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First published online May 4, 2006
Stem Cells Vol. 24 No. 8 August 2006, pp. 1869 -1878
doi:10.1634/stemcells.2005-0430; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Progenitors Systemically Transplanted into Neonatal Mice Localize to Areas of Active Bone Formation In Vivo: Implications of Cell Therapy for Skeletal Diseases

Xujun Wang, Feng Li, Christopher Niyibizi

Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, Pennsylvania, USA

Key Words. Osteogenesis imperfecta • Cell therapy • Progenitors • Mesenchymal stem cells

Correspondence: Christopher Niyibizi, Ph.D., Departments of Orthopedics and Rehabilitation and Biochemistry and Molecular Biology, Division of Musculoskeletal Sciences, Penn State College of Medicine, Mail Code H089, 500 University Drive, Hershey, Pennsylvania 17033, USA. Telephone: 717-531-5649; Fax: 717-531-7583; e-mail: cniyibizi{at}psu.edu

Received September 2, 2005; accepted for publication April 28, 2006.
First published online in STEM CELLS EXPRESS   May 4, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
The potential of cell or gene therapy to treat skeletal diseases was evaluated through analysis of transplanted osteoprogenitors into neonatal homozygous and heterozygous osteogenesis imperfecta mice (oim). The osteoprogenitors used for transplantation were prepared by injection of mesenchymal stem cells (MSCs) marked with the green fluorescent protein (GFP) into normal mice with the subsequent retrieval of the cells at 35 days. The retrieved cells referred to here as osteoprogenitors were expanded in culture and transplanted into the 2-day-old oim mice via the superficial temporal vein. The recipient mice were evaluated at 2 and 4 weeks after cell transplantation. Four weeks after transplantation, tissue sections made from femurs and tibias of oim mice showed that the GFP-positive (GFP+) cells were distributed on the surfaces of the bone spicules in the spongiosa, the area of active bone formation. In the diaphysis, the GFP+ cells were distributed in the bone marrow, on the endosteal surfaces, and also in the cortical bone. Immunofluorescence localization for GFP confirmed that the fluorescence seen in tissue sections was due to the engrafted donor cells, not bone autofluorescence. Gene expression analysis by polymerase chain reaction of the GFP+ cells retrieved from the bones and marrow of the recipient mice demonstrated that the cells from bone were osteoblasts, whereas those from bone marrow were progenitors. These data demonstrate that MSCs delivered systemically to developing osteogenesis imperfecta mice engraft in bones, localize to areas of active bone formation, differentiate into osteoblasts in vivo, and may contribute to bone formation in vivo.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
Stem cells hold promise for the treatment of many diseases, including those with and without genetic links. Studies using human embryonic stem cells are limited because of ethical concerns. However, adult-derived stem cells—particularly those harvested from bone marrow—are believed to possess some characteristics of embryonic stem cells [1, 2]. These cells, referred to as mesenchymal stem cells (MSCs), are found in many tissues and organs, including fat, muscle, and many others [3–7, 8]. MSCs have been shown to give rise to cell phenotypes such as osteoblasts, chondrocytes, adipocytes, neurons, and other differentiated cell types in vitro and, to a lesser extent, in vivo [5, 6, 7, 9, 10, 11].

The contribution of transplanted MSCs to tissue cell phenotypes and the potential for regeneration of the target tissues has not been clearly demonstrated [12, 13]. This is mainly because the biology of MSCs and their engraftment characteristics, differentiation, and survival in vivo are poorly understood. Direct injection of the cells into specific target tissues or organs has shown that these cells may contribute to the tissue cell phenotype and possible repair and regeneration of the target tissues or organs [14–19]. Homing and engraftment of the systemically delivered cells into the skeletal tissues, however, remains highly debated [13]. In some studies in which MSCs were delivered systemically—usually through the tail vein in mice—the cells were shown to engraft in the bones of the recipient mice [20–25]. The number of cells that engraft in the skeletal tissues of recipient animals is very small and is usually detected using sensitive assays that do not reveal the extent of the engraftment or the contribution to the tissues' cell phenotype. Most of the transplanted cells become trapped in organs or tissues of the recipient animals, and few or no cells migrate to the skeletal tissues, especially bone [13, 26–30]. It is not known whether the cells that do migrate to the skeletal tissues give rise to the cell phenotype of the tissues or organs in which they engraft.

