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First published online January 11, 2007
Stem Cells Vol. 25 No. 5 May 2007, pp. 1213 -1221
doi:10.1634/stemcells.2006-0325; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Two Distinct Stem Cell Lineages in Murine Bone Marrow

Yoko Koidea,b, Satoru Morikawaa,c, Yo Mabuchia,g, Yukari Mugurumad,e, Emi Hiratsua,g, Kohji Hasegawab, Makoto Kobayashib, Kiyoshi Andod,e, Kentaro Kinjoa,f, Hideyuki Okanoa,g, Yumi Matsuzakia,g

Departments of aPhysiology,
cDentistry and Oral Surgery, and
fHematology Division, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan;
bDepartment of Periodontology, Showa University Dental School, Tokyo, Japan;
dDivision of Hematology, Department of Medicine and
eResearch Center for Regenerative Medicine, Tokai University School of Medicine, Kanagawa, Japan;
gCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan

Key Words. Hematopoietic stem cell • Mesenchymal stem cell • Transplantation • Lineage relation • Plasticity

Correspondence: Yumi Matsuzaki, M.D., Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Telephone: 03-5363-3117; Fax: 03-5363-3566; e-mail: penguin{at}sc.itc.keio.ac.jp

Received May 29, 2006; accepted for publication January 2, 2007.
First published online in STEM CELLS EXPRESS   January 11, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mesenchymal stem cells (MSC), a distinct type of adult stem cell, are easy to isolate, culture, and manipulate in ex vivo culture. These cells have great plasticity and potential for therapeutic application, but their properties are poorly understood because of their low frequency and the lack of knowledge on cell surface markers and their location of origin. The present study was designed to address the undefined lineage relationship of hematopoietic and mesenchymal stem cells. Genetically marked, highly purified hematopoietic stem cells (HSCs) were transplanted into wild-type animals and, after bone marrow repopulation, the progeny were rigorously investigated for differentiation potential into mesenchymal tissues by analyzing in vitro differentiation into mesenchymal tissues. None/very little of the hematopoietic cells contributed to colony-forming units fibroblast activity and mesenchymal cell differentiation; however, unfractionated bone marrow cells resulted in extensive replacement of not only hematopoietic cells but also mesenchymal cells, including MSCs. As a result, we concluded that purified HSCs have no significant potency to differentiate into mesenchymal lineage. The data strongly suggest that hematopoietic cells and mesenchymal lineage cells are derived from individual lineage-specific stem cells. In addition, we succeeded in visualizing mesenchymal lineage cells using in vivo microimaging and immunohistochemistry. Flow cytometric analysis revealed CD140b (PDGFRβ) could be a specific marker for mesenchymal lineage cells. The results may reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hematopoietic stem cells (HSCs) residing in adult bone marrow possess the unique ability to self-renew and differentiate into multiple lineages [1]. A number of recent studies have suggested that whole bone marrow cells (WBM) or HSCs, upon transplantation into adult recipients, may also transdifferentiate and contribute to the regeneration of a variety of nonhematopoietic lineages in multiple organs [2–8]. These initial findings have provoked extensive follow-up studies; some question the biological significance and even the existence of such transdifferentiation [9–12], and others suggest alternative mechanisms for the observed developmental plasticity of transplanted bone marrow cells [13–17]. In parallel, another line of studies has continued to support and extend the concept of the developmental plasticity of bone marrow-derived cells [18–24].

The apparently conflicting data from these reports may be due to the purity of the "enriched" fraction of HSCs, which becomes crucial when considering the highly proliferative nature of even a single contaminating ectopic stem cell. Bone marrow is composed of a largely heterogeneous population, containing not only HSCs but also stem-like cells that are precursors of nonhematopoietic tissues [25]. The precursors of nonhematopoietic tissues were initially referred to as plastic-adherent cells or colony-forming units fibroblasts (CFU-F), because they readily adhered to culture dishes and formed fibroblast-like colonies [26]. The cells were also referred to as mesenchymal stem cells (MSCs) or mesenchymal progenitor cells [27] because of their ability to differentiate into a variety of nonhematopoietic cells, such as osteocytes, chondrocytes, and adipocytes [28, 29]. The MSCs could thus be considered the most probable candidates for the stem cells that are able to provide circulating progenitors for the repopulation of nonhematopoietic tissues. Alternatively, HSCs themselves or their progeny may have the potential to generate nonhematopoietic cells in a cell fusion manner [30–32].

