Cosmetic Medicine in Japan -東京大学美容外科- トレチノイン(レチノイン酸)療法、アンチエイジング(若返り)
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Human adherent stromal cells isolated from adipose tissue have been shown to have multipotency [1, 2], able to differentiate not only into mesenchymal lineages including endothelial cells [3-6] and cardiomyocytes [7-9] but also into neural cells [2] and hepatocytes [10]. These cells have been referred to by various names, including preadipocytes, vascular stromal cells, adipose-derived mesenchymal progenitor cells, and adipose stromal cells. In this study, we refer to the cells as adipose-derived stem cells (ASCs). The characteristics of ASCs have been extensively studied [11-15], as well as the potential clinical applications of ASCs [3-7, 16]. In addition, clinical trials have already begun involving enhancement of bone and adipose regeneration and angiogenesis [17-20].
Adipose tissue is thought to be a promising source of stem cells because it can be harvested in relatively large quantities (100 mL to > 1 L) using liposuction with minimal morbidity. Although ASCs may be clinically used without cell expansion because of their large quantities, it is of great value to culture and expand ASCs safely and effectively without losing their multipotency for manipulation and further development of cell-based therapies. There have been some reports indicating enhanced proliferation of human ASCs using specific culture media with supplements. It was shown that fibroblast growth factor (FGF)-2 was released by ASCs, enhanced proliferation [21-24], and maintained the adipogenic potential of ASCs [21]. FGF-1 and epidermal growth factor (EGF) were suggested to act as stimulators of both ASC proliferation and differentiation [25-27]. Platelet -derived growth factor (PDGF)-BB [25, 28], tumor necrosis factor (TNF)-α [29], and insulin-like growth factor (IGF)-1 [29] were also shown to promote ASC proliferation, and the former two factors were suggested to have inhibiting effects on ASC differentiation [25, 29]. However, it has not been shown whether ASCs expanded by these methods preserve their multipotency or not.
In this study, we investigated the effects of an endothelial growth medium (EGM-2R, Cambrex, Walkersville, MD) on culturing human ASCs, focusing on proliferation and differentiation potentials. EGM-2 is usually used to support the growth of endothelial cells. In recent studies, EGM-2 has been used for culture of non-endothelial cells [16, 30, 31]. However, ASCs have usually been cultured in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM/F12 medium, and the effects of EGM-2 on ASCs have not been reported.

Materials and methods
Cell isolation and culture
We obtained liposuction aspirates from 12 healthy female donors undergoing liposuction of the abdomen or thighs after informed consent using an IRB-approved protocol. The stromal vascular fraction containing ASCs was isolated from the fatty portion of liposuction aspirates, as previously described [15]. Briefly, the aspirated fat was washed with phosphate buffered saline (PBS) and digested on a shaker at 37oC in PBS containing 0.075% collagenase for 30 min. Mature adipocytes and connective tissues were separated from pellets by centrifugation (800×g, 10 min). The cell pellets were resuspended, filtered with a 100-μm mesh (Millipore, MA, USA), plated at a density of 5×105 nucleated cells/100-mm dish, and cultured at 37°C in an atmosphere of 5% CO2 in humid air. The culture medium was: (1) DMEM (Nissui Pharmaceutical, Tokyo, Japan) containing 10% fetal bovine serum (FBS), or (2) EGM-2 containing 2% FBS. Endothelial basal medium (EBM, Cambrex) is a basal medium for EGM-2. EGM-2 does not contain any animal-derived factors but does contain FGF-2, vascular endothelial growth factor (VEGF), IGF-1, EGF, ascorbic acid, hydrocortisone, GA-1000 (gentamicin and amphotericin-B), and heparin, although the concentration of each agent is not disclosed. Primary cells were cultured for 7 days and were defined as “Passage 0.” The medium was replaced every 3 days. Cells were passaged every week by trypsinization.

<Measurement of doubling time and total cell number>
During cell culture in each medium, doubling time was measured at passages 0, 1, 2, and 3 by seeding ASCs (Passage 0) at a density of 1×105 cells per 10-cm dish. After cells reached the logarithmic growth phase, they were sequentially trypsinized every 48 h and counted with a cell counter (NucleoCounter, Chemometec, Allerod, Denmark). Doubling time was calculated according to the following formula: doubling time = 48 h/log2(N2/N1), where N1 is the first cell count and N2 is the cell count 48 h later. Total cell number after the initiation of culture in each medium was also measured by seeding ASCs (Passage 0) at a density of 1×104 cells per 3.5-cm dish and culturing the cells until they reached the stationary phase.

