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Isolation of multipotent stem cells from mouse adipose tissue
Journal of Dermatological Science (2007) 48, 43—52
www.intl.elsevierhealth.com/journals/jods
Isolation of multipotent stem cells from mouse adipose tissue§ Naoki Yamamoto a,e,*, Hirohiko Akamatsu b, Seiji Hasegawa c, Takaaki Yamada c, Satoru Nakata c, Mahito Ohkuma d, Ei-Ichi Miyachi d, Tohru Marunouchi e, Kayoko Matsunaga b a
Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan b Department of Dermatology, Fujita Health University School of Medicine, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan c Research Laboratories, Nippon Menard Cosmetic Co. Ltd., 2-7 Torimi-cho, Nishi-Ku, Nagoya, Aichi 451-0071, Japan d Department of Physiology, Fujita Health University School of Medicine, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan e Department of Cell Biology, Fujita Health University Institute for Comprehensive Medical Science, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan Received 15 December 2006; received in revised form 23 May 2007; accepted 30 May 2007
KEYWORDS p75 neurotrophin receptor (p75NTR, CD271); Adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells); Mesenchymal stem cell (MSC); CD105; Sca-1; Neuronal cell
Summary Background: Embryonic stem (ES) cells, bone marrow, adipose tissue or other genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. However, there is no specific surface antigen that can be used as a marker of multipotent stem cells. Objective: We tried to isolate and collect putative multipotent stem cells from mouse subcutaneous adipose tissue using the p75 neurotrophin receptor (p75NTR) as a marker. Methods: Adipose tissue was processed for immunostaining using antibodies antiCD90, anti-CD105 and anti-Sca-1 as general mesenchymal stem cell (MSC) markers, and anti-p75NTR, an epithelial stem cell and MSC marker. Subsequently, the expression of cell surface markers in adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells) was examined by flow cytometry (fluorescence-activated cell
§
In accordance with a consensus reached by investigators attending the Second Annual International Fat Applied Technology Society Meeting, they will refer to this adherent cell population as adipose-derived stem cells (ASCs). * Corresponding author at: Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan. Tel.: +81 562 93 2317; fax: +81 562 92 5382. E-mail address: [email protected] (N. Yamamoto). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.05.015
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N. Yamamoto et al. sorting: FACS). Finally, ADSVF cells positive for p75NTR were sorted and cultured to induce their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. Results: Cells positive for several of these markers were found in the deep layers of adipose tissue. Among them, those positive for p75NTR differentiated into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. The rate of differentiation into adipocytes, osteoblasts and neuronal cells was higher for p75NTRpositive cells than for p75NTR-negative cells. Conclusions: p75NTR proved to be a useful marker to isolate adipose tissue-derived stem cells (ASCs). # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Embryonic stem (ES) cells and genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. Because of their multi-differentiation ability and since cells for a specific purpose may be efficiently prepared, stem cells are very useful. However, clinical application of these cells is currently difficult in view of ethical issues as well as the potential risks of carcinogenesis [1]. On the other hand, cells derived from bone marrow, adipose tissue or other tissues have attracted attention nowadays as clinically applicable sources of autografting cells, because these cells can differentiate into various types of cells, including smooth muscle cells, endothelial cells and neuronal cells [2—7]. However, no specific surface antigen that can be used as a marker of multipotent cells has been identified so far. In the present study, we tried to isolate and collect multipotent cells (adipose tissue-derived stem cells: ASCs) from among subcutaneous adipose tissuederived stromal vascular fraction cultured cells (ADSVF cells) [8] of mouse using the p75 neurotrophin receptor (p75NTR), a cell membrane protein, as a marker [9—12]. The p75NTR is a low-affinity nerve growth factor receptor. Recently, a CD number was given to p75NTR, and it is now known as CD271. As we found that ADSVF cells positive for p75NTR differentiated into mature adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells, in this study we compared the rate of differentiation into adipocytes, osteoblasts and neuronal cells obtained using p75NTR-positive cells with that attained using p75NTR-negative cells.
2. Materials and methods 2.1. Animals For the immunohistochemical study and isolation of mouse ADSVF cells, we used 4-week-old male ICR
albino mice (Japan SLC, Inc., Shizuoka, Japan). All procedures were approved by the Education and Research Center for Animal Models of Human Diseases of Fujita Health University.
2.2. Immunohistochemistry Subcutaneous adipose tissue was excised from the abdomen of adult mice. This was fixed with 4% paraformaldehyde and paraffin sections were prepared by the usual method. These sections were processed for H.E. staining and immunostaining using antibodies anti-CD90 (Thy-1), anti-CD105 (Endoglin) (eBioscience, Inc., San Diego, CA) and anti-Sca-1 (Stem cell antigen-1/Ly6) (R&D Systems, Inc., Minneapolis, MN) that are general mesenchymal stem cell (MSC) markers [13—15], and antip75NTR (Chemicon International, Inc., Temecula, CA) that is known to be an epithelial and MSC marker [9—12]. As for the secondary antibodies, anti-rat IgG antibody labeled with Alexa Fluor 488 (Invitrogen Corp., Carlsbad, CA) and anti-rabbit IgG antibody labeled with Alexa Fluor 594 (Invitrogen) were used. DAPI (VECTASHIELD H-1200; Vector Laboratories, Burlingame, CA) was used for nuclear staining. A fluorescence microscope (Power BX-51, Olympus, Tokyo, Japan) was used for observation.