Osteogenesis imperfecta is a genetic brittle bone disease caused by mutations in the genes that encode the polypeptide chains of type I collagen, the major structural protein of bone [31]. Previous studies in mice suggested that MSCs delivered systemically migrate and engraft in the bones and that this approach has potential for the treatment of osteogenesis imperfecta (OI) phenotypes [20]. In fact, clinical trials using whole marrow or cells enriched in MSCs have been attempted in children with osteogenesis imperfecta, and the results suggested that the cells could contribute to the growth and structural integrity of the bones of the recipients [32, 33, 34]. In these studies, however, it was not demonstrated where the transplanted cells were located in vivo, and their participation in bone formation was not demonstrated.

We previously reported that murine MSCs marked with the enhanced green fluorescent protein (eGFP) gene and transplanted into normal neonatal mice migrate and engraft in the bones of the developing mice [35]. We also reported that repeated injection of cells recovered from the bones of the recipient mice into different neonatal mice resulted in the isolation of a population of cells with a predilection to engraft in the bones of the developing mice. In these previous studies, normal mice were used for the cell transplantation, and the number of cells that engrafted in bone was quite low. In addition, the location of the cells in vivo, their phenotype, and their differentiation into tissue-specific manner were not demonstrated.

In this study, we used these eGFP-marked retrieved cells that were kept as frozen stocks to evaluate their engraftment and differentiation in the bones of the developing mouse model of OI. Here, we report the location and differentiation of the eGFP+ cells transplanted in developing homozygous and heterozygous mouse models of OI and the implications for skeletal regeneration.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
Preparation of Osteoprogenitors
The murine MSCs used here were previously isolated from (B6C3Fe a/a) 8-week-old mice and were transduced with a retrovirus carrying eGFP and Zeocin-resistant genes as described previously [35]. The osteoprogenitors used in the present studies were derived from this cell preparation and kept as frozen stocks. The detailed methods for the derivation of the osteoprogenitors used in the present studies are described in detail in the previous publication [35] and are only briefly outlined here.

Briefly, MSCs transduced with a retrovirus carrying eGFP and Zeocin-resistant genes were cultured in a medium supplemented with 25 µg/ml Zeocin with medium changes every 3 days. The selected cells were suspended in saline solution at 5 x 106 cells per ml, and an aliquot of 5 x 104 cells was drawn up in a 0.5-ml syringe equipped with a 30-gauge needle and injected into the 2-day-old mice via the superficial temporal vein [35, 36]. The cells that migrated into the bones were retrieved after 25 days, expanded in culture under Zeocin selection, and reinjected in different neonatal mice. The cells that migrated to bone were again retrieved, expanded in culture under Zeocin selection, and reinjected into neonatal mice. After 35 days, the green fluorescent protein-positive (GFP+) cells were retrieved from bone and expanded in culture under selection for GFP+ cells. Aliquots of the cells were cryopreserved for future use.

Osteogenic Differentiation of the Retrieved Cells
The cryopreserved osteoprogenitors (D35) were revived and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) (v/v), and 50 µg/ml ascorbic acid and expanded in culture. At confluence, the cells were trypsinized, and aliquots of 2 x 104 cells were plated in six-well plates and then cultured in an osteogenic medium. The osteogenic medium consisted of DMEM supplemented with 10% FBS, 50 µg/ml ascorbic acid, 10 mM ß-glycerol phosphate, 10–7 M dexamethasone (Decadron; Merck & Co., Whitehouse Station, NY, http://www.merck.com), and 1% P/S. The cells were maintained in culture, with medium changes every 3 days for 21 days. Then the media were removed, and the cells were rinsed in phosphate-buffered saline (PBS), fixed in 10% formalin, and stained in Alizarin red [37]. Alizarin red S was prepared in distilled water at a concentration of 40 mM and then adjusted to a pH of 4.1. The plates were treated with the Alizarin solution and incubated for 20 minutes at room temperature. After 20 minutes, the plates were rinsed in distilled water and were then examined under light microscope and photographed.

