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Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells
r 2006, Copyright the Authors Differentiation (2006) 74:510–518 DOI: 10.1111/j.1432-0436.2006.00081.x Journal compilation r 2006, International Society of Differentiation
OR IGI N A L A R T IC L E
Hongxiu Ning . Guiting Lin . Tom F. Lue . Ching-Shwun Lin
Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells
Received January 3, 2006; accepted in revised form March 24, 2006
Abstract Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for preclinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70. Key words neuron vascular smooth muscle cells adipose tissue-derived stromal cells differentiation IBMX
Hongxiu Ning Guiting Lin Tom F. Lue Ching-Shwun Lin . ) (* Knuppe Molecular Urology Laboratory Department of Urology, School of Medicine University of California San Francisco, CA 94143-1695, U.S.A. Tel: 11415 353 7205 Fax: 11415 353 9586 E-mail: [email protected] U.S. Copyright Clearance Center Code Statement:
Introduction The adipose tissue contains a stromal vascular fraction (SVF) from which multipotent cells have been isolated (Gronthos et al., 2001; Zuk et al., 2001). These cells are variously called processed lipoaspirate (PLA) cells (Zuk et al., 2001), adipose tissue-derived mesenchymal stem cells (ATSC, AD-MSC) (Song et al., 2005), multipotent adipose-derived stem (MADS) cells (Rodriguez et al., 2005a), adipose tissue-derived stem cells (ASC) (Fujimura et al., 2005), adipose tissue-derived stromal cells (ADSC, ATSC) (Gaustad et al., 2004; Yang et al., 2004; Seo et al., 2005; Hattori et al., 2006), adipose tissuederived adult stem (ADAS) cells (Safford et al., 2002), adipose tissue-derived adult stromal (ADAS) cells (Cowan et al., 2004), and adipose tissue-derived cells (ADC) (Strem et al., 2005b). In this study, the term ‘‘adipose tissue-derived stromal cells (ADSC)’’ will be used. ADSC possess phenotypes and gene expression profiles similar to those of bone marrow stem cells (BMSC) (Gronthos et al., 2001; Zuk et al., 2001, 2002; De Ugarte et al., 2003; Lee et al., 2004; Case et al., 2005; Dicker et al., 2005; Strem et al., 2005a; Wagner et al., 2005). In addition to having the capacity for self-renewal and long-term growth, ADSC are capable of differentiating into diverse cell types including adipocytes (Zuk et al., 2002; Rodriguez et al., 2004, 2005b), osteoblasts (Zuk et al., 2002; Cowan et al., 2004; Cho et al., 2005; Dragoo et al., 2005; Rodriguez et al., 2005a; Yang et al., 2005; Hattori et al., 2006), chondrocytes (Erickson et al., 2002; Zuk et al., 2002; Huang et al., 2004; Betre et al., 2006), hepatocytes (Seo et al., 2005), myocytes (Mizuno et al., 2002; Rodriguez et al., 2005b), cardiomyocytes (Gaustad et al., 2004; Planat-Benard et al., 2004a; Strem et al., 2005b), neurons (Safford et al., 2002, 2004; Zuk et al., 2002; Ashjian et al., 2003; Kang et al., 2003; Tholpady et al., 2003; Yang et al.,
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2004; Case et al., 2005; Fujimura et al., 2005; Jack et al., 2005; Guilak et al., 2006), endothelial cells (PlanatBenard et al., 2004b; Cao et al., 2005), and epithelial cells (Brzoska et al., 2005). Thus, ADSC are not only increasingly accepted as bona fide adult stem cells but also considered to be superior to other types of adult stem cells for future clinical applications (Strem et al., 2005a). Whereas bone marrow can only be obtained in limited quantity because of donor site morbidity, the adipose tissue is usually obtainable in abundance, especially in our increasingly obese society. In addition, clonogenic studies have established that the number of BMSC in bone marrow is approximately 1 in 25,000 to 1 in 100,000 (D’Ippolito et al., 1999; Banfi et al., 2001a, 2001b; Muschler et al., 2001), whereas the average frequency of ADSC in processed lipoaspirate is approximately 2% of nucleated cells (Strem et al., 2005a). Thus, the yield of ADSC from 1 g of fat is approximately 5000 cells, whereas the yield of BMSC is 100– 1000 cells per milliliter of marrow. We are interested in the neuronal potential of ADSC because of our ongoing interest in finding an effective treatment for patients who suffer erectile dysfunction due to injury to the cavernous nerves during surgical and radiation therapies for prostate, bladder, and rectal cancers. We have previously demonstrated the feasibility of using embryonic stem cells (ESC) to treat simulated cavernous nerve injury in rats (Bochinski et al., 2004). However, because of their limited availability and ethical concerns, ESC are presently not a clinically feasible therapeutic venue. On the other hand, the above-mentioned studies suggest ADSC are a viable therapeutic alternative. As such, we set out to isolate rat ADSC and conducted characterization experiments including the induction of neural differentiation. We used a neural induction medium (NIM) that was first reported by Ashjian et al. (2003) and later followed by Fujimura et al. (2005). NIM is consisted of three active ingredients, insulin, indomethacin (INDO), and isobutylmethylxanthine (IBMX), and their choices are explained by Ashjian et al. as follows: (1) Insulin has been shown to promote the maturation of differentiating neocortical cells in rat brains; (2) INDO, an inhibitor of cyclooxygenase, has been shown to promote neural cell survival after ischemic injury to the central nervous system; and (3) IBMX, a phosphodiesterase (PDE) inhibitor, increases intracellular cyclic adenosine monophosphate (cAMP), a neural stimulus. However, these explanations do not indicate why it was necessary to use all three agents. In this study, we determined the effect of each of these agents and their respective solvents. In addition, we included vascular smooth muscle cells (VSMC) in our experiments to test whether NIM-induced changes in cellular morphology and protein expression were specific to ADSC. We report here that IBMX alone was able to induce the differentiation of ADSC into neuron-like cells and VSMC
could be partially induced into neuron-like cells by NIM or IBMX.