2.3. Isolation and culture of mouse adipose tissue Mouse adipose tissue dissected from lymph nodes was cut into fine pieces. These pieces were digested with 0.2% collagenase (Nitta Gelatin, Osaka, Japan) at 37 8C for 30 min and the resultant cell suspension was filtered through a 100 mm mesh (BD Biosciences, San Jose, CA) to remove tissue debris. Then, collagenase was removed by dilution with phosphate-buffered saline solution (PBS, Sigma, St. Louis, MO) and centrifuged twice for 5 min at 260 g each time (himac CF7D2, Hitachi-koki Co. Ltd., Ibaragi, Japan). In this way, most of the matured adipocytes remained in the supernatant. The cell pellet (stromal vascular
Isolation of multipotent cells from mouse adipose tissue fraction [8]) was suspended in 0.83% NH4Cl and incubated to remove contaminating red blood cells. Cells were washed and centrifuged twice with PBS and cultured in Dulbecco’s modified Eagle’s medium/ Ham’s F12 (DMEM/F12) (Sigma) supplemented with 5% fetal bovine serum (FBS, JRH Biosciences, Adelaide, Australia), 2 ng/mL basic fibroblastic growth factor (bFGF, Sigma) and 1% penicillin/streptomycin (Sigma) in 100 mm 20 mm culture dishes at 37 8C in a 5% CO2 humidified incubator. After 4 h, the nonadherent cells were removed and adherent ADSVF cells were cultured for 7 more days.
2.4. Flow cytometric analysis Cultured mouse ADSVF cells were analyzed using fluorescence-activated cell sorting (FACS, cell analyzer) (FACScan, BD Biosciences). In brief, the cells were harvested and incubated for 30 min on ice with 1% BSA in PBS containing the primary antibodies directed against the following cell surface markers: CD29 (Integrin beta-1) [16], TE-7 (fibroblasts marker) (Chemicon), CD31 (PECAM-1), CD34, CD45 (LCA), CD90 (Thy-1), CD105 (Endoglin), CD133 (Prominin-1; AC 133) (eBioscience), Sca-1 (Stem cell antigen-1/Ly6) and p75NTR (CD271). After washing, secondary antibodies Alexa Fluor 488 and PE-Cy5 (for p75NTR labeling, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added, and the cells were incubated for another 30 min on ice. Then, the cells were washed twice before the analysis.
2.5. Sorting of p75NTR-positive cells Mouse ADSVF cells were subjected to fluorescenceactivated cell sorting (FACS, cell sorter) (FACSVantage SE, BD Biosciences) to collect p75NTR-positive and negative cells. The cells were harvested and incubated in 1% BSA-PBS containing anti-p75NTR antibody for 30 min on ice, then anti-rabbit IgG antibody labeled with Alexa Fluor 488 was added and the cells were incubated for another 30 min on ice. The p75NTR-positive or negative cells were then aseptically collected, cultured and used in all subsequent experiments.
2.6. Cell differentiation and immunocytochemistry For cell differentiation, preadipocyte differentiation medium (PT-8000, LONZA Walkersville, Inc., MD) (Sanko Junyaku Co. Ltd., Tokyo), osteoblast differentiation medium (CA417500, TOYOBO Co. Ltd., Osaka), chondrocyte differentiation medium (CC-3225, LONZA), smooth muscle cell proliferating medium (CC-3182, LONZA) and neurobasal medium
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(Invitrogen) that contained 1 mM retinoic acid (Sigma), valproic acid (Sigma) and N-2 supplement (Invitrogen) known to induce differentiation into neuronal cells were used [17]. Simultaneously, p75NTR-negative cells were cultured in the same differentiation media as control.
2.7. Detection of differentiated cells The experiments on differentiation into adipocytes, osteoblasts, chondrocytes and smooth muscle cells were done by adding the specific differentiation medium, but those on differentiation into neuronal cells were done as previously described [17]. ADSVF cells differentiated into mature adipocytes were fixed at 10 days of culture in 4% paraformaldehyde (PFA), examined for morophological changes and stained with Oil Red-O stain (Sigma). Then, stained cells were extracted with isopropanol and absorbance was measured at 520 nm (Versa Max, Molecular Devices Corp., Long Branch, NJ) to assess the content of lipid. The cells differentiated into osteoblasts were fixed at 14 days of culture in 4% PFA and stained with Alizarin red S stain (Wako Pure Chemical Industries, Co. Ltd., Osaka). Cells were observed under a bright field using a CKX41 microscope, and a microscope with a mounted digital camera (DP-71, Olympus) was used to capture 10 random nonoverlapping low-power (200) images of 100 mm 20 mm culture dishes. The capture images were converted into the binary scale, and NIH image software was used to evaluate differentiated osteoblasts for measured area (50 50 pixels) in the captured field. The areas stained with Alizarin red S were expressed as a percentage of the measured area. The cells differentiated into chondrocytes were fixed at 21 days of culture in 4% PFA and cryosections were obtained using a cryostat (CM3050S, Leica Microsystems, Nussloch, Germany) (FINETEC, Tokyo). Then, the cryosections were immunostained with anti-aggrecan antibody (Abcam Limited, Cambridgeshire, UK) and anti-collagen type-2 antibody (LSL Co. Ltd., Tokyo) [18]. To detect the cells differentiated into smooth muscle cells at 14 days, these were immunostained with anti-alfa-smooth muscle actin antibody (alfaSMA) (Lab Vision Corp., Fremont, CA) [11]. The cells differentiated into neuronal cells were fixed at 5 days of culture in 4% PFA and stained with anti-neurofilament M (NF-M) antibody (Chemicon), which is a neuronal cell marker [19,20]. Then, RNA was prepared using a High Pure RNA Isolation Kit (Roche Diagnostics Corp., Indianapolis, IN) at intervals and quantitative Real Time RT-PCR (qRT-PCR) was carried out using PRISM-7900HT (Applied
46 Biosystems, Foster City, CA). The assay ID of the TaqMan primers and probe (Applied Biosystems) used were NF-M: Mm00456201_m1 and GAPDH: Mm99999915_g1.
N. Yamamoto et al. the percentage of CD34 positive cells was low (Fig. 2c), and the common leukocyte antigen CD45 was not observed on ADSVF cells (Fig. 2d). The results of flow cytometric analysis of ADSVF cells isolated from three mice are shown in Table 1.
2.8. Statistical analysis 3.3. Sorting of p75NTR-positive cells Data are presented as the mean value S.D. Differences between two groups were evaluated for statistical significance using Student’s t-test. Differences were considered significant at p < 0.05.
p75NTR-positive cells accounted for 37.8 5.49% of ADSVF cells. Therefore, we separated p75NTR-positive cells from ADSVF cells using FACSVantage SE and succeeded in increasing their population to more than 97%.