Adipogenic Differentiation
For adipogenic differentiation, osteoprogenitors were plated in six-well plates in adipogenic medium at a cell density of 1 x 104 cells per cm2. The adipogenic medium was composed of DMEM supplemented with 10% FBS, 10–6 mM dexamethasone, 50 µM indomethacin, and 0.5 mM isobutyl-methylxanthine [38]. The media were replaced every 3 days until day 14. Adipogenic differentiation was assessed by oil red O staining at 2 weeks after initial adipogenic induction. For oil red staining, the cells were rinsed in PBS and fixed in 10% formalin, followed by incubation of the cells in 2% (w/v) oil red O reagent for 5 minutes at room temperature. The cells were rinsed in 70% ethanol, followed by several changes of distilled water. The cells were observed under a light-inverted microscope and photographed.

Fluorescence-Activated Cell Sorting Analysis
The procedure for fluorescence-activated cell sorting (FACS) analysis described previously by Lee et al. [39] was used. In brief, a total of 2 x 105 cultured osteoprogenitors cells were resuspended in 200 µl of Dulbecco's PBS containing 2% FBS and 0.01% NaN3 and incubated for 30 minutes at 4°C with phycoerythrin (PE)-conjugated murine anti-CD45 or anti-CD105 monoclonal antibodies (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) followed by anti-rat PE-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). The proper isotype-identical Igs served as controls. After staining, the cells were fixed in 2% paraformaldehyde, and quantitative FACS analysis was performed on a FACStar flow cytometer (BD Biosciences, San Diego, http://www.bdbiosciences.com). The cells retrieved from bone and bone marrow of the recipient mice at 4 weeks and selected in a medium supplemented with 25 µg/ml Zeocin were also subjected to FACS analysis to determine whether the concentration of the Zeocin used was 100% lethal to the endogenous cells.

Irradiation and Transplantation of Progenitors into Neonatal Mice
The mouse model of OI used here for cell transplantation was described previously [9, 40, 41]. The heterozygous mice were crossbred to generate the three phenotypes—wild type, heterozygous, and homozygous. The mice were genotyped using DNA extracted from the toe clips following the described methods [42] to distinguish wild-type mice from heterozygous and homozygous mice. Initially, mice were transplanted with the cells without prior irradiation; however, because of low cell engraftment in bone, the subsequent experiments were performed with the sublethally irradiated neonates. For irradiation, the neonatal mice were placed in an irradiation chamber, then sublethally irradiated with 350 rads before cell transplantation using the gamma irradiator GC-200 (MDS Nordion, Ottawa, ON, Canada, http://www.mds.nordion.com). Four hours later, the mice were transplanted with the osteoprogenitors via the superficial temporal vein as described previously [35, 36]. Twenty-five homozygous and 25 heterozygous mice were injected with the GFP+ osteoprogenitors. Three normal mice were also transplanted with the cells after irradiation.

Tracking of the GFP+ Cells Injected in Neonatal Mice
The neonatal mice that received the cells were sacrificed at 14 and 28 days after transplantation. Cells were tracked by gross examination of the harvested tissues under the fluorescent microscope, isolation of the cells from the tissues, or histological examination of the tissue sections made from the femurs and tibias of the recipient mice.

Gross Examination of the Harvested Tissues from the Recipient Mice
Femurs, tibias, forelimbs, and lungs were harvested from the recipient mice at 14 and 28 days after cell injection and were examined under a fluorescent microscope for GFP detection using the Olympus IX71 microscope (Olympus, Melville, NY, http://www.olympusamerica.com). The images were acquired using the Spot SE digital camera (Diagnostic Instruments, Sterling Heights, MI, http://www.diaginc.com). This approach was used as an initial screening to determine which of the tissues or organs contained transplanted cells. The mice that showed GFP+ cells were used for the cell harvest and histological tissue sections. The right femurs and tibias were used for cell harvest, and the left femurs and tibias were used for histological analysis.

