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EMBRYONIC STEM CELLS |
aUpres-Ea3852;
bInserm-Ea3855, Ifr135, Université François Rabelais de Tours and Chru de Tours, Tours, France
Key Words. Stem cells • Mobilization • Blood • Hypoxia • Hematopoiesis
Correspondence: Jorge Domenech, M.D., Ph.D., Laboratoire d'Hématopoïèse, Inserm-ESPRI-EA3855, Faculté de Médecine, 10 Boulevard Tonnellé, 37 032 Tours Cedex, France. Telephone: 33-247-47-47-21; Fax: 33-247-47-69-34; e-mail: domenech{at}med.univ-tours.fr
Received on March 21, 2006;
accepted for publication on May 6, 2006.
First published online in STEM CELLS EXPRESS June 15, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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One of the properties of stem cells is their capacity to home after infusion to the appropriate microenvironment(s) [12, 13]. Certain stem cells are able to exit their production site, circulating into blood before reseeding their target tissues. In the case of hematopoietic stem cells (HSCs), such circulation is essential during development since they migrate from one site to the next (embryonic aorta-gonad-mesonephros region, fetal liver, adult bone marrow). In the adult, HSCs can be mobilized using specific cytokines such as granulocyte colony-stimulating factor (G-CSF). For MSCs, the homing sites and their circulation into peripheral blood (PB) remain a matter of debate. It has been shown that bone marrow, bone, and spleen are sites of engraftment [9, 14, 15]; however, MSCs are found after infusion in multiple tissues, leading to the hypothesis that they can home into, and adjust their differentiation pathway to, diverse tissue microenvironments [16]. Although the existence of circulating MSCs has been proven in fetal and neonatal blood [17–21], their isolation in adult PB depends on the investigators, with reported successes [22–24] and failures [25, 26], which may be related to the low frequency of such cells at steady state. The cogent evidence that MSCs circulate into PB and can be mobilized into the bloodstream using appropriate agents such as cytokines would be of great therapeutic interest since it would allow the collection of MSCs from PB and the eventual manipulation of their subsequent homing to injured tissues.
In this study, we found that MSCs are regularly observed in the circulating blood of rats and showed, to our knowledge for the first time, that the circulating pool is consistently and dramatically increased in animals subjected to chronic hypoxia. Our data provide an in vivo model amenable to analysis of MSC-mobilizing factors.
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MATERIALS AND METHODS |
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All animal investigations were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH [28] and with European directives [29] and approved by the local ethics committee.
Generation of MSCs
MSCs were obtained from rat femoral BM, as previously described [7, 15]. Briefly, bones were cleaned of adherent soft tissue, epiphyses were removed, and marrow was harvested by inserting a 18-gauge syringe needle into one end of the bone shaft and flushing into a 35-mm culture dish with proliferation culture medium consisting of 
-minimal essential medium (-MEM) (Invitrogen, Cergy Pontoise, France, http://www.invitrogen.com) supplemented with 20% (vol/vol) screened fetal calf serum (FCS) (HyClone, Erembodegem, Belgium, http://www.hyclone.com). A single-cell suspension was obtained by passaging through needles of decreasing size. Cells were then centrifuged, and nucleated cells were counted and seeded at a density of 5 x 105 cells per cm2 in culture medium at 37°C in 95% humidified air and 5% CO2. At day 2, all nonadherent cells were removed by changing the medium; thereafter, medium was changed twice a week. The monolayer of adherent cells was trypsinized (0.25% trypsin-EDTA; Invitrogen) when it was half-confluent, resuspended in culture medium, and seeded at 10,000 cells per cm2 (passage 1 [P1]).
Eight- to 12-ml peripheral blood samples were collected in heparinized tubes. Low-density mononuclear cells (MNCs) were separated on Ficoll-Hypaque density gradient (Amersham, Saclay Orsay, France, http://www.amersham.com), counted, and cultured as described for BM; cell density at culture inception was 106 cells per cm2; at P1, density was 10,000 cells per cm2.
Mesenchymal Progenitor Cells
For colony-forming unit fibroblast (CFU-F) assays, BM total cells were plated per triplicate at densities of 5 x 104, 5 x 105, and 5 x 106 cells per 25-cm2 flask in proliferation culture medium, whereas for PB MNCs, cell densities were 105, 106, and 107 cells per 12.5-cm2 flask. The culture medium was changed on day 2, and adherent colonies (>50 cells) deriving from CFU-Fs were counted on days 6–10.