Measurement of the proliferative effect of supplemented growth factors
To examine the proliferative effect of each growth factor supplemented in EGM-2, ASCs were cultured in medium supplemented with a single growth factor (VEGF, EGF, IGF-1, or FGF-2). EBM containing 2% FBS was used as the control medium. ASCs cultured in the control medium (Passage 0) were seeded at a density of 1×104 cells per well in a 6-well plate. Cells were cultured in the control medium (2% FBS), supplemented medium (0.1, 1, or 10 ng/ml of each growth factor) (2% FBS), DMEM (10% FBS), or EGM-2 (2% FBS). The number of cells after 7 days of culture was counted using a cell counter.

Flow cytometry of cultured cells
Cultured cells in each medium were examined for surface marker expression using flow cytometry. The following monoclonal antibodies (MAbs) conjugated to fluorochromes were used: anti-CD29-PE, CD31-PE, CD34-PE, CD45-PE, CD90-PE, CD146-PE (BD Biosciences, San Diego, CA), CD105-PE (Serotec, Oxford, UK), and Flk-1-PE (Techne, Minneapolis, MN). Control MAbs were included for all fluorochromes. Cells were incubated with directly conjugated MAbs for 30 minutes, then washed and fixed in 1% paraformaldehyde. Cells were analyzed using an LSR II (Becton Dickinson, San Jose, CA) flow cytometry system. Data acquisition and analysis were then performed (Cell Quest software, Becton Dickinson). Gates were set based on staining with combinations of relevant and irrelevant MAbs so that no more than 0.1% of cells were positive using irrelevant antibodies.

Induced differentiation of cultured cells
After culture in each medium for 2 weeks, differentiation into the adipogenic, chondrogenic, and osteogenic lineages was examined.
For adipogenic differentiation, cells were incubated for 4 weeks in DMEM containing 10% FBS supplemented with 0.5 mM isobutyl-methylxanthine (Sigma, St. Louis, MO), 1 ?M dexamethasone, 10 ?M insulin (Sigma), and 200 ?M indomethacin. Adipogenic differentiation was visualized with oil red O staining. For quantitative analysis of lipid droplets, we measured Nile Red fluorescence, using AdipoRedTM (Cambrex), with excitation at 485 nm and emission at 535 nm.
For chondrogenic differentiation, cells were incubated for 4 weeks in DMEM containing 1% FBS supplemented with 6.25 ?g/ml insulin, 10 ng/ml TGF-β1, and 50 nM ascorbate-2-phosphate. Chondrogenic differentiation was visualized with Alcian Blue staining. For quantitative analysis, a micromass culture system was used as previously reported [32]. Cells were plated in a 15-ml tube and cultured in the chondrogenic medium for 3 weeks. Then, the diameter of a micromass was measured.
For osteogenic differentiation, cells were incubated for 4 weeks in DMEM containing 10% FBS supplemented with 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, and 10 mM β-glycerophosphate (Nacalai Tesque, Kyoto, Japan). Osteogenic differentiation was visualized with von Kossa staining. For quantitative analysis of total calcium, calcium deposition was evaluated based on the ortho-cresolphthalein complexone (OCPC) method with the Calcium C-Test Wako Kit (Wako Chemicals) according to the manufacturer’s instructions.

Statistical analyses
Results were expressed as mean ± SEM. Welch’s t-test was used to compare each parameter. A value of p < 0.05 was considered significant.
Doubling time and total cell number
Doubling time of ASCs cultured with EGM-2 was significantly shorter than that of cells cultured with DMEM at each passage (19.3 ± 2.1 h vs. 39.8 ± 6.8 h at Passage 0; 15.6 ± 1.1 h vs. 55.1 ± 3.5 h at Passage 1; 20.3 ± 0.7 h vs. 52.0 ± 2.4 h at Passage 2; and 26.5 ± 1.1 h vs. 54.3 ± 6.4 h at Passage 3) (Fig. 1A). Total cell number showed that ASCs cultured with EGM-2 proliferated more rapidly and reached the stationary phase earlier than those cultured with DMEM (40 days vs. 200 days), though the maximum population doubling level of ASCs was similar either when cultured with EGM-2 or DMEM (35?40 with EGM-2 vs. 40?45 with DMEM) (Fig. 1B). Differentiation assays were performed using ASCs cultured with each medium for 2 weeks, and at that stage, ASCs cultured with EGM-2 were supposed to be expanded 105 times (1010 vs. 105) compared to those cultured with DMEM (Fig. 1B).