Materials and methods Isolation of ADSC ADSC were isolated as described by Zuk et al. (2001). Briefly, subcutaneous adipose tissue was excised from the inguinal region of male Sprague–Dawley rats and cut into fine pieces. The cut tissue was added to 3 volumes of phosphate buffered saline (PBS) in a 50ml conical tube, vortexed at full speed for 1 min, and centrifuged at 220 g for 10 min. The adipose tissue was transferred from the upper phase to a fresh tube, and digested in 0.075% collagenase I (Sigma-Aldrich Co., St Louis, MO) for 1 hr at 371C with shaking. The digest was centrifuged at 220 g for 10 min, followed with the removal of the supernatant. Forty milliliters of PBS was added to resuspend the pellet, which was again centrifuged at 220 g for 10 min. Ten milliliters of 160 nM NH4Cl was added to resuspend the pellet for 10 min, followed by the addition of 10 ml of PBS and centrifugation at 220 g for 10 min. Five milliliters of Dulbecco’sModified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, non-selected, lot # 40404, Biomeda Corporation, Foster City, CA) was added to resuspend the pellet, followed by filtration through a 100-mm cell strainer (BD Biosciences, Bedford, MA) into a 10-cm culture dish. After the addition of 5 ml of DMEM (supplemented with 10% FBS), the culture dish was placed in a 5% CO2 incubator for 3–5 days to allow the formation of ADSC colonies, which were then trypsinized and propagated. Isolation of aortic smooth muscle cells VSMC were isolated from rat aorta as described previously (Lin et al., 2004). These cells were confirmed for their smooth muscle identity by indirect immunofluorescence staining with an antismooth muscle myosin heavy chain antibody (Sigma-Aldrich Co.) and Texas red-conjugated secondary antibody. Cells at the 4th passage were used in this study. Antibodies Antibodies used in this study are listed in Table 1. Detection of specific cell markers ADSC were seeded in six-well plates at 1.0 105 cells/well. After overnight, the cells were fixed with ice-cold methanol for 8 min, permeabilized with 0.05% Triton X-100 for 5 min, and blocked with 5% normal horse serum in PBS for 1 hr at room temperature. The cells were then incubated with the primary antibody for 1 hr at room temperature. After washing with PBS three times, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 1 hr at room temperature. After another three washes with PBS, the cells were further stained with 4 0 ,6-diamidino2-phenylindole (DAPI, for nuclear staining) for 5 min and viewed under fluorescence microscope. Adipogenesis ADSC at passage 3 were seeded in six-well plates and cultured in DMEM supplemented with 10% FBS (control medium). After 24 hr, the medium was changed to D-medium (Chang et al., 1981) (kindly provided by Dr. Chang of Michigan State University) supplemented with 10% FBS, 500 mM IBMX (3-isobutyl-1-methylxan-
512 Table 1 Antibodies used in this study Target protein
Target cell type
Supplier
Catalog no.
CD34 STRO-1 PPAR-g Vimentin Myogenin b-III-tubulin Nestin NF-70 S100 CNPase a-SMC-actin Calponin smMHC
Hematopoietic progenitor cells Bone marrow stromal/erythroid precursor cells Adipose cells Mesenchymal cells Skeletal muscle cells Neurogenic precursor cells Various embryonic cells, including neurons Ectodermal precursor cell, neurons Neurons, astrocytes Oligodendrocytes, Schwann cells Smooth muscle cells Smooth muscle cells Smooth muscle cells
Santa Cruz Biotech, Santa Cruz, CA R&D, Minneapolis, MN Abcam, Cambridge, MA Abcam Abcam Abcam Chemicon, Temecula, CA Chemicon Abcam Abcam Sigma-Aldrich Abcam Sigma-Aldrich
sc-7324 MAB1038 ab19481 ab8069 ab1835 ab14545 MAB353 MAB1615 ab8330 ab6319 A-5228 ab700 M-7786
According to data sheet provided by the supplier.
thine, Sigma-Aldrich, I7018), 1 mM dexamethasone (Sigma-Aldrich, D8893) and 1 mM indomethacin (Sigma-Aldrich, I8280). Two days later, the medium was changed to D-medium supplemented with 10% FBS, and 10 mg/ml insulin (Sigma-Aldrich, I1882). One day later, the medium was changed back to the first D-medium, and this induction cycle was repeated for a total of four times. The cells were then examined for the presence of adipocytes by Oil Red O staining.
Myogenesis ADSC were seeded in six-well plates in DMEM with 10% FBS. The next day, the medium was replaced with the induction medium composed of DMEM, 10% FBS, 5% horse serum (Part # SH30074.02, Lot # 1250704, Hyclone, Logan UT) and 50 mM hydrocortisone (Sigma-Aldrich, H0888). The medium was changed every three days and the induction period lasted for 2 weeks. The cells were then examined for the presence of myocytes by immunofluorescence staining for a-smooth muscle actin with antibody purchased from Sigma-Aldrich (A5228).
expression. The results are consistent with previous reports on human ADSC (see Discussion). We also investigated whether P1 ADSC contain lineage-committed progenitor cells. The results shown in Figure 2 indicate that these cells were negative for adipogenic marker PPAR-g, myogenic marker myogenin, and mesodermal precursor cell marker vimentin. Approximately 2% of ADSC were positive for neurogenic marker bIII-tubulin. We further investigated the differentiation potential of these cells. The results shown in Figure 3 indicate that these cells could differentiate into adipocytes (6%), smooth muscle cells (70%), and neurons (100%). In addition, we have continuously cultured one rat ADSC cell line for more than 100 passages (data not shown). Thus, our rat ADSC possessed stem cell characteristics.
