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First published online September 22, 2005
Stem Cells Vol. 24 No. 3 March 2006, pp. 482 -493
doi:10.1634/stemcells.2005-0219; www.StemCells.com
© 2006 AlphaMed Press

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THE STEM CELL NICHE

Characterization of Mesenchymal Stem Cells Isolated from Mouse Fetal Bone Marrow

Xiaoli Wanga,e, Hiroko Hishaa,b,c, Shigeru Taketanid, Yasushi Adachia,b, Qiang Lia, Wenhao Cuia, Yunze Cuia, Jianfeng Wanga, Changye Songa, Tomomi Mizokamia, Satoshi Okazakia, Qing Lia, Tianxue Fana, Hongxue Fane, Zhexiong Lianf, M. Eric Gershwinf, Susumu Ikeharaa,b,c

a First Department of Pathology,
b Regeneration Research Center for Intractable Diseases,
c Department of Transplantation for Regeneration Therapy, Kansai Medical University, Moriguchi City, Osaka, Japan;
d Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan;
e Department of Toxicology, School of Public Health, Jilin University, Changchun, China;
f Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, California, USA

Key Words. Mesenchymal stem cells • Hematopoietic stem cells • Bone marrow cells • PA6 • Mouse

Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: +81-6-6993-9429; Fax: +81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp.

Received on May 16, 2005; accepted for publication on August 30, 2005.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so-called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti-PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6-P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6-P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis-supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6-P cells express a high level of the PA6 molecule, which is reactive with anti-PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemopoiesis in adult mammals is restricted to the bone marrow (BM) cavity in the physiological state. It is well accepted that the maintenance and differentiation of pluripotent hemopoietic stem cells (P-HSCs) are controlled in direct proximity to the endosteal surface, suggesting the presence of a distinct hemopoietic micro-environment or niche for P-HSCs [1]. In mammals, niches regulating stem cells have been well elucidated in hair, gut, and skin, and molecular signals that control the stem cells have already been identified in several reports [2–5]. However, whether the niches indeed interact with the well-characterized mammalian stem cells, hematopoietic stem cells (HSCs) and how the niches control the number of stem cells remain largely unknown, mainly due to the poor elucidation of functional elements that constitute the niche. Since the niche concept was proposed by Schofield in 1978 [6], studies on the roles of stromal cells in supporting HSCs have been based mainly on culture systems that might mimic some features of stem cell niche interaction in vivo. Reticular cells, fibroblasts, adipocytes, endothelial cells and osteoblasts have been considered to be capable of supporting hematopoietic stem /progenitor cells in vitro [7–8]. Recently, two groups have confirmed osteoblasts as definitive regulatory components of the HSC niche in the adult BM [9–10]. However, the identification of other cell types and extracellular matrix molecules close to the endosteal surface may be required to completely define the HSC niche at a cellular and molecular level.

The definitive characteristics of stem cells are their abilities to self-renew and differentiate into one or more specialized lineages [11]. In the adult BM, besides P-HSCs, there is another population of cells, mesenchymal stem cells (MSCs), that meets such criteria. Because of the multilineage potential of MSCs for differentiation and relatively easy isolation, there has been a continuing interest in both biology and therapeutic applications of MSCs from the BM [12–19].

Although MSCs express a set of surface markers, including CD105/SH2, CD73/SH3, CD44, CD90, CD29, and CD106 [20–23], there is no specific marker for direct isolation of MSCs from the BM. Therefore, a single-step purification method using adhesion to cell culture plastic is commonly employed in various species. As a result, a heterogeneous fibroblastic cell population in the phenotype and function is obtained, and MSCs are mainly characterized by their ability to differentiate into multiple mesenchymal lineages, including the osteogenic, chondrogenic, tenogenic, myogenic, adipogenic, and mature stromal lineages [20]. In contrast to human MSCs, which are relatively easy to isolate by their adherence to plastic and can be extensively expanded in culture [17, 24–25], murine MSCs (mMSCs) are far more difficult both to isolate from the BM and to expand in culture [19]. Moreover, they are frequently contaminated by hematopoietic cells [25], which prevents us from studying their basic biology, engraftment, or therapeutic potential in multiple disposable genetic mouse models. Therefore, there is a clear need for novel markers and methods to isolate, enumerate, and detect mMSCs from the BM.

Previously, we established monoclonal antibodies (mAbs) against BM stromal cells [26]. These mAbs were produced by inducing neonatal tolerance in rats using the mouse BM stromal cell lines MC3T3-G2/PA6 (PA6) and PA6-mutant (PA6-M); the former has the capacity to support HSCs, whereas the latter has no such capacity. An anti-PA6 mAb is specific for BM stromal cells, suggesting that this mAb might be applicable as a reagent for selecting or screening of mMSCs from the BM.

In the present study, we isolate PA6-positive BM adherent cells (BMACs) from day-16 mouse fetal BM using the anti-PA6 mAb and characterize this population of cells (FMS/PA6-P). The FMS/PA6-P cells show the fibroblastic feature and can be expanded extensively. They show a multipotentiality for differentiation not only into osteoblasts and adipocytes but also into vascular endothelial cells, indicating that these cells are mMSCs in nature. Furthermore, the FMS/ PA6-P cells express a broad spectrum of cytokines and growth factors regulating hemopoiesis and show a hemopoiesis-supporting capacity similar to BMACs, suggesting that MSCs might be a component of the HSC niche in vivo. The FMS/PA6-P cells express a high level of PA6 molecules reactive with anti-PA6 mAb. However, the cells gradually lose the expression according to their differentiation into osteoblasts and adipocytes, and finally the molecule is no longer detectable after completion of differentiation, indicating that PA6 might serve as a novel marker of mMSCs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice
Seven- to eight-week-old C57BL/6 (B6) mice, pregnant B6 mice with 14-day fetuses, and 8-week-old KSN nude mice were purchased from Shizuoka Experimental Animal Laboratory (Hamamatsu, Japan). All of the mice were maintained in a pathogen-free environment. To study the hemopoiesis-supporting effect of the FMS/PA6-P cell line in vivo, 8- to 12-week-old B6 mice served as donors and 8- to 9-week-old C3H mice served as recipients.