Retrieval of Cells from the Recipient Mice
For cell isolation, marrow was flushed from the right femurs and tibias using a syringe containing DMEM and 20% FBS into 25-mm Petri dishes. The marrow was cultured in DMEM supplemented with 20% FBS and 50 µg/ml ascorbic acid. To isolate cells associated with bone, after a marrow flush, the bones were cut into small pieces and placed in Petri dishes containing DMEM, 20% FBS, and 50 µg/ml ascorbic acid. Soft tissues were minced and treated with trypsin in Hanks' solution (type 1; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 2 mg/ml for 2 hours. This was followed by collagenase treatment with 2 mg/ml in Hanks' solution for 2 hours. After enzyme digest, the 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% (v/v) P/S, and 25 µg/ml Zeocin to select for the GFP+ cells. The isolated cells were characterized for gene expression of known osteoblast- and chondrocyte-specific genes. The cells retrieved from the lung were analyzed for gene expression of lung surfactants A and D [43].

Gene Expression Analysis of the Recovered Cells
Total RNA was extracted from 1 x 106 of the retrieved cells using RNAEasy (Qiagen, Hilden, Germany, http://www1.qiagen.com) per the manufacturer's instructions. The mRNA was reverse-transcribed to cDNA using SuperScript First-Strand Synthesis System for reverse transcription-polymerase chain reaction (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) per the manufacturer's instructions. The cDNA was amplified using a ABI GeneAmp polymerase chain reaction (PCR) System 2400 (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) at 94°C for 30 seconds, 60°C–65°C for 30 seconds, and 72°C for 50 seconds for 30 to 35 cycles, after initial denaturation at 94°C for 5 minutes. All primer sequences that were used were determined using established GenBank sequences and are indicated in Table 1. Triplicate PCRs were amplified using the designed primers, and the ß-actin-designed primers were used as a control for assessing PCR efficiency. The PCR fragments were analyzed by agarose gel electrophoresis. Band intensities were digitally quantified using NIH Image J 1.33 and normalized to that of an internal control, ß-actin.


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  		Table 1. Primers used for the determination of the gene expression of the indicated genes

 
Histological Analysis for GFP
For the histological analysis of the GFP+ cells in vivo, we modified a method described by Harms et al. [44]. The procedure maintains GFP fluorescence throughout decalcification process and reduces the background. Left femurs and tibias harvested from the recipient mice were immediately fixed in the freshly prepared 4% paraformaldehyde in PBS containing 10% sucrose [45] and kept at 4°C for 24 hours in the dark. The bones were then rinsed in PBS and demineralized in 0.5 M EDTA in PBS containing 10% sucrose at 4°C for 48 hours in the dark. The tissues were then washed in distilled water and slowly frozen in cold isopentane cooled on a dry ice bath. Frozen tissues were embedded in optimal cutting temperature compound, and 10-µm sections were cut and mounted on glass slides. The slides were directly observed under a fluorescent microscope (Olympus IX71) without the cover slips, but the sections were kept hydrated in PBS. Images were acquired using the Spot RT SE digital camera. Some tissue sections were stained with H&E, and the images were overlaid with images taken using the filter for GFP detection.

Immunofluorescence
Tissue sections prepared from the femur and tibia of the recipient mice at 4 weeks after cell injection were treated with polyclonal antibodies specific for GFP (Abcam, Cambridge, MA, http://www.abcam.com). For immunofluorescence localization, the tissue sections were treated with cold acetone for 5 minutes and washed three times for 5 minutes each time and then blocked with 10% donkey serum for 1 hour at room temperature. Rabbit anti-GFP primary antibodies were added to the tissue sections (1:250) and incubated overnight. Tissue sections were then washed in PBS Tween 0.05%, followed by addition of the secondary antibody, goat anti-rabbit conjugated to Cy3 (1:500) (Santa Cruz Biotechnology Inc.) for 1 hour. The slides were washed in PBS and observed under a microscope.

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


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
The osteoprogenitor cells used here were prepared during the previous study and preserved in liquid nitrogen [35]. These cells are referred to here as D35 osteoprogenitors. The morphological appearance of the cells, and their gene expression profiles are shown in Figure 1. The original (parental) cells are GFP+ (Fig. 1A), and the morphological appearance of the osteoprogenitors is shown in Figure 1B. Gene expression analysis of the cells by PCR using specific primers for osteoblast- and chondrocyte-specific genes demonstrated that the cells expressed osteopontin and low levels of Sox 9 and Runx2 (Fig. 1C). The cells did not, however, express osteocalcin or osterix, the specific markers of mature osteoblasts [46] (Fig. 1C).