3. Results 3.1. Immunohistochemistry of adipose tissue Mouse adipose tissue from the subcutaneous inguinal region was used. Since the lymph nodes present in the adipose tissue can be easily distinguished (Fig. 1a), they were dissected in advance (Fig. 1b and c). The adipose tissue was stained with H.E. (Fig. 1d and e), anti-CD90 and anti-p75NTR. CD90positive cells (Fig. 1f), p75NTR-positive cells (Fig. 1g) and double positive cells (Fig. 1h) were found to be distributed in the deeper layers of adipose tissue (Fig. 1d), with CD90 and p75NTR double positive cells being observed in the neighborhood of microvessels. Next, adipose tissue was stained with antibody for p75NTR and CD105 or Sca-1. In the same way as CD90-positive cells had been observed, cells positive for CD105 and Sca-1 were observed in the deeper layers of the adipose tissue (Fig. 1i and l). A lot of CD105 or Sca-1 positive cells were also positive for p75NTR (Fig. 1j, k, m, and n).
3.2. Characterization of isolated mouse adipocytes We sorted ADSVF cells by flow cytometry (FACSCan) based on the expression of surface proteins. CD105 positive cells accounted for 48.6% of the cells and CD105 and p75NTR double-positive cells for 15.1%, while p75NTR positive cells accounted for 39.4% (Fig. 2a). ADSVF cells were also analyzed for the expression of Sca-1 and p75NTR. Sca-1 positive cells accounted for 88.8%, and p75NTR positive cells for 33.0%. Furthermore, Sca-1 and p75NTR double-positive cells accounted for 28.9% (Fig. 2b). In other words, CD105 or Sca-1 positive-cells were included among p75NTR-positive or negative cells. We speculated that ADSVF cells had several characteristics. The stroma-associated markers CD29, CD90, CD105 and Sca-1 were expressed in a lot of cells, whereas
3.4. Differentiation of p75NTR positive and negative cells The p75NTR-positive (Fig. 3a) or negative cells were incubated in DMEM/F12 medium supplemented with FBS for 2 days (pre-differentiation) at the cell density of 5 104 cells/mL (Fig. 3b) to induce their differentiation into mature adipocytes, osteocytes, chondrocytes, smooth muscle cells or neuronal cells. Then, we compared the rate of differentiation of p75NTR-positive and negative cells into adipocytes, osteocytes and neuronal cells. The adipocytes derived from p75NTR-positive cells (Fig. 3c) had more lipid droplets than those derived from p75NTR-negative cells (Fig. 3d), and almost all differentiated cells stained positive with Oil RedO stain (Fig. 3e). Absorbance of Oil Red-O in adipocytes derived from p75NTR-positive cells was 0.41 0.292, while in those derived from p75NTRnegative cells it was 0.29 0.051 ( p < 0.05) (Fig. 4a). As for p75NTR-positive differentiated osteocytes, 31.4 7.0% of the microscopic field stained positive with Alizarin red S versus 15.0 3.6% in the culture of p75NTR-negative cells ( p < 0.05) (Figs. 3f, 3g and 4b). Regarding neuronal cells derived from p75NTRpositive cells, the number of cells positive for NF-M per microscope field was higher than that in p75NTR-negative cells cultures (3.2 1.48 versus 1.3 0.52; Figs. 3k, 3l and 4c). Accordingly, the expression of NF-M mRNA in p75NTR-positive cells was significantly higher than in p75NTR-negative cells (23.2 4.53 versus 4.9 1.79; p < 0.05) (Fig. 4d). On the other hand, both p75NTR positive and negative cells differentiated into aggrecan-positive (Fig. 3h) and collagen type 2 (Fig. 3i) positive chondrocytes, and alfa-SMA positive smooth muscle cells (Fig. 3j), with no significant difference in the differentiation rate between p75NTR positive and negative cells.
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Fig. 1 Immunohistochemical staining of mouse subcutaneous adipose tissue for p75NTR and CD90 (Thy-1), CD105 (Endoglin) or Sca-1. (a) Subcutaneous mouse adipose tissue from the inguinal region. (b) Adipose tissue without lymph nodes. (c) H.E. staining of the dissected lymph nodes. (d) H.E. staining of tissue sections including skin and adipose tissue. (e) Adipose tissue in the deep layers (strong magnification of (d)). (f) Staining for CD90. (g) Staining for p75NTR. (h) Staining for CD90 and p75NTR. (i) Staining for CD105. (j) Staining for p75NTR. (k) Staining for CD105 and p75NTR. (l) Staining for Sca-1. (m) Staining for p75NTR. (n) Staining for Sca-1 and p75NTR. A lot of cells positive for several markers (arrow heads) were observed in the deeper layers of adipose tissue and in the neighborhood of microvessels (*) (scale bar, 50 mm).
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Fig. 2 Flow cytometric analysis of mouse ADSVF cells characterized by the expression of MSC markers. ADSVF cells were stained with polyclonal antibodies to p75NTR or monoclonal antibodies to CD105 (a), Sca-1 (b), CD34 (c), and CD45 (d).
4. Discussion During embryogenesis, skin and neurons derive from epithelial cells in the ectoderm, while adipose tissue derives from mesenchymal cells. The mRNA of p75NTR, a marker of epithelial tissue stem cells and MSCs, was detected in adipose cells found in the epithelium, subcutaneous tissue or mesenteric area [21]. Furthermore, it has been shown by FACS analysis that some cells in human adipose tissue express p75NTRs [22—25]. As shown in this study, cells positive for p75NTR and CD90, CD105 or Sca-1 were detected in mouse subcutaneous adipose tissue by immunohistochemical staining, mainly in the deeper layers and in the neighborhood of microvessels. The surface markers of human adipose tissuederived stromal cells (ASCs) have already been reported [23,26]. In this study, we detected mouse ADSVF cells double-positive for p75NTR and MSC markers, and investigated whether they could
differentiate into epithelial (neuronal) and mesenchymal cells, and if these two types of stem cells had some common characteristics. On the other hand, because we were able to remove Table 1
CD29 CD31 CD34 CD45 CD90 CD105 CD133 Sca-1 TE-7
Flow cytometric analysis of ADSVF cells Mean single-positive cells (S.D.)
