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TISSUE-SPECIFIC STEM CELLS

Distribution of Single-Cell Expanded Marrow Derived Progenitors in a Developing Mouse Model of Osteogenesis Imperfecta Following Systemic Transplantation

^aDepartment of Orthopaedics and Rehabilitation, Division of Musculoskeletal Sciences, and
^bDepartment of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA

Key Words. Progenitors • Osteogenesis imperfecta • Stem cell transplantation • Mouse model of osteogenesis imperfecta • Neonatal mice

Correspondence: Christopher Niyibizi, Ph.D., Department of Orthopaedics and Rehabilitation, Division of Musculoskeletal Sciences, Pennsylvania State University College of Medicine, H089, 500 University Drive, Hershey, Pennsylvania 17033, USA. Telephone: 717-531-5649; Fax: 717-531-7583; e-mail: Cniyibizi{at}psu.edu

Received on June 21, 2007; accepted for publication on August 27, 2007.

First published online in STEM CELLS EXPRESS  September 6, 2007.

	ABSTRACT

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

 
We evaluated single-cell-expanded, marrow-derived progenitors for engraftment in a developing mouse model of osteogenesis imperfecta (OI) following systemic transplantation. The present study was initiated to evaluate the potential of mesenchymal stem cells to treat OI. Single-cell-derived progenitors were prepared from marrow stromal cells harvested from normal mice. Selected single-cell-expanded progenitors marked with green fluorescent protein were injected into the neonatal mouse model of OI, and the recipient mice were sacrificed at 2 and 4 weeks following cell transplantation. Examination of the tissues harvested from recipient mice at 2 and 4 weeks after cell transplantation demonstrated that the cells extravasated and engrafted in most of the bones as well as other tissues. Tissue sections made from the tibias and femurs of a selected recipient mouse showed that the cells were distributed in bone marrow, trabecular, and cortical bone as demonstrated by histology and confocal microscopy. The cells that engrafted in the bones of the recipient mouse synthesized and deposited type I collagen composed of 1(I) and 2(I) collagen heterotrimers. Genotyping and gene expression analysis of the cells retrieved from the bones of the recipient mouse at 2 and 4 weeks demonstrated that the cells expressed osteoblast-specific genes, suggesting that the donor cells differentiated into osteoblasts in vivo with no evidence of cell fusion. These data suggest that progenitors infused in developing mice will engraft in various tissues including bones, undergo differentiation, and deposit matrix and form bone in vivo.

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

	INTRODUCTION

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The stromal component of bone marrow is known to contain cells that are referred to as stromal cells or mesenchymal stem cells (MSCs) [1–4]. These cells have generated a lot of interest because of their potential use in regenerative medicine. Direct injection of the MSCs into specific skeletal tissues suggests that these cells may contribute to the tissue cell phenotypes and possible repair and regeneration of the target tissues [5–8]. There is also a great interest in delivery of the cells via the circulatory system for the treatment of the diseases that affect multiple tissues or organs. This is especially attractive for diseases that affect the entire skeleton. This approach has been suggested for the treatment of osteogenesis imperfecta, a brittle bone disease that affects all the skeletal tissues in which type I collagen is synthesized [9–14]. A number of reports in literature using animal models have suggested that MSCs could be transplanted via the circulatory system and that the transplanted cells will migrate and lodge in skeletal tissues, including bones [10, 13, 15]. These studies led to a clinical trial using whole marrow or fractionated MSCs in children with severe forms of osteogenesis imperfecta (OI) [16–18]. The results from the clinical trial showed that the children who received the cell transplant exhibited increased growth velocity, total body mineral content, and fewer fractures [16–18]. These data, however, have not been collaborated in animal models of the disease. Although some studies have shown that MSCs transplanted into animal models will engraft and differentiate into osteoblasts in vivo, distribution of the donor cells and synthesis of bone extracellular matrix in vivo by the donor cells have not been demonstrated [10, 13, 15].

Presently, there are no specific markers available to identify and isolate MSCs from various tissue sources. Studies in mice have generated contradictory results regarding engraftment of MSCs in skeletal tissues following systemic transplantation [19]. The discrepancies in these studies might partly be due to the transplantation of undefined populations of MSCs and also the type of animal models used. This is particularly true for murine MSCs, which are difficult to isolate and to propagate in culture [20].