Neurogenesis ADSC at passages 2–5 were seeded in six-well plates at 40%–60% confluence. After three washes with PBS, the cells were induced with NIM (DMEM supplemented with 500 mM IBMX, 200 mM INDO, and 5 mg/ml insulin) for 1 hr. Alternatively, the cells were induced in DMEM supplemented with each of, or any two of, IBMX, INDO, and insulin. The cells were then examined for the expression of neuronal markers S100, NF70, and nestin with the respective antibodies (anti-NF70 antibody, MAB1615, Chemicon; anti-nestin antibody, MAB353, Chemicon; anti-S100 antibody, ab8330, Abcam), followed by hematoxylin-eosin (HE) staining.
Results Characterization of rat ADSC We examined our rat ADSC for the expression of stem cell markers CD34 and STRO-1. As shown in Figure 1, all cells at passage 1 (P1) expressed both CD34 and STRO-1; however, while 95% of cells (at passage 3 (P3)) continued to express STRO-1, they lost CD34
Fig. 1 Detection of stem cell markers CD34 and STRO-1. Adipose tissue-derived stromal cells at passages 1 and 3 were stained with anti-CD34 or anti-STRO-1 antibody and fluorescein isothiocyanate-conjugated secondary antibodies; green fluorescence indicate positive expressing cells. The presence of cells in all four panels was visualized by nuclear staining with 4 0 ,6-diamidino-2-phenylindole (blue fluorescence).
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tested each of them individually and in combinations (IBMX1INDO, IBMX1insulin, and INDO1insulin). As shown in Figure 5, IBMX induced morphological changes in ADSC similar to those induced by NIM, whereas INDO and insulin had little or no effect. The combination of IBMX with INDO or insulin had same effect as IBMX alone, whereas the combination of INDO and insulin had little or no effect (data not shown). Cells treated with IBMX alone all expressed NF70 (Fig. 6) at a comparable level with those treated with NIM (Fig. 4). Because bone marrow stem cells have been shown to differentiate into oligodendrocytes (Lu et al., 2006), we examined whether ADSC had the same potential. The results in Figure 6 show that IBMX-treated ADSC expressed the oligodendrocyte marker CNPase.
Fig. 2 Detection of lineage-specific markers. Adipose tissue-derived stromal cells (ADSC) at passage 1 were stained with fluorescein isothiocyanate-conjugated secondary antibodies that reacted with primary antibodies for adipogenic marker PPAR-g, myogenic marker myogenin, mesodermal precursor cell marker vimentin, and neurogenic marker b-III-tubulin. Cell nuclei were stained with 4 0 ,6diamidino-2-phenylindole. The results show that early ADSC did not express these markers except for b-III-tubulin that was expressed in approximately 2% of cells.
NIM-induced morphological changes and neuronal marker expression We are interested in the neural differentiation potential of ADSC; therefore, we chose the protocol of Ashjian et al. (2003) to induce such differentiation. Within 1 hr of incubation in NIM, the cytoplasm of ADSC retracted towards the nucleus, forming contracted cell bodies with extended cytoplasmic extensions that were in contact with neighboring cells, thus exhibiting a neuronal morphology (Fig. 3). Immunofluorescence staining showed that the uninduced (control) cells expressed nestin (100%) but not S100 or NF70 (Fig. 4); this is in agreement with previous studies (Safford et al., 2002, 2004; Zuk et al., 2002; Ashjian et al., 2003; Kang et al., 2003; Tholpady et al., 2003; Yang et al., 2004; Case et al., 2005; Fujimura et al., 2005; Jack et al., 2005; Guilak et al., 2006). In contrast, all of the induced cells expressed all of these three neuronal cell markers (Fig. 4). The duration of the neuronal morphology and expression varied from experiment to experiment, possibly because of differences in cell passage numbers, density, age of the donor rats, etc. Cell death could occur after 24 hr, but in some experiments, the neuronal morphology and expression lasted for 5 days (data not shown). Individual effects of IBMX, indomethacin, and insulin NIM contains three active ingredients, namely, IBMX, INDO, and insulin. Because there has been no explanation for why all three compounds are needed, we
Effects of NIM on VSMC Although NIM was able to induce neuron-like morphological changes and neuronal marker expression in ADSC, it was unclear whether these effects were specific for ADSC. As such we isolated aortic VSMC, which all expressed smooth muscle myosin heavy chain (Figs. 7A, 7B), and tested them in NIM. The results showed that uninduced VSMC did not express NF70 (Figs. 7E, 7G), while approximately 50% of induced VSMC assumed neuron-like morphology (Fig. 7D) and were stained positive for NF70 (Figs. 7F, 7H). Further tests with each of the three active ingredients of NIM again demonstrated that IBMX alone was responsible for both the morphological changes and NF70 expression (data not shown).
Discussion Since the first publications in 2001 the number of ADSC papers has been steadily increasing. Most of these papers deal with human ADSC that are not suitable for studies in animal models in respect to autologous stem cell therapy. Because we use rat models principally for our urological research, it is necessary for us to understand the characteristics of rat ADSC. However, to our best knowledge, there have been only two published papers on rat ADSC, but neither reported the characterization of these cells (Yang et al., 2004, 2005). In the present study, we for the first time showed that rat ADSC possessed characteristics similar to human ADSC in that (1) both express the widely recognized stem cell marker STRO-1 (Zuk et al., 2002; Strem et al., 2005a), (2) both express hematopoietic cell marker CD34 in early but not late passages (Mitchell et al., 2006), and (3) both are capable of differentiation into various tissue types in vitro. Based on the identification of these stem-cell characteristics, we proceeded to focus on ADSC neural differentiation.