Mean double-positive cells (S.D.) with p75NTR
93.3 2.5 1.9 0.9 4.6 2.4 0.7 0.2 94.6 2.6 49.7 8.9 1.4 0.6 89.4 4.5 0.8 0.2
46.8 1.3 1.5 0.3 3.7 1.4 0.6 0.1 49.6 1.7 15.5 3.2 1.3 0.4 30.1 1.5 0.7 0.1
Results are expressed as the mean standard deviation (S.D.) of 30,000 ADSVF cells obtained from three mice.
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Fig. 3 Differentiation of p75NTR-positive or negative cells into mature adipocytes, osteocytes, chondrocytes, smooth muscle cells and neuronal cells. (a) After sorting p75NTR-positive cells were immunostained with fluorescent Alexa 488. p75NTR expressed on the cell membrane. (b) The p75NTR-positive cells were cultured for 2 days. p75NTR-positive cells (c) or negative cells (d) differentiated into adipocytes after 10 days. (e) Adipocytes derived from p75NTR-positive cells were stained with Oil Red-O. p75NTR-positive cells (f) or negative cells (g) differentiated into osteocytes were stained with Alizarin red S. The aggregated chondrocytes derived from p75NTR-positive cells were stained with aggrecan (h) and collagen type 2 (i). (j) Smooth muscle cells derived from p75NTR-positive cells were stained with alfa-SMA. Neuronal cells derived from p75NTR-positive cells (k) or negative cells (l) stained positive for NF-M (arrow heads) (scale bar, 100 mm).
low-adhesive cells by changing the culture media of ADSVF cells after incubation for 4 h, we think that it is possible to remove the cells positive for hematopoietic markers (mostly CD45 positive cells), in this way. On another front, the kit MSC Research Tool BoxCD271 (Miltenyi Biotech, Germany) that uses magnetic beads is available in the market to isolate p75NTR-positive adipocytes from human bone marrow [12]. Moreover, it has been reported that human adipose tissue-derived stem cells can be isolated as CD34-positive cells/CD31-negative cells also using magnetic beads [27]. It has been demonstrated that p75NTR-positive cells differentiate into Oil Red-O stain positive adipocytes, Alizarin red S stain positive osteocytes, aggrecan or collagen type-2 positive chondrocytes, alfa-SMA positive smooth muscle cells and NF-M
positive neuronal cells [28], when they were incubated in the respective induction medium. Another report described that among bone marrow derived multipotent MSCs, those positive for p75NTR showed a greater capacity to differentiate into adipocytes and osteoblasts [12]. These findings suggest that the cell membrane protein p75NTR is a useful indicator of ASCs. Furthermore, we have also tried electrophysiologic experiments such as those of Ribera and Spitzer [29] and Ribera [30], as well as those of Ashjian et al. [31] on the induction of cell differentiation into neuronal cells. Our findings indicate that electrophysiological currents are probably mediated by outwardly rectifying K+ channels, but no clear inward currents of Na+ channels, which were measured in neuronal cells, were observed (data not shown). From these observations, we speculated
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Fig. 4 Comparison of primary ADSVF cells with adipocytes, osteocytes and neuronal cells derived from p75NTR-positive or negative cells. (a) In adipocytes, we measured absorbance of Oil Red-O. (b) In osteocytes, we measured the area stained positive with Alizarin red S. (c) Number of neuronal cells that stained positive for NF-M. (d) Expression of NF-M mRNA in neuronal cells derived from p75NTR-positive and p75NTR-negative cells.
that inward currents from Na+ channels could not be measured if the cells differentiated into neuronal cells were not mature cells. Consequently, we reviewed the conditions required for the differentiation into mature neuronal cells. The other important point was self-renewal of stem cells. The subcultured cells (third passage) maintained the expression ratio of p75NTR at about the level observed in primary ADSVF cells. In in vitro cell cultures, stem cells did not maintain the expression of p75NTR. Some cells inherited the selfrenewal capacity character of stem cells while other cells became committed or progenitor cells [32—34]. MSCs were first isolated from bone marrow in 1976 [35,36], in addition to that, multifunctional stem cells that can differentiate into various cell types were reported to be present in the bone marrow of adult humans. Typical examples are multipotent adult progenitor cells (MAPCs) [37,38] and marrow-isolated adult multilineage inducible cells (MIAMI) [39]. MAPCs have been isolated not only from human tissues but also from those of mice
and rats. The result of an experiment using a chimera mouse injected with MAPCs suggested that MAPCs are involved in the formation of various organs [40]. The ASCs dealt with in this study may attract attention as source of clinically applicable autograft cells, as in the case of MAPCs. Since adipose tissue can be collected from the patients themselves, immune rejection is unlikely to occur, and since cells of the patients themselves are used, the ethical problems encountered with ES cells would disappear. However, there are important problems to be solved before ASCs can be applied in a clinical setting [41]. The first may be that ASCs have not been proven to be a kind of stem cell in the strict sense of the word, since these cells have never been shown to undergo self-renewal for a long period. The second is that ASCs must be proven not to transform after differentiation into a specific type of cell, with special attention given to telomerase activity. By further refining the method described in this report, and after more extensive research, ASCs
Isolation of multipotent cells from mouse adipose tissue may become a useful material for the regeneration of specific tissues.
Acknowledgments This work was supported by a grant-in-aid for young scientists: MEXT. KAKENHI 17700341 and MEXT. HAITEKU Grant 2002—2006. This work is now under examination by the Japanese Patent Office to obtain a technical patent.
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52 [33] Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell 1995;80:83—9. [34] Feuerhake F, Sigg W, Hofter EA, Dimpfl T, Welsch U. Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium. Cell Tissue Res 2000;299:47—58. [35] Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568—84. [36] Friedenstein AJ. Precursor cells of mechanocytes. Int Rev Cytol 1976;47:327—59. [37] Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615—25.
