wt/vol) Alizarin red S (Rowley Biochemical Inc., Danvers, MA, USA) at pH 4.2 to detect mineralization.

Total RNA isolation and real-time reverse transcriptionpolymerase chain reaction
Total cellular RNA samples extracted from day 21 monolayer and pellet cultures using Trizol Reagent (Invitrogen Corporation) were reverse-transcribed using random hexamers. Realtime polymerase chain reactions were performed using 10 ng of cDNA and SYBR Green mix (Bio-Rad Laboratories, Inc.). Gene-specific primers (forward/reverse) were designed based on GenBank cDNA sequences and are listed in Table  1: (a) adipogenesis genes: lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor-gamma (PPARγ), (b) osteogenesis genes: alkaline phosphatase (ALP) and osteocalcin (OC), and (c) chondrogenesis genes: collagen type II α1 (COL2) and aggrecan (AGN). Specific transcript levels were normalized by comparison to that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression levels were presented as the fold increase over that of GAPDH, using the formula 2 (ΔCt) , where ΔCt = Ct of target gene -Ct of GAPDH.

Primary mixed lymphocyte reaction
Peripheral blood from healthy human donors was collected into heparinized containers (BD Biosciences), and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation. Mouse splenocytes were isolated in 10 mL of RPMI 1640 medium (Invitrogen Corporation) as described previously. [30]. Responder human PBMCs or splenocytes from CD-1 mice and stimulator human PBMCs or splenocytes from A/J mice were resuspended in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 20 mM HEPES, and 50 μM 2-mercaptoethanol (Invitrogen Corporation). Splenocytes were seeded in triplicates at 1 × 10 5 cells/ 100 μL per well in 96-well round-bottom plates (BD Biosciences). PHA was used at 5 μg/mL as a positive control mitogen to induce T-cell proliferation. MPCs (5 × 10 4 cells unless stated otherwise) were added to obtain a final volume of 300 μL. After 3 days of incubation, 1 μCi/well [ 3 H]-thymidine (GE Healthcare) was added overnight and radioactivity incorporation was determined by liquid scintillation counting. All experiments were performed in triplicates and repeated at least twice.
Interferon-gamma assay IFN-γ in culture supernatants was quantified using a commercially available enzyme-linked immunosorbent assay kit (R&D Systems, Inc.) according to the manufacturer's protocol.

Statistics
Data from the proliferation, real-time reverse transcription- Table 1 Reverse transcription-polymerase chain reaction primers for differentiation-specific gene expression analysis

Genes
Primer sequences ( polymerase chain reaction (RT-PCR), and IDO activity assays were analyzed statistically using the Student t test, with statistical significance set at a P value of less than 0.05.

Cell viability, proliferation, and clonogenicity
The cell yield from each tonsil ranged from 1 to 5 × 10 9 , with the majority being non-adherent and likely of hematopoietic origin. After multiple buffer washes and subsequent medium changes, approximately 0.1% to 1% of the isolated cells were found to be adherent. Cell colonies from processed tonsillar specimens began to appear approximately 5 to 10 days after initial plating. Three different cell morphologies were generally observed: (  plasmic lipid droplets were noticeable as early as 1 week of adipogenic induction, as visualized by Oil red O staining (Figure 3a). mRNA expression of LPL and PPARγ was detected by quantitative RT-PCR (Table 1) after 21 days of induction and revealed a similar expression level of the two markers in T-MPCs and BM-MPCs (Figure 3b). Interestingly, expression of adipogenic markers was significantly stronger in higher passages of T-MPCs (P4 and P5) than in lower passages (P2 and P3) (data not shown).

Osteogenesis
Upon osteogenic induction, the morphology of both T-MPCs and BM-MPCs changed from spindle-shaped to flattened and spread. Quantitative RT-PCR analysis showed lower levels of OC and ALP mRNA in T-MPCs compared with BM-MPCs (Figure 3b). Osteoblastic phenotype was also detected based on positive staining for ALP (not shown) and Alizarin red S (Figure 3a).