In a previous report, we demonstrated that single-cell transplantation of cells with the strongest Hoechst 33342 dye efflux activity (Tip-SP) and the CD34– c-Kit+ Sca-1+ Lin– (CD34– KSL) phenotype resulted in more than 90% engraftment activity and long-term multilineage hematopoietic reconstitution [33]. Therefore, CD34– KSL Tip-SP cells represent a highly purified HSC population.

In the present study, to address whether HSCs and/or their progeny have the capacity to differentiate into mesenchymal cells (MCs), we examined the differentiation and expansion of bone marrow-derived cells, using a syngenic mouse model and primary CFU-F assays to quantitate the numbers of MSCs in bone marrow reconstituted with genetically marked purified HSCs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
C57BL/6 mice and B6.SJL-Ptprca Pep3b/BoyJ (C57BL/6-Ly5.1; Ly5.1) mice 8–10 weeks old were purchased from CLEA Japan, Inc. (Tokyo, Japan, http://www.clea-japan.com/). C57BL/6 background CAG-EGFP transgenic mice that ubiquitously express enhanced green fluorescent protein (EGFP) under the control of the CAG promoter [34] were kindly provided by Dr. Jun-ichi Miyazaki (Osaka University, Osaka, Japan) and were bred in our animal facility. The mice were bred and maintained under specific pathogen-free conditions according to protocols approved by the Laboratory Animal Research Facility (LARC) of the Keio University School of Medicine. All studies were approved by LARC and adhered strictly to Keio University School of Medicine guidelines for the use and care of experimental animals. All transgenic mice used in this study were heterozygous for the transgene.

Isolation of CD34– cKit+ Sca-1+ Lineage– Tip-SP Cells (HSCs)
The bone marrow (BM) cells suspended at 1 x 106 cells per milliliter in calcium- and magnesium-free Hanks' balanced salt solution supplemented with 2% fetal calf serum, 10 mM HEPES, and 1% penicillin/streptomycin (HBSS+) were incubated with 5 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 60 minutes at 37°C. After washing, cells were resuspended in ice-cold HBSS+ at 107 cells per milliliter and then stained for 30 minutes on ice with various monoclonal antibodies (i.e., biotinylated CD34, allophycocyanin (APC)-conjugated c-kit, phycoerythrin (PE)-conjugated Sca-1, and PE-Cy5 conjugated lineage markers [Gr-1, Mac-1, B220, CD3, TER119]). Biotinylated antibodies were visualized with PharRed (APC-Cy7) conjugated streptavidin. All of these reagents were purchased from e-Bioscience (San Diego, http://www.ebioscience.com/). Cell sorting was performed on a triple laser MoFlo (Dako Colorado, Inc., Fort Collins, CO, http://www.dakousa.com) using Summit software. Hoechst 33342 was excited at 350 nm, and fluorescence emission was detected using 405/BP30 and 570/BP20 optical filters against Hoechst blue and Hoechst red, respectively, and a 555 nm long-pass dichroic mirror (Omega Optical Inc., Brattleboro, VT, http://www.omegafilters.com/) to separate emission wavelengths. Both Hoechst blue and red fluorescence were shown on a linear scale. After collecting 105 events, the side population (SP) was defined as described previously [35], and additional gates were defined as positive for Sca-1 and c-kit and negative for CD34 and lineage markers according to the isotype control fluorescence intensity. Populations of CD34– KSL Tip-SP cells were routinely prepared with 99% purity by this method. Single CD34– KSL Tip-SP cells derived from CAG-EGFP transgenic animals were sorted directly into separate wells of a 96-well plate containing 100 µl of HBSS+ using a CyClone automated cell deposition unit.

HSC Transplantation to B6 Mice
CD34– KSL Tip-SP cells or 5 x 106 whole BM cells either from CAG-EGFP transgenic mice or Ly5.1 congenic mice were injected intravenously into the retro-orbital plexus of anesthetized recipient mice that had been irradiated with a lethal dose (10.5 Gy for C57BL/6, 9.5 Gy for CAG-EGFP transgenic mice). Single HSC or 100 HSCs were transplanted along with 2 x 105 whole BM cells as radioprotective cells from recipient strain mice into each recipient animal.

Lineage Analysis
Three months after BM transplantation, peripheral blood samples were prepared from recipient mice, erythrocyte-depleted using Ficoll-Paque (GE Healthcare, Chalfont St Giles, Buckinghamshire, U.K., http://www.gehealthcare.com), and stained with the following reagents: PE-anti-Mac-1, PE-anti-Gr-1, APC-anti-CD3, APC-anti-B220, and APC-anti-Ter119 (e-Bioscience). Dual-laser fluorescence-activated cell sorting analysis was performed using FACSCalibur (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Donor BM-derived cells were determined by the fluorescent intensity of EGFP compared with wild-type cells. The mean percentage with SD of EGFP+ cells in the peripheral mononuclear cells of the mice transplanted with WBM was 87 ± 3 and that of the mice transplanted with a single CD34– KSL Tip-SP cells was 33 ± 29, respectively. We then chose the mice with more than 70% of their peripheral blood cells originating from the donor at 12 weeks after transplantation for the following experiments.

