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Effects of Rehmannia glutinosa oligosaccharide on human adipose-derived mesenchymal stem cells in vitro
Life Sciences 91 (2012) 1323–1327
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Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Effects of Rehmannia glutinosa oligosaccharide on human adipose-derived mesenchymal stem cells in vitro Yu Zhang a, b, 1, Yuhong Wang c, 1, Lei Wang a, Yanqin Zhang a, Yihong Qin a, Ting Chen a, Weidong Han d, Guanghui Chen a,⁎ a
Department of Cardiology, Chinese PLA General Hospital, Beijing 100853, China Nankai University School of Medicine, Nankai University, Tianjin 300071, China Department of Emergency, The General Hospital of Beijing Military Command, Beijing 100700, China d Institute of Basic Medicine Science, Chinese PLA General Hospital, Beijing 100853, China b c
a r t i c l e
i n f o
Article history: Received 1 June 2012 Accepted 11 October 2012 Keywords: Human adipose-derived mesenchymal stem cells Rehmannia glutinosa oligosaccharide Cell proliferation Apoptosis VEGF HGF
a b s t r a c t Aims: Adipose-derived mesenchymal stem cells (ADMSCs) are considered as a good cell source for regenerative medicine with their self-renew capacity, multilineage differentiation and immunomodulatory potency. Based on this background, the aim of this study was to evaluate the influence of Rehmannia glutinosa oligosaccharide (RGO), a traditional Chinese medicine, on human ADMSCs' proliferation, H2O2-induced apoptosis, and secretion of Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in vitro. Main methods: Human ADMSCs were isolated and cultured in vitro. Then flow cytometry was carried out to characterize the cells, and MTT assay was performed to detect the proliferation. H2O2-induced apoptosis was evaluated by flow cytometry. VEGF and HGF production were detected by ELISA kits. Key findings: Human ADMSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The results also indicate that RGO can promote the proliferation and alleviate H2O2-induced apoptosis of human ADMSCs. The mechanism of RGO's protective effect may involve the up-regulation of VEGF and HGF. Significance: The present study indicates that RGO may increase the viability and proliferative capacity and alleviate H2O2-induced apoptosis of human ADMSCs via the paracrine release of VEGF and HGF. These results indicate that RGO application will enhance stem cell viability and improve their effects in cell therapy. © 2012 Elsevier Inc. All rights reserved.
Introduction Mesenchymal stem cells (MSCs), originating from bone marrow, adipose tissue, umbilical cord blood and many other tissues, are multipotent cells that can differentiate into multi-lineage cell types, including osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, neurons and epithelial cells (Badri et al., 2011; Deng et al., 2005; Moreno et al., 2010). Among different types of mesenchymal stem cells, adipose-derived mesenchymal stem cells (ADMSCs) seem to be one of the most promising candidates for cell therapy for a variety of reasons (Zuk et al., 2001; Gimble et al., 2007; Tobita et al., 2011). ADMSCs are stromal cells that exhibit self-renew capacity, ability to undergo multilineage differentiation and immunomodulatory potency (Hoke et al., 2009; Pénicaud and Casteilla, 2004). The ease of harvest and quantity obtained make these sources most practical for experimental and possible clinical applications. However, adult stem cells undergo fewer replicative cycles in comparison with embryonic stem cells, which may restrict harvesting of a ⁎ Corresponding author. Tel./fax: +86 10 6693 6453. E-mail address: [email protected] (G. Chen). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.10.015
sufficient amount of stem cell biomass for an adequate therapy (Wagner et al., 2009). Thus, to reach a sufficient number of ADMSCs for clinical use, it is necessary to culture expand the cells for several weeks, and the efficiency of postimplantation survival and proliferative capacity of ADMSCs is not satisfactory. In recent years, numerous specific culture media have been proposed to optimize the self-renewal and increase the expanding efficacy of stem cells. In some approach, stem cell proliferation is promoted by the nutritional supplementation of culture media with rice culture additives (Steele et al., 2010), blueberry and green tea extracts, or Vitamin D3, et al. (Sanberg et al., 2008). There is a growing body of evidence supporting the hypothesis that paracrine mechanisms mediated by factors released by stem cells play an essential role in the reparative process (Gnecchi et al., 2008). Paracrine factors, including growth factors or cytokines, mediate multiple mechanisms such as increased blood flow to ischemic tissue, reduction in apoptosis, regulation of inflammatory response, recruiting endogenous stem cells to regenerate injured tissue, and promoting neovascularization (Rehman et al., 2004; Planat-Bénard et al., 2010). Evidence suggests that vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) may be the two crucial paracrine factors (Rodrigues et al., 2010; Song et al., 2007). It is also known that angiogenesis and arteriogenesis typically involve mediators such as VEGF and
