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TISSUE-SPECIFIC STEM CELLS |
Department of Cell Biology, Beijing Institute of Basic Medical Sciences, Beijing, China
Key Words. Mesenchymal progenitor cells • Mouse • Compact bone • Immunosuppression
Correspondence: Ning Mao, M.D., Department of Cell Biology, Beijing Institute of Basic Medical Sciences, Beijing 100850, China. Telephone: 8610-66931318; Fax: 8610-68213039; e-mail: maoning{at}nic.bmi.ac.cn
Received May 17, 2005;
accepted for publication November 13, 2005.
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ABSTRACT |
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H2d), suggestive of the immunosuppressive effects in vivo. The findings demonstrate that muMPCs obtained with this simple protocol are similar in property to their marrow counterparts, and thus, the protocol described here could be used for further investigations in mouse physiological and pathological models.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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MPC culture was first introduced by Friedenstein et al. and modified by others, using the physical propensity of MPCs to adhere to plastic flasks [9–11]. This methodology has been widely employed for bone marrow MPC propagation from human beings [1], rats [12], pigs [13], rabbits [14], cats [15], and dogs [16]. However, the standard method of plastic adherence has failed to yield relatively homogenous MPCs from murine bone marrow, mainly because of hematopoietic cell contamination [17].
Several groups have reported their independent work on murine MPC (muMPC) purification from bone marrow by magnetic selection [18], retroviral infection [19], or unique culture systems [17, 20–22]. In our own previous work, muMPC culture was developed by addition of bone fragments with mouse bone marrow cells in the presence of basic fibroblast growth factor (basic FGF) and bone fragment-conditioned medium [23]. Furthermore, mouse colony-forming unit-fibroblast (CFU-F), a strong in vitro correlative of MPCs, has been successfully enriched by Simmons et al., who clearly identify the femoral bone itself as a richer source of muMPCs than the marrow plug within it [24, 25]. Based on these findings and those from other investigators demonstrating that MPCs from human compact bones are similar in property to their counterpart in bone marrow [26] and that human bone-derived MPC culture can be developed by adherent culture of either enzyme-treated bone fragments or the released cells [27–31], we postulate that murine counterparts are also able to be purified with these simple methods. To test this hypothesis, mouse bones were digested with collagenase and the released cells or the remaining bone fragments were propagated as previously reported [23, 26]. Interestingly, muMPC culture could be developed only by inoculation of digested bone chips other than the suspending cells. These putative muMPCs were homogenous in surface epitopes, possessed the classic tridifferentiation potential, inhibited in vitro splenocyte proliferation, and maintained hematopoietic cells in long-term culture as effectively as mouse primary stromal cells. Furthermore, muMPC delivery could prolong the survival time of skin grafts transplanted across the MHC barrier (H2b H2d). These findings add more proof that compact bones are an alternative source of muMPCs, and the protocol described in the present study provides novel cues for further investigations in mouse pathological models.
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MATERIALS AND METHODS |
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Culture and Expansion of muMPCs
The femurs and tibiae were collected from 2- to 3-week-old C57BL/6 female mice. The epiphyses were removed, bone marrow was flushed out, and the bone cavities were washed thoroughly by drawing and expelling with a syringe. The compact bones were excised into chips of about 1 mm3 into plastic culture dishes, suspended in 
-modified minimal essential medium (-MEM) containing 10% fetal bovine serum (FBS) from selected lots in the presence of 1 mg/ml of collagenase II (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and incubated for 2 hours at 37°C with shaking at a speed of 200 rpm. The released cells were aspirated, and the bone fragments were washed three times with -MEM, followed by incubation in -MEM containing 10% selected FBS at 37°C in 5% CO2. Medium was changed every 3–4 days. The adherent cells were harvested by trypsin digestion and passaged at an initial density of 1,500 cells per cm2 thereafter. The cells at the third passage were used for the following experiments except otherwise described. Also, the released cells after digestion were propagated separately in the same culture medium in some experiments.