Adherent Cell Culture (CFU-F: Fibroblast Colony Formation Assay)
Twelve weeks after the transplantation, the recipients with >70% chimerism were sacrificed, and the bone marrow cells were flushed from femurs and tibias to prepare single cell suspensions. After the erythrocytes were removed with Ficoll-Paque, bone marrow mononuclear cells were seeded on fibronectin-coated 96-well plates at a density of 1–2 x 105 cells per well, in Dulbecco's modified Eagle's medium supplemented with 20% FBS and antibiotics, and incubated at 37°C with 5% CO2. After 3 days, nonadherent cells were removed, and the medium was replaced. The cultures were maintained with medium changes every 3–4 days for 2 weeks.

Immunostaining
The adherent cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, then stained with a primary rabbit antibody against EGFP (1:500 dilution; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). EGFP expression was visualized using the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000 dilution; Molecular Probes). Each sample was also stained with Hoechst 33342 (1:1,000 dilution; Sigma Aldrich). The fixed slides were examined using confocal microscopy (Axiovert 100; Carl Zeiss, Inc., Thornwood, NY, http://www.zeiss.com/).

Differentiation Assays
To induce adipocyte differentiation, the subconfluent cells were cultured with three cycle of adipogenic induction medium/ adipogenic maintenance medium (Cambrex Bio Science Walkersville., Inc., Walkersville, MD, http://www.cambrex.com). Each cycle consisted of feeding the subconfluent cells with induction medium for 3 days followed by 1–3 days of culture in maintenance medium. After 14 days, these cells were fixed with 4% paraformaldehyde for 15 min, and stained with oil red O (MutoPure Chemicals, Tokyo, http://www.mutokagaku.com).

To induce osteoblast differentiation, the subconfluent cells were cultured with osteogenic differentiation medium (Cambrex Bio Science Walkersville., Inc.) for 14 days. After 14 days, these cells were fixed with 4% paraformaldehyde for 15 min, stained with alkaline phosphatase (Histofine; Nichirei, Tokyo, Japan, http://www.nichirei.co.jp/english/index.html), and microscopically examined (Axiovert 100; Carl Zeiss, Inc.).

Chondrogenic differentiation was induced by harvesting the cells with trypsinization, after which 5 x 106 cells were pelleted on the bottom of a 15-ml conical tube. To this pellet, 100 µl of MSC medium was added and, after incubation overnight, the medium was changed to 100 µl of Differentiation Basal Medium Chondrogenic, supplemented with Chondrogenic SingleQuots (Cambrex Bio Science Walkersville., Inc.). After 3 weeks of culture, cell clumps were harvested, washed in 4% paraformaldehyde, and stained with Alcian blue.

Tissue Processing and Immunofluorescent Staining
Mice were anesthetized with pentobarbital sodium and then perfused with 4% paraformaldehyde (PFA) in PBS. Bones were excised, immersion fixed with PFA overnight, infiltrated with sucrose, embedded in 4% carboxymethyl cellulose in H2O, and frozen in liquid nitrogen. Longitudinal sections (6 µm) cut through the center of bones were obtained and stored at –80°C until staining. Before addition of the primary antibody, non-specific antibody binding was blocked by incubating slides with 5% serum from animals in which secondary antibodies were raised. After incubation with the primary antibody, slides were incubated with species-specific secondary antibodies conjugated with fluorochromes (Alexa Fluor 594; Molecular Probes). Slides were mounted in glycerol/PBS containing diazabicyoctane and examined using a confocal microscope (LSM 510 META; Carl Zeiss, Inc.). Cell nuclei were visualized by staining with TOTO-3 (Molecular Probes). Enzyme immunohistochemistry was visualized as brown products of the diaminobenzidine.

Antibodies
The antibodies used for immunostaining were rabbit polyclonal anti-GFP (1:500; MBL, Nagoya, Japan, http://www.mbl.co.jp/e/index.html) and guinea pig polyclonal anti-vimentin (1:400; PROGEN Biotechnik GmbH, Heidelberg, Germany, http://www.progen.de).