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HGF. Tissue levels of VEGF are significantly increased in infarcted hearts that are treated with ADMSCs (Gnecchi et al., 2006). HGF has been shown to have anti-apoptotic effects in acute myocardial infarction (MI) (Anderson et al., 2008). Rehmannia glutinosa oligosaccharide (RGO) is component isolated from the R. glutinosa Libosch., a traditional Chinese medicinal herb, which was already reported to play a role in the cell regeneration, growth, development and immune regulatory response of the body (Sano et al., 2007; Zhang et al., 2004). We hypothesized that RGO could be beneficial for cell therapies by stimulating proliferation and promoting paracrine mechanisms of human ADMSCs (hADMSCs). In this study, we aimed to evaluate the influence of RGO on the proliferation and the H2O2-induced apoptosis of hADMSCs, and investigate RGO's effect on the paracrine mechanisms through the secretion of VEGF and HGF in hADMSCs. Materials and methods The present study was performed in accordance with Declaration of Helsinki and the guidelines of the Ethic Committee of Chinese PLA (People's Liberty Army) General Hospital, Beijing, China. Tissue sampling Human abdominal subcutaneous adipose tissue was obtained from patients (under 30 years-old) undergoing abdominal non-tumor surgery procedures in the Department of General Surgery of Chinese PLA General Hospital, Beijing, China. The patients gave their written informed consent. Isolation and culture of hADMSCs Twenty grams freshly isolated human adipose tissue from abdominal surgery was washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed adipose tissue was cut into fragments less than 1 mm3 and then digested with trypsin (2.5 g/L)-EDTA (0.3 g/L) (Sigma, St. Louis, USA) and collagenase (3 g/L) for 30 min at 37 °C with gentle agitation. Enzyme activity was neutralized with Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS). After filtration through 25-μm filters and centrifugation for 6 min at 1500 g, the cellular pellet was resuspended in DMEM containing 10% FBS, counted with a hemocytometer, plated at 1 × 10 5/mL in tissue culture plates and incubated overnight in DMEM containing 10% FBS with 0.5% CO2 at 37 °C. After incubation for 24 h, the culture media was changed to remove residual nonadherent red cells. The cells were daily observed under an inverted phase-contrast microscope and were passaged after 70–80% confluence. The culture media was changed every 2–3 days. Flow cytometric characterization For cell surface antigen immune-phenotyping, third passaged hADMSCs were detached and fixed for 30 min in 2% paraformaldehyde. The fixed cells were washed in flow cytometry buffer (FBS, Biolegend) and incubated for 30 min in FBS containing the following antibodies: FITC-conjugated anti-CD90, FITC-conjugated anti-CD45, FITCconjugated anti-CD31, PE-conjugated antiCD-34, PE/Cy7-conjugated anti-CD29 at a concentration of 2 μg/mL at 4 °C for 30 min. The cells that stained with FITC- or PE-labeled IgG were considered as negative controls. Then, fluorescence-activated cell sorting (FACS) analysis was performed on a BD FACS Callibur cytometry. MTT assay Cells in logarithmic growth phase were seeded in 96-well plates at densities of 3 × 10 3/well and were incubated with different concentration of RGO (0, 1, 10, 100 and 400 mg/L, respectively) in a triplicate
pattern. After the cells were cultured for 1, 3, 5, 7, 9, 11 days, respectively, 20 μL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added to each well which has already contained 100 μL culture medium and incubated for 4 h at 37 °C. After removing the culture medium, 200 μL DMSO was added to each well and the plates were shaken for 10 min. Then the optical density (OD) value was measured at A490 nm. Finally the growth curve was drawn after data collection of A490 nm at each time point. The assay was repeated 3 times. Flow cytometric analysis for apoptosis Cells were divided into three groups: control, H2O2, and RGO + H2O2 groups. Cells of control group were incubated in culture medium without RGO or H2O2. Cells of H2O2 group were incubated in culture medium containing 0.1 mmol/L H2O2. Cells of RGO+ H2O2 group were incubated in culture medium containing 200 mg/L RGO and 0.1 mmol/L H2O2. All the three groups were incubated for 1, 6 and 12 h, respectively. At each time point, cells were collected, centrifuged and resuspended in 1× binding buffer supplemented with 5 μL Annexin V-FITC and 10 μL PI. After 15 min of incubation at room temperature in the dark, the cells were then analyzed for FACS with BD FACS Callibur cytometry. ELISA detection of VEGF and HGF Third passaged hADMSCs were seeded in 6-well plates at densities of 1 × 105/well and were incubated with different concentration of RGO (0, 1, 10, 100 and 400 mg/L, respectively). After incubation for 72 h, the supernatant of the cell culture medium was collected. VEGF and HGF in the supernatant were detected by commercial enzymelinked immunosorbent assay (ELISA) kits. Assays were carried out according to the manufacturer's instructions. Statistical analysis Results are presented as mean ± standard error of the mean. SPSS 17.0 was used to perform one-way analysis of variance (ANOVA) and F-test. A P-value of b0.05 was considered significant. Results Morphology and phenotypic characterization of hADMSCs We isolated a mesenchymal cell population from human adipose tissue. During the first day after plating, hADMSCs adhered to the plastic surfaces of tissue culture plates, and displayed a spindle-shaped or fibroblast-like morphology (Fig. 1 A and B). These cells needed to be passaged after achieving a confluence of 80–90%. For phenotypic characterization, third passaged hADMSCs were assessed by flow cytometric analysis, indicating hADMSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45 (Fig. 2). Effect of RGO on the proliferation of hADMSCs We found that RGO promoted the proliferation of hADMSCs in a concentration-dependent manner. The cells were treated with different concentrations (0–400 mg/L) of RGO, and their effects on cells growth were observed. From Fig. 3, we can make a conclusion that the most adaptable concentration of RGO to stimulate the proliferation of hADMSCs was 100 mg/L (Fig. 3) and the level of hADMSCs proliferation treated with 400 mg/L of RGO was also higher than that of untreated. Low concentration of RGO (1 or 10 mg/L) did not have significant effect on the growth of hADMSCs.
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Fig. 3. Growth curve showed the effect of RGO on the proliferation of hADMSCs. The most suitable concentration of RGO for hADMSCs growth is 100 mg/L. ⁎P b 0.05; ⁎⁎P b 0.01 compared with the control group.