Hematopoietic Progenitor Cells
BM and PB MNCs were assayed for colony-forming units-granulocyte-macrophage (CFU-GM), colony-forming units-macrophage (CFU-M), colony-forming units-erythroid (CFU-E), burst-forming units-erythroid (BFU-E) and colony-forming units-mixed lineage (CFU-Mix) in semisolid culture medium. Briefly, MNCs were cultured in Iscove's modified Dulbecco's medium (Invitrogen) containing 1% (wt/vol) methylcellulose (Sigma-Aldrich, Saint Quentin Fallavier, France, http://www.sigmaaldrich.com), 1% (wt/vol) bovine serum albumin (Sigma-Aldrich), 1 x 10–4 M ß-mercaptoethanol (Sigma-Aldrich), and 30% FCS (Invitrogen). Cells were plated at 2.5 x 104 cells per milliliter for BM and 2.5 x 105 cells per milliliter for PB in 35-mm plastic dishes containing human G-CSF (100 IU/ml; AbCys, Paris, France, http://www.abcysonline.com), rat granulocyte-macrophage colony-stimulating factor (1,000 IU/ml; AbCys), rat stem cell factor (5 IU/ml; AbCys), rat interleukin-3 (1 IU/ml; AbCys), and rat erythropoietin (10 ng/ml; R&D Systems, Lille, France, http://www.rndsystems.com). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere, and colonies were scored on day 4 for CFU-E (aggregates >50 hemoglobinized cells), and on day 10 for CFU-GM, CFU-M (aggregates >50 nonhemoglobinized cells), BFU-E (aggregates >200 hemoglobinized cells), and CFU-Mix (aggregates >50 hemoglobinized and nonhemoglobinized cells).
MSC Immunophenotype
Membrane antigen expression on BM- and PB-derived MSCs was determined at P2 by flow cytometry with a FACSCalibur flow cytometer (Becton, Dickinson and Company, Le-Pont-de-Claix, France, http://www.bdbiosciences.com) using a 488-nm argon laser. Cells from single-cell suspension were incubated for 60 minutes at 4°C with monoclonal antibodies (Abs) against rat antigens, including CD31 (clone TLD-3A12; Serotec, Cergy St. Christophe, France, http://www.serotec.com), CD44 (clone OX-50; Serotec), CD45 (clone MRC OX-1; Serotec), CD54 (clone 1A29; Serotec), CD73 (clone 5F/B9; Becton, Dickinson and Company) and CD90 (clone MRC OX-7; Serotec). Irrelevant isotype-identical Abs (clone F8–11-13; Serotec) served as negative control. Specific and unspecific Ab binding was detected with a secondary phycoerythrin-labeled anti-mouse Ab (Serotec). Samples were analyzed by collecting 10,000 events using Cell-Quest software (Becton, Dickinson and Company).
MSC Differentiation
P2 BM- and PB-derived MSC differentiation potential was evaluated as follows.
Adipogenic Induction.
Cells were cultured for 14 days in -MEM containing 10% (vol/vol) FCS, 100 µM isobutyl methylxanthine (Sigma-Aldrich), 60 µM indomethacin (Sigma-Aldrich), 1 µg/ml insulin (Sigma-Aldrich), and 0.5 µM hydrocortisone (Sigma-Aldrich), with medium changes every 3 days. Adipogenic differentiation was shown by cellular accumulation of large (
5 µm in diameter) lipid vacuoles that were stained with oil red O (Sigma-Aldrich) and counterstained with 4,6-diamidino-2-phenylindole (AbCys).
Osteogenic Induction.
Cells were cultured for 21 days in -MEM containing 20% (vol/vol) FCS, 0.1 µM dexamethasone (Sigma-Aldrich), 2 mM ß-glycerophosphate (Sigma-Aldrich), and 150 µM ascorbic acid (Invitrogen); medium was changed every 3 days. Mineralization areas were revealed by von Kossa's stain.
Chondrogenic Induction. Cells at 80% confluence were trypsinized with 0.05% (vol/vol) trypsin-EDTA and resuspended in low-glucose DMEM containing 1 mM dexamethasone (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen), 1x insulin-transferrin-selenium (Invitrogen), 17 mM ascorbic acid (Invitrogen), 35 mM proline (Sigma-Aldrich), and 10 ng/ml transforming growth factor ß1 (R&D Systems). Viable cells were counted and seeded at a density of 5 x 105 cells per pellet in 15-ml conical tubes. Cells were gently centrifuged to the bottom of the tubes and allowed to form compact cell pellets, then incubated in a humidified atmosphere at 37°C with 5% CO2 with medium changes every 3 days. After 21 days in culture, pellets were embedded in paraffin. Cartilage glycosaminoglycans were detected by staining with Safranin O (Sigma-Aldrich).