N. Yamamoto et al. [38] Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291—302. [39] D’Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004;117:2971—81. [40] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41—9. [41] Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035—9.
www.intl.elsevierhealth.com/journals/jods
Isolation of multipotent stem cells from mouse adipose tissue§ Naoki Yamamoto a,e,*, Hirohiko Akamatsu b, Seiji Hasegawa c, Takaaki Yamada c, Satoru Nakata c, Mahito Ohkuma d, Ei-Ichi Miyachi d, Tohru Marunouchi e, Kayoko Matsunaga b a
Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan b Department of Dermatology, Fujita Health University School of Medicine, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan c Research Laboratories, Nippon Menard Cosmetic Co. Ltd., 2-7 Torimi-cho, Nishi-Ku, Nagoya, Aichi 451-0071, Japan d Department of Physiology, Fujita Health University School of Medicine, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan e Department of Cell Biology, Fujita Health University Institute for Comprehensive Medical Science, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan Received 15 December 2006; received in revised form 23 May 2007; accepted 30 May 2007
KEYWORDS p75 neurotrophin receptor (p75NTR, CD271); Adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells); Mesenchymal stem cell (MSC); CD105; Sca-1; Neuronal cell
Summary Background: Embryonic stem (ES) cells, bone marrow, adipose tissue or other genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. However, there is no specific surface antigen that can be used as a marker of multipotent stem cells. Objective: We tried to isolate and collect putative multipotent stem cells from mouse subcutaneous adipose tissue using the p75 neurotrophin receptor (p75NTR) as a marker. Methods: Adipose tissue was processed for immunostaining using antibodies antiCD90, anti-CD105 and anti-Sca-1 as general mesenchymal stem cell (MSC) markers, and anti-p75NTR, an epithelial stem cell and MSC marker. Subsequently, the expression of cell surface markers in adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells) was examined by flow cytometry (fluorescence-activated cell
§
In accordance with a consensus reached by investigators attending the Second Annual International Fat Applied Technology Society Meeting, they will refer to this adherent cell population as adipose-derived stem cells (ASCs). * Corresponding author at: Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 1-98 Kutsukake-cho, Toyoake, Aichi 470-1192, Japan. Tel.: +81 562 93 2317; fax: +81 562 92 5382. E-mail address: [email protected] (N. Yamamoto). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.05.015
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N. Yamamoto et al. sorting: FACS). Finally, ADSVF cells positive for p75NTR were sorted and cultured to induce their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. Results: Cells positive for several of these markers were found in the deep layers of adipose tissue. Among them, those positive for p75NTR differentiated into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. The rate of differentiation into adipocytes, osteoblasts and neuronal cells was higher for p75NTRpositive cells than for p75NTR-negative cells. Conclusions: p75NTR proved to be a useful marker to isolate adipose tissue-derived stem cells (ASCs). # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Embryonic stem (ES) cells and genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. Because of their multi-differentiation ability and since cells for a specific purpose may be efficiently prepared, stem cells are very useful. However, clinical application of these cells is currently difficult in view of ethical issues as well as the potential risks of carcinogenesis [1]. On the other hand, cells derived from bone marrow, adipose tissue or other tissues have attracted attention nowadays as clinically applicable sources of autografting cells, because these cells can differentiate into various types of cells, including smooth muscle cells, endothelial cells and neuronal cells [2—7]. However, no specific surface antigen that can be used as a marker of multipotent cells has been identified so far. In the present study, we tried to isolate and collect multipotent cells (adipose tissue-derived stem cells: ASCs) from among subcutaneous adipose tissuederived stromal vascular fraction cultured cells (ADSVF cells) [8] of mouse using the p75 neurotrophin receptor (p75NTR), a cell membrane protein, as a marker [9—12]. The p75NTR is a low-affinity nerve growth factor receptor. Recently, a CD number was given to p75NTR, and it is now known as CD271. As we found that ADSVF cells positive for p75NTR differentiated into mature adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells, in this study we compared the rate of differentiation into adipocytes, osteoblasts and neuronal cells obtained using p75NTR-positive cells with that attained using p75NTR-negative cells.
2. Materials and methods 2.1. Animals For the immunohistochemical study and isolation of mouse ADSVF cells, we used 4-week-old male ICR
albino mice (Japan SLC, Inc., Shizuoka, Japan). All procedures were approved by the Education and Research Center for Animal Models of Human Diseases of Fujita Health University.
2.2. Immunohistochemistry Subcutaneous adipose tissue was excised from the abdomen of adult mice. This was fixed with 4% paraformaldehyde and paraffin sections were prepared by the usual method. These sections were processed for H.E. staining and immunostaining using antibodies anti-CD90 (Thy-1), anti-CD105 (Endoglin) (eBioscience, Inc., San Diego, CA) and anti-Sca-1 (Stem cell antigen-1/Ly6) (R&D Systems, Inc., Minneapolis, MN) that are general mesenchymal stem cell (MSC) markers [13—15], and antip75NTR (Chemicon International, Inc., Temecula, CA) that is known to be an epithelial and MSC marker [9—12]. As for the secondary antibodies, anti-rat IgG antibody labeled with Alexa Fluor 488 (Invitrogen Corp., Carlsbad, CA) and anti-rabbit IgG antibody labeled with Alexa Fluor 594 (Invitrogen) were used. DAPI (VECTASHIELD H-1200; Vector Laboratories, Burlingame, CA) was used for nuclear staining. A fluorescence microscope (Power BX-51, Olympus, Tokyo, Japan) was used for observation.