Effect of RGO on the H2O2-induced apoptosis To assess whether RGO has an effect on the H2O2-induced apoptosis, we performed flow cytometric analysis. As shown in Fig. 4, treatment with 0.1 mmol/L H2O2 significantly induced apoptosis in hADMSCs, and H2O2 induced more apoptosis at 12 h (58.72 ± 6.31%) than that at 6 h (25.87 ± 3.02%) and 1 h (14.35 ± 2.10%). RGO alleviated the H2O2-induced apoptosis (8.13 ± 0.94% at 1 h, 15.24 ± 1.87% at 6 h, 29.67 ± 4.26% at 12 h), and RGO's protective effects were more significant at 12 h than that at 6 h. Control group without treatment with H2O2 showed slightly apoptosis (0.08 ± 0.02% at 1 h, 0.15 ± 0.04% at 6 h, and 0.81 ± 0.09% at 12 h). Effect of RGO on production of VEGF and HGF in hADMSCs
Fig. 1. The morphology of hADMSCs in A (40×) and B (200×). When cultured in vitro, the cells showed a spindle-shaped or fibroblast-like morphology.
We determined whether treatment with RGO could promote the formation of VEGF and HGF in hADMSCs. Cells were treated with different concentrations (0–400 mg/L) of RGO for 72 h, and the generated VEGF and HGF in the supernatant of cell culture medium were
Fig. 2. Flow cytometry analysis of CD marker expression on third passaged hADMSCs. These cells tested negative for hematopoietic markers (CD31, CDE34 and CD45), but positive for mesenchymal-specific markers (CD29 and CD90).
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Discussion
Fig. 4. Evaluation of H2O2-induced apoptosis in hADMSCs. Apoptotic rate detected by flow cytometry. Values were calculated from three independent experiments. ⁎⁎⁎P b 0.01 compared with control group; ###P b 0.01 compared with H2O2 group.
measured using ELISA kits. As shown in Fig. 5, low concentration of RGO (1 or 10 mg/L) did not have significant effect on secretion of VEGF or HGF in hADMSCs. Human ADMSCs treated with 100 mg/L of RGO produced highest level of VEGF and HGF, and those treated with 400 mg/L of RGO secreted slightly but significant higher level of VEGF and HGF compared to controls. RGO promoted the generation of VEGF and HGF in hADMSCs in a concentration-dependent manner and the most suitable concentration of RGO was 100 mg/L.
Fig. 5. Concentration of VEGF (A) and HGF (B) detected by ELISA. We determined the levels of VEGF and HGF in the supernatant of hADMSCs culture medium after 72 h culturing in each group. ⁎⁎⁎Pb 0.01 compared with control group; ###Pb 0.05 compared with hADMSCs treated with 400 mg/L of RGO.
Cell therapy seems to be a promising treatment for various diseases, such as Parkinson's disease, myocardial infarction, and liver cirrhosis et al. Adipose tissue is an attractive cell source for stem cell therapy, because it is relatively easy to harvest, available in sufficient quantities, and yields a significantly high number of uncommitted stem cells (Bai et al., 2010). Today, the uses for ADMSCs in tissue repair/regeneration are quite impressive (Zuk, 2010). The international Society for Cell Therapy (ISCT) has come up with the minimal set of standard criteria to identify mesenchymal stem cells (MSCs) (Dominici et al., 2006): (1) plastic adherent ability; (2) expression of CD73, CD90, and CD105 and lack the expression of CD14, CD19, CD31, CD34, CD45, and HLA-DR surface molecules; (3) tripotential mesodermal differentiation capability into osteoblasts, chondrocytes, and adipocytes and (4) immunomodulatory functions. In the present study, hADMSCs were isolated from human abdominal subcutaneous adipose tissues and were cultured in DMEM containing 10% FBS. The cells adhered to the plastic surfaces of tissue culture plates, and displayed fibroblast-like morphology. Characterization of hADMSCs is mainly based on the expression of CD markers. Human ADMSCs expressed CD29 and CD90, both of which are considered a marker for MSCs (Haynesworth et al., 1992). In contrast, no expression of the hematopoietic lineage markers CD31, CD34, and CD45 was observed in hADMSCs in our study. Despite the encouraging results regarding the therapeutic potential of hADMSCs to treat diseases (Yamada et al., 2006), many obstacles are in the way of broad clinical application. One of the major issues is the low efficiency of cell expansion and postimplantation survival. Studies revealed that R. glutinosa oligosaccharide (RGO), component isolated from the R. glutinosa Libosch., was already reported to play a role in cell regeneration (Sano et al., 2007). Also, RGO had the effect of promoting proliferation of hemopoietic progenitors in SAMP8 mice (Fujun et al., 1998). In the present study, we found that RGO promoted the proliferation of hADMSCs in a concentration-dependent manner, and treatment with 100 mg/L of RGO induced significantly highest level of proliferation. In a certain range, the proliferation ability of cells is rising following the increase of RGO concentration, but if the range was exceeded, it may exert a less powerful effect. Thus, according to the present findings, 100 mg/L of RGO can be recommended to induce proliferation of hADMSCs. We also found RGO decreased H2O2-induced apoptosis in hADMSCs. It is known that cell apoptosis plays an important role in the pathogenesis of organ dysfunctions (Zhao, 2004). Oxidative stress-induced reactive oxygen species (ROS) can lead to apoptosis (Curtin et al., 2002; Chandra et al., 2000). As one of ROS, H2O2 is highly reactive and toxic and thus capable of leading to cell apoptosis (Mittler 2002). Our present results show that oxidative stress in the form of H2O2 treatment led to enhanced ROS induced apoptosis in hADMSCs, and H2O2 induced more apoptosis at 12 h than that at 6 h and 1 h. RGO decreased H2O2-induced apoptosis in hADMSCs as indicated by flow cytometric analysis, and RGO's protective effects were more significant at 12 h than that at 6 h. The present results demonstrate that RGO promoted the secretion of VEGF and HGF in hADMSCs in a concentration-dependent manner, and hADMSCs treated with 100 mg/L of RGO produced highest level of VEGF and HGF. The mechanisms underlying the protective effect of RGO might involve up-regulation of paracrine factors, such as VEGF and HGF. VEGF can stimulate the stem-cell recruitment through binding to VEGF receptor 1, which is considered as a potential target to reduce inflammation by preventing the recruitment and activation of inflammatory cells (Eriksson and Alitalo, 2002). HGF plays an important role in many biological processes such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. HGF was also found having the effects in enhancing the recruitment of progenitor and homing of stem cell to the injured liver for tissue targeting and repair (Kollet et al., 2003) and functioning as immune regulator in brain system (Okunishi et al.,