RNA Extraction and Reverse Transcription–Polymerase Chain Reaction Analysis of Gene Expression
Total RNA was collected from the cells that were induced to differentiate into osteoblastic and adipocytic lineages as detailed above, using the RNeasy Protect Mini Kit (QIAGEN, Hilden, Germany, http://www.qiagen.com). Standard reverse transcription reactions were performed with 100 ng of total RNA using the StrataScript First Strand cDNA Synthesis Kit (Stratagene, La Jolla, CA, http://www.stratagene.com) according to the manufacturer's instructions. Subsequent polymerase chain reaction (PCR) was performed with the following solution: 1 µl of cDNA, 2 µl of 10x PCR buffer, 1.6 µl of dNTP, 1 µl of each primer pair, and 0.1 µl of Taq DNA polymerase (TaKaRa Ex Taq; Takara Bio Inc., Otsu, Japan, http://www.takara.co.jp). The following primers were used to detect osteoblastic differentiation: osteocalcin (GenBank accession number NM_007541), 5'-GATGATGACGATGATGATGACGATGGA-3' and 5'-AGGCTGGCTTTGGAACTTGCTTGAC-3'; osteopontine (GenBank accession number AF515708), 5'-GATGATGACGATGATGATGACGATGGA-3' and 5'- AGGCTGGCTTTGGAACTTGCTTGAC-3'. Detection of adipogenic differentiation was performed with the following primers: peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), 5'-AACTGCAGGGTGAAACTCTGGGAGATTCTCC-3' and 5'-GGATTCAGCAACCATTGGGTCAGCTCT-3'; murine adipsin 5'-GATGATGACGATGATGATGACGATGGA-3' and 5'-AGGCTGGCTTTGGAACTTGCTTGAC-3'. The following primer pair was used for detection of EGFP expression: 5'-TGAACCGCATCGAGCTGAAGGG-3' and 5'-TCCAGCAGGACCATGTGATC-3'; and as a positive control, the primer pair for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-AGGTTGTCTCCTGCGACTTCA-3' and 5'-CCAGGAAATGAGCTTGACAAAG-3', was used. Amplification conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 98°C for 15 seconds, annealing at 59°C (osteocalcin), 58°C (osteopontin), 55°C (PPAR{gamma}), 61°C (adipsin), 65°C (EGFP), and 60°C (GAPDH) for 30 seconds (osteocalcin, osteopontin, PPAR{gamma}, EGFP, and GAPDH) or 45 seconds (adipsin), extension at 72°C for 45 seconds (osteocalcin and osteopontin) or 30 seconds (PPAR{gamma}, adipsin, EGFP and GAPDH), and a final polymerization at 72°C for 7 minutes (PPAR{gamma}, adipsin, EGFP, and GAPDH). Each PCR was performed in triplicate and under linear conditions. The products were analyzed on 2% agarose gel and visualized by ethidium bromide staining.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
CD34– KSL Tip-SP cells (HSCs) were isolated from CAG-EGFP transgenic mice as described in Materials and Methods and transplanted into lethally irradiated wild-type animals with 1 x 105 recipient type EGFP negative WBM cells (supplemental online Fig. S1A). The frequency of donor-derived mononuclear cells in the peripheral blood was assessed by flow cytometry 3 months after transplantation. Mice with more than 70% of their peripheral blood cells originating from the donor and multilineage engraftment for >12 weeks were sacrificed, and bone marrow cell suspensions were prepared.

Representative lineage analysis data are shown in Figure 1. Bone marrow cells from animals transplanted with a single EGFP-positive (EGFP+) HSC revealed donor-derived cells in all hematopoietic lineages such as CD3/B220-positive lymphocytes (Fig. 1A), Gr-1/Mac-1-positive myeloid cells (Fig. 2B), and Ter119-positive erythroid cells (Fig. 1C). In addition to mature cells, the immature SP cell fraction also contained EGFP+ cells (Fig. 1D). Therefore, the recipient animals were fully reconstituted from a single hematopoietic cell (Fig. 2A, leftmost graph). We also performed another series of bone marrow transplantation, and hematopoietic cells were also repopulated by EGFP+ 5 x 106 WBM transplantation (Fig. 2A, second from left).


Figure 1
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  		Figure 1. Representative flow cytometric analysis of bone marrow cells from a mouse engrafted with a single hematopoietic stem cell. Three months after transplantation, substantial donor-derived EGFP+ multilineage hematopoietic engraftment is present: T/B lymphocytes (A), myeloid cells (B), and CD45– erythroid cells (C). EGFP-positive cells in the subset of side population cells (D). Abbreviation: EGFP, enhanced green fluorescent protein.