In the present study, we have used a neonatal mouse model of OI to assess whether systemic delivery of MSCs into the developing mouse model of OI (oim) leads to the engraftment, differentiation, and deposition of the bone extracellular matrix by the transplanted cells in vivo. Because most studies use undefined cell populations of MSCs for transplantation, we have isolated single-cell-expanded progenitors from bone marrow and assessed their migration, engraftment, differentiation, and synthesis of extracellular matrix in vivo. The cells were transplanted into the neonatal mice because treatment of the disease would require early intervention to reduce the development of further skeletal abnormalities. We report here that single-cell-expanded progenitors transplanted into a developing OI mouse model migrate, engraft, differentiate, and deposit bone extracellular matrix within the bones of the recipient mice.

	MATERIALS AND METHODS

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Isolation and Culture of Bone Marrow-Derived Mesenchymal Stem Cells
Murine MSCs were isolated from (B6C3Fe a/a) 8-week-old mice as described previously [12]. Briefly, femurs were harvested from 8-week-old mice, stripped of periosteum, placed in Petri dishes, cut at both ends, split open, and scraped gently with a needle to release the cells from the bone surface as well as bone marrow. The bones were placed in Petri dishes containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (vol/vol) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 50 µg/ml ascorbic acid. After 5 days in culture, the medium and the bones were removed from the Petri dishes, and the adherent cells were fed with new medium supplemented with 20% FBS, 1% penicillin/streptomycin (vol/vol), and 50 µg/ml ascorbic acid and were then maintained in culture until confluence.

Transduction of the Cells with a Retrovirus Carrying Green Fluorescent Protein-Zeocin cDNA
For cell tracking, the cells were transduced with a retroviral vector carrying the enhanced green fluorescence protein (eGFP)-Zeocin^r genes using methods described previously [12]. The retrovirus was prepared and characterized as described [21, 22]. Briefly, the cells were plated in six-well plates in DMEM until they reached 60% confluence. The cells were then treated with 1 ml of a high-titer DFG-eGFP retrovirus in DMEM supplemented with 10% FBS. After 24 hours, the medium containing the retroviral vector was replaced with new medium, and the retroviral vector was again added, as described above, two more times. After 24 hours, the medium was replaced with new medium supplemented with 25 µg/ml Zeocin to select for the green fluorescent protein (GFP)-positive cells (GFP+), and the cells were expanded in culture.

Isolation of Single-Cell Expanded MSCs
The GFP+ cells were serially diluted and plated onto 96-well plates (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) at a final density of 30 cells per 96-well plate to generate wells that contained single cells. The wells that contained only single cells were maintained in culture for expansion.

In Vitro Differentiation of the Single-Cell Expanded Progenitors
The selected single-cell-expanded MSCs are referred to here as single-cell-expanded progenitor cells (SPCs). The SPCs were isolated and characterized for differentiation toward osteogenic and adipogenic differentiation in vitro and potential to lodge in skeletal tissues following systemic transplantation.

Osteogenic Differentiation

Alkaline Phosphatase Activity Assay.   The alkaline phosphatase (ALP) activity of the SPCs was assessed after treatment with human recombinant bone morphogenetic protein 2 (hrBMP-2) using methods described previously [23]. ALP activity was assessed after 2 days, and aliquots of 40 µl of the lysates were used for the ALP activity assay using the Sigma-Aldrich ALP assay kit following the manufacturer's protocol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com).

Alizarin Red S Staining.   The SPCs were plated in six-well plates and cultured in an osteogenic medium. The osteogenic medium consisted of -Minimal essential medium (Invitrogen) supplemented with 10% FBS, 50 µg/ml ascorbic acid, 10 mM β-glycerol phosphate, 10^–7 M dexamethasone, and 1% penicillin/streptomycin (P/S). The cells were maintained in culture with medium changes every 3 days for 21 days. After 21 days, the media were removed, and the cells were rinsed in phosphate-buffered saline (PBS), fixed in 10% formalin, and stained with alizarin red S [24]. The plates were treated with the alizarin red solution and incubated for 5 minutes at room temperature. After 5 minutes, the plates were rinsed in distilled water and were then examined under a light microscope and photographed.