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2005). Therefore, our results suggested that RGO may promote the stem cells recruitment and regulate the immune response indirectly by increasing secretion of VEGF and HGF as beneficial paracrine factors. Conclusions In conclusion, this study provides better understanding of RGO's effect on hADMSCs. The present study clearly demonstrated for the first time that RGO is a useful element to promote the proliferation of human ADMSCs in a concentration-dependent manner, and alleviate H2O2-induced apoptosis in human ADMSCs. Moreover, we identified part of the mechanism underlying these processes—RGO' protective effect may involve the paracrine mechanisms through the up-regulation of VEGF and HGF. By combining RGO with stem cell therapy, one may be able to enhance stem cell function and viability and result in further therapeutic benefits. RGO may be considered as a useful drug to improve the results of stem cell therapy in the future clinical application. Conflict of interest statement The authors declare that they have no conflicts of interests.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30471924) and the Chinese National High-tech R&D Programme (863 Programme, 2011AA020101). The authors are grateful to the Department of General Surgery, Institute of Basic Medicine Science and Cardiology Lab of Chinese PLA General Hospital for assistance. References Anderson C, Heydarkhan-Hagvall S, Schenke-Layland K, Yang J, Jordan M, Kim J, et al. The role of cytoprotective cytokines in cardiac ischemia/reperfusion injury. J Surg Res 2008;148:164–71. Badri L, Walker NM, Ohtsuka T, Wang Z, Delmar M, Flint A, et al. Epithelial interactions and local engraftment of lung-resident mesenchymal stem cells. Am J Respir Cell Mol Biol 2011;45:809–16. Bai X, Yan Y, Song YH, Seidensticker M, Rabinovich B, Metzele R, et al. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. Eur Heart J 2010;31(4):489–501. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med 2000;29:323–33. Curtin JF, Donovan M, Cotter TG. Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 2002;265:49–72. Deng W, Han Q, Liao L, Li C, Ge W, Zhao Z, et al. Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue. Tissue Eng 2005;11:110–9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–7. Eriksson U, Alitalo K. VEGF receptor 1 stimulates stem-cell recruitment and new hope for angiogenesis therapies. Nat Med 2002;8(8):775–7.
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Fujun L, Xiunan Z, Jianfang T, Xiangbin R, Yongxiang Z, Lin Q, et al. Effect of Rehmannia glutinosa oligosaccharide on hemopoietic progenitors in senescence-accelerated mouse P8 subseries. Chin J Pharmacol Toxicol 1998;12:127–9. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–60. Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 2006;6:661–9. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res 2008;103(11):1204–19. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992;13(1): 69–80. Hoke NN, Salloum FN, Loesser-Casey KE, Kukreja RC. Cardiac regenerative potential of adipose tissue-derived stem cells. Acta Physiol Hung 2009;96(3):251–65. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 2003;112(2):160–9. Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 2002;7: 405–10. Moreno R, Martinez-Gonzalez I, Rosal M, Farwati A, Gratacos E, Aran JM. Characterization of mesenchymal stem cells isolated from the rabbit fetal liver. Stem Cells Dev 2010;19:1579–88. Okunishi K, Dohi M, Nakagome K, Tanaka R, Mizuno S, Matsumoto K, et al. A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function. J Immunol 2005;175(7):4745–53. Pénicaud L, Casteilla L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 2004;94(2):223–9. Planat-Bénard V, Menard C, André M, Puceat M, Perez A, Garcia-Verdugo JM, et al. Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Res Ther 2010;1(4):32. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove C, Bovenkerk J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004;109:1292–8. Rodrigues M, Griffith LG, Wells A. Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Res Ther 2010;1(4):32. Sanberg DC, Sanberg P, Bickford P, Shytle DR, Tan J. Combined effects of nutrients on proliferation of stem cells. US Patent 2008;7442394. Sano K, Asanuma-Date K, Arisaka F, Hattori S, Ogawa H. Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology 2007;17(7):784–94. Song YH, Gehmert S, Sadat S, Pinkernell K, Bai X, Matthias N, et al. VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes. Biochem Biophys Res Commun 2007;354:999-1003. Steele AM, Smith MD, Boonchuen S. Use of rice-derived products in a universal cell culture medium. US Patent 2010;0035327. Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives. Discov Med 2011;11:160–70. Wagner W, Bork S, Horn P, Krunic D, Walenda T, Diehlmann A, et al. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One 2009;4:e5846. Yamada Y, Wang XD, Yokoyama S, Fukuda N, Takakura N. Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium. Biochem Biophys Res Commun 2006;342(2):662–70. Zhang R, Zhou J, Jia Z, Zhang Y, Gu G. Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. J Ethnopharmacol 2004;90(1):39–43. Zhao ZQ. Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol 2004;4(2):159–65. Zuk PA. The adipose-derived stem cell: looking back and looking ahead. Mol Biol Cell 2010;21:1783–7. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28.