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Fig. 3 Differentiation of rat adipose tissue-derived stromal cells (ADSC) into adipocytes, smooth muscle cells, and neurons. ADSC were induced to differentiate into these three cell types as described in Materials and methods. Oil Red O staining was used to identify adipocytes with red-stained fats in the cytoplasm (upper right panel). Immunofluorescence staining with anti-smooth muscle a-actin and anti-calponin antibodies was performed to identify smooth
muscle cells (green fluorescence, middle panels). Nuclear staining with 4 0 ,6-diamidino-2-phenylindole (blue fluorescence) was done to reveal cellular presence. For neurogenesis following induction with neural induction medium, cell morphological changes were visualized by phase contrast microscopy (lower panels). Their expression of neuronal markers is presented in Fig. 4.
Experimental protocols previously used to induce ADSC neural differentiation were largely adopted from earlier studies on BMSC neural differentiation. These protocols generally relied on exposing cells to retinoic acid (RA) and cytokine cocktails (Sanchez-Ramos et al., 2000; Kim et al., 2002; Case et al., 2005), butylated hydroxyanisole, di-methyl-sulfoxide (DMSO) (Woodbury et al., 2000; Safford et al., 2002; Woodbury et al., 2002; Zuk et al., 2002; Yaghoobi et al., 2005; Guilak et al., 2006), and agents that elevate intracellular cAMP levels (Deng et al., 2001; Ashjian et al., 2003; Suon et al., 2004; Fujimura et al., 2005). While the choice of some of these agents was rationalized in a limited number of studies, there has been a lack of explanation for why it was necessary to use multiple compounds in the induction cocktail. As such, in the present
study we tested each of the three components in the induction cocktail used by Ashjian et al. (2003) and by Fujimura et al. (2005). The results showed that while insulin and INDO were moderately effective, IBMX alone was sufficient for full induction. The inductive effect of IBMX on BMSC neural differentiation was first reported by Deng et al. (2001), and it was believed that IBMX induced neural differentiation by raising intracellular cAMP levels. This interpretation was supported by a later study in which both IBMX and another cAMP-elevating agent, forskolin, were shown to be highly effective in inducing BMSC neural differentiation (Suon et al., 2004). Recently, Kim et al. (2005) further reported that forskolin induces BMSC neural differentiation via activation of the ERK/ MAPK signaling pathway. Specifically they demon-
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Fig. 4 Detection of neuronal markers in neural induction medium (NIM)-treated adipose tissue-derived stromal cells (ADSC). Passage 4 ADSC were incubated in control medium (left panels) or NIM (right panels) for 5 hr and then stained for neuronal markers
S100, NF70, and nestin. 4 0 ,6-diamidino-2-phenylindole staining (data not shown), which overlapped with the fluorescein isothiocyanate staining, indicates all induced cells expressed these markers.
strated that blockade of ERK phosphorylation with a MAPK inhibitor abrogated cAMP-mediated morphological changes and induction of neuronal proteins. While there have been numerous reports on the in vitro neural differentiation of BMSC and ADSC, few if any have demonstrated or discussed whether the associated morphological and gene expression changes are the unique properties of these particular cell types. As such, we included VSMC in this study, and for the first time, we showed that a portion of VSMC could also undergo neuron-like morphological changes and express neuronal protein NF70. In addition, we showed that these changes were the results of IBMX treatment. Ashjian et al. (2003) reported that neural differentiation of human ADSC began 3 days after induction and peaked at the 9th or 10th day. However, using the same induction medium (NIM) and protocol, we observed rapid morphological changes in our rat ADSC in 1 hr. Fujimura et al. (2005) also noted rapid induction of neural differentiation in NIM-treated mouse ADSC. Zuk et al. (2002), using a different induction protocol, also observed rapid neural differentiation in human ADSC. Similar observations of rapid induction of neural differentiation in BMSC following various treat-
ments have also been reported (Woodbury et al., 2000; Kim et al., 2002, 2005; Suon et al., 2004; Yaghoobi et al., 2005). Indeed, the rapidity of the morphological transformation and expression of neuronal proteins in BMSC following treatment with DMSO had raised doubts about the authenticity of the observed neural differentiation. In two consecutive papers, Lu et al. (2004) and Neuhuber et al. (2004) contended that the neuron-like morphological changes in the DMSO-treated BMSC were due to disruption of the cytoskeleton rather than true differentiation. They also argued that the increased immunohistochemical staining of neuronal proteins was due to an increase of antigen levels per unit area, as opposed to an overall increase of protein concentrations. Although the above-mentioned concerns were addressed specifically to DMSO-treated BMSC, they could possibly be generalized to include studies using other inducing agents and other cell types (such as ADSC). Indeed, two recent studies that used nonDMSO inducing agents have responded to these concerns. Kim et al. (2005) reported that forskolin-induced BMSC neural differentiation was accompanied by increases of neuronal proteins NSE and NF200 as deter-
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Fig. 5 Effects of neural induction medium (NIM) components and their solvents on adipose tissue-derived stromal cells (ADSC). Photographs in the right panel are ADSC treated with isobutylmethylxanthine (IBMX), indomethacin (INDO), or insulin. Those in the left are ADSC treated with the respective solvents for IBMX, INDO, and insulin. The concentration for each compound and solvent was identical to that in NIM. All cells were stained with Hematoxylin–Eosin.
mined by western blotting (instead of immunohistochemistry). More importantly, they demonstrated that
Fig. 6 Detection of NF70 and CNPase expression in isobutylmethylxanthine (IBMX)-treated adipose tissue-derived stromal cells. Cells were incubated in control (left panel) or IBMX-containing medium (right panel) for 5 hr and then stained for neuronal marker NF70 and oligodendrocyte marker CNPase. 4 0 ,6-diamidino-2-phenylindole staining (data not shown), which overlapped with the fluorescein isothiocyanate staining, indicates all induced cells expressed these markers.