Purification of PA6-Positive BMACs (FMS/PA6-P)
The femurs, tibias, and humeri of B6 fetuses on day 16 of gestation were removed and diced. The resultant bone pieces were then inoculated in a 25-cm2 flask containing Dulbecco’s modified Eagle’s medium (DMEM) (low glucose, LG) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2 in air. Half the medium in the culture flask was replaced with the same volume of fresh medium every 3 days. When the adherent cells reached 70–80% confluency, they were collected by trypsin-EDTA treatment and were subcultured with 2 x 105 cells/ flask (25 cm2) until the third passage.

Fetal BMACs of the third passage were incubated with anti-PA6 mAbs [26] and then incubated with phycoerythrin (PE)-anti-rat IgG (Serotec Ltd., Oxford, U.K., http://www.serotec.com). PA6-positive cells were then sorted using a fluorescence-activated cell sorter (EPICS ALTRA, Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). PA6-positive cells (100,000) were cultured in a flask containing DMEM (LG) supplemented with 10% FBS and leukemia inhibiting factor (LIF, 10 ng/ml, Chemicon, Temecula, CA, http://www.chemicon.com).

Phenotypic Analyses of FMS/PA6-P Cells
The FMS/PA6-P cells and BMACs of the third passage were collected by trypsin-EDTA treatment and then stained with fluorescein isothiocyanate (FITC)-anti-ICAM-1, VCAM-1, CD11c, Mac-1, Sca-1, CD45, PE-anti-CD11a, CD31, and vascular endothelial growth factor receptor 2 (VEGFR-2) (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). They were also stained with anti-PA6, CD44, and VLA-4 mAbs, followed by PE-anti-rat IgG. The stained cells were analyzed using a FACScan (BD Biosciences, San Diego, http://www.bdbiosciences.com).

In Vitro Differentiation Assays
Osteogenic differentiation was induced by culturing the FMS/ PA6-P cells for 5 weeks in differentiation medium: 10% FBS in DMEM supplemented with 50 µg/ml ascorbic acid (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 10 mM ß-glycerophosphate (Sigma-Aldrich), and 0.01 µM dexamethasone (Sigma-Aldrich). The medium was refreshed every 3 days. A change of the PA6 expression on thus-treated cells was analyzed every week using a FACScan. Mineralized deposits were visualized by von Kossa staining.

To induce adipogenic differentiation, the FMS/PA6-P cells were cultured for 2 to 4 weeks in differentiation medium: 10% FBS in {alpha}-MEM supplemented with 4.5 g/l glucose, 1 µM dexamethasone (Sigma-Aldrich) and 5 µg/ml insulin (Sigma-Aldrich). The medium was refreshed every 3 days. Adipocytes were easily discerned from the undifferentiated cells by phase-contrast microscopy. To further confirm their identity, cells were washed twice with phosphate-buffered saline, fixed in 10% formalin neutral buffer solution for 30 minutes at room temperature, and rinsed with distilled water. They were then stained with Oil Red O (6 parts 0.6% Oil Red O dye in isopropanol, and 4 parts water) for 1 hour and washed with distilled water. The PA6 expression on the FMS/PA6-P cells was also analyzed by FACS every week during the culture of induction.

In Vivo Differentiation Assay
To investigate the ability of the FMS/PA6-P cells to differentiate into vascular endothelial cells, we first prepared a murine model of hindlimb ischemia. On day 0, unilateral hindlimb ischemia was induced by ligating the right main femoral arteries and veins of KSN nude mice (H-2Kd) irradiated by 4 Gy 20–24 hours beforehand. The FMS/PA6-P cells (2 x 106/mouse, H-2Kb) were injected into the mice 16 hours after the ligation. Ten days after injection, the skeletal muscles (bilateral gastrocnemius muscles) were removed, embedded in optimal cutting temperature compound (Miles Scientific, Elkhardt, IN), and snap-frozen in liquid nitrogen. The 3-µm sections were stained with PE-anti-CD31 (platelet and endothelial cell adhesion molecule) or VEGFR-2/Flk-1 mAb, and then blocked by normal rat immunoglobulins. These sections were further stained with FITC-anti-H-2Kb or H-2Kd or H-2Kk. The sections stained with FITC-anti-H-2Kk served as a negative control. The stained samples were examined on a confocal laser scanning microscope (LSM-GB200, Olympus, Tokyo, Japan, http://www.olympus.com) equipped with a 20x objective lens. The samples were visualized using a band pass F490-560 filter after excitation at 488 nm for FITC and a high-pass TR 610 filter after excitation at 568 nm for PE.