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted from undifferentiated (control) and differentiated BM- and PB-derived MSCs using Trizol reagent (Invitrogen). A total of 100 ng of RNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using SuperScript One-Step RT-PCR with Platinum Taq system (Invitrogen). Polymerase chain reaction was carried out with primers specific for rat acidic ribosomal phosphoprotein large P0 (Rplp0), lipoprotein lipase (Lpl), peroxisome proliferator-activated receptor 
2 (Pparg2), bone -carboxyglutamate protein 2 (Bglap2), runt-related transcription factor 2 (Runx2), pro-1(II) collagen (Col2a1), and pro-1(X) collagen (Col10a1) (Table 1). Amplified cDNA fragments were electrophoresed through a 2% (wt/vol) agarose gel, stained by ethidium bromide, and photographed under an ultraviolet light transilluminator.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Generation of MSCs Under Normoxic and Hypoxic Conditions
PB cells from normoxic and hypoxic rats, set in culture in parallel to BM cells from normoxic rats, formed, by the third week, a homogeneous layer of cells that closely resembled BM MSCs (Fig. 2A).
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4 integrin chain), and CD49f (6 integrin chain) (data not shown). The cell surface antigen expression pattern of PBN- and PBH-derived cells was therefore comparable to that of BM MSCs. To assess the capacity of PBN- and PBH-derived adherent cells to differentiate into mesenchymal lineages, we cultured the cells in adipogenic, osteogenic, and chondrogenic induction media for 14–21 days. We checked by specific stainings for the presence of cytoplasmic neutral lipid vacuoles, the formation of mineralized areas, and the accumulation of cartilage glycosaminoglycans. As shown in Figure 3A, PBN- and PBH-derived cells were positive for all specific markers, similarly to BM MSCs differentiated using identical protocols.
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These results show that PB-derived adherent cells collected from normoxic and hypoxic rats display a trilineage-differentiation potential comparable to that of BM MSCs. Taken together, our results (CFU-Fs, differentiation potential, and immunophenotype) indicate that blood-derived adherent cells are bona fide MSCs.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Several studies have been carried out to isolate MSCs in humans and in animals from adult PB using culture conditions equivalent to those used for BM-derived MSCs. Some studies concluded that cells similar to BM MSCs are present in PB from humans and animals [20, 22–24], whereas other studies led to opposite conclusions [25, 26]. The discrepancy of results may be due to the relatively low frequency of such cells, making their detection all the more dependent on the models and the methods used, including species, age, previous treatment, or means of enrichment [reviewed in 32]. In the present study, we report that low levels of multipotent MSCs were detected in the PB of adult rats at steady state. Moreover, we show, to our knowledge for the first time, that the circulating blood MSC pool is dramatically increased in hypoxic animals.
Mechanisms of MSC mobilization into the bloodstream are unknown. They are probably different from those involved in growth factor-induced HSC mobilization, although some reports indicate that MSCs can be detected directly or indirectly in PB grafts after such mobilization procedure [24, 33, 34]. However, this mechanism is probably a rare event since, in our experience (unpublished data), as in that of others [25], CFU-Fs are not detected in the blood of large series of patients after G-CSF infusion. The mobilizing effect of hypoxia observed in our study concerned selective by CFU-Fs, since total circulating HPC level was not significantly increased. The BM CFU-F frequency was unchanged, suggesting that hypoxia either favors MSC egress from the BM into the bloodstream without significantly increasing the BM pool or induces the mobilization of MSCs from other non-BM sources.
Recent studies have demonstrated that long-term cultures of MSCs under an atmosphere of low oxygen that closely approximates documented in vivo oxygen tension enabled these cells to optimally proliferate, to differentiate [35], and to survive after infusion in vivo [36]. These findings indicate that MSC integrity and function can be preserved under hypoxic conditions, but the in vivo effect of chronic hypoxia on MSC mobilization had not been explored in these studies. Although the role of increased levels of erythropoietin cannot be excluded from our study (evidenced by increased hematocrit and hemoglobin levels), the dissociation observed in PB between increased CFU-Fs and unchanged numbers of erythroid progenitor cells suggests that another mechanism is at play. Chronic hypoxia could induce MSC mobilization through a number of direct or indirect mechanisms, including modification of local factors involved in the maintenance of MSCs within BM or other tissues, increased PB concentration of chemotactic factors specific for MSCs and/or increased vascular permeability favored by the neoangiogenesis described in animal models similar to ours [37]. A recent study has shown that hypoxia increases the in vitro migration capacity of MSCs, a phenomenon dependent on metalloproteinases (MMPs) [38]. In addition, hypoxia has been reported to stimulate expression of MMP-2 and vascular endothelial growth factor in endothelial cells [39, 40]. Our animal model provides the opportunity to test in vivo the role of such factors in the mobilization process of MSCs. Determination of the critical factors responsible for this process is of clinical importance since they could be used further to trigger at will the mobilization of endogenous MSCs without the cumbersome cell culture expansion step, a potential source of contamination or transformation.
In conclusion, we report that few MSCs circulate in the PB under stationary conditions in rats. Large amounts of MSCs can be mobilized (an increase of almost 15-fold) under hypoxic conditions. These data demonstrate that MSCs can be mobilized into the PB via stimuli distinct from those involved in hematopoietic stem cell mobilization.
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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