Fig. 7 Neural induction medium (NIM)-induced morphological changes and NF70 expression in vascular smooth muscle cells (VSMC). VSMC were first verified for smMHC expression (A, Cells stained with anti-smMHC antibody; B, Cells stained with 4 0 ,6-diamidino-2-phenylindole (DAPI)). VSMC were then incubated in control medium (C, E, G) or NIM (D, F, H) for 5 hr and then stained with HE (C, D), anti-NF70 antibody (E, F), or DAPI (G, H; DAPI images merged with E, F).
blockade of ERK phosphorylation with a MAPK inhibitor abrogated cAMP-mediated morphological changes and induction of neuronal proteins. In another study Fujimura et al. (2005) used electron microscopic analysis to show that NIM-induced ADSC, but not control cells, possessed microtubules in the soma and processes, thus providing strong evidence that the neurally induced cells had neuronal structures. While in vitro neural differentiation of ADSC is still controversial, a promising in vivo study has been reported by Kang et al. (2003). In this study human ADSC injected into the lateral ventricle of rat brain were able to migrate to multiple sites in the brain, and approximately 2% of the injected cells remained in the brain at 5–14 days after injection. In addition, the authors stated that the cells persisted in the migration sites for at least 30 days after injection. When ADSC were injected into the brain of a rat model of ischemic brain injury, more cells were found to migrate into the injured
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area of the brain cortex. Furthermore, approximately 4% and 9% of the injected cells were found to express neural markers MAP2 and GFAP, respectively. More importantly, ADSC transplantation in the injured brain resulted in significant recovery of motor and somatosensory behavior, suggesting its usefulness in clinical applications. We are interested in ADSC for its potential to treat degenerative diseases such as erectile dysfunction and stress urinary incontinence. Therefore, in addition to this in vitro study, we have examined ADSC’s differentiation potential in vivo by injecting them into various urological organs in rats. The results showed that ADSC were able to differentiate into various tissue types including nerves (manuscript in preparation), thus supporting the above-mentioned study by Kang et al. that ADSC are indeed capable of neural differentiation. Acknowledgments This work was supported by grants from the California Urology Foundation, the Rock Foundation, and the National Institutes of Health. We would like to thank Dr. ChiaCheng Chang of Michigan State University for providing the adipocyte-inducing medium.
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518 Kim, S.S., Choi, J.M., Kim, J.W., Ham, D.S., Ghil, S.H., Kim, M.K., Kim-Kwon, Y., Hong, S.Y., Ahn, S.C., Kim, S.U., Lee, Y.D. and Suh-Kim, H. (2005) cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK. Neuroreport 16:1357–1361. Lee, R.H., Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C. and Jung, J.S. (2004) Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 14:311–324. Lin, G., Chow, S., Lin, J., Wang, G., Lue, T.F. and Lin, C.S. (2004) Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells. J Cell Biochem 92:104–112. Lu, J., Moochhala, S., Moore, X.L., Ng, K.C., Tan, M.H., Lee, L.K., He, B., Wong, M.C. and Ling, E.A. (2006) Adult bone marrow cells differentiate into neural phenotypes and improve functional recovery in rats following traumatic brain injury. Neurosci Lett 398:12–17. Lu, P., Blesch, A. and Tuszynski, M.H. (2004) Induction of bone marrow stromal cells to neurons: differentiation, transdifferentiation, or artifact? J Neurosci Res 77:174–191. Mitchell, J.B., McIntosh, K., Zvonic, S., Garrett, S., Floyd, Z.E., Kloster, A., Halvorsen, Y.D., Storms, R.W., Goh, B., Kilroy, G., Wu, X. and Gimble, J.M. (2006) Immunophenotype of human adipose derived cells: temporal changes in stromal- and stem cellassociated markers. Stem Cells 24:376–385. Mizuno, H., Zuk, P.A., Zhu, M., Lorenz, H.P., Benhaim, P. and Hedrick, M.H. (2002) Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 109:199–209; discussion 210–191. Muschler, G.F., Nitto, H., Boehm, C.A. and Easley, K.A. (2001) Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 19:117–125. Neuhuber, B., Gallo, G., Howard, L., Kostura, L., Mackay, A. and Fischer, I. (2004) Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype. J Neurosci Res 77:192–204. Planat-Benard, V., Menard, C., Andre, M., Puceat, M., Perez, A., Garcia-Verdugo, J.M., Penicaud, L. and Casteilla, L. (2004a) Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 94:223–229. Planat-Benard, V., Silvestre, J.S., Cousin, B., Andre, M., Nibbelink, M., Tamarat, R., Clergue, M., Manneville, C., SaillanBarreau, C., Duriez, M., Tedgui, A., Levy, B., Penicaud, L. and Casteilla, L. (2004b) Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 109:656–663. Rodriguez, A.M., Elabd, C., Amri, E.Z., Ailhaud, G. and Dani, C. (2005a) The human adipose tissue is a source of multipotent stem cells. Biochimie 87:125–128. Rodriguez, A.M., Elabd, C., Delteil, F., Astier, J., Vernochet, C., Saint-Marc, P., Guesnet, J., Guezennec, A., Amri, E.Z., Dani, C. and Ailhaud, G. (2004) Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochem Biophys Res Commun 315:255–263. Rodriguez, A.M., Pisani, D., Dechesne, C.A., Turc-Carel, C., Kurzenne, J.Y., Wdziekonski, B., Villageois, A., Bagnis, C., Breittmayer, J.P., Groux, H., Ailhaud, G. and Dani, C. (2005b) Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med 201:1397–1405. Safford, K.M., Hicok, K.C., Safford, S.D., Halvorsen, Y.D., Wilkison, W.O., Gimble, J.M. and Rice, H.E. (2002) Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 294:371–379.