Analyses of mRNA Expression for Adipogenic and Osteoblastic Markers, Cytokines, and Growth Factors on FMS/PA6-P Cells
Total cellular RNA was extracted using TRIzol reagent (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com). The isolated RNA (2 µg) dissolved in diethylpyrocarbonate-H2O was then employed in reverse transcriptase (RT), in which the reactive solution (20 µl) contained 200 units of monkey murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 40 units of recombinant ribonuclease inhibitor (Invitrogen), and 50 pmol of random nonamer (Takara Shuzo Co., Ltd., Otsu, Japan, http://www.takara.co.jp) in an appropriate RT buffer. The RT solution was incubated at 30°C for 10 minutes and then at 42°C for 70 minutes. The reaction was terminated by heating at 99°C for 5 minutes. Generated cDNA were amplified in PCR using recombinant Taq DNA polymerase (Toyobo, Osaka, Japan, http://www.toyobo.co.jp). The expression of adipogenic and osteoblastic markers, cytokines, and growth factors was assayed with previously defined primers [27–31].

Purification of HSCs
Bone marrow cells (BMCs) were collected from B6 mice that had been treated with 5-fluorouracil (150 mg/kg) 3 or 4 days before sacrifice. Low density (LD) BMCs were purified by discontinuous density gradient centrifugation using Percoll (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). The LD cells (1.066 < {rho} <1.077) were incubated with mAb (rat IgG class) mixtures against lineage markers (Mac-1, Gr-1, B220, CD4, CD8, TER119, and CD71) and then incubated twice with sheep anti-rat IgG-conjugated immuno-beads (Ibs) (Dynal Inc., Oslo, Norway, http://www.dynal.no) with gentle agitation at a 3:1 bead/cell ratio. Ibs-rosetted cells were removed using a magnetic particle concentrator. Remaining nonrosetted cells were considered as Lin– cells, among which Sca-1+ cells were contained at a concentration of 26%. Sca-1+ cells were separated from Lin– BMCs using a magnetic cell separation system (MACS, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany, http://www.miltenyibiotec.com). Sca-1+ Lin– cells were considered to be HSCs.

Long-Term Culture of HSCs on FMS/PA6-P Cells or BMACs of the Third Passage
Monolayers of the FMS/PA6-P cells or BMACs of the third passage were prepared in flask and irradiated (20 Gy), and then the HSCs (1.3 x 105 in 8 ml of 10% FBS Iscove’s modified Dulbecco’s medium) were inoculated. Each week, the medium in the flask containing nonadherent cells was completely removed and replaced with fresh medium. The non-adherent cells were then counted and stained with anti-Sca-1, -Mac-1, -Gr-1, and -TER119 mAbs (all from BD Bioscience Pharmingen). The stained cells were analyzed using a FACScan.

Intravenous or Portal Vein Pathway Injection
Three thousand HSCs alone or together with 5 x 105 FMS/ PA6-P cells were injected into 9.5-Gy-irradiated recipient mice (C3H) via the portal vein (PV) or intravenous (IV) route. PV injection was performed using a slightly modified version of the method described previously [32].

Detection of Donor-Derived Hemopoietic Cells in Hemopoietic Organs
Splenic and hepatic mononuclear cells (HMNCs) were prepared [33] and then stained with PE-anti-H-2Kb, biotinylated anti-H-2Kk, and FITC-anti-CD45 mAbs, followed by streptavidin-RED670 (Gibco-BRL). The cells with the immunophenotype of CD45+/H-2Kb+ were categorized as donor-derived hematopoietic cells. The stained cells were analyzed by using the FACScan.

Detection of Donor-Derived Stromal Cells
To prepare the BM-derived stromal cells, the femurs, tibias, and humeri of the recipient mice were cultured in a flask containing DMEM (LG) with 10% FBS. The medium containing nonadherent cells was removed and replaced by fresh medium every week. Three weeks later, nonadherent cells were extensively removed, and adherent cells were collected by trypsin-EDTA treatment. To detect donor-derived stromal cells, the adherent cells were stained with anti-PA6 mAbs, followed by PE-anti-rat IgG. After blocking with normal rat Igs, the cells were further stained with FITC-anti-H-2Kb or anti-H-2Kk mAb, and analyzed by a FACScan. The adherent cells stained with the iso-type-matched Igs served as a negative control.

Statistics
Statistical differences in survival rates were analyzed by a log-rank test. Statistical differences in other experiments were analyzed by Student’s two-tailed t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Morphological Characterization of FMS/PA6-P Cells
A few PA6-positive cells were detected in fetal BMACs of initial culture. However, the percentage of the PA6-positive cells increased along with the passages. The PA6-positive cells were therefore sorted from the third passage of fetal BMACs and cultured in a flask containing DMEM supplemented with 10% FBS and LIF (10 ng/ml), which are required for the successful expansion of rodent MSCs [34]. Interestingly, although the cells were dormant for ~2 months, several cells showing the same morphology began to proliferate thereafter and formed several large colonies 2 weeks later. These cells were then subcultured into 25 passages and were named FMS/ PA6-P. The FMS/PA6-P cells showed a homogeneous fibroblastoid feature (Fig. 1AGo), although fetal BMACs showed a heterogeneous morphology (data not shown). However, the morphology of the FMS/PA6-P cells changed after they differentiated into osteoblasts (Fig. 1BGo) and adipocytes (Fig. 1CGo). The FMS/PA6-P cells proliferated rapidly with a doubling time of 1.5 days and stopped dividing at confluency. The FMS/PA6-P cells exhibited continuous growth and retained a stable morphology for more than 25 passages.