Flow Cytometry
Adherent cells at the indicated passages were retrieved by trypsin digestion and aliquots of 1 x 106 cells were labeled with fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies against mouse CD11b, CD29, CD31, CD34, CD44, CD45, CD105, and stem cell antigen-1 (Sca-1) (all products from eBio-Science, San Diego, http://www.ebioscience.com) for 30 minutes at room temperature in the dark. After washing twice, events were acquired by FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), and data analysis was conducted with WinMDI 2.9 software (Joseph Trotter, The Scripps Institute, La Jolla, CA) after gating for the designated population.
Multipotent Differentiation Assay
Osteogenic differentiation was induced by osteoinductive medium as previously described in our laboratory [23]. A histochemical kit (Sigma) was used to assess alkaline phosphatase (ALP) activity according to the manufacturer’s protocol.
An aggregate culture system was used according to the protocol described for chondrogenic differentiation of marrow muMPCs [22]. Briefly, aliquots of 2 x 105 cells were pelleted and cultured in serum-free inductive medium containing 10ng/ml recombinant human transforming growth factor ß-3 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and 500 ng/ml recombinant human bone morphogenetic protein-6 (BMP-6) (R&D Systems). Pellet cultures were maintained for 21 days, with medium changes every 3–4 days. The pellets were fixed in paraformaldehyde, embedded in paraffin, and sectioned for toluidine blue staining.
To assess adipogenic capacity, cells were propagated to confluence and maintained in -MEM containing 10% FBS, 10–7 M dexamethasone, and 10 µg/ml insulin (Sigma-Aldrich). Intracellular lipid droplets were generally evident after 5 days, and lipid accumulation was further identified by in situ oil red-O staining as routinely described [23, 32].
Maintenance of CFU-F Formation and Multipotential Differentiation
Aliquots (100 cells per well) of muMPCs at the indicated passages were seeded in 12 replicates into a 24-well tissue culture plate and were maintained in culture for 12 days. All visible colonies larger than 5 mm in diameter were counted after Giemsa staining. For evaluating the multiple differentiation capacities, trilineage-associated transcripts in cells after dexamethasone induction were detected by reverse transcription-polymerase chain reaction (RT-PCR), with that of hypoxanthine phosphoribosyl-transferase (HPRT) as a control for mRNA quality.
RT-PCR
Cells at different passages were detached and plated into plastic flasks at a density of 1,000 cells per cm2. Culture was maintained for 2 weeks in -MEM containing 10% FBS in the presence of 10–8 M dexamethasone for inducing nonspecific differentiation as previously described [33]. After 2 weeks, total cellular RNA was extracted using TRIZOL reagent (Gibco, Grand Island, NY, http://www.invitrogen.com) and mRNA was reverse-transcribed with 15-mer poly-(d)-T (Promega; Madison, WI, http://www.promega.com) for cDNA amplification. Primers used were as those reported previously [20]. PCR products were separated on a 2% agarose gel containing 1 µg/ml ethidium bromide and visualized under UV light.