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OR IGI N A L A R T IC L E
Hongxiu Ning . Guiting Lin . Tom F. Lue . Ching-Shwun Lin
Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells
Received January 3, 2006; accepted in revised form March 24, 2006
Abstract Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for preclinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70. Key words neuron vascular smooth muscle cells adipose tissue-derived stromal cells differentiation IBMX
Hongxiu Ning Guiting Lin Tom F. Lue Ching-Shwun Lin . ) (* Knuppe Molecular Urology Laboratory Department of Urology, School of Medicine University of California San Francisco, CA 94143-1695, U.S.A. Tel: 11415 353 7205 Fax: 11415 353 9586 E-mail: [email protected] U.S. Copyright Clearance Center Code Statement:
Introduction The adipose tissue contains a stromal vascular fraction (SVF) from which multipotent cells have been isolated (Gronthos et al., 2001; Zuk et al., 2001). These cells are variously called processed lipoaspirate (PLA) cells (Zuk et al., 2001), adipose tissue-derived mesenchymal stem cells (ATSC, AD-MSC) (Song et al., 2005), multipotent adipose-derived stem (MADS) cells (Rodriguez et al., 2005a), adipose tissue-derived stem cells (ASC) (Fujimura et al., 2005), adipose tissue-derived stromal cells (ADSC, ATSC) (Gaustad et al., 2004; Yang et al., 2004; Seo et al., 2005; Hattori et al., 2006), adipose tissuederived adult stem (ADAS) cells (Safford et al., 2002), adipose tissue-derived adult stromal (ADAS) cells (Cowan et al., 2004), and adipose tissue-derived cells (ADC) (Strem et al., 2005b). In this study, the term ‘‘adipose tissue-derived stromal cells (ADSC)’’ will be used. ADSC possess phenotypes and gene expression profiles similar to those of bone marrow stem cells (BMSC) (Gronthos et al., 2001; Zuk et al., 2001, 2002; De Ugarte et al., 2003; Lee et al., 2004; Case et al., 2005; Dicker et al., 2005; Strem et al., 2005a; Wagner et al., 2005). In addition to having the capacity for self-renewal and long-term growth, ADSC are capable of differentiating into diverse cell types including adipocytes (Zuk et al., 2002; Rodriguez et al., 2004, 2005b), osteoblasts (Zuk et al., 2002; Cowan et al., 2004; Cho et al., 2005; Dragoo et al., 2005; Rodriguez et al., 2005a; Yang et al., 2005; Hattori et al., 2006), chondrocytes (Erickson et al., 2002; Zuk et al., 2002; Huang et al., 2004; Betre et al., 2006), hepatocytes (Seo et al., 2005), myocytes (Mizuno et al., 2002; Rodriguez et al., 2005b), cardiomyocytes (Gaustad et al., 2004; Planat-Benard et al., 2004a; Strem et al., 2005b), neurons (Safford et al., 2002, 2004; Zuk et al., 2002; Ashjian et al., 2003; Kang et al., 2003; Tholpady et al., 2003; Yang et al.,
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2004; Case et al., 2005; Fujimura et al., 2005; Jack et al., 2005; Guilak et al., 2006), endothelial cells (PlanatBenard et al., 2004b; Cao et al., 2005), and epithelial cells (Brzoska et al., 2005). Thus, ADSC are not only increasingly accepted as bona fide adult stem cells but also considered to be superior to other types of adult stem cells for future clinical applications (Strem et al., 2005a). Whereas bone marrow can only be obtained in limited quantity because of donor site morbidity, the adipose tissue is usually obtainable in abundance, especially in our increasingly obese society. In addition, clonogenic studies have established that the number of BMSC in bone marrow is approximately 1 in 25,000 to 1 in 100,000 (D’Ippolito et al., 1999; Banfi et al., 2001a, 2001b; Muschler et al., 2001), whereas the average frequency of ADSC in processed lipoaspirate is approximately 2% of nucleated cells (Strem et al., 2005a). Thus, the yield of ADSC from 1 g of fat is approximately 5000 cells, whereas the yield of BMSC is 100– 1000 cells per milliliter of marrow. We are interested in the neuronal potential of ADSC because of our ongoing interest in finding an effective treatment for patients who suffer erectile dysfunction due to injury to the cavernous nerves during surgical and radiation therapies for prostate, bladder, and rectal cancers. We have previously demonstrated the feasibility of using embryonic stem cells (ESC) to treat simulated cavernous nerve injury in rats (Bochinski et al., 2004). However, because of their limited availability and ethical concerns, ESC are presently not a clinically feasible therapeutic venue. On the other hand, the above-mentioned studies suggest ADSC are a viable therapeutic alternative. As such, we set out to isolate rat ADSC and conducted characterization experiments including the induction of neural differentiation. We used a neural induction medium (NIM) that was first reported by Ashjian et al. (2003) and later followed by Fujimura et al. (2005). NIM is consisted of three active ingredients, insulin, indomethacin (INDO), and isobutylmethylxanthine (IBMX), and their choices are explained by Ashjian et al. as follows: (1) Insulin has been shown to promote the maturation of differentiating neocortical cells in rat brains; (2) INDO, an inhibitor of cyclooxygenase, has been shown to promote neural cell survival after ischemic injury to the central nervous system; and (3) IBMX, a phosphodiesterase (PDE) inhibitor, increases intracellular cyclic adenosine monophosphate (cAMP), a neural stimulus. However, these explanations do not indicate why it was necessary to use all three agents. In this study, we determined the effect of each of these agents and their respective solvents. In addition, we included vascular smooth muscle cells (VSMC) in our experiments to test whether NIM-induced changes in cellular morphology and protein expression were specific to ADSC. We report here that IBMX alone was able to induce the differentiation of ADSC into neuron-like cells and VSMC
could be partially induced into neuron-like cells by NIM or IBMX.