 


Figure 2
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  		Figure 2. In vitro colony-forming unit fibroblast (CFU-F) study for bone marrow cells derived from individual transplanted animals. (A): Results representative of flow cytometric analysis for peripheral mononuclear cells. Relative cell numbers are plotted as EGFP-positive cells (green line) or Ly5.1-positive cells (red line) of mice transplanted with hematopoietic stem cells, whole bone marrow cells, or side population cells, as indicated. (B): Identification of bone marrow-derived adherent cells. Confocal microscopic results are shown for Hoechst 33342 (A–E), EGFP (F–G), and merged images (K–O). Bars indicate 200 µm. (C): Individual adherent cells obtained from each animal transplanted with a different cell source were cultured in adipogenic or osteogenic media for 2 weeks. As indicated, the accumulated lipid vacuoles were stained by oil red O (top) or osteogenic induction plates were stained for alkaline phosphatase (bottom). Bars indicate 200 µm. Abbreviations: ALP, alkaline phosphatase; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; HSC, hematopoietic stem cells; SP, side population; WBM, whole bone marrow.

 
We then attempted to elucidate whether these repopulated bone marrow cells have the potential to differentiate into MCs/MSCs. The remaining bone marrow cells from the same recipients were cultured on a plastic dish in a medium promoting fibroblastic growth, because MCs are referred to as plastic adherent and fibroblast-like cells [27]. After 2 weeks of culture, we observed a large number of adherent cells in each group (Fig. 2Ba, 2Bb). The mean percentage of cells with mesenchymal phenotype (CD45– Ter119–) was 97% ± 2% (supplemental online Fig. S2).

However, adherent cells from the single HSC transplanted group were mostly negative for EGFP (Fig. 2Bf), in contrast to the majority of cells obtained from the WBM transplanted group, which were EGFP+ (Fig. 2Bg). A small number of EGFP+ cells in the single HSC transplanted group was also expressed CD11b (supplemental online Fig. S3); therefore, it is presumable that the cells may represent monocytes or macrophages. It is noteworthy that the adherent cells derived from isolated SP cell-transplanted animals revealed a heterogeneous profile. In some cases, a large portion of the adherent cells were positive for EGFP (Fig. 2Bh), whereas in other cases, there were quite a few EGFP+ cells (data not shown). Such a heterogeneous phenotype may reflect the cell populations included among SP cells. The SP fraction of bone marrow cells are considered heterogeneous [33, 36, 37] and may potentially include the population considered to be enriched in HSCs [35], MSCs, or multipotent cells that are more primitive than HSC [38]. It is plausible that these nonhematopoietic cells in the SP fraction might be responsible for the SP-subpopulation-derived fibroblast-like cell growth observed in this assay.

The quantitative results shown in Table 1 also indicate significant differences among these three groups. The adherent cells from the single HSC-transplanted group, or even a hundred HSC-transplanted groups, were mainly negative for EGFP expression, whereas the chimerism of donor-derived hematopoietic cells was essentially equivalent among all groups (Fig. 2A and Table 1). These findings suggest that the transplanted HSCs were readily involved in the production of hematopoietic cells, whereas a remarkably small number of the progeny had the capacity to replace MCs and furthermore that the adherent cells were mostly radio-resistant recipient-derived. To verify this, we performed a reverse combination of bone marrow transplantation (supplemental online Fig. S1B). HSCs or WBM were prepared from C57BL/6-Ly5.1 congenic mice (Ly5.1) and transplanted into CAG-EGFP (Ly5.2) transgenic recipients (Ly5.1 HSC/WBM transplanted group) according to a protocol similar to the one used in this study.


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  		Table 1. Quantitative adherent cell assay results for bone marrow cells from animals transplanted with each individual cell source

 
Three months after transplantation, the peripheral blood cells were analyzed to confirm the engraftment of Ly5.1-positive donor derived cells in both HSC- and WBM-transplanted animals. The mean percentage of donor-derived cells is shown in Table 1. The peripheral blood cells indicated that more than 80% were Ly5.1-positive donor-derived cells and residual cells expressing EGFP that were surviving hematopoietic cells (data not shown). We then prepared a bone marrow cell suspension from transplanted animals for the adherence assay. The attached cells from the Ly5.1 HSC-transplanted group were clearly the reciprocal of those in the EGFP+ HSC transplanted group (Fig. 2Bd–2Bi). Although the majority of peripheral blood cells were Ly5.1-positive but EGFP-negative, the adherent cells were largely positive for EGFP, whereas there was no discrepancy between the peripheral chimerism and frequency of adherent cells in the WBM-transplanted group (Fig. 2A and Table 1).