Figure 1
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  		Figure 1. Morphology of undifferentiated and differentiated FMS/ PA6-P cells. (A): Undifferentiated FMS/PA6-P cells showing homogeneous fibroblastoid morphology. May-Giemsa staining (10x). (B): The FMS/PA6-P cells (ninth passage) were incubated in osteogenic medium for 5 weeks, and mineralized deposits were detected by von Kossa staining (10x). (C): The FMS/PA6-P cells (ninth passage) were incubated in adipogenic medium for 4 weeks. As early as after 1 week, lipid droplets were detectable, and lipid accumulation then increased along with the inductive periods. After 4 weeks, lipid accumulation was stained with Oil Red O, and then cells were counterstained with Mayer (20x).

 
Phenotypic Characterization of FMS/PA6-P Cells
As shown in Fig. 2AGo, BMACs of the third passage reacted with anti-Mac-1 (25.01%), CD45 (27.92%), CD11a (11.34%), and CD11c (12.73%) mAbs, suggesting the presence of hematopoietic cells in this population. Moreover, they also reacted with anti-CD31 (14.98%) and VEGFR-2/FLK1 (2.05%), suggesting that endothelial cells were also contained in the BMACs of the third passage. Furthermore, the percentages of cells positive for ICAM-1 (64.20%), VCAM-1 (46.67%), CD44 (29.63%), VLA-4 (29.33%), and Sca-1 (77.15%) were much lower in the BMACs of the third passage than those in FMS/PA6-P cells (Fig. 2BGo). In contrast, as shown in Fig. 2BGo, the FMS/PA6-P cells of the 16th passage were negative for the hemopoietic lineage panel of antibodies: Mac-1, CD45, CD11a, and CD11c. They were also negative for the endothelial markers: VEGFR-2/FLK1 and CD31. However, they reacted with anti-VCAM-1 (92.08%), ICAM-1 (73.60%), CD44 (52.60%), VLA-4 (43.11%), and Sca-1 (90.44%) mAbs, which are characteristics of MSCs. Most strikingly, a high expression of PA6 molecules (96.76%) was observed on the FMS/PA6-P cells, although only 23.37% of the BMACs of the third passage showed positive staining. These findings suggest that, in contrast to the BMACs, the FMS/ PA6-P cells are homogeneous and possess antigens found frequently on MSCs.


Figure 2
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  		Figure 2. Flow cytometric analyses of FMS/PA6-P cells. Bone marrow adherent cells (BMACs) of the third passage (A) and the FMS/PA6-P cells of the 16th passage (B) were collected by trypsin-EDTA treatment and stained with fluorescein isothiocyanate (FITC)-anti-ICAM-1, VCAM-1, CD11c, Mac-1, Sca-1, and CD45 and phycoerythrin (PE)-anti-CD11a, CD31, and VEGFR-2. They were also stained with anti-PA6, CD44, and VLA-4 monoclonal antibodies (mAbs), followed by PE-anti-rat IgG. The stained cells were analyzed using a FACScan. The FMS/PA6-P cells reacted with anti-PA6, CD44, Sca-1, VCAM-1, ICAM-1 and VLA-4 mAbs, but not with mAbs against surface markers of hematolymphoid cells and endothelial cells. The broken line indicates the cells stained with isotype-matched control Abs. Abbreviations: VEGFR-2, vascular endothelial growth factor receptor 2.

 
Differentiation Ability of FMS/PA6-P Cells

Differentiation into Osteoblastic Lineage.   To induce osteoblastic differentiation, the FMS/PA6-P cells were cultured in an osteoinductive medium as described in Materials and Methods. In the initial culture, no mineralized cell was detected (data not shown). Three weeks after culturing, some mineralized cells were found using von Kossa staining. Five weeks later, cells positive for von Kossa staining were widely distributed throughout the flask (SlideFlask, Nunc, Rochester, NY, http://www.nuncbrand.com) (Fig. 1BGo). To further confirm the osteoblastic differentiation, the expression of core-binding factor {alpha}1 (Cbfa-1) [35] (a transcription factor that is involved in controlling osteoblastic differentiation by regulating the expression of multiple genes in osteoblasts), alkaline phosphatase (ALP), and osteocalcin (osteoblast-specific marker) in the FMS/PA6-P cells was analyzed by RT-polymerase chain reaction (PCR) before and after induction. The FMS/PA6-P cells constitutively expressed cbfa-1 and ALP under basic culture conditions. In contrast, osteocalcin, a marker for advanced osteoblastic differentiation was not detected. After 5-week culture in the osteoinductive medium, Cbfa-1 and ALP mRNA levels were unchanged or slightly increased, whereas osteocalcin mRNA were strongly induced (Fig. 3AGo), confirming the osteoblastic differentiation of the FMS/PA6-P cells.


Figure 3
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  		Figure 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the expression of transcriptional factors and lineage-specific markers in the FMS/PA6-P cells before and after differentiation. (A): Expression of Cbfa-1, ALP, and osteocalcin after induction to differentiate into osteogenic lineage. Before induction (1); 5 weeks after induction (2). (B): Expression of PPAR{gamma}2 and aP2 after induction to differentiate into adipogenic lineage. Before induction (1); 2 to 4 weeks after induction (2–4). Abbreviations: Cbfa-1, core-binding factor {alpha}1; ALP, alkaline phosphatase; PPAR{gamma}2, peroxisome proliferation-activated receptor {gamma} 2; aP2, adipocyte lipid-binding protein; GAPDH, glyceralde-hyde-3-phosphate dehydrogenase.