Materials and methods Isolation of ADSC ADSC were isolated as described by Zuk et al. (2001). Briefly, subcutaneous adipose tissue was excised from the inguinal region of male Sprague–Dawley rats and cut into fine pieces. The cut tissue was added to 3 volumes of phosphate buffered saline (PBS) in a 50ml conical tube, vortexed at full speed for 1 min, and centrifuged at 220 g for 10 min. The adipose tissue was transferred from the upper phase to a fresh tube, and digested in 0.075% collagenase I (Sigma-Aldrich Co., St Louis, MO) for 1 hr at 371C with shaking. The digest was centrifuged at 220 g for 10 min, followed with the removal of the supernatant. Forty milliliters of PBS was added to resuspend the pellet, which was again centrifuged at 220 g for 10 min. Ten milliliters of 160 nM NH4Cl was added to resuspend the pellet for 10 min, followed by the addition of 10 ml of PBS and centrifugation at 220 g for 10 min. Five milliliters of Dulbecco’sModified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, non-selected, lot # 40404, Biomeda Corporation, Foster City, CA) was added to resuspend the pellet, followed by filtration through a 100-mm cell strainer (BD Biosciences, Bedford, MA) into a 10-cm culture dish. After the addition of 5 ml of DMEM (supplemented with 10% FBS), the culture dish was placed in a 5% CO2 incubator for 3–5 days to allow the formation of ADSC colonies, which were then trypsinized and propagated. Isolation of aortic smooth muscle cells VSMC were isolated from rat aorta as described previously (Lin et al., 2004). These cells were confirmed for their smooth muscle identity by indirect immunofluorescence staining with an antismooth muscle myosin heavy chain antibody (Sigma-Aldrich Co.) and Texas red-conjugated secondary antibody. Cells at the 4th passage were used in this study. Antibodies Antibodies used in this study are listed in Table 1. Detection of specific cell markers ADSC were seeded in six-well plates at 1.0 105 cells/well. After overnight, the cells were fixed with ice-cold methanol for 8 min, permeabilized with 0.05% Triton X-100 for 5 min, and blocked with 5% normal horse serum in PBS for 1 hr at room temperature. The cells were then incubated with the primary antibody for 1 hr at room temperature. After washing with PBS three times, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 1 hr at room temperature. After another three washes with PBS, the cells were further stained with 4 0 ,6-diamidino2-phenylindole (DAPI, for nuclear staining) for 5 min and viewed under fluorescence microscope. Adipogenesis ADSC at passage 3 were seeded in six-well plates and cultured in DMEM supplemented with 10% FBS (control medium). After 24 hr, the medium was changed to D-medium (Chang et al., 1981) (kindly provided by Dr. Chang of Michigan State University) supplemented with 10% FBS, 500 mM IBMX (3-isobutyl-1-methylxan-
512 Table 1 Antibodies used in this study Target protein
Target cell type
Supplier
Catalog no.
CD34 STRO-1 PPAR-g Vimentin Myogenin b-III-tubulin Nestin NF-70 S100 CNPase a-SMC-actin Calponin smMHC
Hematopoietic progenitor cells Bone marrow stromal/erythroid precursor cells Adipose cells Mesenchymal cells Skeletal muscle cells Neurogenic precursor cells Various embryonic cells, including neurons Ectodermal precursor cell, neurons Neurons, astrocytes Oligodendrocytes, Schwann cells Smooth muscle cells Smooth muscle cells Smooth muscle cells
Santa Cruz Biotech, Santa Cruz, CA R&D, Minneapolis, MN Abcam, Cambridge, MA Abcam Abcam Abcam Chemicon, Temecula, CA Chemicon Abcam Abcam Sigma-Aldrich Abcam Sigma-Aldrich
sc-7324 MAB1038 ab19481 ab8069 ab1835 ab14545 MAB353 MAB1615 ab8330 ab6319 A-5228 ab700 M-7786
According to data sheet provided by the supplier.
thine, Sigma-Aldrich, I7018), 1 mM dexamethasone (Sigma-Aldrich, D8893) and 1 mM indomethacin (Sigma-Aldrich, I8280). Two days later, the medium was changed to D-medium supplemented with 10% FBS, and 10 mg/ml insulin (Sigma-Aldrich, I1882). One day later, the medium was changed back to the first D-medium, and this induction cycle was repeated for a total of four times. The cells were then examined for the presence of adipocytes by Oil Red O staining.