2.3. Isolation and culture of mouse adipose tissue Mouse adipose tissue dissected from lymph nodes was cut into fine pieces. These pieces were digested with 0.2% collagenase (Nitta Gelatin, Osaka, Japan) at 37 8C for 30 min and the resultant cell suspension was filtered through a 100 mm mesh (BD Biosciences, San Jose, CA) to remove tissue debris. Then, collagenase was removed by dilution with phosphate-buffered saline solution (PBS, Sigma, St. Louis, MO) and centrifuged twice for 5 min at 260 g each time (himac CF7D2, Hitachi-koki Co. Ltd., Ibaragi, Japan). In this way, most of the matured adipocytes remained in the supernatant. The cell pellet (stromal vascular
Isolation of multipotent cells from mouse adipose tissue fraction [8]) was suspended in 0.83% NH4Cl and incubated to remove contaminating red blood cells. Cells were washed and centrifuged twice with PBS and cultured in Dulbecco’s modified Eagle’s medium/ Ham’s F12 (DMEM/F12) (Sigma) supplemented with 5% fetal bovine serum (FBS, JRH Biosciences, Adelaide, Australia), 2 ng/mL basic fibroblastic growth factor (bFGF, Sigma) and 1% penicillin/streptomycin (Sigma) in 100 mm 20 mm culture dishes at 37 8C in a 5% CO2 humidified incubator. After 4 h, the nonadherent cells were removed and adherent ADSVF cells were cultured for 7 more days.
2.4. Flow cytometric analysis Cultured mouse ADSVF cells were analyzed using fluorescence-activated cell sorting (FACS, cell analyzer) (FACScan, BD Biosciences). In brief, the cells were harvested and incubated for 30 min on ice with 1% BSA in PBS containing the primary antibodies directed against the following cell surface markers: CD29 (Integrin beta-1) [16], TE-7 (fibroblasts marker) (Chemicon), CD31 (PECAM-1), CD34, CD45 (LCA), CD90 (Thy-1), CD105 (Endoglin), CD133 (Prominin-1; AC 133) (eBioscience), Sca-1 (Stem cell antigen-1/Ly6) and p75NTR (CD271). After washing, secondary antibodies Alexa Fluor 488 and PE-Cy5 (for p75NTR labeling, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added, and the cells were incubated for another 30 min on ice. Then, the cells were washed twice before the analysis.
2.5. Sorting of p75NTR-positive cells Mouse ADSVF cells were subjected to fluorescenceactivated cell sorting (FACS, cell sorter) (FACSVantage SE, BD Biosciences) to collect p75NTR-positive and negative cells. The cells were harvested and incubated in 1% BSA-PBS containing anti-p75NTR antibody for 30 min on ice, then anti-rabbit IgG antibody labeled with Alexa Fluor 488 was added and the cells were incubated for another 30 min on ice. The p75NTR-positive or negative cells were then aseptically collected, cultured and used in all subsequent experiments.
2.6. Cell differentiation and immunocytochemistry For cell differentiation, preadipocyte differentiation medium (PT-8000, LONZA Walkersville, Inc., MD) (Sanko Junyaku Co. Ltd., Tokyo), osteoblast differentiation medium (CA417500, TOYOBO Co. Ltd., Osaka), chondrocyte differentiation medium (CC-3225, LONZA), smooth muscle cell proliferating medium (CC-3182, LONZA) and neurobasal medium
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(Invitrogen) that contained 1 mM retinoic acid (Sigma), valproic acid (Sigma) and N-2 supplement (Invitrogen) known to induce differentiation into neuronal cells were used [17]. Simultaneously, p75NTR-negative cells were cultured in the same differentiation media as control.
2.7. Detection of differentiated cells The experiments on differentiation into adipocytes, osteoblasts, chondrocytes and smooth muscle cells were done by adding the specific differentiation medium, but those on differentiation into neuronal cells were done as previously described [17]. ADSVF cells differentiated into mature adipocytes were fixed at 10 days of culture in 4% paraformaldehyde (PFA), examined for morophological changes and stained with Oil Red-O stain (Sigma). Then, stained cells were extracted with isopropanol and absorbance was measured at 520 nm (Versa Max, Molecular Devices Corp., Long Branch, NJ) to assess the content of lipid. The cells differentiated into osteoblasts were fixed at 14 days of culture in 4% PFA and stained with Alizarin red S stain (Wako Pure Chemical Industries, Co. Ltd., Osaka). Cells were observed under a bright field using a CKX41 microscope, and a microscope with a mounted digital camera (DP-71, Olympus) was used to capture 10 random nonoverlapping low-power (200) images of 100 mm 20 mm culture dishes. The capture images were converted into the binary scale, and NIH image software was used to evaluate differentiated osteoblasts for measured area (50 50 pixels) in the captured field. The areas stained with Alizarin red S were expressed as a percentage of the measured area. The cells differentiated into chondrocytes were fixed at 21 days of culture in 4% PFA and cryosections were obtained using a cryostat (CM3050S, Leica Microsystems, Nussloch, Germany) (FINETEC, Tokyo). Then, the cryosections were immunostained with anti-aggrecan antibody (Abcam Limited, Cambridgeshire, UK) and anti-collagen type-2 antibody (LSL Co. Ltd., Tokyo) [18]. To detect the cells differentiated into smooth muscle cells at 14 days, these were immunostained with anti-alfa-smooth muscle actin antibody (alfaSMA) (Lab Vision Corp., Fremont, CA) [11]. The cells differentiated into neuronal cells were fixed at 5 days of culture in 4% PFA and stained with anti-neurofilament M (NF-M) antibody (Chemicon), which is a neuronal cell marker [19,20]. Then, RNA was prepared using a High Pure RNA Isolation Kit (Roche Diagnostics Corp., Indianapolis, IN) at intervals and quantitative Real Time RT-PCR (qRT-PCR) was carried out using PRISM-7900HT (Applied
46 Biosystems, Foster City, CA). The assay ID of the TaqMan primers and probe (Applied Biosystems) used were NF-M: Mm00456201_m1 and GAPDH: Mm99999915_g1.
N. Yamamoto et al. the percentage of CD34 positive cells was low (Fig. 2c), and the common leukocyte antigen CD45 was not observed on ADSVF cells (Fig. 2d). The results of flow cytometric analysis of ADSVF cells isolated from three mice are shown in Table 1.