 

Differentiation into Adipocytic Lineage.   To induce adipocytic differentiation, the FMS/PA6-P cells were cultured in a adipo-inductive medium as described in Materials and Methods. One prominent characteristic of cell differentiation into adipocytes is the accumulation of triglyceride-containing vesicles in the cell cytosol. In the tissue culture, intracellular fat droplets were observed by phase-contrast light microscopy (data not shown) and were chemically stained by Oil Red O (Fig. 1CGo). As early as 1 week later, lipid droplets were detectable, and lipid accumulation thereafter increased along with the inductive periods. To further confirm the adipocytic differentiation, the expression of peroxisome proliferation-activated receptor {gamma} 2 (PPAR{gamma}2, transcription factor), which is induced during the early phase of adipocyte differentiation and is thought to be absolutely required for adipocyte differentiation [36–38], and adipocyte lipid-binding protein (aP2), which is a downstream target of PPAR{gamma}2 activation and is the most widely used adipocyte differentiation marker [39–40], were analyzed by RT-PCR before and after induction. The FMS/PA6-P cells in basic culture conditions constitutively expressed PPAR{gamma}2 (Fig. 3BGo). In contrast, no aP2 was detectable. After a 2-week induction, PPAR{gamma}2 mRNA levels increased significantly and maintained a high expression level during the following induction (Fig. 3BGo). The adipocytic differentiation of the FMS/PA6-P cells was further confirmed by determining the expression levels of the aP2 gene. The expression of aP2 in the FMS/PA6-P cells increased during inductive periods (Fig. 3BGo).

Differentiation into Endothelial Cell Lineage.   Next, we attempted to induce the FMS/PA6-P cells to differentiate into vascular endothelial cells. We first established a murine model of hindlimb ischemia, and the FMS/PA6-P cells (2 x 106) were then injected into the model mice. Ten days later, the skeletal muscles (bilateral gastrocnemius muscles) were removed, and the formation of donor-derived vascular endothelial cells was observed as described in Materials and Methods. Donor-derived cells were stained by FITC-anti-H-2Kb mAb (Fig. 4A and 4DGo, cells colored green), and vascular endothelial cells were stained by PE-anti-CD31 (endothelial-specific adhesion molecule) mAb (Fig. 4BGo, cells, colored red) or VEGFR-2 mAb (Fig. 4EGo, cells colored red). Both CD31 and VEGFR-2 are expressed on vascular endothelial cells, and their signaling is critical in both physiological and pathological angiogenesis [41]. Both donor-derived CD31- and VEGFR2-positive cells (Figs. 4C and 4FGo, cells colored yellow) were found in the blood vessels of mice with hindlimb ischemia, suggesting the differentiation of the FMS/PA6-P cells into vascular endothelial cells.


Figure 4
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  		Figure 4. Differentiation of FMS/PA6-P cells in vivo. A murine model of hindlimb ischemia was made by ligating the right main femoral arteries and veins of 4Gy-irradiated KSN nude mice (H-2Kd). Sixteen hours later, FMS/PA6-P cells (ninth passage, 2 x 106/mouse, H-2Kb) were injected. Ten days after the injection, the formation of donor-type vascular endothelial cells was observed as described in Materials and Methods. Donor-derived cells were stained by fluorescein isothiocyanate (FITC)-anti-H-2Kb monoclonal antibody (mAb) (A and D, cells colored green). Vascular endothelial cells were stained by phycoerythrin (PE)-anti-CD31 (B, cells colored red) or vascular endothelial growth factor receptor 2 (VEGFR-2) mAb (E, cells colored red). Donor-derived vascular endothelial cells were double-positive for H-2Kb and CD31 (C, cells colored yellow) or VEGFR-2 (F, cells colored yellow) (x20).

 
mRNA of Cytokine and Growth Factor in FMS/PA6-P Cells
We used RT-PCR to detect the steady-state levels of mRNAs for various cytokines and growth factors in the FMS/PA6-P cells or BMACs of the third passage (Fig. 5Go). These cytokines and growth factors are as follows: ligand for c-kit (stem cell factor, SCF) and flt3/flk2 ligand (Flt3-L), both of which are known to transduce signals crucial for HSC proliferation and regarded as key factors for triggering self-renewal of HSCs [42–44]; basic fibroblast growth factor (bFGF) [45–46], which positively regulates hemopoiesis; granulocyte colony-stimulating factor (G-CSF); LIF [47]; MIP-1{alpha} [48]; transforming growth factor-ß1 (TGF-ß1) [48–49], which negatively regulates hemopoiesis; and insulin-like growth factor I (IGF-I), which regulates both proliferation and differentiation of hematopoietic cells. As shown in Figure 5Go, although bFGF, TGF-ß1, and MIP-1{alpha} expression was lower, whereas IGF-1 expression was higher in the FMS/PA6-P cells than in the BMACs of the third passage, there was no difference in the expression of G-CSF, Flt3-L, LIF, and SCF between these two populations of cells when maintained under normal culture conditions.