We also investigated the mesenchymal multilineage differentiation potential of the adherent cells obtained from each transplanted group into adipocytes, chondrocytes, and osteoblasts. Except for the formation of chondroblasts, all other differentiation lineages could be detected in our cultures. When subjected to adipogenic or osteogenic differentiation conditions, the adherent cell populations were cultured in either an adipo-inductive or osteo-inductive medium as described in Materials and Methods. After a 3-week adipocytic differentiation period, intracellular lipid droplets were observed in each group and were chemically stained with oil red O (Fig. 2C, top row). To further confirm the adipocytic differentiation, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) analysis before and after induction of adherent cells prepared from EGFP-positive HSC or WBM-transplanted recipients. The mRNA expressions of both PPAR{gamma} and adipsin, which are the most widely used adipocyte differentiation markers, were undetectable in undifferentiated cells, whereas the expression of both markers was induced during the differentiation period (Fig. 3A).


Figure 3
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  		Figure 3. Reverse transcriptase-polymerase chain reaction analysis of the expression of transcriptional factors and lineage-specific markers. (A): Expression of Adipsin, peroxisome proliferation-activated receptor {gamma}, and enhanced green fluorescent protein before or 3 weeks after induction of adipogenic lineage differentiation. (B): Expression of osteopontin, osteocalcin, and EGFP before or 3 weeks after induction of osteogenic lineage differentiation. Abbreviations: D, after induction; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehide-3-phosphate dehydrogenase; HSC, hematopoietic stem cells; PPAR{gamma}, peroxisome proliferation-activated receptor {gamma}; U, before induction; WBM, whole bone marrow.

 
After 3 weeks of osteogenic induction, osteocytic cells evidenced by the increased expression of alkaline phosphatase were observed in each group (Fig. 2C, bottom row). Differentiation was further demonstrated by RT-PCR analysis of the osteoblast-specific markers. The undifferentiated adherent cells from WBM-transplanted animals produced osteocalcin in very low levels, but the osteopontin mRNA was not. After a 3-week induction period, osteocalcin and osteopontin mRNA levels significantly increased (Fig. 3B). Consequently, the cells were not only adherent but also retained their potential for mesenchymal lineage functions.

In addition, EGFP mRNA was detectable in the WBM transplanted group, both undifferentiated and differentiated. The adhered donor WBM cells, which expressed EGFP mRNA, had the adipogenic and osteogenic differentiation abilities. On the other hand, EGFP was not detected in the HSC transplanted group. These findings suggest that the adipocytes and osteocytes induced in the HSC transplanted group were most likely differentiated from recipient bone marrow, implying that WBM cells, but not HSCs, have the characteristics associated with mesenchymal lineage functions.

Our data indicate that transplantation of HSC does not positively take part in the replacement of MCs even after the lethal irradiation and that the MSCs apparently derive from endogenous stem cells that are resistant to irradiation. In contrast, an excess of unfractionated bone marrow cells will lead to replacement of certain populations of MCs and/or MSCs as described in a previous study [39].

The bone marrow cells of CAG-EGFP transgenic mice (Ly5.2) transplanted with Ly5.1+ HSCs theoretically consist of two distinct populations, which are EGFP-negative hematopoietic cells derived from HSCs or nonhematopoietic cells derived from endogenous EGFP+ bone marrow cells. Moreover, nonhematopoietic cells are expected to be more concentrated in the population of recipient origin than that of the donor origin cells, because of their radiation-resistant nature, as already shown. Therefore, we then performed immunohistochemical assays to determine the localization of the recipient derived EGFP+ cells.

Figure 4 shows the expression of EGFP in bone marrow and the surrounding bone tissue. The majority of EGFP+ cells were located throughout the bone marrow cavity (Fig. 4Aa). A portion of the cells was located in the perivascular region (Fig. 4Ac) or on the bone surface (Fig. 4Ad, 4Ae). In addition, the recipient animal indicated a 70% chimera of donor cells; therefore, not only MCs but also the hematopoietic cells were partially positive for EGFP and, in most cases, the hematopoietic cells had colonized as a cluster (Fig. 4Ae). Fluorescence imaging also showed the reticular distribution of EGFP+ cells and that the majority of the cells had typical morphology (i.e., abundant cytoplasm and a spindle or elongated shape) (Fig. 4Ba, 4Bb). Interestingly, the perivascular cells were mostly EGFP-positive (Fig. 4Bc, 4Bd). The cells located on the bone surface featured a large cytoplasm and spindle-shape in osteoblasts (Fig. 4Be–4Bg). In vivo imaging of the chimeric bone marrow also presented EGFP+ MCs localization. (A supplemental movie is available online. Recipient derived cells were visualized with EGFP expression and hematopoietic cells were stained with a CD45-PE antibody.)