Proliferative effect of each growth factor
VEGF, EGF, and IGF-1 showed no significant proliferative effect on ASCs cultured in EBM containing 2% FBS. FGF-2 at a density of 0.1, 1, or 10 ng/ml significantly promoted proliferation of ASCs compared to control. However, the proliferative effect of FGF-2 was much less than that of EGM-2 containing all of the growth factors, indicating a synergistic effect of supplemented growth factors (Fig. 2).

Flow cytometry
Flow cytometry of ASCs cultured in DMEM and EGM-2 showed no significant differences except CD 105 at passages 1, 2, and 3 (Table 1). Both cell populations uniformly expressed mesenchymal markers (CD29 and CD90) and were devoid of a hematopoietic cell marker CD45. Expressions of CD34 (stem cell marker), CD31 (endothelial cell marker), CD146 (endothelial and mural cells marker), and Flk-1 (VEGFR-2) were similar in both cell populations, and CD34 expression of ASCs markedly decreased at passage 1 in both media (Fig. 3).

Differentiation capacity
Both cell populations cultured in DMEM and EGM-2 for 2 weeks had similar capacities to differentiate into adipogenic, chondrogenic, and osteogenic lineages. No morphological differences between the two cell populations were observed during and after differentiation (Fig. 4A). Quantitative analyses (lipid droplets in adipogenic differentiation, micromass diameter in chondrogenic differentiation, and total calcium content in osteogenic differentiation) also showed no significant differences between the two cell populations (Fig. 4B).
In this study, EGM-2 expanded ASCs very rapidly while preserving their multipotency for at least 2 weeks; the proliferative efficiency of EGM-2 was 105 times of that of DMEM in the first 2 weeks. A doubling time of ASCs shorter than that shown in this study (15?20 hours) has not been reported previously in the literature. EGM-2 contains 2% FBS and various growth factors including FGF-2, VEGF, IGF-1, and EGF. The highly boosting effects of EGM-2 on ASC proliferation are suggested to result from supplemented growth factors and other unknown synergistic effects, as discussed below. ASCs cultured with EGM-2 proliferated much more rapidly and reached the stationary phase earlier than those cultured with DMEM (40 days vs. 200 days), although the maximum population doubling levels were similar between the two culture media (Fig. 1B). The results suggest that a majority of ASCs may have a limited capacity of self renewal.
Serum concentrations can affect proliferation activity of ASCs. We previously reported that the doubling time of ASCs cultured with 15% FBS was significantly shorter than that of cells cultured with 10% FBS, although the culture media was M199 supplemented with FGF-1 in that study [15]. FBS is made through coagulations of fetal bovine whole blood; thus, it is supposed to contain not only IGF-1, which is regularly present in serum including platelet-poor plasma-derived serum, but also platelet-derived cytokines such as PDGF and EGF [our unpublished data, in submission]. In the present study, however, the doubling time of ASCs cultured in DMEM was significantly longer than that for ASCs cultured in EGM-2, in spite of the higher FBS concentration (10%) in DMEM compared to EGM-2 (2%). This result suggests that the growth factors added to EGM-2 have greater effects on the proliferation activity of ASCs than do serum concentrations. In fact, a recent report suggested that platelet-derived growth factors (not designated but assumed to be PDGF and EGF) may reduce proliferation activity and adipogenic differentiation capacity of ASCs [23].
Among growth factors contained in EGM-2, supplementation with IGF-1, EGF, or VEGF did not significantly promote proliferation activity of ASCs in EBM culture containing 2% FBS. The growth factors have some proliferative effects on ASCs when added to serum-free media or SPPP, as reported previously [5, 23, 29]; however, any effects in this study using a low concentration of FBS with IGF-1 and EGF were subtle or masked. In this study, only FGF-2 showed a statistically significant promoting effect on ASC proliferation, which has been suggested in previous studies [21-24]. The results strongly suggest that FGF-2 is a critical growth factor for supplementation of serum-containing culture media. A previous study suggested that FGF-2 plays a critical role in self renewal of ASCs [21]. It was also shown that FGF-2 added to SPPP increased proliferation activity and adipogenic differentiation capacity [23]. Another study reported efficient proliferation of ASCs transfected with the FGF-2 gene [33]. However, in 7 days, EGM-2 expanded ASCs significantly and several-fold more compared to FGF-2-supplemented EBM (Fig. 2), and thus the effect of EGM-2 on ASC proliferation cannot be explained solely based on the influence of FGF-2 alone. It is likely that synergistic effects of various growth factors and other factors contributed to the exceptional efficiency.
In our study, the character of ASCs expanded with EGM-2 did not appear to change significantly. ASCs cultured with EGM-2 preserved differentiation capacities similar to those with DMEM at least into three mesenchymal lineages: adipogenic, chondrogenic, and osteogenic. In addition, flow cytometry of both populations showed no significant differences except for CD105 and presented no increase of differentiation markers such as CD31, suggesting that ASCs remain in an undifferentiated and proliferating state. These results suggest that EGM-2 accelerates expansion of ASCs mainly by facilitating proliferation of undifferentiated cells.
Recent reports have shown that ASCs can differentiate into endothelial cells in vitro under certain culture conditions using endothelial growth media and also in vivo [3-6]. ASCs may be essentially common progenitors of adipocytes and vascular cells [5]. In most of the in vitro studies, a semisolid medium like methylcellulose or Matrigel was used, which may be key to endothelial differentiation of ASCs [34]. Although EGM-2 containing VEGF was originally a medium for expanding endothelial cells and ASCs express Flk-1, a VEGF receptor, endothelial cell marker expression of ASCs was not enhanced by EGM-2 in our study using cell culture on a plastic dish. In addition, hypoxic conditions have various influences on ASCs [35-38], one of which is enhancing ASC secretion of angiogenic factors such as VEGF and HGF [35]. In the studies showing endothelial differentiation of ASCs in vivo, ASCs were transplanted to the ischemic hindlimb or under other ischemic conditions [4-6], so that a hypoxic condition may be an important factor in endothelial differentiation in vivo.
A number of preclinical studies with human ASCs have been reported; in most, undifferentiated ASCs were used, rather than ASCs differentiated into a specific lineage, although the functional mechanism of transplanted ASCs varied among studies. Transplanted ASCs survive as undifferentiated cells and act as tissue-specific progenitors or provider cells of soluble factors in some studies [3, 7, 16]. In others, transplanted ASCs differentiated into a specific lineage such as bone and vessels according to the circumstances of recipient sites [3, 7, 17]. In the therapeutic use of ASCs, expansion of undifferentiated cells, rather than their differentiation into a specific lineage, is likely of great importance in the processing of the cells before transplantation. In clinical practice, a safer and more rapid expansion method is required in view of time and cost requirements. EGM-2 does not contain animal-derived factors, and the FBS used in this study can be easily replaced with autologous serum or human allogenic serum. The present expansion method with EGM-2 has an exceptional efficiency and lays the groundwork for establishing a practical route to mega-expansion of ASCs for clinical applications.