Contents lists available at SciVerse ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Effects of Rehmannia glutinosa oligosaccharide on human adipose-derived mesenchymal stem cells in vitro Yu Zhang a, b, 1, Yuhong Wang c, 1, Lei Wang a, Yanqin Zhang a, Yihong Qin a, Ting Chen a, Weidong Han d, Guanghui Chen a,⁎ a
Department of Cardiology, Chinese PLA General Hospital, Beijing 100853, China Nankai University School of Medicine, Nankai University, Tianjin 300071, China Department of Emergency, The General Hospital of Beijing Military Command, Beijing 100700, China d Institute of Basic Medicine Science, Chinese PLA General Hospital, Beijing 100853, China b c
a r t i c l e
i n f o
Article history: Received 1 June 2012 Accepted 11 October 2012 Keywords: Human adipose-derived mesenchymal stem cells Rehmannia glutinosa oligosaccharide Cell proliferation Apoptosis VEGF HGF
a b s t r a c t Aims: Adipose-derived mesenchymal stem cells (ADMSCs) are considered as a good cell source for regenerative medicine with their self-renew capacity, multilineage differentiation and immunomodulatory potency. Based on this background, the aim of this study was to evaluate the influence of Rehmannia glutinosa oligosaccharide (RGO), a traditional Chinese medicine, on human ADMSCs' proliferation, H2O2-induced apoptosis, and secretion of Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in vitro. Main methods: Human ADMSCs were isolated and cultured in vitro. Then flow cytometry was carried out to characterize the cells, and MTT assay was performed to detect the proliferation. H2O2-induced apoptosis was evaluated by flow cytometry. VEGF and HGF production were detected by ELISA kits. Key findings: Human ADMSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The results also indicate that RGO can promote the proliferation and alleviate H2O2-induced apoptosis of human ADMSCs. The mechanism of RGO's protective effect may involve the up-regulation of VEGF and HGF. Significance: The present study indicates that RGO may increase the viability and proliferative capacity and alleviate H2O2-induced apoptosis of human ADMSCs via the paracrine release of VEGF and HGF. These results indicate that RGO application will enhance stem cell viability and improve their effects in cell therapy. © 2012 Elsevier Inc. All rights reserved.
Introduction Mesenchymal stem cells (MSCs), originating from bone marrow, adipose tissue, umbilical cord blood and many other tissues, are multipotent cells that can differentiate into multi-lineage cell types, including osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, neurons and epithelial cells (Badri et al., 2011; Deng et al., 2005; Moreno et al., 2010). Among different types of mesenchymal stem cells, adipose-derived mesenchymal stem cells (ADMSCs) seem to be one of the most promising candidates for cell therapy for a variety of reasons (Zuk et al., 2001; Gimble et al., 2007; Tobita et al., 2011). ADMSCs are stromal cells that exhibit self-renew capacity, ability to undergo multilineage differentiation and immunomodulatory potency (Hoke et al., 2009; Pénicaud and Casteilla, 2004). The ease of harvest and quantity obtained make these sources most practical for experimental and possible clinical applications. However, adult stem cells undergo fewer replicative cycles in comparison with embryonic stem cells, which may restrict harvesting of a ⁎ Corresponding author. Tel./fax: +86 10 6693 6453. E-mail address: [email protected] (G. Chen). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.10.015
sufficient amount of stem cell biomass for an adequate therapy (Wagner et al., 2009). Thus, to reach a sufficient number of ADMSCs for clinical use, it is necessary to culture expand the cells for several weeks, and the efficiency of postimplantation survival and proliferative capacity of ADMSCs is not satisfactory. In recent years, numerous specific culture media have been proposed to optimize the self-renewal and increase the expanding efficacy of stem cells. In some approach, stem cell proliferation is promoted by the nutritional supplementation of culture media with rice culture additives (Steele et al., 2010), blueberry and green tea extracts, or Vitamin D3, et al. (Sanberg et al., 2008). There is a growing body of evidence supporting the hypothesis that paracrine mechanisms mediated by factors released by stem cells play an essential role in the reparative process (Gnecchi et al., 2008). Paracrine factors, including growth factors or cytokines, mediate multiple mechanisms such as increased blood flow to ischemic tissue, reduction in apoptosis, regulation of inflammatory response, recruiting endogenous stem cells to regenerate injured tissue, and promoting neovascularization (Rehman et al., 2004; Planat-Bénard et al., 2010). Evidence suggests that vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) may be the two crucial paracrine factors (Rodrigues et al., 2010; Song et al., 2007). It is also known that angiogenesis and arteriogenesis typically involve mediators such as VEGF and