Figure 4
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  		Figure 4. Immunohistochemical staining against EGFP of bone marrow from an Ly5.1 hematopoietic stem cells (HSC)-transplanted CAG-EGFP transgenic recipient. Bone marrow section from a recipient EGFP transgenic animal with 60% Ly5.1+ peripheral blood leukocytes at 3 months after transplantation. (Aa): Panoramic view of entire bone marrow cavity shows the distribution of EGFP+ (recipient type) cells. Bar indicates 100 µm. (Ab): Anti-EGFP staining of bone marrow section from a wild-type animal. (Ac): Magnified view shows the majority of EGFP+ cells to have a spindle shape and to be larger than the round hematopoietic cells. (Ad): EGFP+ hematopoietic cell cluster. The area boxed in (Ad) is magnified x1.5 (Ae). Bars indicate 100 µm (Ab–Ad). (B): Confocal micrograph of bone section from a CAG-EGFP transgenic mouse transplanted with Ly5.1 congenic EGFP– HSCs. The areas boxed (Ba, Bc, Be) are magnified x2.5 in (Bb, Bd, Bf, Bg), respectively. (Ba, Bb): The characteristic reticular distribution of EGFP+ cells. Bar indicates 50 µm in (Ba). (Bc, Bd): Spindle-shaped EGFP+ cells surrounding vessels. (Be–Bg): EGFP expression of osteoblast-like cells. Bars indicate 20 µm in (Bb–Bd, Bf, Bg). (C): Expression of Vimentin (red) in surviving endogenous EGFP+ (green) cells or engrafted EGFP– cells. Bar indicates 20 µm. Abbreviations: EGFP, enhanced green fluorescent protein; TB, trabecular bone; v, blood vessel.

 
Vimentin, known as a fibroblast marker [40], was expressed on only a portion of the EGFP+ cells (Fig. 4C). Therefore, we then performed flow cytometric analysis to determine the mesenchymal lineage-specific surface antigen of recipient-derived EGFP+ cells with flow cytometry. Bone marrow cells derived from Ly5.1+ EGFP– HSC transplanted EGFP+ recipient animals were stained with various antibodies, as shown in Table 2. In Figure 5A, Ly5.1+ donor HSC-derived cells were gated out, and the fluorescent intensity of various antigen expressions of recipient-derived EGFP+ cells, both Ly5.2-positive hematopoietic cells or Ly5.2-negative nonhematopoietic cells, are shown in Figure 5B.


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  		Table 2. Monoclonal antibody list

 


Figure 5
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  		Figure 5. In vivo phenotype of hematopoietic and nonhematopoietic cells. Bone marrow cells were prepared from the animals described in Figure 1B and labeled with monoclonal antibodies shown in Table 2. Cells were analyzed by flow cytometry. (A): Expression profile for CD45 and EGFP in Ly5.1 (CD45.1)-negative cells (region indicated in the upper plot) were further divided into a CD45 ± population. (B): Fluorescent intensity of individual samples. Red line, CD45+ hematopoietic cells; blue line, CD45– nonhematopoietic cells. Abbreviation: EGFP, enhanced green fluorescent protein.

 
CD49e and CD105, known as conventional MSC markers [41, 42], were expressed in both hematopoietic and nonhematopoietic cells. Some of the nonhematopoietic cells expressed Sca-1, Tie-2, Notch1, Flk-1, and CD34. In the vast majority of hematopoietic and nonhematopoietic cells, neither CD15, CD150, CD133, nor CD117 (c-Kit) appeared. It is interesting that most of the nonhematopoietic cells tested CD140b (PDGRβ)-positive, but not the hematopoietic cells. Immunohistostaining indicated that the cells obtained by the standard mesenchymal cell culture method exhibited the expression of CD140b (supplemental online Fig. S4). As a result, CD140b would be a panspecific marker for mesenchymal lineage cells.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
There is accumulating evidence showing that the adult bone marrow may contain progenitor or stem cells that can differentiate into various tissues, beyond their original differentiating and regenerative potential for the tissues in which they reside [7, 18, 20, 24, 43]. In adult bone marrow, two kinds of multipotent stem cells have been identified. They are classified into HSCs [44] and MSCs [25]. Although it was assumed that HSCs give rise only to hematopoietic cells, recent reports suggest that they may have a broader potential to differentiate into various cell types through myeloid intermediates [32, 45, 46].