Long-Term Culture of Hematopoietic Cells on muMPC or Stromal Feeders
Stromal feeder layers for supporting maintenance of long-term culture-initiating cell (LTC-IC) were established as previously described with some modifications [34]. Briefly, mononucleated cells from adult female C57/BL bone marrow were separated by Ficoll density gradient centrifugation and were seeded in LTC medium (Myelocult M5300; Stem Cell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com) containing 10–6 M hydrocortisone. Nonadherent cells were removed by thorough washing after 24 hours, and culture was maintained until an adherent layer formed. The adherent cells were retrieved by trypsin/EDTA digestion and seeded into a gelatin-precoated 96-well culture plate at 2 x 104 cells per well. Cells were irradiated (20 Gy) to eliminate hematopoietic activity without affecting the ability of the stroma to support hematopoiesis. muMPCs were also collected, plated, and irradiated as described above. Lineage-negative (Lin–) cells from adult C57BL/6 bone marrow were prepared by labeling mononuclear cells with a cocktail of antibodies against CD3, CD4, CD8, Ter-119, Mac-1, and Gr-1 (eBioScience) and subsequently reacted with immunomagnetic beads (DynalBiotech; Brown Deer, WI, http://www.dynalbiotech.com) and separated by a magnetic particle concentrator (Dynal MPC-S) per the manufacturer’s instructions. In a typical experiment, about 10% of marrow mononuclear cells were harvested as Lin– cells. Graded number of enriched Lin– cells (6,000, 3,000, 1,500, 750, and 375 cells per well) were overlaid in 12 replicates per dilution onto the adherent cell layers and maintained in LTC medium supplemented with 10–6 M hydrocortisone in humidified 5% CO2 atmosphere at 33°C. Culture was continued for 5 weeks with weekly changes of half of the medium, followed by cell collecting by trypsinization. All the cells from an individual well were plated in MethoCult 3434 (Stem Cell Technologies) into one dish for determination of colony-forming cells (CFCs). Seven days later, the dishes were scored as positive or negative for the presence or absence of CFCs. Frequency of LTC-IC was calculated, and the data were further statistically analyzed with the L-Calc software (Stem Cell Technologies).
In Vitro Splenocyte Proliferation Assays
Graded numbers of muMPCs (104, 5 x 103, 103, and 0 per well) were seeded in a gelatin-precoated 96-well culture plate and maintained at 37°C for 6 hours before 20 Gy 
-irradiation. Mononuclear spleen cells from C57BL/6 mice were prepared by Ficoll density gradient centrifugation, and aliquots of 2 x 105 cells were added into each well and cultivated in the presence of 20 µg/ml Con A or 2 x 104 irradiated splenocytes from BALB/c. The total volume was 200 µl per well. In mitogen stimulation experiments, cells were maintained at 37°C for 72 hours and pulsed with 5 µCi of 3H-thymidine deoxyribonucleoside/ml for an additional 8-hour culture. In mixed lymphocyte reaction experiments, 3H-TdR (1 µCi/well) was pulsed for 18 hours after a 5-day culture. Cells were harvested onto glass fiber filters, and radioactivity was measured on a Wallac Microbeta Trilux 1450-02P (PerkinElmer, Wellesley, MA, http://perkinelmer.com). The results are expressed as mean cpm for at least triplicate cultures.
Tail Skin Grafting
muMPCs (1 x 106) from female C57/BL were injected intravenously into BALB/c mice, and sex-matched skin grafting was conducted as described elsewhere [35]. Briefly, full-thickness tail skin (around 0.5 x 0.5 cm2) from C57/BL (allografting) or BALB/c (syngeneic grafting) was transferred to the sides of the recipient’s tail, from which an equivalent amount of skin had been removed. Each mouse received two allografts and one syngeneic graft. The grafts were protected with a glass tube and monitored daily by visual inspection. A graft was considered rejected when more than 90% was necrotic, and grafts transferred to the tail on which the syngeneic skin had not survived were screened out for further analysis. On day 10, some of the skin grafts were removed for histological examination after H&E staining.
Statistical Analysis
Data were presented as the mean ± standard deviation and analyzed with Student’s t test. Data from skin grafting were analyzed with the log-rank test. A p value less than .05 was considered statistically significant.
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RESULTS |
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), although CD45 negative cells gradually overgrew with passaging. Third, most of the adherent cells presented ALP activity, as evaluated by histochemical staining (data not shown). These results were consistent in several independent experiments.
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). Also, some small colonies were evident apart from the fragments after 72 hours (Fig. 2C), at which time, culture medium was changed after removal of nonadherent cells and tissue debris. Typically, cells reached –80% of confluence within 5 days (Fig. 2D). The cells proliferated and grew rapidly and could be serially passaged twice weekly at a split ratio of 1:4 or 1:3 before the 10th passage; however, some of the cells became flattened and polygonal with further subculturing, and cell senescence was usually obvious in culture at passage 15.