Adipogenic Differentiation
For adipogenic differentiation, the SPCs were plated in 24-well plates in adipogenic medium at a cell density of 5 x 10³ cells per well. The adipogenic medium was composed of DMEM with high glucose supplemented with 10% FBS, 0.1 mM indomethacin, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 10^–6 M dexamethasone [25]. The media were replaced every 3 days for 14 days. Adipogenic differentiation was assessed by oil red O staining at 3 weeks after initial adipogenic induction. For oil red O staining, the cells were rinsed in PBS and fixed in 10% formalin followed by incubation of the cells in 2% (wt/vol) oil red O reagent for 5 minutes at room temperature. The cells were rinsed in isopropanol followed by several changes of distilled water and were then examined under a light microscope and photographed.

Fluorescence-Activated Cell Sorting Analysis for Cell Surface Markers
Fluorescence-activated cell sorting analysis was performed on cultured SPCs. The procedure for fluorescence-activated cell sorting (FACS) staining was described previously [14, 26]. Antibodies used for FACS analysis were phycoerythrin-conjugated anti-CD13, anti-CD34, anti-CD44, anti-CD73, anti-CD90, anti-CD117, and unconjugated antibodies against CD105 (all from BD Biosciences, San Diego, http://www.bdbiosciences.com) and CD146 (Chemicon, Temecula, CA, http://www.chemicon.com).

Transplantation of the SPCs into the Neonatal oim Mice
The Institutional Animal Care and Use Committee of Penn State University College of Medicine approved all animal procedures. The oim used here for cell transplantation was described previously [27, 28]. Prior to cell injection, neonatal oim mice were sublethally irradiated with 350 rads using the gamma irradiator GC-200 (MDS Nordion, Ottawa, http://www.mds.nordion.com). Four hours after irradiation, the mice were injected with 5 x 10⁴ eGFP-positive SPCs via the superficial temporal vein as described previously [12, 29].

Tracking of the GFP-Positive SPCs Injected into Neonatal Mice
The neonatal mice that received the cells were sacrificed at 2 and 4 weeks after cell transplantation. Cells were tracked by gross examination of the harvested tissues under the fluorescent microscope, isolation of the cells from the tissues, and histological examination of the tissue sections made from the tissues harvested from the recipient mice as described previously [14].

Retrieval of Cells from the Recipient Mice
For cell retrieval, the methods described previously were followed [14]. The whole bones after marrow flush were cut into small pieces and placed in Petri dishes containing DMEM, 20% FBS, and 50 µg/ml ascorbic and were maintained as tissue explants. Soft tissues were minced into small pieces and treated with trypsin in Hanks' balanced salt (HBSS) solution at 2 mg/ml for 2 hours. This was followed by collagenase treatment (type 1 collagenase; Sigma-Aldrich) at 2 mg/ml in Hanks' balanced salt solution for 2 hours. The enzyme digests were filtered through a nylon membrane, and the cells were plated in Petri dishes. All cells were maintained in culture in DMEM, supplemented with 20% FBS, 50 µg/ml ascorbic acid, 1% (vol/vol) P/S. After 1 week of culture, 25 µg/ml Zeocin was added to select for the GFP+ cells.

FACS Analysis and Sorting for GFP+ Cells Retrieved from the Various Tissues of the Recipient Mice
The cells retrieved from various tissues and organs of the recipient mice at 4 weeks were expanded in culture in presence of Zeocin for 7 days. The expanded cells were sorted by FACS for the GFP+ donor cells prior to their use for genotyping and gene expression analysis.

Gene Expression Analysis of the Retrieved Cells
Total RNA was extracted from 1 x 10⁶ cells of the GFP+ cells retrieved from various tissues and sorted by FACS. The RNA was extracted using RNeasy (Qiagen, Valencia, CA, http://www.qiagen.com) per the manufacturer's instructions. The mRNA was reverse-transcribed to cDNA using SuperScript First-Strand Synthesis System for reverse transcriptase-polymerase chain reaction (PCR) (Invitrogen) per the manufacturer's instructions. cDNA was amplified using an Eppendorf Thermal Cycler at 94°C for 30 seconds, 60°C–65°C for 30 seconds, and 72°C for 50 seconds for 30–35 cycles, after initial denaturation at 94°C for 5 minutes. All the primer sequences that were used were determined using the established GenBank sequences and are indicated in supplemental online Table 1. Triplicate PCRs were amplified using primers designed for β-actin as a control for assessing PCR efficiency.