The present study was designed to rigorously determine the differentiation potency of highly purified HSCs by analyzing the in vitro differentiation to mesenchymal tissues after bone marrow reconstitution with isolated HSC. Although a single HSC showed appreciable level of hematopoietic engraftment activity, MCs were basically derived from recipient cells. In contrast, bone marrow cells substantially contributed to mesenchymal repopulation when recipient animals were transplanted with unfractionated BM cells.

In contrast to our data, in numerous studies where bone marrow cells were infused, as in clinical transplantations, donor-type MSC could not be detected [47, 48] or could be detected only in a part of the recipient group [49, 50]. However, in more recent studies, applying gene-tagged cells in a syngenic murine transplantation model, donor-derived MSCs were detected [39, 51, 52]. Therefore, it is likely that this discrepancy can be fully explained by the fact that the previous studies concerned allogeneic transplantation techniques rather than syngeneic ones. Reyes et al. have reported, after syngenic bone marrow transplants into lethally irradiated recipient mice, that MAPCs were of donor origin but stromal cells were of host origin. The discrepancy of this and previous syngenic animal model, including our own data, may be explained by the difference in the experimental design, because Reyes et al. used MAPC culture conditions instead of a CFU-F assay.

In addition, we observed that MSC chimerism in BM was relatively higher than we expected when WBM were transplanted. The frequency of CFU-F in bone marrow is 1–5 per 1 x 104 (our unpublished observations and Rombouts et al. [39]). When 5 x 106 bone marrow cells were transplanted, the fraction contained about 1,000 CFU-F. In a previous report [39], after a lethal irradiation dose, the frequency of CFU-F was reduced to 1/1,000; that means 3–7 x 104 CFU-Fs were still surviving in the recipient animals (per pair of tibias and femurs). Thus, the initial ratio of donor/host MSCs after transplant was 1:3–40. This contrasted with the 95.3% ± 4.4% donor chimerism observed in the present study. The observed differences are unexplained at the moment; however, it may suggest that either the donor and host cells competed with unequal strength for the niches in the bone marrow immediately after transplantation or the radiation did not physically kill the cells and left them alive in their niche but led to a kind of arrest accounting for a delayed regeneration of host MSCs compared with the freshly isolated donor MSCs. The latter situation is likely to explain our result showing the outgrowth of the radio-resistant, recipient-derived MSCs in the bone marrow of the hosts and their contribution to the regeneration of the MSC compartment to preirradiation levels after HSC transplantation.

We recently reported the contribution of bone marrow-derived cells to the remodeling of ischemic cardiac muscle [53] and injured artery [54], whereas highly purified HSC and its progeny rarely contribute to it. As a result, both in vivo and in vitro studies strongly suggested that nonhematopoietic tissue regeneration is mainly mediated by their own lineage stem cell but not by hematopoietic cells, including mature blood cells, progenitors, and HSCs.

Previous reports suggest that HSCs or their progeny adopt a tissue-specific phenotype by cell fusion, but not by transdifferentiation [12, 13, 16, 31]. Others suggested that nonhematopoietic developmental potential of an HSC could be reactivated by local cues [55]. Our data do not rule out either possibility; however, we seldom detected HSC-derived mesenchymal cells even under in vitro culture conditions. Consequently, we believe that only under rare circumstances would HSC/progeny change the cell fate in response to the microenvironment or cell fusion. For this reason, selection of cells from a suitable cell source is important in terms of cell therapy for a particular disease. Although the mechanism involved in the regeneration of damaged tissue is complex, it would still be worthwhile to identify the major cell population involved in the process. The cell population, which is considered to be MSCs, or even stem cells that are more primitive than HSCs, is believed to play a role in the regeneration of tissues distributed throughout the body. However, the physiological phenotype and lineage of such stem/progenitor cells in the bone marrow remain unknown. Because we have shown that HSCs are not involved in nonhematopoietic tissue regeneration, the next objective would be to further characterize the different populations of stem cells in the bone marrow.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We sincerely and deeply thank Sakura Motion Picture Co. (Tokyo, http://www.sakuraeiga.com) for providing an excellent in vivo microimaging technique. We also thank Dr. Shigeto Shimmura, Dr. Hiroyuki Katoh, and Lawrence Lein for proofreading the manuscript and Miyuki Ogawara and Takayuki Ohkawa for technical assistance. This work was supported in part by grants from Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation; from the Ministry of Education, Science, and Culture of Japan (to H.O. and Y.M.); and a grant-in-aid from the 21st Century COE program of the Ministry of Education, Science, and Culture of Japan to Keio University.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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