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, more than 90% of the cells expressed Sca-1 (a murine hematopoietic and mesenchymal stem/progenitor cell marker), CD29 (ß1-integrin), and CD44 (receptor for hyaluronate and osteopontin). Meanwhile, they were homogenously negative for CD34 (non-specific marker for progenitors of mouse hematopoietic, endothelial, and mesenchymal lineages), CD45 (pan-hematopoietic marker), CD11b (monocyte marker), and CD31 (endothelial cell marker), demonstrating that the culture was devoid of hematopoietic and endothelial cell lineages. However, cells seemed heterogeneous in CD105 (endoglin) expression in that the positive proportion of the passage 3 cells was around 70%.
To address whether antigenic profile of these putative muMPCs varied with increasing population doubling, cells at different passages were collected for flow cytometric analysis. As shown in Figure 1, the proportion of CD105+ cells decreased progressively, and no CD105 expression was evident from the 11th passage. It is also of interest that reduction in CD29 and increase in CD34 expression was observed on cells at late passages.
Multilineage Differentiation
The commonly used standard for identifying MPCs is their inherent capacity to differente into osteoblasts, chondroblasts, and adipoblasts under the appropriate conditions [1]. To test whether the cells met these criteria, the passage cells were maintained in matched inductive medium, and cultures in muMPC medium served as controls. Although the cells revealed specific differentiation into osteocytes and adipocytes after induction as assessed by ALP and oil red-O staining, none of the controls displayed characteristics of differentiative phenotypes (Fig. 3A–3D). In complement to the results of histochemical analysis, the cells after induction exhibited mRNA expression of osteocalcin (OCN) and peroxisome proliferator-activated receptor 2 (PPAR2) (Fig. 4), which are molecular hallmarks of differentiated osteoblasts and adipocyte, respectively. On the other hand, little evidence of proteoglycan secretion was observed in pellet culture for chondrogenic induction (data not shown), which is consistent with the previous findings in marrow MPCs from BALB/c and C57/BL mice [22]. Moreover, transcripts for collagen II were detectable in dexamethasone-induced cells, as assessed by sensitive RT-PCR, and similar constitutive expression of HPRT mRNA was observed in treated and untreated cells (Fig. 4). Therefore, the cells prepared with this protocol are designated muMPCs herein.
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, the expressions of OCN, PPAR2, and COL II were evident in differentiated cells at passages 3, 5, 7, and 9, but not passages 11 and 13, at which the positivity was exclusively observed for OCN. It is noteworthy that OCN mRNA was also detectable in control cells at passage 11 and 13, indicating of spontaneous differentiation along the osteocytic lineage.
Maintenance of CFU-F Formation
To observe whether the proliferation features could be sustained with serial passaging, CFU-F frequencies of the cultured cells at passage 1, 3, 5, 7, 9, 11, and 13 were evaluated. As indicated in Fig. 5, the CFU-F frequency remained relatively constant in culture before passage 11, around 6% (p = .3515, .8582, .7897, .4975, and .1653 for P3, P5, P7, P9, and P11, respectively, compared with that of P1). However, it decreased significantly at passage 13 (around 2%) (p < .0001 compared with that of P1), and CFU-F could not be detected from cells at passage 15.
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muMPCs Inhibited In Vitro Lymphocyte Proliferation Triggered by Con A or Allogeneic Cellular Stimuli
It has been well documented that human or murine MPCs suppress in vitro T cell activation stimulated by nonspecific mitogens or allogeneic lymphocytes [4–6, 23]. To observe whether muMPCs defined in this study displayed similar features, cells of graded numbers were cocultured with splenocytes from the same strain (C57BL/6) in the presence of Con A. Consistent with previous reports, muMPCs inhibited Con A-stimulated splenocyte proliferation in an MPC dose-dependent manner (Fig. 6A). The proliferation activity was completely restrained if the MPC to splenocyte ratio exceeded 1:40, although the majority of the splenocytes in culture were viable, as evaluated by trypan blue exclusion test (data not shown). Further, muMPCs suppressed lymphocyte activation by allogeneic cellular stimuli in one-way mixed lymphocyte reaction, and likewise, the suppressive effect exhibited itself in a dose-dependent way (Fig. 6B).