Figure 5
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  		Figure 5. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of cytokines and growth factors in the FMS/PA6-P cells and bone marrow adherent cells (BMACs). (A): FMS/PA6-P cells. (B): BMACs of the third passage. Total RNA of the FMS/PA6-P was extracted, and RT and PCR reactions were performed as described in Materials and Methods. Although bFGF, TGF-ß1, and MIP-1{alpha} expressions were lower and IGF-1 expression was higher in FMS/PA6-P cells than those in BMACs of the third passage, there were no differences in the expression of G-CSF, Flt3-L, LIF, and SCF between these two populations of cells. Abbreviations: M, marker; LIF, leukemia inhibiting factor; TGF-ß1, transforming growth factor ß1; SCF, stem cell factor; Flt3-L, Flt-3 ligand; bFGF, basic fibroblast growth factor; MIP-1{alpha}, macrophage inflammatory protein1 {alpha}; IGF-1, insulin-like growth factor type 1; G-CSF, granulocyte-colony stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Long-Term Hemopoiesis-Supporting Capacity of FMS/PA6-P Cells In Vitro
We next focused on the long-term hemopoiesis-supporting capacity of the FMS/PA6-P cells. When the HSCs of B6 mice were cocultured with the FMS/PA6-P layer without cytokines, they "crawled" under the stromal layer and then proliferated. The proliferating cells showed a cobblestone-like appearance and were referred to as "cobblestone area-forming" cells (data not shown). Hematopoiesis was maintained for up to 26 weeks when HSCs were cocultured with the FMS/PA6-P cells in a flask (Fig. 6AGo). In contrast, BM-adherent cell layers detached from the flask surface at 19 weeks, and the HSC culture could no longer be continued (Fig. 6AGo). Both FMS/PA6-P cells and BMACs facilitated the production of not only Sca-1+ cells (Fig. 6BGo) but also myeloid and erythroid lineage cells (Mac-1+, Gr-1+, and TER119+) (Fig. 6CGo) for 26 and 19 weeks, respectively, indicating that the FMS/PA6-P cells are able to promote the proliferation of hemopoietic cells from primitive to mature cells without adding any exogenous cytokines. These findings suggest that the FMS/PA6-P cell line mimics the in vivo hematopoietic microenvironment and can support long-term hematopoiesis in vitro.


Figure 6
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  		Figure 6. Hemopoiesis-supporting capacity of FMS/PA6-P cells in vitro. (A): The numbers of nonadherent cells per flask. (B, C): Continuous production of Sca-1+, Gr-1+, Mac-1+, and TER119+ cells. Hemopoiesis was maintained for up to 26 weeks on the FMS/PA6-P cells. In contrast, bone marrow (BM) adherent cell layers detached from the flask surface at 19 weeks, and the HSC culture could then no longer be continued. Abbreviations: HSC, hemopoietic stem cell; Sca-1, stem cell antigen-1.

 
Long-Term Hemopoiesis-Supporting Capacity of FMS/PA6-P Cells In Vivo

Analyses of Donor-Derived Hemopoietic Cells.   We have previously reported that hemopoietic reconstituting ability is better when BM stromal cells are portalvenously injected than when they were intravenously injected. Therefore, to confirm the hematopoiesis-supporting effect of the FMS/ PA6-P cells in vivo, 3 x 103 HSCs alone or HSCs (3 x 103) plus FMS/PA6-P cells (5 x 105) were portalvenously injected. When injected with HSCs alone (Fig. 7AGoGo), hardly any donor-derived cells were detected in the spleen and liver until 10 days after the treatment, although a very low number of donor-derived cells (<0.3%) were detected in the BM. Moreover, the percentage of donor-derived cells in these hemopoietic organs increased only slightly until 14 days after the treatment. However, in the mice injected with HSCs plus FMS/PA6-P cells (Fig. 7BGo), the percentages of donor-derived cells gradually increased up to 10 days and thereafter rapidly increased, and reached nearly 30% in the BM by 14 days after the injection. The absolute numbers of donor-derived cells in each tissue of the mice injected with HSCs plus FMS/PA6-P cells also increased markedly compared with those in the mice injected with HSCs alone: 10.3 times in the BM, 23.2 in the spleen, and 14.6 in the liver at day 14 after injection.


Figure 7
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  		Figure 7. Long-term hemopoiesis-supporting capacity of FMS/PA6-P cells in vivo. Percentages of donor-derived cells in C3H mice treated with HSCs alone (A) or HSCs plus FMS/PA6-P cells (B) by PV pathway. BMCs, HMNCs, and splenic cells were collected from the recipient mice at the days indicated on the x-axis. The percentages of donor-type cells were obtained using FACS analysis. The results are expressed as the mean ± SD of 5 to 6 mice. In mice that received HSCs plus FMS/PA6-P cells, a significantly higher number of donor-type BMCs, splenic cells, and HMNCs were detected at 14 days after BMT compared with the mice that received HSCs alone (HSCs plus FMS/ PA6-P vs. HSCs alone: BMCs [*] and splenic cells [**]: p < .05; HMNCs [***]: p < .01). Moreover, a marked higher number of donor-type splenic cells was detected at 7 and 10 days in mice that received HSCs plus FMS/PA6-P cells than those in mice that received HSCs alone (p < .05). (C): The FMS/PA6-P cells migrate into recipient bone marrow (BM) and support hematopoiesis. Bone pieces were collected from recipient mice treated with HSCs plus FMS/PA6-P cells or HSCs alone on days 7, 10, and 14 after BMT. The cells were cultured for 3 weeks, and the adherent cells were collected. The cells were stained with anti-PA6 monoclonal antibody (mAb) followed by phycoerythrin (PE)-anti-rat IgG. They were further stained with fluorescein isothiocyanate (FITC)-anti-H-2Kb mAb. The results are expressed as the mean ± SD of 5 or 6 mice. (**, p < .01; ***, p < .05).