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HGF. Tissue levels of VEGF are significantly increased in infarcted hearts that are treated with ADMSCs (Gnecchi et al., 2006). HGF has been shown to have anti-apoptotic effects in acute myocardial infarction (MI) (Anderson et al., 2008). Rehmannia glutinosa oligosaccharide (RGO) is component isolated from the R. glutinosa Libosch., a traditional Chinese medicinal herb, which was already reported to play a role in the cell regeneration, growth, development and immune regulatory response of the body (Sano et al., 2007; Zhang et al., 2004). We hypothesized that RGO could be beneficial for cell therapies by stimulating proliferation and promoting paracrine mechanisms of human ADMSCs (hADMSCs). In this study, we aimed to evaluate the influence of RGO on the proliferation and the H2O2-induced apoptosis of hADMSCs, and investigate RGO's effect on the paracrine mechanisms through the secretion of VEGF and HGF in hADMSCs. Materials and methods The present study was performed in accordance with Declaration of Helsinki and the guidelines of the Ethic Committee of Chinese PLA (People's Liberty Army) General Hospital, Beijing, China. Tissue sampling Human abdominal subcutaneous adipose tissue was obtained from patients (under 30 years-old) undergoing abdominal non-tumor surgery procedures in the Department of General Surgery of Chinese PLA General Hospital, Beijing, China. The patients gave their written informed consent. Isolation and culture of hADMSCs Twenty grams freshly isolated human adipose tissue from abdominal surgery was washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed adipose tissue was cut into fragments less than 1 mm3 and then digested with trypsin (2.5 g/L)-EDTA (0.3 g/L) (Sigma, St. Louis, USA) and collagenase (3 g/L) for 30 min at 37 °C with gentle agitation. Enzyme activity was neutralized with Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS). After filtration through 25-μm filters and centrifugation for 6 min at 1500 g, the cellular pellet was resuspended in DMEM containing 10% FBS, counted with a hemocytometer, plated at 1 × 10 5/mL in tissue culture plates and incubated overnight in DMEM containing 10% FBS with 0.5% CO2 at 37 °C. After incubation for 24 h, the culture media was changed to remove residual nonadherent red cells. The cells were daily observed under an inverted phase-contrast microscope and were passaged after 70–80% confluence. The culture media was changed every 2–3 days. Flow cytometric characterization For cell surface antigen immune-phenotyping, third passaged hADMSCs were detached and fixed for 30 min in 2% paraformaldehyde. The fixed cells were washed in flow cytometry buffer (FBS, Biolegend) and incubated for 30 min in FBS containing the following antibodies: FITC-conjugated anti-CD90, FITC-conjugated anti-CD45, FITCconjugated anti-CD31, PE-conjugated antiCD-34, PE/Cy7-conjugated anti-CD29 at a concentration of 2 μg/mL at 4 °C for 30 min. The cells that stained with FITC- or PE-labeled IgG were considered as negative controls. Then, fluorescence-activated cell sorting (FACS) analysis was performed on a BD FACS Callibur cytometry. MTT assay Cells in logarithmic growth phase were seeded in 96-well plates at densities of 3 × 10 3/well and were incubated with different concentration of RGO (0, 1, 10, 100 and 400 mg/L, respectively) in a triplicate
pattern. After the cells were cultured for 1, 3, 5, 7, 9, 11 days, respectively, 20 μL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added to each well which has already contained 100 μL culture medium and incubated for 4 h at 37 °C. After removing the culture medium, 200 μL DMSO was added to each well and the plates were shaken for 10 min. Then the optical density (OD) value was measured at A490 nm. Finally the growth curve was drawn after data collection of A490 nm at each time point. The assay was repeated 3 times. Flow cytometric analysis for apoptosis Cells were divided into three groups: control, H2O2, and RGO + H2O2 groups. Cells of control group were incubated in culture medium without RGO or H2O2. Cells of H2O2 group were incubated in culture medium containing 0.1 mmol/L H2O2. Cells of RGO+ H2O2 group were incubated in culture medium containing 200 mg/L RGO and 0.1 mmol/L H2O2. All the three groups were incubated for 1, 6 and 12 h, respectively. At each time point, cells were collected, centrifuged and resuspended in 1× binding buffer supplemented with 5 μL Annexin V-FITC and 10 μL PI. After 15 min of incubation at room temperature in the dark, the cells were then analyzed for FACS with BD FACS Callibur cytometry. ELISA detection of VEGF and HGF Third passaged hADMSCs were seeded in 6-well plates at densities of 1 × 105/well and were incubated with different concentration of RGO (0, 1, 10, 100 and 400 mg/L, respectively). After incubation for 72 h, the supernatant of the cell culture medium was collected. VEGF and HGF in the supernatant were detected by commercial enzymelinked immunosorbent assay (ELISA) kits. Assays were carried out according to the manufacturer's instructions. Statistical analysis Results are presented as mean ± standard error of the mean. SPSS 17.0 was used to perform one-way analysis of variance (ANOVA) and F-test. A P-value of b0.05 was considered significant. Results Morphology and phenotypic characterization of hADMSCs We isolated a mesenchymal cell population from human adipose tissue. During the first day after plating, hADMSCs adhered to the plastic surfaces of tissue culture plates, and displayed a spindle-shaped or fibroblast-like morphology (Fig. 1 A and B). These cells needed to be passaged after achieving a confluence of 80–90%. For phenotypic characterization, third passaged hADMSCs were assessed by flow cytometric analysis, indicating hADMSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45 (Fig. 2). Effect of RGO on the proliferation of hADMSCs We found that RGO promoted the proliferation of hADMSCs in a concentration-dependent manner. The cells were treated with different concentrations (0–400 mg/L) of RGO, and their effects on cells growth were observed. From Fig. 3, we can make a conclusion that the most adaptable concentration of RGO to stimulate the proliferation of hADMSCs was 100 mg/L (Fig. 3) and the level of hADMSCs proliferation treated with 400 mg/L of RGO was also higher than that of untreated. Low concentration of RGO (1 or 10 mg/L) did not have significant effect on the growth of hADMSCs.
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Fig. 3. Growth curve showed the effect of RGO on the proliferation of hADMSCs. The most suitable concentration of RGO for hADMSCs growth is 100 mg/L. ⁎P b 0.05; ⁎⁎P b 0.01 compared with the control group.
