HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0567v1 26/2/330    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tomiyama, K. Articles by Marra, K. G. Search for Related Content PubMed PubMed Citation Articles by Tomiyama, K. Articles by Marra, K. G.

TISSUE-SPECIFIC STEM CELLS

Characterization of Transplanted Green Fluorescent Protein⁺ Bone Marrow Cells into Adipose Tissue

^aThomas E. Starzl Transplantation Institute, Department of Surgery,
^bDepartment of Cell Biology and Physiology,
^dDivision of Plastic Surgery, Department of Surgery, and
^eDepartment of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
^cMcGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania, USA

Key Words. Adipose • Chimera • Stem cell • Green fluorescent protein

Correspondence: Correspondence: Kacey G. Marra, Ph.D., University of Pittsburgh, 200 Lothrop Street, BST 1655E, Pittsburgh, Pennsylvania 15261, USA. Telephone: 412-383-8924; Fax: 412-648-2821; e-mail: marrak{at}upmc.edu

Received on July 17, 2007; accepted for publication on October 25, 2007.

First published online in STEM CELLS EXPRESS  November 1, 2007.

	ABSTRACT

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

 
Following transplantation of green fluorescent protein (GFP)-labeled bone marrow (BM) into irradiated, wild-type Sprague-Dawley rats, propagated GFP⁺ cells migrate to adipose tissue compartments. To determine the relationship between GFP⁺ BM-derived cells and tissue-resident GFP^– cells on the stem cell population of adipose tissue, we conducted detailed immunohistochemical analysis of chimeric whole fat compartments and subsequently isolated and characterized adipose-derived stem cells (ASCs) from GFP⁺ BM chimeras. In immunohistochemistry, a large fraction of GFP⁺ cells in adipose tissue were strongly positive for CD45 and smooth muscle actin and were evenly scattered around the adipocytes and blood vessels, whereas all CD45⁺ cells within the blood vessels were GFP⁺. A small fraction of GFP⁺ cells with the mesenchymal marker CD90 also existed in the perivascular area. Flow cytometric and immunocytochemical analyses showed that cultured ASCs were CD45^–/CD90⁺/CD29⁺. There was a significant difference in both the cell number and phenotype of the GFP⁺ ASCs in two different adipose compartments, the omental (abdominal) and the inguinal (subcutaneous) fat pads; a significantly higher number of GFP^–/CD90⁺ cells were isolated from the subcutaneous depot as compared with the abdominal depot. The in vitro adipogenic differentiation of the ASCs was achieved; however, all cells that had differentiated were GFP^–. Based on phenotypical analysis, GFP⁺ cells in adipose tissue in this rat model appear to be of both hematopoietic and mesenchymal origin; however, infrequent isolation of GFP⁺ ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation might be minor.

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

	INTRODUCTION

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

 
The distribution of cells within adipose tissue has been investigated, with the primary cell type present in adipose tissue being adipocytes. However, as fat is a vascularized tissue, other cell types that reside in the adipose tissue compartment include endothelial cells, macrophages, smooth muscle cells, and fibroblasts. The determination of a population of cells other than adipocytes was first made in the mid-1970s. In 1975, Stiles et al. documented a morphologically different phenotype of cells in human adipose tissue [1]. In 1976, Dardick et al. determined that these cells can differentiate into adipocytes [2]. Several groups began studying these cells, termed "preadipocytes" [3–9]. It was in 2001 that the plasticity of these cells was reported by Zuk et al. [10]. Initial reports demonstrated the mesenchymal plasticity of the cells, followed by several reports on the transdifferentiation of these seemingly mesenchymal stem cells into both ectodermal and endodermal phenotypes [11–16]. The identification of a stem cell population in adipose tissue has led to an extensive and rapid growth of research in the fields of regenerative medicine and adipose biology.