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BALB/c) with or without muMPC pretransfusion. The results showed that muMPC delivery gave rise to a significant survival prolongation of allogeneic skin grafts (10.13 ± 1.51 vs. 11.95 ± 2.92 days, n = 26 and 20, respectively; p = .029) (Fig. 7, left). Histological examination on day 10 showed intact epidermis in grafts from muMPC-treated mice, although obvious leukocyte infiltration was evident in the subcutaneous layer, further proving the inhibitory activities that muMPCs exert on the ongoing immune response in vivo.
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DISCUSSION |
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In fact, the presence of mesenchymal stem or stem-like cells in murine compact bone has been previously reported by Short et al., who enriched muMPCs successfully from enzyme-released cells by immunodepletion of Lin– hematopoietic cells and further FACS selection of Sca-1+ cells [25]. Also, a relatively simple method of isolating human bone-derived MPCs by inoculation of enzyme-released cells for developing an adherent culture has been well described [26]. To test the postulate that MPCs residing in mouse compact bones could also be purified with this simple methodology, the suspended cells from murine long bone fragments after collagenase digestion were cultured in our initial experiments. Although most of the adherent cells were fibroblastoid in morphology, the presence of CD45 positive cells was evident in culture as evaluated by flow cytometry, denoting the presence of hematopoietic cell contamination. This finding is not surprising, as hematopoietic cells reside along the bone surface, and enzyme digestion could release them with other cell types from the densely structural arrangement. The proliferation and/or differentiation of the contaminated hematopoietic cells could be further supported by osteoblasts and/or muMPCs in culture [38], yielding a heterogeneous cell population in adherence. Hence, it seems impractical to isolate and culture expand pure muMPCs by cultivation of the released cells because they were not homogenous per se, and some of them, mainly osteoblasts in the cell suspension, possessed the propensity to adhere to plastic flasks. The high-level ALP expression supports this possibility and indicates that some of them may represent preferably as osteoblasts or mesenchymal progenitors at hierarchically late stage (data not shown).
In the present study, therefore, muMPC culture was initiated by inoculation of the remaining bone fragments after digestion as described for purification of human bone-derived MPCs [31]. In contrast, an adherent cell layer yielded within several days, which seemed much earlier than was observed in human MPC culture [31]. This cell population comprises morphologically homogenous fibroblast-like cells, and the population doublings could reach at least 30x with successive subculture. Flow cytometric analysis showed that these cells were positive for CD29, CD44, Sca-1, and CD105 but negative for hematopoietic and endothelial cell markers, including CD11b, CD45, and CD31, which is consistent with results reported in the literature for their counterparts from human and murine bone marrow and is thus expected of mesenchymal progenitors. Furthermore, the capacity of these cells to differentiate along osteogenic, chondrogenic, and adipogenic pathways in response to classic stimuli was identified. The cells underwent osteogenic differentiation as verified by cell ALP positivity and expression of osteocalcin specific for differentiated osteoblasts. In vitro chondrogenesis was revealed by the expression of collagen type II gene, although cells could secrete little proteoglycan in pellet culture system. The presence of intracellular lipid droplets and PPAR- expression proved the differentiation competence into adipocytic lineage. The results here are in agreement with the routinely accepted standard for MPC identification. Therefore, it would be reasonable to conclude that these cells arising from the propagation of digested bone fragments enable themselves to be defined as bona fide muMPCs.