Real-Time Reverse Transcriptase-Polymerase Chain Reaction
For analysis of the ratio of GFP+ donor cells in the bones of the recipient mice, genomic DNA from the tibias of the recipient mice was isolated using DNeasy tissue kit (Qiagen). PCR assays for the mouse albumin and EGFP gene were performed using a 25-µl reaction mixture that contained 12.5 µl of SYBR Green PCR Master Mix (Qiagen), 200 ng of DNA template, and forward and reverse primers. The mouse albumin forward primer was 5'-GAAAACCAGGCGACTATCTCCA-3'; mouse albumin reverse primer was 5'-TGCACACTTCCTGGTCCTCA-3'; EGFP gene forward primer was 5'-TCCAGGAGCGCACCATCTT-3'; and the EGFP reverse primer was 5'-TGCCGTTCTTCTGCTTGTCG-3. The relative quantitative value of the target EGFP gene was normalized to an endogenous control albumin gene and calculated with the comparative C_T method [30].

Genotyping of the Cells Retrieved from the Various Tissues of the Recipient Mice
A genotyping approach that is used to distinguish the three mice genotypes (wild-type, heterozygous, and homozygous) based on the absence of the G deletion at nucleotide 3,983 in the pro2(I) collagen gene was used to assess the potential of donor cells to fuse with the endogenous cells in vivo [27, 31]. In this approach, a PCR strategy dependent on the differential hybridization of two similar and distinguishing primers, the wild-type and oim primers, was used [31]. Total genomic DNA was isolated from the 1 x 10⁶ of the GFP+ cells retrieved from bone, heart, lung, and ribs and used for genotyping. The genotyping procedure is fully described by Saban and King [31].

Histological Analysis
For histological analysis of the GFP+ cells in vivo, a method described previously was used [14, 32]. Briefly, tissues harvested from the recipient mice were immediately fixed in freshly prepared 4% paraformaldehyde in PBS, containing 10% sucrose [33] and kept at 4°C for 24 hours in the dark. After 24 hours, the bones were rinsed in PBS and then demineralized in 0.5M EDTA in PBS containing 10% sucrose at 4 °C for 48 hours in the dark. The bone samples were washed in PBS and slowly frozen in cold isopentane cooled on a dry ice bath. Frozen tissues were embedded in Tissue-Tek optimal cutting temperature Compound, and 10-µm sections were cut and mounted on glass slides. The slides were directly observed under a fluorescent microscope without the coverslips, but the sections were kept hydrated in PBS to reduce autofluorescence. Images were acquired using the Spot RT SE digital camera (Diagnostics Instruments Inc., Sterling Heights, MI, http://www.diaginic.com). Some tissue sections were stained by a modified Masson trichrome [34] and also with hematoxylin and eosin.

Immunofluorescence for eGFP
Cryosections prepared from the femurs and tibias of the recipient mice at 4 weeks after cell injection were fixed in cold acetone for 5 minutes and treated with 10% goat serum, followed by treatment with a polyclonal antibody specific for GFP (1:500; Abcam, Cambridge, MA, http://www.abcam.com). For visualization, the sections were treated with the secondary rabbit anti-rat antibodies conjugated with Cy3 at a concentration of 1:1,000 (Chemicon).

Collagen Analysis in the Bones of the Recipient Mice
To determine whether the cells that engrafted in the bones of the recipient mice synthesized type I collagen composed of 1(I) and 2(I) heterotrimers, the bones from the recipient mice were powdered, suspended in 0.5 M acetic acid, and treated with pepsin. The collagens were extracted from the bones at 4°C for 18–24 hours, centrifuged, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein Microsequencing by Mass Spectrometry
Protein bands migrating in the position of 2(I) chains on SDS-PAGE gels were excised and subjected to mass spectrometry after pepsin digestion. The tryptic peptides identified by mass spectrometry were compared with the published mouse pro2(I) sequences.