Although the plasticity and potential of the stem cells derived from the adipose tissue compartment remain under intense scrutiny, the origin of the stem cells is unclear. One possibility is that the cells are derived from the bone marrow. Recently, Crossno et al. reported that progenitor cells originating from the bone marrow can contribute to an increase in adipocyte number [17]. Their in vivo mouse study demonstrated that exposure to a high-fat diet or treatment with the thiazolidinedione rosiglitazone for 3 weeks promoted the trafficking of circulating bone marrow-derived progenitor cells into adipose tissue. This was evidenced by the appearance of green fluorescent protein (GFP)⁺ multilocular adipocytes.

To test the hypothesis that adipose-derived stem cells (ASCs) are derived from the bone marrow, we identified a population of GFP-positive cells, as well as GFP^– cells, from the adipose compartments of bone marrow (BM) chimeric rats. There was migration of the GFP⁺ cells to two different adipose compartments (the omental and the inguinal fat pad), as identified by flow cytometry and immunohistochemistry. In addition to whole adipose tissue analysis, ASCs were isolated from the two different adipose compartments and cultured to passage 3. Our results indicate that GFP⁺ cells in adipose tissue in this rat model appear to be of both hematopoietic and mesenchymal origin; however, infrequent isolation of GFP⁺ ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation may be a rare event.

	MATERIALS AND METHODS

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

 
In Vivo Model
GFP-transgenic and wild-type (WT) Sprague-Dawley rats were obtained from Japan SLC, Inc. (Hamamatsu, Japan, http://www.jslc.co.jp). The expression of GFP was under the control of the cytomegalovirus enhancer and the chicken β-actin promoter derived from an expression vector, pCAGGS [18, 19]. Animals were maintained in laminar flow cages in a specific pathogen-free animal facility at the University of Pittsburgh. All procedures in this experiment were performed according to the guidelines of the Council on Animal Care at the University of Pittsburgh and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals.

Bone marrow cells (BMCs) were obtained from 8–12-week-old GFP rats by flushing the tibias and femurs with RPMI 1640 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 25 mM HEPES buffer, 2 mM L-glutamine, and 10 µg/ml gentamicin (all from Life Technologies, Grand Island, NY, http://www.lifetech.com). Flow cytometric analysis showed that 69.7% ± 5.1% of GFP BMCs were GFP-positive, whereas 30% did not express GFP. A similar observation was reported in the BMCs from the GFP mouse [20]. Furthermore, the majority of GFP⁺ BMCs were CD45⁺, whereas the GFP^– BMCs were CD45^–.

Unfractionated BMCs (2 x 10⁸ cells per animal) with >95% viability in a trypan blue exclusion test were intravenously injected into WT rat recipients after 9.5 Gy of whole body irradiation (¹³⁷Cs source). Tacrolimus (0.5 mg/kg/day; a gift from Astellas Pharma Inc., Tokyo, http://www.astellas.com) was given for 7 days after BMC infusion. The successful creation of a GFP chimera was confirmed by detecting GFP⁺ peripheral blood mononuclear cells (PBMCs) with flow cytometry in the blood. The percentage of GFP⁺ PBMCs in WT recipients quickly increased and reached >95% by 100 days [21]. At 116–402 days after transplantation, subcutaneous and intra-abdominal fat tissues were obtained from 26 GFP radiation chimeras and used in this study.

Cell Characterization Using Immunofluorescence Microscopy
For immunocytochemistry analysis, cultured ASCs were fixed and processed for immunofluorescence as described [22] using the antibodies listed in Table 1. For immunohistochemistry, adipose tissue was removed from GFP⁺ bone marrow chimeric rats and immersion-fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for several hours. Fixed tissue was stored in PBS at 4°C until it was processed. Tissue was then cut into approximately 1-mm³ pieces and stained in situ. Tissue was mixed on a Nutator instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) during all procedures, and rinses were 15 minutes each. Tissue pieces were rinsed three times in PBS and then rinsed three times in PBS containing 0.5% BSA and 0.15% glycine (PBG buffer). Tissue was permeabilized in PBGT buffer containing 0.1% Triton X-100 (PBG) for 2 hours at room temperature and then blocked in 5% nonimmune goat serum in PBGT buffer (PBG with 0.05% Triton X-100) for 2 hours at room temperature. Primary antibodies (Table 1) diluted in PBGT buffer were added to tissue overnight at 4°C. Tissue was rinsed four times in PBGT buffer, and then fluorescently tagged after secondary and phalloidin (Table 1), diluted in PBGT buffer, were added to the sections overnight at 4°C. Tissue was rinsed three times in PBGT buffer and three times in PBS, and then nuclei were stained using 0.01% Hoechst dye (bis benzimide) in PBS for 10 minutes. Following a wash in PBS, the tissue was coverslipped using gelvatol (23 g of poly[vinyl alcohol] average molecular weight 30,000–70,000), 50 ml of glycerol, 0.1% sodium azide to 100 ml of PBS) and viewed on a Fluoview 1000 confocal microscope (Olympus, Center Valley, PA, http://www.olympus-global.com). Confocal stacks were processed using Imaris image analysis software (Bitplane, St. Paul, MN, http://www.bitplane.com).