It is noteworthy, however, that muMPCs maintained in the defined medium at increasing passages progressively lost their multiple differentiation potentials, accompanied by a decrease in CFU-F contents. Cells at passages 11 and 13 could not differentiate along chondroblast and adipoblast lineages; at these points, however, OCN mRAN transcripts could be detected both in control and dexamethasone-treated cells. This phenomenon can also be observed in the culture expansion of human marrow MPCs. The differentiation potential for chondrogenesis, adipogenesis, and, subsequently, osteogenesis disappears with increasing cell doubling [2]. These findings indicate the need for further optimization of culture systems for the maintenance of multipotentials during extensive ex vivo expansion by means of, perhaps, addition of growth factors such as FGF-2, platelet-derived growth factor BB, and epidermal growth factor as observed in marrow muMPCs [17]. Yet further investigations might be needed to clarify the effects of growth factors on the proliferation and differentiation of MPCs. Evidence in a recent study has shown that a supplement of FGF-2 in human MPC culture favors their osteogenic [39, 40] or chondrogenic potential [41].
Furthermore, we have found phenotypic changes of muMPCs with successive subcultures, in accordance with the progressive loss of multiple potentials. The expression of CD105, a vascular-specific TGF-ß coreceptor and a molecular indicator for MPCs and long-term hematopoiesis reconstructing cells, decreases dramatically, whereas that of CD34 increases progressively, and CD29 expression is evident at passage 13. Interestingly, Sca-1, a surface marker for mesenchymal or hematopoietic stem/progenitor cells, is highly expressed as marrow MPCs from the same mouse strain [22] and remains unchanged with passaging. Previous knockout studies have demonstrated that CD105 is actively involved in angiogenesis [42] and that Sca-1 plays a critical role in the self-renewal of stem/progenitor cells from both hematopoietic and mesenchymal lineages [43, 44]. Meanwhile, CD34 expression has been shown on human [45] and murine [22] marrow MPCs, which is observed on bone-derived muMPCs defined in this study exclusively at late passages. Interestingly, both marrow [22] and bone-derived muMPCs from the same strain (C57/BL) seem not display in vitro chondrogenic potential in aggregate cultures in spite of the discrepancy in CD34 expression. Hence, further detailed investigations might be needed to clarify the exact association between phenotypic characteristics and differentiation potential of muMPCs.
Accumulating data have proven that human marrow or placenta-derived MPCs support in vitro hematopoiesis by means of secretion of cytokines and direct contact with hematopoietic progenitors, in that MPCs enhance ex vivo expansion of CD34+ cells in the presence of cytokine cocktail and sustain LTC-IC for 5 weeks in the absence of additive cytokines [7, 8, 36, 37]. In this study, we have shown that feeder layers of muMPCs maintain week 5 LTC-IC as effectively as those of primary stromal cells. This finding denotes that muMPCs might be a crucial cellular component for hematopoietic niches within compact bone and play an active role in the control and regulation of proliferation and differentiation of hematopoietic stem/progenitor cells in this unique niche [38, 46].
Here, we also demonstrate that compact bone-derived muMPCs suppress in vitro lymphocyte proliferation elicited by mitogen or allogeneic splenocyte, which is consistent with the previous reports on murine marrow MPCs [23]. In an attempt to further expand our knowledge about whether bone muMPCs exert similar action in vivo, we employed a skin grafting model across MHC disparity to evaluate the regulatory effects of muMPC transfer on immune alloreactivity. The results showed that muMPCs significantly prolong the survival time of skin grafts between mice across strains (C57BL/6BALB/c), which is consistent with the results in a baboon model [47] and suggestive of the suppressive effect of muMPCs on the ongoing immune process in vivo. These findings provide further support for the clinical use of MPCs in settings such as the prevention of graft versus host disease in allogeneic bone marrow transplantation.
Taken together, our data here demonstrate that muMPCs share similar phenotypic and functional properties with their marrow counterparts, including plastic adherence, fibroblast-like morphology, phenotypic characteristics, tridifferentiation potential in appropriate conditions, hematopoietic support, and immune suppression both in vitro and in vivo. This study provides new clues for further investigation on mesenchymal progenitors in murine physiological and pathological models.
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ACKNOWLEDGMENTS |
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DISCLOSURES |
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REFERENCES |
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