2.8. Statistical analysis 3.3. Sorting of p75NTR-positive cells Data are presented as the mean value S.D. Differences between two groups were evaluated for statistical significance using Student’s t-test. Differences were considered significant at p < 0.05.
p75NTR-positive cells accounted for 37.8 5.49% of ADSVF cells. Therefore, we separated p75NTR-positive cells from ADSVF cells using FACSVantage SE and succeeded in increasing their population to more than 97%.
3. Results 3.1. Immunohistochemistry of adipose tissue Mouse adipose tissue from the subcutaneous inguinal region was used. Since the lymph nodes present in the adipose tissue can be easily distinguished (Fig. 1a), they were dissected in advance (Fig. 1b and c). The adipose tissue was stained with H.E. (Fig. 1d and e), anti-CD90 and anti-p75NTR. CD90positive cells (Fig. 1f), p75NTR-positive cells (Fig. 1g) and double positive cells (Fig. 1h) were found to be distributed in the deeper layers of adipose tissue (Fig. 1d), with CD90 and p75NTR double positive cells being observed in the neighborhood of microvessels. Next, adipose tissue was stained with antibody for p75NTR and CD105 or Sca-1. In the same way as CD90-positive cells had been observed, cells positive for CD105 and Sca-1 were observed in the deeper layers of the adipose tissue (Fig. 1i and l). A lot of CD105 or Sca-1 positive cells were also positive for p75NTR (Fig. 1j, k, m, and n).
3.2. Characterization of isolated mouse adipocytes We sorted ADSVF cells by flow cytometry (FACSCan) based on the expression of surface proteins. CD105 positive cells accounted for 48.6% of the cells and CD105 and p75NTR double-positive cells for 15.1%, while p75NTR positive cells accounted for 39.4% (Fig. 2a). ADSVF cells were also analyzed for the expression of Sca-1 and p75NTR. Sca-1 positive cells accounted for 88.8%, and p75NTR positive cells for 33.0%. Furthermore, Sca-1 and p75NTR double-positive cells accounted for 28.9% (Fig. 2b). In other words, CD105 or Sca-1 positive-cells were included among p75NTR-positive or negative cells. We speculated that ADSVF cells had several characteristics. The stroma-associated markers CD29, CD90, CD105 and Sca-1 were expressed in a lot of cells, whereas
3.4. Differentiation of p75NTR positive and negative cells The p75NTR-positive (Fig. 3a) or negative cells were incubated in DMEM/F12 medium supplemented with FBS for 2 days (pre-differentiation) at the cell density of 5 104 cells/mL (Fig. 3b) to induce their differentiation into mature adipocytes, osteocytes, chondrocytes, smooth muscle cells or neuronal cells. Then, we compared the rate of differentiation of p75NTR-positive and negative cells into adipocytes, osteocytes and neuronal cells. The adipocytes derived from p75NTR-positive cells (Fig. 3c) had more lipid droplets than those derived from p75NTR-negative cells (Fig. 3d), and almost all differentiated cells stained positive with Oil RedO stain (Fig. 3e). Absorbance of Oil Red-O in adipocytes derived from p75NTR-positive cells was 0.41 0.292, while in those derived from p75NTRnegative cells it was 0.29 0.051 ( p < 0.05) (Fig. 4a). As for p75NTR-positive differentiated osteocytes, 31.4 7.0% of the microscopic field stained positive with Alizarin red S versus 15.0 3.6% in the culture of p75NTR-negative cells ( p < 0.05) (Figs. 3f, 3g and 4b). Regarding neuronal cells derived from p75NTRpositive cells, the number of cells positive for NF-M per microscope field was higher than that in p75NTR-negative cells cultures (3.2 1.48 versus 1.3 0.52; Figs. 3k, 3l and 4c). Accordingly, the expression of NF-M mRNA in p75NTR-positive cells was significantly higher than in p75NTR-negative cells (23.2 4.53 versus 4.9 1.79; p < 0.05) (Fig. 4d). On the other hand, both p75NTR positive and negative cells differentiated into aggrecan-positive (Fig. 3h) and collagen type 2 (Fig. 3i) positive chondrocytes, and alfa-SMA positive smooth muscle cells (Fig. 3j), with no significant difference in the differentiation rate between p75NTR positive and negative cells.
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Fig. 1 Immunohistochemical staining of mouse subcutaneous adipose tissue for p75NTR and CD90 (Thy-1), CD105 (Endoglin) or Sca-1. (a) Subcutaneous mouse adipose tissue from the inguinal region. (b) Adipose tissue without lymph nodes. (c) H.E. staining of the dissected lymph nodes. (d) H.E. staining of tissue sections including skin and adipose tissue. (e) Adipose tissue in the deep layers (strong magnification of (d)). (f) Staining for CD90. (g) Staining for p75NTR. (h) Staining for CD90 and p75NTR. (i) Staining for CD105. (j) Staining for p75NTR. (k) Staining for CD105 and p75NTR. (l) Staining for Sca-1. (m) Staining for p75NTR. (n) Staining for Sca-1 and p75NTR. A lot of cells positive for several markers (arrow heads) were observed in the deeper layers of adipose tissue and in the neighborhood of microvessels (*) (scale bar, 50 mm).
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Fig. 2 Flow cytometric analysis of mouse ADSVF cells characterized by the expression of MSC markers. ADSVF cells were stained with polyclonal antibodies to p75NTR or monoclonal antibodies to CD105 (a), Sca-1 (b), CD34 (c), and CD45 (d).
4. Discussion During embryogenesis, skin and neurons derive from epithelial cells in the ectoderm, while adipose tissue derives from mesenchymal cells. The mRNA of p75NTR, a marker of epithelial tissue stem cells and MSCs, was detected in adipose cells found in the epithelium, subcutaneous tissue or mesenteric area [21]. Furthermore, it has been shown by FACS analysis that some cells in human adipose tissue express p75NTRs [22—25]. As shown in this study, cells positive for p75NTR and CD90, CD105 or Sca-1 were detected in mouse subcutaneous adipose tissue by immunohistochemical staining, mainly in the deeper layers and in the neighborhood of microvessels. The surface markers of human adipose tissuederived stromal cells (ASCs) have already been reported [23,26]. In this study, we detected mouse ADSVF cells double-positive for p75NTR and MSC markers, and investigated whether they could
differentiate into epithelial (neuronal) and mesenchymal cells, and if these two types of stem cells had some common characteristics. On the other hand, because we were able to remove Table 1
CD29 CD31 CD34 CD45 CD90 CD105 CD133 Sca-1 TE-7
Flow cytometric analysis of ADSVF cells Mean single-positive cells (S.D.)