Effect of RGO on the H2O2-induced apoptosis To assess whether RGO has an effect on the H2O2-induced apoptosis, we performed flow cytometric analysis. As shown in Fig. 4, treatment with 0.1 mmol/L H2O2 significantly induced apoptosis in hADMSCs, and H2O2 induced more apoptosis at 12 h (58.72 ± 6.31%) than that at 6 h (25.87 ± 3.02%) and 1 h (14.35 ± 2.10%). RGO alleviated the H2O2-induced apoptosis (8.13 ± 0.94% at 1 h, 15.24 ± 1.87% at 6 h, 29.67 ± 4.26% at 12 h), and RGO's protective effects were more significant at 12 h than that at 6 h. Control group without treatment with H2O2 showed slightly apoptosis (0.08 ± 0.02% at 1 h, 0.15 ± 0.04% at 6 h, and 0.81 ± 0.09% at 12 h). Effect of RGO on production of VEGF and HGF in hADMSCs
Fig. 1. The morphology of hADMSCs in A (40×) and B (200×). When cultured in vitro, the cells showed a spindle-shaped or fibroblast-like morphology.
We determined whether treatment with RGO could promote the formation of VEGF and HGF in hADMSCs. Cells were treated with different concentrations (0–400 mg/L) of RGO for 72 h, and the generated VEGF and HGF in the supernatant of cell culture medium were
Fig. 2. Flow cytometry analysis of CD marker expression on third passaged hADMSCs. These cells tested negative for hematopoietic markers (CD31, CDE34 and CD45), but positive for mesenchymal-specific markers (CD29 and CD90).
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Discussion
Fig. 4. Evaluation of H2O2-induced apoptosis in hADMSCs. Apoptotic rate detected by flow cytometry. Values were calculated from three independent experiments. ⁎⁎⁎P b 0.01 compared with control group; ###P b 0.01 compared with H2O2 group.
measured using ELISA kits. As shown in Fig. 5, low concentration of RGO (1 or 10 mg/L) did not have significant effect on secretion of VEGF or HGF in hADMSCs. Human ADMSCs treated with 100 mg/L of RGO produced highest level of VEGF and HGF, and those treated with 400 mg/L of RGO secreted slightly but significant higher level of VEGF and HGF compared to controls. RGO promoted the generation of VEGF and HGF in hADMSCs in a concentration-dependent manner and the most suitable concentration of RGO was 100 mg/L.
Fig. 5. Concentration of VEGF (A) and HGF (B) detected by ELISA. We determined the levels of VEGF and HGF in the supernatant of hADMSCs culture medium after 72 h culturing in each group. ⁎⁎⁎Pb 0.01 compared with control group; ###Pb 0.05 compared with hADMSCs treated with 400 mg/L of RGO.
Cell therapy seems to be a promising treatment for various diseases, such as Parkinson's disease, myocardial infarction, and liver cirrhosis et al. Adipose tissue is an attractive cell source for stem cell therapy, because it is relatively easy to harvest, available in sufficient quantities, and yields a significantly high number of uncommitted stem cells (Bai et al., 2010). Today, the uses for ADMSCs in tissue repair/regeneration are quite impressive (Zuk, 2010). The international Society for Cell Therapy (ISCT) has come up with the minimal set of standard criteria to identify mesenchymal stem cells (MSCs) (Dominici et al., 2006): (1) plastic adherent ability; (2) expression of CD73, CD90, and CD105 and lack the expression of CD14, CD19, CD31, CD34, CD45, and HLA-DR surface molecules; (3) tripotential mesodermal differentiation capability into osteoblasts, chondrocytes, and adipocytes and (4) immunomodulatory functions. In the present study, hADMSCs were isolated from human abdominal subcutaneous adipose tissues and were cultured in DMEM containing 10% FBS. The cells adhered to the plastic surfaces of tissue culture plates, and displayed fibroblast-like morphology. Characterization of hADMSCs is mainly based on the expression of CD markers. Human ADMSCs expressed CD29 and CD90, both of which are considered a marker for MSCs (Haynesworth et al., 1992). In contrast, no expression of the hematopoietic lineage markers CD31, CD34, and CD45 was observed in hADMSCs in our study. Despite the encouraging results regarding the therapeutic potential of hADMSCs to treat diseases (Yamada et al., 2006), many obstacles are in the way of broad clinical application. One of the major issues is the low efficiency of cell expansion and postimplantation survival. Studies revealed that R. glutinosa oligosaccharide (RGO), component isolated from the R. glutinosa Libosch., was already reported to play a role in cell regeneration (Sano et al., 2007). Also, RGO had the effect of promoting proliferation of hemopoietic progenitors in SAMP8 mice (Fujun et al., 1998). In the present study, we found that RGO promoted the proliferation of hADMSCs in a concentration-dependent manner, and treatment with 100 mg/L of RGO induced significantly highest level of proliferation. In a certain range, the proliferation ability of cells is rising following the increase of RGO concentration, but if the range was exceeded, it may exert a less powerful effect. Thus, according to the present findings, 100 mg/L of RGO can be recommended to induce proliferation of hADMSCs. We also found RGO decreased H2O2-induced apoptosis in hADMSCs. It is known that cell apoptosis plays an important role in the pathogenesis of organ dysfunctions (Zhao, 2004). Oxidative stress-induced reactive oxygen species (ROS) can lead to apoptosis (Curtin et al., 2002; Chandra et al., 2000). As one of ROS, H2O2 is highly reactive and toxic and thus capable of leading to cell apoptosis (Mittler 2002). Our present results show that oxidative stress in the form of H2O2 treatment led to enhanced ROS induced apoptosis in hADMSCs, and H2O2 induced more apoptosis at 12 h than that at 6 h and 1 h. RGO decreased H2O2-induced apoptosis in hADMSCs as indicated by flow cytometric analysis, and RGO's protective effects were more significant at 12 h than that at 6 h. The present results demonstrate that RGO promoted the secretion of VEGF and HGF in hADMSCs in a concentration-dependent manner, and hADMSCs treated with 100 mg/L of RGO produced highest level of VEGF and HGF. The mechanisms underlying the protective effect of RGO might involve up-regulation of paracrine factors, such as VEGF and HGF. VEGF can stimulate the stem-cell recruitment through binding to VEGF receptor 1, which is considered as a potential target to reduce inflammation by preventing the recruitment and activation of inflammatory cells (Eriksson and Alitalo, 2002). HGF plays an important role in many biological processes such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. HGF was also found having the effects in enhancing the recruitment of progenitor and homing of stem cell to the injured liver for tissue targeting and repair (Kollet et al., 2003) and functioning as immune regulator in brain system (Okunishi et al.,