Figure 1
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  		Figure 1. Morphological appearance and gene expression profile of the original cells and the D35 progenitors. (C): By polymerase chain reaction analysis, the original cells (A) express type IIA collagen gene but the D35 (B) express low levels of BMP-2, which is not expressed by the original cells. Original magnification x100. Abbreviations: BMP, bone morphogenetic protein; Ori, original.

 
Characteristics of the D35 Progenitors
The D35 progenitors used here for transplantation possessed MSC characteristics. The cells deposited calcium in the matrix when cultured in osteogenic medium, as demonstrated by the intense Alizarin red staining (Fig. 2A). The cells also differentiated toward adipogenic lineage when they were cultured in adipogenic medium (Fig. 2B). FACS analysis to identify markers of hematopoietic cell phenotype demonstrated that the cells were devoid of hematopoietic cells, as demonstrated by the absence of cells expressing CD45 antigen (Fig. 2C). The cells also displayed high levels of CD105 antigen (Fig. 2D). These properties are consistent with the cells described previously [35].


Figure 2
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  		Figure 2. Characteristics of the D35 progenitors. (A): Alizarin red S staining. (B): Oil red O staining. (C, D): Fluorescence-activated cell sorting analysis for CD45 and CD105, respectively. The cells are CD45-negative, but they express high levels of CD 105 antigen, as shown in (D). Original magnification (A, B) x200.

 
Transplantation of GFP+ Cells into Neonatal Mice
The GFP+ D35 progenitors were transplanted into the homozygous or heterozygous neonatal mice via the superficial temporal vein at 2 days after birth [35, 36]. The temporal vein is visible in newborn mice before 4 days after birth. Gross examination of the bones at 14 days for GFP fluorescence demonstrated that the cells were present in the femurs and tibias of the recipient mice (data not shown). At 28 days after transplantation, tibias, femurs, and forelimbs harvested from the recipient homozygous or heterozygous mice showed that GFP+ cells were present in all the bones. This approach was used to ascertain the mice that contained GFP+ cells for further analysis. Histological examination of the tissue sections made from the femurs harvested from the recipient mice at 28 days after cell transplantation demonstrated that the donor GFP+ cells were located on the bone spicules in the spongiosa, just below the growth plate in the epiphysis (Fig. 3A). Immunofluorescence localization for GFP in the tissue section shown in Figure 3A, using a polyclonal antibody specific for GFP, confirmed that the GFP+ cells seen in the femur tissue sections represent the donor cells and that this fluorescence is not the result of bone autofluorescence (Fig. 3B; overlay image, Fig. 3C). The data clearly indicate that the GFP+ cells are located on the surfaces of newly formed bone of the developing mice. The cells in the spongiosa are located on the surfaces of the bone spicules, suggesting that they are actively participating in the bone formation.


Figure 3
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  		Figure 3. Green fluorescent protein-positive (GFP+) donor cell distribution and immunofluorescence for GFP in femur tissue sections of a heterozygous mouse. (A): GFP+ cells in the spongiosa. (B): Immunofluorescence for GFP. (C): Overlay image of (A) and (B). (D): GFP+ cells on the endosteal surface (white arrows), cortical bone (red arrow), and bone marrow (yellow arrows). Original magnification x100. Abbreviations: B, bone; FH, femoral head; GP, growth plate; M, marrow.

 
In the bone cavity of the same femoral tissue section, GFP+ cells were distributed on the bone surfaces in the endosteum (Fig. 3D, white arrows), and some cells are also present in the bone marrow and cortical bone (Fig. 3D, yellow and red arrows, respectively). The GFP+ cells seen in the cortical bone are probably osteocytes, but this was not investigated further. Tissue sections made from the tibia of the homozygous recipient mice at 28 days demonstrated findings similar to the femurs.