Chondrogenesis
The chondrogenic potential of BM-MPCs and T-MPCs was evaluated in high-density pellet cultures maintained in serumfree chondrogenic medium. After 3 weeks of culture, matrix sulfated proteoglycan accumulation in chondrogenic cultures was detectable by Alcian blue staining (Figure 3a). Quantitative RT-PCR analysis revealed a significant increase of AGN and COL2 expression in both T-MPCs and BM-MPCs, although the increase in COL2 expression was significantly lower in T-MPCs pellets compared with BM-MPCs ( Figure  3b).

Inhibition of proliferation of alloreactive T cells
The MLR was used to test the immunosuppressive properties of T-MPCs and BM-MPCs. Initially, using human PBMCs from healthy donors as responding cells and PHA as a mitogen, the addition of BM-MPCs and T-MPCs both inhibited the PHAinduced proliferative response of PBMCs (Figure 4a). In the MLR, BM-MPCs and T-MPCs were also seen to suppress the proliferation of responder PBMCs, elicited by allogeneic PBMCs (Figure 4a). In parallel, the level of IFNγ, which reflected T-cell proliferation, showed a decrease in the MLR proportionally in the presence of MPCs isolated from both tissues, but to a significantly lower extent with T-MPCs, supporting a less potent suppressive activity of these cells (Figure 4b).
Proliferation suppression by T-MPCs was dose-dependent and was partially reversed at a T-MPC-to-responder cell ratio of 1:5, suggesting a potent effector mechanism (Figure 4c). Indeed, similar to BM-MPCs, T-MPC suppression of T-cell proliferation was dose-dependent for PHA stimulation as well as in an MLR. However, the immunomodulatory activity of T-MPCs was significantly less pronounced than that of BM-MPCs. The immunosuppressive activities of human BM-MPCs and T-MPCs also crossed the species barrier. When CD-1 murine splenocytes were stimulated with allogeneic A/J splenocytes, dose-dependent inhibition of the proliferative response was seen when xenogeneic human BM-MPCs and T-MPCs were added (Figure 4d).

Involvement of interferon-gamma and indoleamine 2,3dioxygenase in MPC-mediated immunosuppression MPC immunosuppression was recently shown to require IFNγ (produced by T cells and natural killer [NK] cells) to stimulate
IDO production by MPCs, which in turn inhibits the proliferation of activated T or NK cells. [13]. Also, treatment with neutralizing anti-IFN-γR antibody completely abrogated the immunosuppressive effect of MPCs. Analysis of BM-MPCs and T-MPCs showed that both cell types expressed IFN-γR1, with a substantially higher level in the former (Figure 5a). Incubation with IFN-γ resulted in induction of IDO activity in both cell populations, with a lower level in T-MPCs (Figure 5b). Furthermore, the IFN-γ-induced IDO activity was completely suppressed by neutralizing anti-IFN-γR1 antibody. These findings thus strongly suggested that the immunsuppressive activities of both BM-MPCs and T-MPCs were mediated via IDO activity, with BM-MPCs being more active. This differential inhibitory ability correlated with a reduced capacity of T-MPCs to decrease IFN-γ secretion as well as to induce their IDO activity as compared with BM-MPCs (Figure 5b).