	RESULTS

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Characterization of the Selected Single-Cell Expanded Progenitors
Single-cell cloning by limited dilution resulted in the isolation of eight SPCs. All eight SPCs were initially characterized for cell surface marker expression, differentiation toward osteogenic and adipogenic cell lineages in vitro, and distribution in developing mice following systemic transplantation. All the progenitors demonstrated migration to bone with variable efficiencies (data not shown). One clone that demonstrated a higher efficiency of lodging into skeletal tissues following transplantation into developing mice was selected for further characterization. Figure 1 shows the morphological appearance, cell surface antigen expression, and differentiation toward osteogenic and adipogenic cell lineages in vitro of the selected SPCs. Figure 1A shows a single cell in a six-well plate, and Figure 1B shows morphological appearance of the expanded SPCs in comparison with the original mixed cell population (MSCs) (Fig. 1C). The expanded progenitors consisted of a population of small spindle-shaped cells. Fluorescence-activated cell sorting using multiple surface epitopes demonstrated that the single-cell-expanded progenitors were CD34-, CD45-, CD90-, CD117-, and CD146-negative (Fig. 1D). The cells expressed high levels of CD13 and CD44 and moderate CD73. Interestingly, the cells expressed low levels of CD105 (Fig. 1D). Expression of these surface antigens was maintained throughout the cell expansion in vitro.

View larger version (51K): [in this window] [in a new window]   Figure 1. Single-cell-expanded progenitors. (A): A GFP+ single cell. (B): SPCs at 30 population doublings. (C): Mixed population of MSCs. (D): CD markers of the SPCs and the MSCs. (E): Alkaline phosphatase activity of the SPCs. (F): Alizarin red S staining of the SPCs. (G): Gene expression of the SPCs after 21 days of culture in OM. (H): Adipogenesis of the SPCs. (I): Expression of the adipocyte-specific genes by the SPCs. Original magnification (A, B, C, F, H), x200. The control cells were not cultured in OM or AM, respectively. Abbreviations: AM, adipogenic medium; BMP, bone morphogenetic protein; CD, cluster of differentiation; GFP, green fluorescent protein; LPL, lipoprotein lipase; OCN, osteocalcin; OM, osteogenic medium; OPN, osteopontin; OSX, osterix; PPAR , peroxisome proliferator activated receptor; SPC, single-cell-expanded progenitor cell.

Transplantation of the Selected Progenitors into Developing OI Mice and Histological Analysis
The distribution of the progenitors following systemic transplantation into developing neonatal OI mice was assessed. The cells transplanted into the developing OI mice extravasated into several skeletal tissues, mostly all the bones of the recipient mice. The donor cells were present in all the femurs, tibias, fore limbs, scapulas, and ribs of the recipient mice (Table 1). All the recipient mice showed the presence of the donor cells in the lungs (Table 1). In bone, cell engraftment was variable in different mice; the engraftment ranged from 0.3% to 28% of total cells in a given bone chip of different recipient mice as determined by quantitative PCR for the GFP at 4 weeks following cell transplantation (Table 1). Tissue sections made from the tibia of the recipient mouse showed that the GFP+ cells were distributed in the entire tibia, the ends of the bones toward the knee joint and toward the foot demonstrating higher concentration of the donor cells (Fig. 2B). Immunofluorescence staining for GFP in the equivalent tibia tissue section showed that the GFP fluorescence was due to the engrafted cells, not autofluorescence (Fig. 2C). Overlay of the images in Figure 2B and 2C showed that the immunofluorescence staining for GFP was in precisely the same location as the GFP fluorescence, confirming that the observed fluorescence in tibia was the result of the engrafted GFP+ donor cells, not autofluorescence (Fig. 2D). This was further confirmed by the absence of immunofluorescence staining for GFP in a tissue section from a tibia of a mouse that did not receive the cells (Fig. 2A). Tissue sections made from the femur of the recipient mouse showed similar findings (Fig. 2E–2J). In the femur, the GFP+ donor cells were present in whole bone; in the diaphysis, cells were present in the metaphysis and diaphysis toward the knee joint in this particular mouse (Fig. 2E, 2H). Immunofluorescence staining for GFP and the overlay image confirmed that the GFP fluorescence in the femur tissue sections is in precisely the same location as the immunofluorescence staining for GFP (Fig. 2F, 2I). These data reinforce the conclusion that the donor cells colonized the entire tibias and femurs of this particular recipient mouse. Tissue sections made from a recipient mouse with approximately 2% donor cells are shown in Figure 2K and 2L for the femur and tibia, respectively. The donor cells are located at the ends of the bones (Fig. 2K, 2L).