Isolation and Culture of Rat ASCs
Adipose tissue was excised from both the omentum (i.e., abdominal) and the inguinal (i.e., subcutaneous) fat pads of naïve (e.g., nonchimeric, GFP⁺) and chimeric rats and underwent enzymatic digestion by 0.075% collagenase II (Worthington Biochemical, Lakewood, NJ, http://www.worthington-biochem.com) in Hanks' balanced saline solution for 60 minutes at 37°C with shaking. Digested tissue was filtered and centrifuged, and erythrocytes were removed by treatment with erythrocyte lysis buffer. The remaining cells were transferred to tissue culture flasks with Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, http://www.invitrogen.com) plus supplemental Ham's F-12 medium (F12) (Gibco), and after an attachment period of 6 hours, nonadherent cells were removed by a PBS (Gibco) wash. Attached cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Gibco), 0.1 µM dexamethasone (Sigma-Aldrich), 1% penicillin-streptomycin (Gibco), and 1.25 mg/l amphotericin B (Gibco) and expanded in vitro until passage 3.

Adipocytic Differentiation and Histochemical Assays
ASCs (passage 3) were seeded into six-well tissue culture plates (5 x 10⁵ cells per well) and grown until subconfluence (1–2 days) in DMEM/F12. Medium was then replaced with adipogenic or control medium. Adipogenic medium (AM) consisted of DMEM/F12 supplemented with 33 µM biotin, 0.5 µM insulin, 17 µM D-pantothenic acid, 0.2 nM dexamethasone, 1 µM ciglitazone, 0.2 nM triiodothyronine, and 10 mg/l transferrin. Control medium was identical to the DMEM/F12 used during the expansion stage. After cells were grown to 100% confluence, adipogenic induction medium with 3-isobutyl-1-methylxanthine (540 µM) was added for 2 days. Then, for 12 days, the medium was changed every 48 hours with adipogenic medium. Cells were washed with PBS with EDTA twice and fixed with 10% buffered formalin for 10 minutes. Cells were then washed with distilled H₂O twice, and stained with oil red O (20 ml of stock solution consisting of 30 mg of oil red O powder in 60 ml of 2-propanol [0.5%]). Next, 13.3 ml of H₂O was added, and the cells were incubated for 30 minutes at room temperature. Cells were washed with H₂O to remove debris. The resultant positive red stain was evaluated via light microscopy.

Cell Surface Expression Using Flow Cytometry
Cultured ASCs (passage 3) were analyzed by flow cytometry for their surface marker expression. Antibodies used in this study are listed in Table 1. For flow cytometry, cultured ASCs were washed and incubated with monoclonal antibodies at 4°C for 30 minutes. After three washes with PBS, ASCs were further incubated for 30 minutes with secondary antibodies as needed. Stained cells were fixed in 1% paraformaldehyde and analyzed on an LSR II instrument (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), and data were analyzed using FACSDiva software (BD Biosciences). Isotype-matched nonspecific antibodies were used for the control.

Statistical Analysis
Unless otherwise specified, the results are reported as mean ± SD. t tests were conducted to assess differences among treatment groups. Statistical significance was set at p .05.