The donor GFP+ cells were lined on the bone spicules in the spongiosa just below the growth plate at the knee joint (Fig. 4A). These data suggest that they were participating in the bone formation of the recipient mice. Some of the tissue sections made from tibias at 28 days after cell transplantation were stained with hematoxylin, and the resulting images were overlaid with the images taken under GFP detection (Fig. 4B). Examination of the marrow cavity in the diaphysis of the same tissue sections showed that some GFP+ cells were distributed on the endosteal surfaces, and some were also present in bone marrow (Fig. 4C). In Figure 4C, the GFP image was overlaid with the image taken under bright field to demonstrate the location of the GFP+ donor cells. Immunofluorescence localization for GFP in a tissue section made from the tibia of a different OI recipient mouse confirmed that the fluorescence observed in the tissue sections is associated with the donor cells (Fig. 4D, 4E). The data again show that the cells are located on the bone spicules in the active areas of bone formation.


Figure 4
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  		Figure 4. Green fluorescent protein-positive (GFP+) donor cell distribution and immunofluorescence for GFP in tibia tissue sections from a homozygous mouse. (A): GFP+ cells in spongiosa (white arrows). (B): Overlay of (A) with the same image stained with hematoxylin and eosin. (C): Overlay image of GFP with image taken under bright field. GFP+ cells on endosteal surface (white arrows), cortical bone and bone marrow (red arrow). GFP+ cells in spongiosa from a tibia of a different osteogenesis imperfecta mouse (D) and GFP immunofluorescence (E). Original magnification (A–C) x100; original magnification (D, E) x40. Abbreviations: FH, femoral head; GP, growth plate; B, bone; M, marrow.

 
The engraftment of the progenitors differed between the homozygous and heterozygous mice; of the 25 homozygous mice that received the cells, 14 died either immediately or a few days after cell injection. Nine of the surviving mice demonstrated the presence of the cells in bone or bone marrow. In other mice, either the cells were present in lung tissue, or there were no cells present in any tissues. In contrast, 25 heterozygous mice were injected, and 14 survived for analysis. Three of the 14 surviving mice demonstrated presence of the cells in bone. These data are summarized in Tables 2and 3.


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  		Table 2. Tissues and organs in which the donor cells were detected in homozygous mice

 


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  		Table 3. Tissues and organs in which the donor cells were detected in heterozygous mice

 
These data clearly show that the cells have preference to engraft in the bones of homozygous mice. The level of cell engraftment in heterozygous mice and homozygous mice that demonstrated cells in bone, however, did not appear to be different. Only three normal mice were injected with the cells, and there were few or no cells detected in the normal mice after 4 weeks of cell injection. Because of the small number of normal mice used here, no specific conclusion can be derived from these studies.

The left femurs and tibias were used for the tissue sections, and the right femurs and tibias of the same mice were used for the cell isolation. Figure 5 shows the morphological appearance of the donor cells retrieved from bone, bone marrow, and lung tissue (Fig. 5A, 5B, and 5C, respectively) at 28 days after cell transplantation into a homozygous mouse. As can be seen in these figures, the cells retrieved from bone, bone marrow, and lung exhibit distinct morphological appearances. The cells recovered from bone have the characteristic osteoblast morphological appearance (Fig. 5A). The donor cells recovered from the bone marrow are much smaller and more rounded than the cells recovered from bone or lung (Fig. 5B). The cells retrieved from the lung have more of a fibroblastic morphological appearance (Fig. 5C). These data suggest that cells that engraft in different tissues exhibit distinct morphological appearances, indicating that the cells differentiate into the cell phenotypes of the tissues or organs that they engraft.


Figure 5
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  		Figure 5. Morphological appearance and gene expression of cells retrieved from tissues of recipient mice. (A): Cells retrieved from bone. (B): Cells retrieved from bone marrow. (C): Cells retrieved from lung. (D): Gene expression analysis of retrieved cells. Original magnification x100. Abbreviation: BMP, bone morphogenetic protein.

 
Gene expression analysis showed that the cells retrieved from bone express osteoblast-specific genes, whereas those from bone marrow express genes characteristic of osteoprogenitors phenotype (Fig. 5D). The cells recovered from bone marrow expressed osteopontin, Sox 9, Runx2, and low levels of Osterix in addition to the type II A and type I collagen genes. The cells recovered from bone expressed high levels of osteocalcin and osterix, specific markers of mature osteoblasts [46] (Fig. 5D). These data clearly demonstrate that the GFP+ cells retrieved from bone are osteoblasts, and those recovered from bone marrow are osteoprogenitors, as they do not express osterix or osteocalcin, the markers of mature osteoblasts [46]. Surprisingly, the cells recovered from the lung expressed genes for lung surfactants A and D (Fig. 5D). The data suggest that the cells that migrated into the lung may have differentiated into lung epithelial cells, but this has not been confirmed [43].