Discussion
The purpose of this study was twofold: (a) to assess the existence of MPCs in human palatine tonsils and to characterize their phenotype, and (b) to determine and compare the differentiation potential and immunomodulatory activity of these cells (T-MPCs) to those of the well-characterized MPCs isolated from BM (BM-MPCs). The results showed that human palatine tonsils contained a multipotent MPC population. By means of standard procedure, T-MPCs can be successfully isolated and expanded in vitro. The initial cell population (post tonsil digest) was contaminated with non-fibroblastoid tissue culture adherent cell types; specifically, monocytes remained attached to the polystyrene flasks even after extensive trypsinization, while epitheloid cells did not survive under the expansion medium conditions used to grow BM-MPCs. Only the fibroblastoid cell population remained after two passages. The proliferation profile of T-MPCs in our study was significantly different from that of BM-MPCs, with an average population doubling time of 37 hours compared with 58 hours for BM-MPCs. This discrepancy between T-MPCs and BM-MPCs observed in this study is probably in part age-related as it is well documented that BM-MPCs from older donors have a slower proliferation rate at the initial passage up to cell senescence [31,32]. Since the BM-MPCs used here are from old donors whereas the T-MPCs are derived from children, our observation is thus consistent with the previous age-related observations, although intrinsic differences between BM-MPCs and T-MPCs cannot be ruled out.
The CD surface epitope profile of T-MPCs was characterized by immunofluorescence and flow cytometry. Confocal microscopy revealed that both BM-MPCs and T-MPCs expressed CD29, CD44, CD90, and CD105, but hematopoietic surface markers, including CD14, CD34, and CD45, were absent.  [33] found no difference in osteogenic and adipogenic differentiation capacity between MPCs from young and old donors. However, an age-related loss of both chondrogenic and osteogenic potential of MPCs has also been reported [31,34]. In our study, we did not observe age-related greater differentiation potential expected for T-MPCs from young donors. We speculate that the lower level of expression of particular markers of differentiation could be the result of prior in vivo exposure of T-MPCs to high concentrations of inflammatory cytokines, which are characteristically present in this type of tissue source [35]. In this study, all T-MPCs were obtained from patients undergoing tonsillectomy as a result of chronic tonsillitis. Chronic bacterial infection in the tonsils results in the production of local antibodies, a shift of B-and T-cell ratios, and production of large amounts of pro-inflammatory cytokines, including tumor necrosis factoralpha (TNF-α). Recently, the addition of TNF-α to human MPCs was shown to suppress the osteogenic mediuminduced morphological change from spindle to cuboidal shape and also ALP enhancement. [36]. To address this issue, we are currently analyzing the levels of pro-inflammatory cytokine in T-MPC culture medium. In support of our theory that exposure to pro-inflammatory cytokines suppressed the differentiation capacity of T-MPCs, passage 5 T-MPCs showed significantly enhanced differentiation potential for all three lineages when compared with passage 2 cells of the same patients; this characteristic is contrary to what is observed with BM-MPCs (that is, reduced differentiation capacity is seen with increasing passage number). We speculate that increased passaging not only eliminates tissue-plastic adherent inflammatory cells (monocytes), but also aids in reducing pro-inflammatory cytokine production by cells outside their original environment that might suppress differentiation.
Recently, MPCs have been shown to display immunosuppressive properties both in vitro and in vivo [10,30,37,38]. MPC inhibition of T-cell proliferation stimulated by allogeneic T cells or non-specific mitogenic stimuli [30,37] affects the expression of activation markers, antigen-specific proliferation (both for naive and memory T cells), cytotoxic T-lymphocyte formation, IFN-γ production by Th1 cells, and interleukin-4 production by Th2 cells. [39,40]. The ability to decrease IFN-γ production, characteristic of the potent suppressive effect of MPCs on T-cell proliferation, is present but less pronounced for T-MPCs compared with BM-MPCs. This finding corroborates with the relatively reduced suppression of IFN-γ, a measure of the proliferative activity of the T-cell population in the assay in the presence of T-MPCs versus BM-MPCs. Interestingly, it was recently reported that IFN-γ level affects MPC function; that is, the dual roles of MPCs as antigen-presenting cells or as immune-suppressor cells depend on IFN-γ levels. Chan and colleagues [41] showed that the antigen-presenting characteristic of MPCs occurs within a narrow window of IFNγ level (10 U/mL), whereas at 100 U/mL of IFN-γ, MPCs are immunosuppressive. Though speculative, our observations are consistent with the reported low level of IFN-γ (25.6 ± 7.9 IU/ mL; P < 0.05) produced by tonsillar mononuclear cells compared with the level recorded in the BM sera (41 ± 23 IU/mL; P < 0.001), which could explain, in part, the different immunosuppressive characteristics between BM-MPCs and T-MPCs [42][43][44].