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2005). Therefore, our results suggested that RGO may promote the stem cells recruitment and regulate the immune response indirectly by increasing secretion of VEGF and HGF as beneficial paracrine factors. Conclusions In conclusion, this study provides better understanding of RGO's effect on hADMSCs. The present study clearly demonstrated for the first time that RGO is a useful element to promote the proliferation of human ADMSCs in a concentration-dependent manner, and alleviate H2O2-induced apoptosis in human ADMSCs. Moreover, we identified part of the mechanism underlying these processes—RGO' protective effect may involve the paracrine mechanisms through the up-regulation of VEGF and HGF. By combining RGO with stem cell therapy, one may be able to enhance stem cell function and viability and result in further therapeutic benefits. RGO may be considered as a useful drug to improve the results of stem cell therapy in the future clinical application. Conflict of interest statement The authors declare that they have no conflicts of interests.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30471924) and the Chinese National High-tech R&D Programme (863 Programme, 2011AA020101). The authors are grateful to the Department of General Surgery, Institute of Basic Medicine Science and Cardiology Lab of Chinese PLA General Hospital for assistance. References Anderson C, Heydarkhan-Hagvall S, Schenke-Layland K, Yang J, Jordan M, Kim J, et al. The role of cytoprotective cytokines in cardiac ischemia/reperfusion injury. J Surg Res 2008;148:164–71. Badri L, Walker NM, Ohtsuka T, Wang Z, Delmar M, Flint A, et al. Epithelial interactions and local engraftment of lung-resident mesenchymal stem cells. Am J Respir Cell Mol Biol 2011;45:809–16. Bai X, Yan Y, Song YH, Seidensticker M, Rabinovich B, Metzele R, et al. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. Eur Heart J 2010;31(4):489–501. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med 2000;29:323–33. Curtin JF, Donovan M, Cotter TG. Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 2002;265:49–72. Deng W, Han Q, Liao L, Li C, Ge W, Zhao Z, et al. Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue. Tissue Eng 2005;11:110–9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–7. Eriksson U, Alitalo K. VEGF receptor 1 stimulates stem-cell recruitment and new hope for angiogenesis therapies. Nat Med 2002;8(8):775–7.
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Fujun L, Xiunan Z, Jianfang T, Xiangbin R, Yongxiang Z, Lin Q, et al. Effect of Rehmannia glutinosa oligosaccharide on hemopoietic progenitors in senescence-accelerated mouse P8 subseries. Chin J Pharmacol Toxicol 1998;12:127–9. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–60. Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 2006;6:661–9. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res 2008;103(11):1204–19. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992;13(1): 69–80. Hoke NN, Salloum FN, Loesser-Casey KE, Kukreja RC. Cardiac regenerative potential of adipose tissue-derived stem cells. Acta Physiol Hung 2009;96(3):251–65. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 2003;112(2):160–9. Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 2002;7: 405–10. Moreno R, Martinez-Gonzalez I, Rosal M, Farwati A, Gratacos E, Aran JM. Characterization of mesenchymal stem cells isolated from the rabbit fetal liver. Stem Cells Dev 2010;19:1579–88. Okunishi K, Dohi M, Nakagome K, Tanaka R, Mizuno S, Matsumoto K, et al. A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function. J Immunol 2005;175(7):4745–53. Pénicaud L, Casteilla L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 2004;94(2):223–9. Planat-Bénard V, Menard C, André M, Puceat M, Perez A, Garcia-Verdugo JM, et al. Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Res Ther 2010;1(4):32. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove C, Bovenkerk J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004;109:1292–8. Rodrigues M, Griffith LG, Wells A. Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Res Ther 2010;1(4):32. Sanberg DC, Sanberg P, Bickford P, Shytle DR, Tan J. Combined effects of nutrients on proliferation of stem cells. US Patent 2008;7442394. Sano K, Asanuma-Date K, Arisaka F, Hattori S, Ogawa H. Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology 2007;17(7):784–94. Song YH, Gehmert S, Sadat S, Pinkernell K, Bai X, Matthias N, et al. VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes. Biochem Biophys Res Commun 2007;354:999-1003. Steele AM, Smith MD, Boonchuen S. Use of rice-derived products in a universal cell culture medium. US Patent 2010;0035327. Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives. Discov Med 2011;11:160–70. Wagner W, Bork S, Horn P, Krunic D, Walenda T, Diehlmann A, et al. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One 2009;4:e5846. Yamada Y, Wang XD, Yokoyama S, Fukuda N, Takakura N. Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium. Biochem Biophys Res Commun 2006;342(2):662–70. Zhang R, Zhou J, Jia Z, Zhang Y, Gu G. Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. J Ethnopharmacol 2004;90(1):39–43. Zhao ZQ. Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol 2004;4(2):159–65. Zuk PA. The adipose-derived stem cell: looking back and looking ahead. Mol Biol Cell 2010;21:1783–7. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28.