Mean double-positive cells (S.D.) with p75NTR
93.3 2.5 1.9 0.9 4.6 2.4 0.7 0.2 94.6 2.6 49.7 8.9 1.4 0.6 89.4 4.5 0.8 0.2
46.8 1.3 1.5 0.3 3.7 1.4 0.6 0.1 49.6 1.7 15.5 3.2 1.3 0.4 30.1 1.5 0.7 0.1
Results are expressed as the mean standard deviation (S.D.) of 30,000 ADSVF cells obtained from three mice.
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Fig. 3 Differentiation of p75NTR-positive or negative cells into mature adipocytes, osteocytes, chondrocytes, smooth muscle cells and neuronal cells. (a) After sorting p75NTR-positive cells were immunostained with fluorescent Alexa 488. p75NTR expressed on the cell membrane. (b) The p75NTR-positive cells were cultured for 2 days. p75NTR-positive cells (c) or negative cells (d) differentiated into adipocytes after 10 days. (e) Adipocytes derived from p75NTR-positive cells were stained with Oil Red-O. p75NTR-positive cells (f) or negative cells (g) differentiated into osteocytes were stained with Alizarin red S. The aggregated chondrocytes derived from p75NTR-positive cells were stained with aggrecan (h) and collagen type 2 (i). (j) Smooth muscle cells derived from p75NTR-positive cells were stained with alfa-SMA. Neuronal cells derived from p75NTR-positive cells (k) or negative cells (l) stained positive for NF-M (arrow heads) (scale bar, 100 mm).
low-adhesive cells by changing the culture media of ADSVF cells after incubation for 4 h, we think that it is possible to remove the cells positive for hematopoietic markers (mostly CD45 positive cells), in this way. On another front, the kit MSC Research Tool BoxCD271 (Miltenyi Biotech, Germany) that uses magnetic beads is available in the market to isolate p75NTR-positive adipocytes from human bone marrow [12]. Moreover, it has been reported that human adipose tissue-derived stem cells can be isolated as CD34-positive cells/CD31-negative cells also using magnetic beads [27]. It has been demonstrated that p75NTR-positive cells differentiate into Oil Red-O stain positive adipocytes, Alizarin red S stain positive osteocytes, aggrecan or collagen type-2 positive chondrocytes, alfa-SMA positive smooth muscle cells and NF-M
positive neuronal cells [28], when they were incubated in the respective induction medium. Another report described that among bone marrow derived multipotent MSCs, those positive for p75NTR showed a greater capacity to differentiate into adipocytes and osteoblasts [12]. These findings suggest that the cell membrane protein p75NTR is a useful indicator of ASCs. Furthermore, we have also tried electrophysiologic experiments such as those of Ribera and Spitzer [29] and Ribera [30], as well as those of Ashjian et al. [31] on the induction of cell differentiation into neuronal cells. Our findings indicate that electrophysiological currents are probably mediated by outwardly rectifying K+ channels, but no clear inward currents of Na+ channels, which were measured in neuronal cells, were observed (data not shown). From these observations, we speculated
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Fig. 4 Comparison of primary ADSVF cells with adipocytes, osteocytes and neuronal cells derived from p75NTR-positive or negative cells. (a) In adipocytes, we measured absorbance of Oil Red-O. (b) In osteocytes, we measured the area stained positive with Alizarin red S. (c) Number of neuronal cells that stained positive for NF-M. (d) Expression of NF-M mRNA in neuronal cells derived from p75NTR-positive and p75NTR-negative cells.
that inward currents from Na+ channels could not be measured if the cells differentiated into neuronal cells were not mature cells. Consequently, we reviewed the conditions required for the differentiation into mature neuronal cells. The other important point was self-renewal of stem cells. The subcultured cells (third passage) maintained the expression ratio of p75NTR at about the level observed in primary ADSVF cells. In in vitro cell cultures, stem cells did not maintain the expression of p75NTR. Some cells inherited the selfrenewal capacity character of stem cells while other cells became committed or progenitor cells [32—34]. MSCs were first isolated from bone marrow in 1976 [35,36], in addition to that, multifunctional stem cells that can differentiate into various cell types were reported to be present in the bone marrow of adult humans. Typical examples are multipotent adult progenitor cells (MAPCs) [37,38] and marrow-isolated adult multilineage inducible cells (MIAMI) [39]. MAPCs have been isolated not only from human tissues but also from those of mice
and rats. The result of an experiment using a chimera mouse injected with MAPCs suggested that MAPCs are involved in the formation of various organs [40]. The ASCs dealt with in this study may attract attention as source of clinically applicable autograft cells, as in the case of MAPCs. Since adipose tissue can be collected from the patients themselves, immune rejection is unlikely to occur, and since cells of the patients themselves are used, the ethical problems encountered with ES cells would disappear. However, there are important problems to be solved before ASCs can be applied in a clinical setting [41]. The first may be that ASCs have not been proven to be a kind of stem cell in the strict sense of the word, since these cells have never been shown to undergo self-renewal for a long period. The second is that ASCs must be proven not to transform after differentiation into a specific type of cell, with special attention given to telomerase activity. By further refining the method described in this report, and after more extensive research, ASCs
Isolation of multipotent cells from mouse adipose tissue may become a useful material for the regeneration of specific tissues.
Acknowledgments This work was supported by a grant-in-aid for young scientists: MEXT. KAKENHI 17700341 and MEXT. HAITEKU Grant 2002—2006. This work is now under examination by the Japanese Patent Office to obtain a technical patent.
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