	RESULTS

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

 
Immunohistochemistry of Rat Adipose Tissue
Whole fat tissue from the chimera was assessed for the markers described in Table 1 using immunofluorescence microscopy. Results indicate GFP⁺ cells were evenly scattered around the 70–100-µm adipocytes (Fig. 1A). Most GFP⁺ cells had a mesenchymal morphology and were observed to be smooth muscle actin (SMA)-positive. The SMA signal in the GFP⁺ cells was equivalent to that observed in the pericytes surrounding the blood vessels. In Figure 1B, partial confocal stack shows a GFP⁺ adipocyte in the BM chimera. The GFP⁺ cytoplasmic labeling of the positive adipocyte surrounds the lipidic portion of the cell. This was observed only once in six different chimeric animals examined. GFP⁺ blood cells were also identified within the F-actin⁺ blood vessels (Fig. 1C). Transmission electron microscopy revealed that the typical GFP⁺ cell within the adipose tissue integrated between adipocytes possessed large quantities of rough endoplasmic reticulum (Fig. 1D). Immuno-transmission electron microscopy analysis of LRWhite acrylic-embedded chimeric adipose tissue indicated that these cells were GFP⁺ when stained for the GFP protein (Fig. 1E).

View larger version (82K): [in this window] [in a new window]   Figure 1. Evaluation of the distribution and ultrastructure of bone marrow-derived green fluorescent protein (GFP)+ cells within the fat of a radiation chimera rat 165 days following bone marrow transplantation. (A): Tissue was stained for smooth muscle actin (SMA) (red), F-actin (blue), and the nucleus (Hoechst dye, cyan), in addition to collecting the endogenous fluorescence of GFP (green). Single slice confocal microscopy of the tissue shows that GFP+ cells were evenly scattered around the 70–100-µm adipocytes (some labeled as reference). Most GFP+ cells were observed to be SMA-positive (arrows) as more clearly shown in the black and white rendition of the red channel in the inset (A). The SMA signal in the GFP+ cells was equal to that observed in the pericytes surrounding the blood vessels (arrowhead). (B): Partial confocal stack showing a GFP+ adipocyte in the bone marrow chimera. This is a rare event and was observed only once in six chimeric animals examined (tissue counterstained for F-actin [rhodamine phalloidin, red] and nucleus [Hoechst dye, blue]). GFP signal is cytoplasmic and does not partition into the lipidic inclusion of the adipocyte. (C): Expanded x-y projection from (B) at axes delineated by the horizontal and vertical lines, indicating the GFP+ blood cells within the F-actin+ blood vessels (arrows). The GFP+ cytoplasmic labeling of the positive adipocyte surrounds the lipidic portion of the cell (*). (D): Transmission electron micrograph of the typical GFP+ cell within the adipose tissue, showing these cells, integrated between adipocytes, possess large quantities of rough endoplasmic reticulum. (E): Immuno-transmission electron microscopy analysis of LRWhite acrylic-embedded chimeric adipose tissue indicated that these cells were GFP+ when stained for the GFP protein (arrows indicate secondary antibody 5-nm gold particles). Abbreviation: A, adipocyte.

View larger version (19K): [in this window] [in a new window]   Figure 2. Adipose tissue from green fluorescent protein (GFP)+ chimeric rats was stained for CD90 (Cy3, red) and counterstained for F-actin (blue). Areas within the tissue show variation in colocalization of CD90 with the GFP bone marrow marker. Top left panel, arrows indicate dual signal, arrowhead indicates GFP–/CD90+ cell. Bottom panels indicate black and white rendition of red channel. Top right panel shows an area without apparent colocalization of CD90 and GFP signal.

View larger version (44K): [in this window] [in a new window]   Figure 3. Adipose tissue from GFP+ chimeric rats was stained for CD45 (Cy5, blue) and counterstained for F-actin (red). Top left panel shows single optical slice confocal image, and top right panel shows the black and white rendition of the Cy5 channel, accentuating the CD45+ cells. Arrows indicate the cells that are both GFP+/CD45+, and arrowheads indicate the cells that are GFP+/CD45–. In the top right panel, there are also a few GFP–/CD45+ cells in this field with this phenotype. The bottom panels are higher magnification of the top panels. Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein.