FACS analysis was performed on the cells from the frozen stocks of the cells retrieved from the bone and bone marrow shown in Figure 5. The FACS analysis data showed that the cells retrieved from bone and bone marrow and selected in a medium supplemented with Zeocin were devoid of endogenous cells (Fig. 6A, 6B). These data clearly demonstrate that the Zeocin concentration used here was 100% lethal to the endogenous cells. Because there were no retrieved cells available before selection in a medium supplemented with Zeocin for analysis, FACS analysis was performed using cells from a new experiment, in which cells were retrieved from a tibia of a recipient homozygous mouse at 2 weeks after cell injection. At this point, the data showed that the donor cells made up approximately 1% of the total cells harvested from bone (Fig. 6C).


Figure 6
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  		Figure 6. Fluorescence-activated cell sorting (FACS) analysis of cells retrieved from bones of recipient mice. (A, B): FACS analysis profile of bone- and marrow-retrieved cells, respectively, after selection in a medium supplemented with 25 µg/ml of Zeocin. (C): FACS analysis of unselected cells retrieved from tibias of D35 cell recipient mouse at 2 weeks after cell injection.

 
Collagen Analysis
Analysis of the collagens harvested from the bones of the recipient mice after pepsin extraction did not reveal any presence of type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers. The data suggest that although the cells that were delivered systemically differentiated into osteoblasts in vivo, they were not sufficient in number to synthesize detectable type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers by the pepsin-extraction procedure used here. In contrast, when a sufficient number of these cells were directly infused into the femur cavities of OI mice, type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers was detected after pepsin extraction of the collagens from the femurs of the recipient mice (unpublished data). These data suggest that the transplanted cells are capable of synthesizing bone extracellular matrix in vivo if a sufficient number of cells are supplied.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosures
 Acknowledgments
 References
 
The data presented in this article have clearly demonstrated that murine progenitors will migrate and incorporate into the bones of the developing homozygous and heterozygous OI mouse model, differentiate into osteoblasts, and appear to participate in the bone formation of the recipient mice in vivo. The cells used here for transplantation were generated by repeated injection of the cells recovered from the bones of the recipient mice into the developing neonatal mice. This approach generated a population of progenitors that exhibited predilection to migrate and engraft in the bones of the developing mice.

In our previous publication, we showed that these cells migrated to bone, and no cells were detected in the lungs at 2 weeks after cell injection [35]. The present studies used cells from the frozen stocks of cells that were previously prepared. Because the cells were revived and expanded further in culture before they were used for transplantation, it appears that they may have undergone some changes. Indeed, we have observed that extended passaging of the cells in culture results in the loss of the cells' ability to migrate and engraft in bone (unpublished observation). Compared with the original (parental) cells, the D35 progenitors expressed low levels of osterix and bone morphogenetic protein (BMP-2) genes that were not detectable in the original cells. This property may explain the predilection of the cells' ability to migrate and engraft in the bones of the developing mice. The cells, however, still exhibited potential to differentiate into other cell phenotypes of mesenchymal lineages, including chondrocytes and adipocytes.

As far as we aware, this is the first report to clearly demonstrate histologically the location and compartmentalization of MSCs in bone when the cells are delivered systemically into developing mice. Most of the previous studies on systemic delivery of MSCs in animal models have used sensitive methods (for example, PCR) to detect the presence of the cells in bone [20–25]. In this article, we have shown that MSCs systemically transplanted into neonatal mice migrate and engraft in the bones and participate in the bone formation in vivo. The cells migrated and engrafted in all the bones examined, femurs, tibias, and even the forelimbs of the developing mice. Some of the transplanted cells differentiated into osteoblasts and osteocytes in vivo, whereas other cells remained in the bone marrow as progenitors. These findings are of great interest because they suggest that donor cells that remain in bone marrow may serve as a reservoir for osteoblasts during bone growth and repair. In utero cell transplantation has been attempted in animal models, and the data have shown that a large number of donor cells will distribute into different tissues and organs and that they differentiate into different cell phenotypes in vivo. The cell phenotype in which the cells differentiate is detected by the tissue or organ in which the cells engraft [47]. These previous data and our present findings suggest that systemic transplantation of MSCs into developing animals may lead to the migration and engraftment of the cells into the developing animals' tissues and organs.