View larger version (46K): [in this window] [in a new window]   Figure 2. Distribution of GFP+ cells in tibias and femurs of recipient osteogenesis imperfecta (OI) mice at 4 weeks post-transplantation. (A): Tibia section from a control OI mouse. (B): Tibia section from a recipient mouse. (C): Immunofluorescence staining for GFP. (D): Overlay of (B, C) images. (E, H): Femur sections of a recipient mouse. (F, I): Immunofluorescence staining for GFP. (G, J): Overlay images. (K, L): Distribution of GFP+ donor cells in a femur and tibia of a mouse with approximately 2% cell engraftment. Tissues were harvested from the recipient mice at 4 weeks after single-cell-expanded progenitor cell transplantation into the 2-day-old OI mouse. Immunofluorescence staining for GFP was visualized by a secondary antibody conjugated to Cy3. The control tissue section was from a mouse that did not receive the cells, but the section was treated with both primary and secondary antibodies. Original magnification in all images, x40. Abbreviation: GFP, green fluorescent protein.

View larger version (81K): [in this window] [in a new window]   Figure 3. Higher magnifications and confocal microscopy. (A): A higher-magnification image of an area of the tibia tissue section shown in Figure 2B. White arrow, cells on bone surface, presumed to be osteoblasts. (B): Equivalent image stained with H&E. (C): Overlay of (A, B) images. (D): An area of a tibia from the recipient mouse showing GFP+ cells. (G): The boxed area in (D) examined under confocal microscopy showing GFP+ rounded cells in bone. (E, H): Images from (D, G) stained with DAPI. (F): Overlay of (E, D) images. (I): Overlay of the confocal images (G, H) to show the location of the donor cells and the endogenous cells in bone. Shown are endogenous cells (yellow arrows), donor cells (red arrows), marrow (green arrowheads), muscle (white arrowheads), periosteum (yellow arrowheads), and bone (blue arrowheads). Original magnifications, x400 (A–C), x100 (D–F), and x400 (G–I). The GFP used here is not nuclear-localized, although it appears to cover the nucleus in the overlay image. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

View larger version (97K): [in this window] [in a new window]   Figure 4. Modified trichrome staining, histology, and collagen analysis in the bones of the mouse that received the single-cell-expanded progenitor cells (SPCs) at 4 weeks following transplantation. (A): Stained tibia section of a control OI mouse. (B): Stained tibia section from the recipient mouse. (C): Equivalent section stained with H&E. (D): Stained femur section of a control mouse. (E): Stained femur section of the recipient mouse. (F): Equivalent section stained with H&E. (G): SDS-polyacrylamide gel electrophoresis of the collagens extracted from the indicated mice. (H): Amino acid-deduced seq. of the peptides derived from the protein band excised from the gel of collagens harvested from the SPC recipient mouse. The recipient mouse synthesized 2(I) chains. Original magnification for all tissue sections, x40. The control mouse did not receive the cells. Abbreviations: OI, osteogenesis imperfecta; Seq., sequence. Boxed area in C is equivalent to Figure 2B.

View larger version (38K): [in this window] [in a new window]   Figure 5. Retrieval, fluorescence-activated cell sorting (FACS) analysis, and sorting of the GFP+ cells from the various tissues of the recipient osteogenesis imperfecta (OI) mouse 4 weeks following cell transplantation. (A): Bone chips in culture from the tibia of the OI mouse transplanted with the GFP+ single-cell-expanded progenitor cells. (B–E): Morphological appearances of the cells retrieved from various tissues. (F): FACS analysis based on GFP expression by the cells retrieved from various tissues of the recipient mouse. The FACS analysis profile showed that the GFP+ cells retrieved from various tissues and expanded in culture in presence of Zeocin were more than 99% GFP+. Red arrow, bone chip from a mouse that did not receive the cells; green arrow, bone chip from a recipient mouse. Abbreviation: GFP, green fluorescent protein.