View larger version (56K): [in this window] [in a new window]   Figure 4. Adipose cells derived from tissue. (A): Light micrograph of isolated cells from adipose tissue at passage 3. (B): Corresponding image with green fluorescent protein (GFP)+. (C): Light micrograph of adipose stem cells that had undergone adipogenesis. (D): Corresponding image with GFP+.

Surface Marker Expression of ASCs
ASCs were isolated and cultured from subcutaneous and abdominal fat tissues obtained from GFP⁺ radiation chimeras at 116–402 days after bone marrow transplantation. Surface marker expression of cultured ASCs was analyzed by flow cytometry. In the naïve (e.g., GFP⁺, nonchimera) rats, nearly all ASCs obtained from subcutaneous and abdominal fat tissues of naïve animals were CD45^– and CD29⁺. The majority of the ASCs also expressed CD90, but they did not express CD31, CD106, or CD133 (Fig. 5A). Subsequently, CD90 and CD29 were used to determine the origin of ASCs in experiments of GFP radiation chimera. Mitchell et al. demonstrated that ASCs are positive for both CD90 and CD29, with expression increasing to >90% at passage 4 [24]. The findings were confirmed with immunocytochemistry of ASCs (Fig. 5B).

View larger version (92K): [in this window] [in a new window]   Figure 5. Surface marker expression of cultured passage 3 adipose-derived stem cells (ASCs) from naïve green fluorescent protein (GFP)+ rats. (A): Representative plots of ASCs obtained by culturing the subcutaneous and abdominal fat tissues obtained from naïve animals. Flow cytometric analysis showed that cultured ASCs expressed CD29 and CD90. (B): Cells expressed CD29 and CD90 (red) in vitro, whereas the majority of these cells were negative for CD31, CD45, CD106, and CD133. Blue is F-actin stain and green is endogenous GFP signal. Insets are the black and white rendition of the red channel for each of these markers.

View larger version (29K): [in this window] [in a new window]   Figure 6. Surface marker expression of cultured passage 3 adipose-derived stem cells (ASCs) from a green fluorescent protein (GFP)+ radiation chimera. (A): ASCs obtained from fat tissues of a radiation chimera were analyzed by flow cytometry. CD29- and/or CD90-expressing ASCs were mostly GFP-negative; however, small numbers of GFP-positive bone marrow-derived ASCs were detected. Of note, ASCs from the abdominal fat and subcutaneous fat differ in the expression of CD90. Data are mean ± SD of three experiments; *, p .05. (B): Representative scattergrams showing GFP+ CD29+/CD90+ ASCs. The intensity of the expression of CD29 and CD90 on GFP+ ASCs was lower than that expressed on GFP– ASCs. The abdominal and subcutaneous fats were obtained from a radiation chimera at 161 days after bone marrow transplantation. Abbreviation: GFP, green fluorescent protein.

	DISCUSSION

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

The migration of stem cells after transplantation has been studied previously, including a study by ten Hove et al., who determined that donor-derived cells (from blood) were found in liver tissue specimens after allogeneic stem cell transplantation in nine female patients [31]. Migration of stem cells into the liver had previously been shown to be a rare event. For example, in a study of sex-mismatched liver transplant recipients, Ng et al. reported that although recipient cells constituted up to 50% of all cells in the liver allograft, most cells demonstrated macrophage/Kupffer cell differentiation, with only 1.6% showing hepatocytic differentiation [32]. In 2002, Körbling et al. reported that recipients of peripheral blood stem cells showed donor-derived hepatocytes up to 7% in liver tissue specimens endothelium by bone-marrow-derived cells [33]. Recently, Nakao et al. reported that infused bone marrow stem cells engraft into both the allogeneic marrow environment and the syngeneic extramarrow environment (intestinal graft) [34].