The homozygous mice used here synthesize and deposit type I collagen in bone and other tissues composed of {alpha}1(I) homotrimers. Analysis of the collagens from the bones of the recipient mice by pepsin digestion at 2 and 4 weeks after transplantation did not reveal the presence of type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers. The failure to detect expression of type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers can be explained by the fact that although the donor cells differentiated into osteoblasts in vivo, they were not in sufficient numbers to synthesize detectable type I collagen in vivo. This conclusion is based on our unpublished data, which demonstrate that infusion of a large number of D35 cells (1 x 106 cells) into OI mice femurs leads to the synthesis of detectable type I collagen composed of {alpha}1(I) and {alpha}2(I) heterotrimers in vivo. The synthesized collagen was easily detected on SDS-PAGE after pepsin extraction of the collagens from the femurs of the recipient mice. These unpublished data suggest that if a sufficient number of D35 cells can be delivered into the bones of the developing mice, they are capable of synthesizing and depositing normal bone extracellular matrix in vivo.

It is likely that the transplanted cells underwent proliferation in vivo. In the present report, only 5 x 104 cells were initially transplanted into the neonatal mice, yet many GFP+ cells were seen in the bones of some recipient mice. This finding was not consistent. In some mice, there were extremely low numbers of donor cells present, whereas in others, the cells migrated and engrafted in the bones with high efficiency. The explanation for this variability in cell engraftment is not clear: perhaps immunological response to the transplanted cells by different recipient mice contributed to the varying levels of cell engraftment in different mice. The progenitors, however, exhibited higher incidence of engrafting in the bones of the neonatal homozygous mice than in the heterozygous mice (Tables 2, 3). The reason for this is not clear, but it may relate to the fact that the homozygous neonatal mice have less bone and therefore more space for the cells to occupy. The level of cell engraftment in OI and heterozygous did not appear to differ, however, as demonstrated by the distribution of the donor cells in tissue sections made from the femurs and tibias of the heterozygous and homozygous mice.

In the present study, cell engraftment in the bones of the recipient mice was enhanced by prior irradiation. Our studies are in agreement with a recent study in which the authors demonstrated that engraftment of cells in bones of the recipient mice was enhanced by prior irradiation of the mice [48]. But a large number of mice died before or immediately after cell transplantation. The high mortality may relate to the entrapment of the donor cells in the blood vessels of the recipient mice or the irradiation regiment used prior to cell infusion. The most likely explanation for high mortality is related to the number of cells that were administered. Transplantation of higher concentrations of cells (more than 5 x 104 cells) significantly increased the mortality of the cell recipients. The rate of mortality was equal for both heterozygous and homozygous mice.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosures
 Acknowledgments
 References
 
In summary, the present data suggest that a sufficient number of cells will engraft in the bones of developing animals with skeletal diseases if delivered systemically. The cells that migrate to skeletal tissues differentiate into osteoblasts and may contribute to the bone formation in vivo. The data also suggest that if a sufficient number of cells can be delivered systemically, they have the potential to synthesize bone extracellular matrix in vivo. These data, taken together, suggest that the development of cell therapies for skeletal disease will require isolation of cells with predilection to migrate and engraft in the skeletal tissues in high numbers. The contradictory reports in literature regarding engraftment of MSCs delivered systemically into skeletal tissues may relate to the inability to isolate cells with propensity for migration and engraftment in bone. The results, therefore, suggest that treatment of skeletal diseases using cell therapy will require isolation of defined cell populations that will migrate and engraft in the skeletal tissues with high efficiency.


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


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
References
 
This work was supported in part by NIH Grant R01 AR049688 and a grant from Children's Brittle Bone Foundation. We thank Drs. Paul Robbins and Zhibao Mi of the University of Pittsburgh for the generous gift of the DFG-eGFP-Zeocin retrovirus.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 

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