View larger version (43K): [in this window] [in a new window]   Figure 6. Genotyping and gene expression analysis of the cells retrieved from the various tissues of the recipient OI mouse at 4 weeks following cell transplantation. (A): A genotyping profile of the WT, OI, and Hetero mice using genomic DNA harvested from the toe clips. (B): Results from the genotyping of green fluorescent protein (GFP)+ cells from the lung, bone, heart, and the ribs of the recipient mouse. The genotyping profile of the cells retrieved from various tissues demonstrated a WT genotype, suggesting that the donor cells retrieved from OI recipient mouse tissues did not fuse with the endogenous cells. (C): Expression profile of specific genes by the cells retrieved from the indicated tissues at 4 weeks. The GFP+ cells were retrieved from the respective tissues as described in Materials and Methods and were then subjected to fluorescence-activated cell sorting prior to genotyping and gene expression analyses. Abbreviations: BMP, bone morphogenetic protein; bp, base pairs; Hetero, heterozygous; OCN, osteocalcin; OI, osteogenesis imperfecta; OPN, osteopontin; OSX, osterix; SP, surfactant protein; SPC, single-cell-expanded progenitor cell; WT, wild-type.

	DISCUSSION

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

The conventional methods for isolating bone marrow-derived stromal cells involve flushing the marrow from the bones followed by plating cells on tissue culture plates. The cells that adhere to the plates are believed to represent the MSCs. In this report, we modified this approach; to make sure that potential progenitors were not excluded in the preparation, cells were scraped from the bone surfaces and cultured with a mixture of the marrow cells. This approach yields cells that expressed high levels of alkaline phosphatase activity upon treatment with hrBMP-2. The SPCs expressed some of the markers that are believed to characterize MSCs [38–40]. The cells were negative for CD90 and CD146 and expressed low levels of CD105, the markers attributed to MSCs. The finding that the SPCs were negative or expressed low levels of these markers suggests that the cells isolated here may represent lineage committed progenitors or a unique population of MSCs in the bone or marrow with the described characteristics. The former may be the correct explanation because the cells expressed low levels of osteoblasts differentiation markers Runx2 and osteopontin, indicating that they may represent committed progenitors. Because they expressed low levels of Runx2 under basal conditions, we have called these cells progenitors. The cells exhibit the potential to give rise to osteoblasts as well as adipocytes in vitro and showed the potential to give rise to other cell types in vivo. These data suggest that the cells are at least bipotential.

The level of engraftment of the SPCs in different mice was variable; in some mice the level of engraftment was very high and in others it was very low. Although the level of engraftment was variable, in all cases, the donor cells were present in the bones of the recipients. The variability in bone engraftment among different mice is not clear; all the experimental mice were of the same age when they received the cells. The variability in engraftment may be related to technical errors in cell injection or differences in immune response elicited by different mice to the transplanted cells. All the mice were sublethally irradiated prior to cell injection; perhaps lethal irradiation of the recipients would generate more consistency in cell engraftment among different mice.

In summary, we have demonstrated that the SPCs engrafted into various skeletal tissues, including the bones of the developing OI mice, extravasated, differentiated into osteoblasts, synthesized bone extracellular matrix, and made bone in vivo. This is the first demonstration, as far as we are aware, that progenitors delivered systemically will engraft and deposit bone in vivo. Transplantation of these cells, however, was more efficient in developing OI mice than in adult mice. Because of the variability of cell engraftment in different mice, approaches to increasing cell engraftment in the bones will be needed before this approach can be used to treat bone diseases using progenitors.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Drs. Robbins and Mi of the University of Pittsburgh for the generous gift of the DFG-eGFP-Zeocin retrovirus. This work was supported by NIH grant number R01 AR049688. FACS analysis and cell sorting was performed at the Penn State College of Medicine Core Facility.

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