Bone marrow-derived mesenchymal stem cells have been used to promote engraftment of peripheral mononuclear blood cells in transplanted animals [35–41]. In 2005, Kim et al. reported the examination of adipose-derived mesenchymal stem cells to promote engraftment of hematopoietic stem cell transplantation in NOD/SCID mice, indicating that ASCs could be used as an alternative to bone marrow stem cells [35]. Planat-Benard et al. demonstrated that mouse adipose-derived stem cells, human adipose-derived stem cells, and even dedifferentiated adipocytes all induce significant neovascularization when injected into the muscles of an ischemic mouse hind limb model (immunodeficient strains were used with human donor cells) [42].

Although the potential of ASCs is exciting, it is important to determine the origin of ASCs, as these cells could potentially cure hematopoietic malignancies that are as yet untreatable. We have observed a GFP⁺ population of cells residing in the adipose tissue compartments after GFP⁺ bone marrow transplantation. We conducted detailed immunohistochemical analysis of chimeric whole fat compartments and subsequently isolated, characterized, and cultured ASCs from GFP⁺ BM chimeras. The immunohistochemistry analysis of fat tissue revealed that a large fraction of GFP⁺ cells in adipose tissue were strongly positive for CD45 and smooth muscle actin (Fig. 1). The CD45⁺/SMA⁺ cells were evenly scattered around the adipocytes and blood vessels, whereas CD45⁺ leukocytes within the vessels were GFP⁺ (Fig. 3). A small fraction of GFP⁺/CD90⁺ cells also existed in the perivascular area (Fig. 2), but their presence was highly variable within the tissue.

In addition to whole fat analysis, ASCs were isolated, characterized, and cultured from two different adipose tissue compartments: the omental (abdominal) and the inguinal (subcutaneous) fat pads. Flow cytometric and immunocytochemical analyses showed that cultured ASCs were CD45^–/CD90⁺/CD29⁺ (Fig. 5). A significantly higher number of GFP^–/CD90⁺ cells were isolated from the subcutaneous depot as compared with the abdominal depot (Fig. 6). Furthermore, the in vitro adipogenic differentiation of ASCs was achieved; however, all cells that had differentiated were GFP^– (Fig. 4).

Based on phenotypical analysis, GFP⁺ cells in adipose tissue in this rat model appear to be of both hematopoietic and mesenchymal origin; however, infrequent isolation of GFP⁺ ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation is a rare event. This could also be due to the initial transplantation of a bone marrow cell population with a limited mesenchymal stem cell population or engraftment. Numerous previous studies have used the bone marrow transplantation model to examine the differentiation of BMCs into the parenchyma of various tissues; however, the bone marrow stroma and mesenchymal stem cells have been shown to remain of the host phenotype after experimental and clinical bone marrow transplantation [43–46], suggesting limited engraftment capability of donor BMCs into host bone marrow stroma compartment after transplantation. Nevertheless, both hematopoietic and mesenchymal GFP⁺ cells were identified in the adipose tissue of radiation chimeras in this study, and the result may indicate the differentiation of hematopoietic stem cells into mesenchymal cell populations. Thus, although improved engraftment of BMCs into mesenchymal compartment might increase BM-derived ASCs, the study demonstrates that in spite of the plasticity of hematopoietic stem cells, they appear to have a limited contribution to ASC generation. Future studies to further understand the cell behavior after migration and the effect of body mass index include treating the rats with a high-fat diet. This study provides further insight into both the frequency of ASCs in two different rat depots and the stem cell migration into the adipose tissue compartments.

	CONCLUSION

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

 
These data increase our understanding of the origin of mesenchymal stem cells residing in adipose tissue by providing further insight into the migration of bone marrow cells into adipose tissue compartments. Both hematopoietic and mesenchymal stem cells were identified as migrating from the bone marrow into both adipose compartments; however, infrequent isolation of GFP⁺ ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation might be minor. This could be correlated to the initial population of mesenchymal stem cells in the transplanted bone marrow cells. It was also found that the GFP^– mesenchymal stem cell population was significantly higher in the subcutaneous depot as compared with the abdominal depot, indicating that the inguinal fat pad may be a better source of ASCs. Although in this rat study, the presence of a small population (5%) of GFP⁺ ASCs suggests that ASCs could be recruited from the bone marrow, if necessary, a significant GFP⁺ mesenchymal stem cell population originating from the bone marrow was not identified. Additional studies can be conducted to identify alternative ASC origins, such as pericytes.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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

 
We gratefully acknowledge Dr. Masaru Okabe (Osaka University, Osaka, Japan) for providing GFP rats and Dr. Fengli Guo, Marc Rubin, and Mara Sullivan for electron microscopy technical assistance. This work was supported by Grant NIH RO1 CA76541 (to D.B.S.).