While the immunosuppressive mechanisms of MPCs remain to be clarified, mediation by soluble factors, such as IFN-γ, is strongly suggested. First, it was reported that MPCs immunosuppressed peripheral blood CRTH2 -CD4 + T cells that produce IFN-γ, but did not affect the proliferation of purified CRTH2 + CD4 + T cells unable to produce IFN-γ [13,45]. Furthermore, fetal MPCs, normally non-immunosuppressive in an MLR, when exposed to IFN-γ for 7 days, inhibit lymphocyte proliferation at a magnitude similar to that seen with adult MPCs. [38,46,47]. IFN-γ induction of the suppressive effect of MPCs on cell proliferation has been suggested to be related in part to the enhancement of IDO activity [12,13]. IFN-γ/ receptor binding leads to subsequent endocytosis and IFN-γ nuclear localization sequence (NLS)-guided binding to the IFN-γ-activated sequence (GAS) response element in the promoter region of IFN-γ-activated genes, such as IDO. [48]. This pathway involving IFN-γ, IFN-γR, and IDO is supported by a study showing complete abrogation of the suppressive potential of MPCs and the ability of IFN-γ to stimulate IDO activity upon treatment with a neutralizing antibody to IFN-γR. [13]. Furthermore, the response to IFN-γ is related and proportional to the level of its receptor; that is, increased IFN-γR expression results in increased IFN-γ signaling and enhanced IDO gene activation. [49]. Our findings are thus consistent with these reports in that the less pronounced immunosuppressive activity of T-MPCs compared with BM-MPCs is associated with a significant fourfold lower expression of IFN-γR1. Also, treatment with neutralizing antibody to IFN-γR1 completely blocked the IFN-γ-stimulated IDO activity in BM-MPCs and T-MPCs. This study suggests, for the first time, a correlation between IFN-γR1 expression level in MPCs from two different tissue sources and their immunosuppressive potency.
The secondary lymphoid organs -lymph nodes, spleen, tonsils, and Peyer's patches -are the sites where immune responses against microbes or antigens are initiated. B lymphocytes that continuously recirculate through the blood and secondary lymphoid organs to encounter antigens or that specifically recognize and respond to antigens located in the tonsils become activated and undergo clonal expansion and somatic hypermutation, leading to differentiation into memory and plasma cells. [50]. The identification of MPCs in tonsils raises interesting questions about their immunosuppressive functions and their effects on B-cell biology in this lymphoid organ. Few studies have addressed the effects of BM-MPCs on B-lymphocyte functions. It has been reported that MPCs inhibit B-lymphocyte proliferation [51][52][53] and that this activity requires soluble factors [54] such as IFN-γ. [13]. It is noteworthy that B-cell differentiation requires a close association with stromal cells [55,56], and MPCs have been recently shown to give rise to a fully functional population of B-cell supportive fibroblastic reticular cells (FRCs) [57], found associated with the follicular dendritic cells (FDCs) within secondary lymphoid organs, where they play a key role in the initiation and maintenance of efficient immune response. FDCs have been suggested to differentiate from stromal precursors of mesenchymal origin upon interaction with lymphotoxin α1β2 produced by activated B cells. [58]. BM-MPCs have also been shown to acquire a complete FRC phenotype in response to a combination of TNF-α and lymphotoxin-α1β2 [57]. The importance of environmental influences, particularly those related to inflammation, on the immunosuppressive properties of MPCs has been previously stated. [59]. Indeed, the controversy concerning modulation of B-cell functions by MPCs may reflect the differentiation state of MPCs (that is, into FRCs or FDCs) and/or may be the result of microenvironmental signals. Thus, MPC effects on the immune system are modulated not only by cell-to-cell interactions, but also by environmental factors that shape their phenotype and their functions. Accordingly, the functional differences between BM-MPCs and T-MPCs, notably their immunosuppressive potency, most likely reflect their previous environment in situ.
MPCs appear to bypass immune rejection, thus making them attractive candidates for allogeneic transplantation. However, in vivo, the behavior of the transplanted MPCs is expected to be influenced by their exposure to immune cells and mediators. Another challenging consideration for the clinical use of MPCs and notably T-MPCs, which are isolated from tonsil tissue frequently infected and infused with inflammatory mediators, is to predict how the host tissues will affect the properties of the MPCs. Future studies are necessary to better understand the impact of the inflammatory microenvironment on MPCs for their application in transplantation protocols.