 

Figure 7
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  		Figure 7. cont. (D): Survival rate in C3H mice treated by various methods. Thirty percent of recipient mice transplanted with HSCs plus FMS/PA6-P cells via the PV could survive >110 days, whereas all mice transplanted with HSCs alone died within 7 weeks (***, p < .05). Abbreviations: HSC, hematopoietic stem cell; PV, portal vein; BMC, bone marrow cell; HMNC, hepatic mononuclear cell; BMT, bone marrow transplantation.

 
The donor-derived cells with common leukocyte antigen (CD45+) were next analyzed in the hemopoietic organs. As shown in Table 1Go, high percentages of donor-derived cells were detected in the BM, spleen, and liver of the mice injected with HSCs plus FMS/PA6-P cells 14 days after the injection. In contrast, the mice injected with HSCs alone showed markedly low percentages of donor-type CD45+ cells, especially in the spleen and liver. These findings indicate that the FMS/PA6-P cells significantly facilitate the early engraftment and continuous proliferation and differentiation of donor cells.


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  		Table 1. Analyses of surface antigens on donor-derived cells in C3H mice injected with HSCs plus FMS/PA6-P cells or HSCs alone

 

Analyses of Donor-Derived Stromal Cells.   We have recently found that a major histocompatibility complex restriction exists between HSCs and stromal cells [50] and that donor-derived stromal cells are required for successful allogeneic BM transplantation (BMT) [33, 51–54]. Here, we have found that a large number of donor-derived PA6-positive stromal cells can be detected in the BM on day 7 after the injection of HSCs plus FMS/PA6-P cells via the portal vein (PV), although only a very small number of donor-derived PA6-positive stromal cells can be detected in the BM until day 14 after the injection of HSCs alone via the PV (Fig. 7CGo). This finding suggests that the injected HSCs (Sca-1+ Lin– cells) contain a small number of PA6+ stromal cells. This is reasonable, because most stromal cells also have Sca-1 antigen. Anyway, it is clearly indicated that the FMS/PA6-P cells are not only trapped efficiently in the recipient liver by the PV injection, but also migrate into the BM, where they construct the hemopoietic microenvironments and support hemopoiesis.

Long-Term Reconstitution.   All mice injected with the cells via the IV route died within 21 days, even when the FMS/PA6-P cells (5 x 105) were injected along with the HSCs. Such early death is caused by an injection of a low number of HSCs (3000 cells/mouse). In contrast, 30% of the recipient mice injected with HSCs (3 x 103) plus FMS/PA6-P cells (5 x 105) via the PV survived for more than 4 months. However, when HSCs (3 x 103) alone were injected into the recipient via the PV, only 1 of 19 mice survived for 46 days (Fig. 7DGo). Fifteen weeks after BMT, almost all the lymphoid and myeloid lineage cells were donor-type cells in mice injected with HSCs plus FMS/PA6-P cells via the PV (data not shown).

Reduction of PA6 Expression on FMA/PA6-P Cells After Differentiating into Osteoblastic or Adipocytic Lineage
The FMS/PA6-P cells are capable of differentiating not only into osteoblasts and adipocytes, but also into vascular endothelial cells, indicating that the cells are mMSCs in nature. Because the FMS/PA6-P cells are characteristic of a high level of PA6 expression, we then analyzed kinetics of the PA6 expression on FMS/PA6-P cells during the course of differentiation. As shown in Fig. 8Go, the PA6 expression gradually decreased on the FMS/ PA6-P cells along with the induction, indicating that PA6 antigen is only present on undifferentiated mMSCs and disappears once the cells embark upon differentiation. These findings suggest that PA6 antigen is specific for mMSCs and is applicable to the isolation of mMSCs.


Figure 8
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  		Figure 8. Time course of PA6 expression in the FMS/PA6-P cells induced to differentiate into osteoblastic or adipocytic lineage. Each week after induction, the PA6 expression in the FMS/PA6-P cells was analyzed by fluorescence-activated cell sorting. The PA6 antigens disappeared once the FMS/PA6-P cells differentiated into osteoblasts or adipocytes. (A): FMS/PA6-P cells induced to differentiate into osteoblastic lineage. (B): FMS/PA6-P cells induced to differentiate into adipocytic lineage.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Very recently, it has been found that osteoblasts lining trabecular bones are major components of the HSC niche in the BM [55]. However, there is a possibility that other nonhematopoietic mesenchymal cells and extracellular matrix molecules close to the endosteal surface constitute the HSC niche. The candidates for such cells include vascular endothelial cells, myoepithelial cells, or unidentified mesenchymal cells. In the present study, we have shown the possibility that MSCs (FMS/PA6-P) are one component of the HSC niche.

The FMS/PA6-P cells show homogeneously fibroblastic morphology (Fig. 1AGo) and lack hematolymphoid and endothelial cell markers (Fig. 2BGo). They also express common MSC markers, including VLA-4, CD44, and VCAM-1 (Fig. 2BGo). Moreover, the cells can differentiate not only into osteoblasts and adipocytes in vitro (Figs. 1B, 1CGo, 3Go) but also into vascular endothelial cells in vivo (Fig. 4Go), and these cells retain their differentiative potential following extensive subcultivation in vitro (data not shown). Collectively, based on their morphologies, phenotypes, and differentiation potentials in vitro and in vivo, we conclude that the FMS/PA6-P cells are MSCs.