	REFERENCES

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

 

1. Stiles J, Francendese A, Masoro E. Influence of age on size and number of fat cells in the epididymal depot. Am J Physiol 1975;229:1561–1568.[Abstract/Free Full Text]

2. Dardick I, Poznanski W, Waheed I et al. Ultrastructural observations on differentiating human preadipocytes cultured in vitro. Tissue Cell 1976;8:561–571.[CrossRef][Medline]

3. Hausman GJ, Richardson RL. Newly recruited and pre-existing preadipocytes in cultures of porcine stromal-vascular cells: Morphology, expression of extracellular matrix components, and lipid accretion. J Anim Sci 1998;76:48–60.[Abstract/Free Full Text]

4. Niesler CU, Siddle K, Prins JB. Human preadipocytes display a depot-specific susceptibility to apoptosis. Diabetes 1998;47:1365–1368.[Abstract]

5. Maslowska MH SA, MacLean LD, Cianflone K. Regional differences in triacylglycerol synthesis in adipose tissue and in cultured preadipocytes. J Lipid Res 1993;34:219–228.[Abstract]

6. Kirkland JL, Hollenberg CH, Gillon WS. Age, anatomic site, and the replication and differentiation of adipocyte precursors. Am J Physiol 1990;258:C206–C210.[Medline]

7. Tchkonia T, Giorgadze N, Pirtskhalava T et al. Fat depot origin affects adipogenesis in primary cultured and cloned human preadipocytes. Am J Physiol Regul Integr Comp Physiol 2002;282:R1286–R1296.[Abstract/Free Full Text]

8. Hausman GJ, Richardson RL. Adipose tissue angiogenesis. J Anim Sci 2004;82:925–934.[Abstract/Free Full Text]

9. Hausman G, Hausman D. Search for the preadipocyte progenitor cell. J Clin Invest 2006;116:3103–3106.[CrossRef][Medline]

10. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211–228.[CrossRef][Medline]

11. Safford KM, Hicok KC, Safford SD et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002;294:371–379.[CrossRef][Medline]

12. Seo M, Suh S, Bae Y et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun 2005;328:258–264.[CrossRef][Medline]

13. Taléns-Visconti R, Bonora A, Jover R et al. Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage. Toxicol In Vitro 2007;21:324–329.[CrossRef][Medline]

14. Tholpady SS, Katz AJ, Ogle RC. Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec A Discov Mol Cell Evol Biol 2003;272:398–402.[CrossRef][Medline]

15. Kang SK, Lee DH, Bae YC et al. Improvement of neurological deficits by intracerebral transplantation of human adipose-derived stromal cells after cerebral ischemia in rats. Exp Neurol 2003;183:355–366.[CrossRef][Medline]

16. Safford KM, Safford SD, Gimble JM et al. Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells. Exp Neurol 2004;187:319–328.[CrossRef][Medline]

17. Crossno J, Majka S, Grazia T et al. Rosiglitazone promotes development of a novel adipocyte population from bone marrow-derived circulating progenitor cells. J Clin Invest 2006;116:3220–3228.[CrossRef][Medline]

18. Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.[CrossRef][Medline]

19. Ito T, Suzuki A, Imai E et al. Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. J Am Soc Nephrol 2001;12:2625–2635.[Abstract/Free Full Text]

20. Kawakami N, Sakane N, Nishizawa F et al. Green fluorescent protein-transgenic mice: Immune functions and their application to studies of lymphocyte development. Immunol Lett 1999;70:165–171.[CrossRef][Medline]

21. Toyokawa H, Nakao A, Stolz D et al. 3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury. Lab Invest 2006;86:72–82.[CrossRef][Medline]