We are very grateful to Ayako Kurata, Akiko Matsuura, and Satomi Kawarasaki for their technical assistance.

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Figure legends

Fig. 1. (A) Doubling time at passages 0?3. Doubling time of ASCs cultured with EGM-2 was significantly shorter than for those cultured with DMEM at each passage.
(B) Total cell number and population doubling level after the initiation of culture with DMEM or EGM-2. ASCs cultured in EGM-2 proliferated more rapidly and reached the stationary phase earlier than those cultured in DMEM.

Fig. 2. Cell number after 7 days of culture in EBM (2% FBS) supplemented with one of the following growth factors, VEGF, EGF, IGF-1, or FGF-2 (n=3). FGF-2 at a density of 0.1, 1, or 10 ng/ml significantly promoted proliferation of ASCs compared to control medium. The numbers of cells cultured in DMEM (10% FBS) or EGM-2 (2% FBS) are also indicated. *: p < 0.05.

Fig. 3. Representative results of flow cytometry at Passage 0 and Passage 1. No significant differences were observed between the two cell populations cultured in DMEM and EGM-2 except for CD 105 at Passage 1.

Fig. 4. (A) Microscopic results of cell differentiation. Both cell populations cultured in DMEM and EGM-2 for 2 weeks had similar capacities to differentiate into adipogenic, chondrogenic, and osteogenic lineages. Adipogenic, chondrogenic, and osteogenic differentiations were visualized with oil red O staining, Alcian Blue staining, and von Kossa staining, respectively. Scale bar = 100 μm.
(B) Quantitative analyses of cell differentiation. Differentiation potentials were evaluated by lipid droplet contents (adipogenic), micromass diameter (chondrogenic), and total calcium contents (osteogenic). No statistical significances were observed (adipogenic, p = 0.31: chondrogenic, p = 0.68, and osteogenic, p = 0.55). NS: no significant difference.

Table 1.
Flow cytometry analyses of cell surface marker antigens. Expression of mesenchymal markers (CD29, CD90, CD105), endothelial markers (CD31, CD146, Flk-1), a stem cell marker (CD34), and a hematopoietic marker (CD45) of ASCs cultured with DMEM or EGM-2 was quantitatively examined at passages 0?3.


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