RT-PCR analyses revealed that the FMS/PA6-P cells express a broad spectrum of cytokines and growth factors associated with hematopoiesis (Fig. 5Go) and indeed show good hematopoiesis-supporting capacity both in vitro and in vivo (Figs. 6Go and 7GoGo). Our previous report has clearly shown that endosteal cells present on the surface of BM are positive for PA6 antigen [26]. Moreover, the FMS/PA6-P cells are characteristic of a high expression of PA6 antigen. These findings indicate that the FMS/PA6-P cells are one component of the HSC niche in vivo.

Currently, there is great interest in using MSCs as therapeutic agents to treat many kinds of diseases, including osteogenesis imperfecta, spinal cord injury, stroke, and myocardial infarction. The characterization of mMSCs provides an essential foundation for elucidating the fate, distribution, and therapeutic benefit of MSCs. However, the purification of mMSCs has been only poorly reported, and the engraftment or therapeutic potential of mMSCs has been evaluated in vivo only by administering the plastic adherent cell fraction of the BM to experimental animals. Because this population includes hematopoietic cells possessing an appreciable engraftment potential in vivo, it is difficult to examine the contribution of mMSCs in the therapeutic studies. mAbs provide a useful means of isolating a defined population of cells from heterogeneous components. In recent years, although there is a lack of specific cell surface markers to strictly identify human MSCs, enrichment strategies have been developed based on selection of cells positive for the NGF receptor [56], STRO-1 antibody-recognized antigen [57–59], and the SH-2, -3, and -4 antibody-recognized antigens [22–23]. However, no specific surface markers have been found for mMSCs. Here, we succeeded in isolating and enriching mMSCs having a high expression level of PA6 using anti-PA6 mAb. Moreover, the PA6 antigens disappeared once the FMS/PA6-P cells had differentiated into osteoblasts or adipocytes (Fig. 8Go). These findings suggest that the PA6 molecule might be applicable in selection and characterization of mMSCs from the BM. Very recently, we have succeeded in identifying the PA6 molecule as neural cell adhesion molecule [60]. Our next research will focus on studies to further explore its role in the biological control of MSC differentiation and to determine its application as a cell surface marker of mMSCs.

It has been reported that the expression of lineage-specific markers is controlled by key transcriptional factors; for example, a forced expression of Cbfa-1 induces osteoblast-specific gene expression in nonosteoblastic cells in vitro and in vivo [35, 61], and ectopic expression of PPAR{gamma}2 can stimulate adipose differentiation of fibroblastic cell lines in a lipid-dependent manner [62]. In accordance with these results, Cbfa-1-deficient mice do not share osteoblastic differentiation and are unable to calcify their cartilaginous skeleton. In mouse embryos, Cbfa-1 is expressed on a common progenitor for the osteoblasts and chondrocytes in the mesenchymal condensations. However, Cbfa-1 remains only on cells of the osteoblastic lineage at a later period of development [35]. These findings suggest that Cbfa-1 expression might be used to identify a common progenitor for the osteoblasts and chondrocytes. Seshi et al. [63] reported that each human BM stromal cell of Dexter-type culture coexpresses genes specific for various mesenchymal cell lineages, including adipocytes, osteoblasts, fibroblasts, and muscle cells, and they postulated that human BM stromal cells are composed of a single cell type (designated multidifferentiated mesenchymal progenitor cells, MPCs), although the MPCs might include various types of stromal cells. Our undifferentiated FMS/PA6-P cells coexpress these two key transcriptional factors (Cbfa-1 and PPAR{gamma}2) but do not express lineage-specific markers (osteocalcin and aP2), confirming that the FMS/PA6-P have a multi-potentiality. We will also investigate their ability to differentiate into other mesenchymal lineages and even into ectodermal or endodermal lineages.

In conclusion, we have shown that the FMS/PA6-P cells are MSCs in nature and are one component of the HSC niche. We have also shown that PA6 antigen can be used as a novel marker for isolating mMSCs from the BM. The FMS/PA6-P cells will be of great advantage in further investigating the HSC niche and the biology and therapeutic application of MSCs and in finding a new marker of both mouse and human MSCs.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We thank S. Miura for conducting fluorescence-activated cell sorting, Y. Tokuyama and M. Shinkawa for preparing tissue sections, and Hilary Eastwick-Field and K. Ando for preparing the manuscript. This work was supported by a grant from Haiteku Research Center of the Ministry of Education; a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology; a grant-in-aid for scientific research (B) 11470062; grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221; Health and Labor Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology); The 21st Century Center of Excellence Program (Project Leader); The Ministry of Education, Culture, Sports, Science and Technology; a grant from the Department of Transplantation for Regeneration Therapy (Sponsored by Otsuka Pharmaceutical Company, Ltd., Tokyo, Japan); a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.; and a grant from Japan Immunoresearch Laboratories Co., Ltd (Gunma, Japan).


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 References
 

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J. Ma, M. Shi, J. Li, B. Chen, H. Wang, B. Li, J. Hu, Y. Cao, B. Fang, and R. C. Zhao
Senescence-unrelated impediment of osteogenesis from Flk1+ bone marrow mesenchymal stem cells induced by total body irradiation and its contribution to long-term bone and hematopoietic injury
Haematologica, July 1, 2007; 92(7): 889 - 896.
[Abstract] [Full Text] [PDF]

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Q. Li, H. Hisha, R. Yasumizu, T.-X. Fan, G.-X. Yang, Q. Li, Y.-Z. Cui, X.-L. Wang, C.-Y. Song, S. Okazaki, et al.
Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantation: Comparison of Intravenous and Intrabone Marrow Routes
Stem Cells, May 1, 2007; 25(5): 1186 - 1194.
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