22. Stolz D, Zamora R, Vodovotz Y et al. Peroxisomal localization of inducible nitric oxide synthase in rat hepatocytes. Hepatology 2002;36:81–93.[CrossRef][Medline]

23. Wack KE, Ross MA, Zegarra V et al. Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver. Hepatology 2001;33:363–378.[CrossRef][Medline]

24. Mitchell J, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. STEM CELLS 2006;24:376–385.[Abstract/Free Full Text]

25. Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279–4295.[Abstract/Free Full Text]

26. Gimble JM, Guilak F. Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Biol 2003;58:137–160.[Medline]

27. Dragoo JL, Choi JY, Lieberman JR et al. Bone induction by BMP-2 transduced stem cells derived from human fat. J Orthop Res 2003;21:622–629.[CrossRef][Medline]

28. Awad HA, Halvorsen YD, Gimble JM et al. Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells. Tissue Eng 2003;9:1301–1312.[CrossRef][Medline]

29. Nathan S, Das De S, Thambyah A et al. Cell-based therapy in the repair of osteochondral defects: A novel use for adipose tissue. Tissue Eng 2003;9:733–744.[CrossRef][Medline]

30. Ashjian PH, Elbarbary AS, Edmonds B et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 2003;111:1922–1931.[Medline]

31. ten Hove W, Verspaget H, Barge R et al. Liver chimerism after allogeneic blood stem cell transplantation. Transplant Proc 2007;39:231–236.[CrossRef][Medline]

32. Ng I, Chan K, Shek W et al. High frequency of chimerism in transplanted livers. Hepatology 2003;38:989.[Medline]

33. Körbling M, Katz R, Khanna A et al. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 2002;346:738–746.[Abstract/Free Full Text]

34. Nakao A, Toyokawa H, Kimizuka K et al. Simultaneous bone marrow and intestine transplantation promotes marrow-derived hematopoietic stem cell engraftment and chimerism. Blood 2006;108:1413–1420.[Abstract/Free Full Text]

35. Kim S, Cho H, Kim Y et al. Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice. Biochem Biophys Res Commun 2005;329:25–31.[CrossRef][Medline]

36. Angelopoulou M, Novelli E, Grove J et al. Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 2003;31:413–420.[CrossRef][Medline]

37. Maitra B, Szekely E, Gjini K et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004;33:597–604.[CrossRef][Medline]

38. Laughlin M, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815–1822.[Abstract/Free Full Text]

39. Nolta J, Hanley M, Kohn D. Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: Analysis of gene transduction of long-lived progenitors. Blood 1994;83:3041–3051.[Abstract/Free Full Text]

40. Anklesaria P, Kase K, Glowacki J et al. Engraftment of a clonal bone marrow stromal cell line in vivo stimulates hematopoietic recovery from total body irradiation. Proc Natl Acad Sci U S A 1987;84:7681–7685.[Abstract/Free Full Text]

41. Almeida-Porada G, Porada C, Tran N et al. Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 2000;95:3620–3627.[Abstract/Free Full Text]

42. Planat-Benard V, Silvestre JS, Cousin B et al. Plasticity of human adipose lineage cells toward endothelial cells: Physiological and therapeutic perspectives. Circulation 2004;109:656–663.[Abstract/Free Full Text]

43. Simmons P, Przepiorka D, Thomas E et al. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature 1987;328:429–432.[CrossRef][Medline]

44. Rieger K, Marinets O, Fietz T et al. Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation. Exp Hematol 2005;33:605–611.[CrossRef][Medline]

45. Cilloni D, Carlo-Stella C, Falzetti F et al. Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation. Blood 2000;96:3637–3643.[Abstract/Free Full Text]

46. Pozzi S, Lisini D, Podesta M et al. Donor multipotent mesenchymal stromal cells may engraft in pediatric patients given either cord blood or bone marrow transplantation. Exp Hematol 2006;34:934–942.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0567v1 26/2/330    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tomiyama, K. Articles by Marra, K. G. Search for Related Content PubMed PubMed Citation Articles by Tomiyama, K. Articles by Marra, K. G.