Myogenesis ADSC were seeded in six-well plates in DMEM with 10% FBS. The next day, the medium was replaced with the induction medium composed of DMEM, 10% FBS, 5% horse serum (Part # SH30074.02, Lot # 1250704, Hyclone, Logan UT) and 50 mM hydrocortisone (Sigma-Aldrich, H0888). The medium was changed every three days and the induction period lasted for 2 weeks. The cells were then examined for the presence of myocytes by immunofluorescence staining for a-smooth muscle actin with antibody purchased from Sigma-Aldrich (A5228).
expression. The results are consistent with previous reports on human ADSC (see Discussion). We also investigated whether P1 ADSC contain lineage-committed progenitor cells. The results shown in Figure 2 indicate that these cells were negative for adipogenic marker PPAR-g, myogenic marker myogenin, and mesodermal precursor cell marker vimentin. Approximately 2% of ADSC were positive for neurogenic marker bIII-tubulin. We further investigated the differentiation potential of these cells. The results shown in Figure 3 indicate that these cells could differentiate into adipocytes (6%), smooth muscle cells (70%), and neurons (100%). In addition, we have continuously cultured one rat ADSC cell line for more than 100 passages (data not shown). Thus, our rat ADSC possessed stem cell characteristics.
Neurogenesis ADSC at passages 2–5 were seeded in six-well plates at 40%–60% confluence. After three washes with PBS, the cells were induced with NIM (DMEM supplemented with 500 mM IBMX, 200 mM INDO, and 5 mg/ml insulin) for 1 hr. Alternatively, the cells were induced in DMEM supplemented with each of, or any two of, IBMX, INDO, and insulin. The cells were then examined for the expression of neuronal markers S100, NF70, and nestin with the respective antibodies (anti-NF70 antibody, MAB1615, Chemicon; anti-nestin antibody, MAB353, Chemicon; anti-S100 antibody, ab8330, Abcam), followed by hematoxylin-eosin (HE) staining.
Results Characterization of rat ADSC We examined our rat ADSC for the expression of stem cell markers CD34 and STRO-1. As shown in Figure 1, all cells at passage 1 (P1) expressed both CD34 and STRO-1; however, while 95% of cells (at passage 3 (P3)) continued to express STRO-1, they lost CD34
Fig. 1 Detection of stem cell markers CD34 and STRO-1. Adipose tissue-derived stromal cells at passages 1 and 3 were stained with anti-CD34 or anti-STRO-1 antibody and fluorescein isothiocyanate-conjugated secondary antibodies; green fluorescence indicate positive expressing cells. The presence of cells in all four panels was visualized by nuclear staining with 4 0 ,6-diamidino-2-phenylindole (blue fluorescence).
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tested each of them individually and in combinations (IBMX1INDO, IBMX1insulin, and INDO1insulin). As shown in Figure 5, IBMX induced morphological changes in ADSC similar to those induced by NIM, whereas INDO and insulin had little or no effect. The combination of IBMX with INDO or insulin had same effect as IBMX alone, whereas the combination of INDO and insulin had little or no effect (data not shown). Cells treated with IBMX alone all expressed NF70 (Fig. 6) at a comparable level with those treated with NIM (Fig. 4). Because bone marrow stem cells have been shown to differentiate into oligodendrocytes (Lu et al., 2006), we examined whether ADSC had the same potential. The results in Figure 6 show that IBMX-treated ADSC expressed the oligodendrocyte marker CNPase.
Fig. 2 Detection of lineage-specific markers. Adipose tissue-derived stromal cells (ADSC) at passage 1 were stained with fluorescein isothiocyanate-conjugated secondary antibodies that reacted with primary antibodies for adipogenic marker PPAR-g, myogenic marker myogenin, mesodermal precursor cell marker vimentin, and neurogenic marker b-III-tubulin. Cell nuclei were stained with 4 0 ,6diamidino-2-phenylindole. The results show that early ADSC did not express these markers except for b-III-tubulin that was expressed in approximately 2% of cells.
NIM-induced morphological changes and neuronal marker expression We are interested in the neural differentiation potential of ADSC; therefore, we chose the protocol of Ashjian et al. (2003) to induce such differentiation. Within 1 hr of incubation in NIM, the cytoplasm of ADSC retracted towards the nucleus, forming contracted cell bodies with extended cytoplasmic extensions that were in contact with neighboring cells, thus exhibiting a neuronal morphology (Fig. 3). Immunofluorescence staining showed that the uninduced (control) cells expressed nestin (100%) but not S100 or NF70 (Fig. 4); this is in agreement with previous studies (Safford et al., 2002, 2004; Zuk et al., 2002; Ashjian et al., 2003; Kang et al., 2003; Tholpady et al., 2003; Yang et al., 2004; Case et al., 2005; Fujimura et al., 2005; Jack et al., 2005; Guilak et al., 2006). In contrast, all of the induced cells expressed all of these three neuronal cell markers (Fig. 4). The duration of the neuronal morphology and expression varied from experiment to experiment, possibly because of differences in cell passage numbers, density, age of the donor rats, etc. Cell death could occur after 24 hr, but in some experiments, the neuronal morphology and expression lasted for 5 days (data not shown). Individual effects of IBMX, indomethacin, and insulin NIM contains three active ingredients, namely, IBMX, INDO, and insulin. Because there has been no explanation for why all three compounds are needed, we
Effects of NIM on VSMC Although NIM was able to induce neuron-like morphological changes and neuronal marker expression in ADSC, it was unclear whether these effects were specific for ADSC. As such we isolated aortic VSMC, which all expressed smooth muscle myosin heavy chain (Figs. 7A, 7B), and tested them in NIM. The results showed that uninduced VSMC did not express NF70 (Figs. 7E, 7G), while approximately 50% of induced VSMC assumed neuron-like morphology (Fig. 7D) and were stained positive for NF70 (Figs. 7F, 7H). Further tests with each of the three active ingredients of NIM again demonstrated that IBMX alone was responsible for both the morphological changes and NF70 expression (data not shown).
Discussion Since the first publications in 2001 the number of ADSC papers has been steadily increasing. Most of these papers deal with human ADSC that are not suitable for studies in animal models in respect to autologous stem cell therapy. Because we use rat models principally for our urological research, it is necessary for us to understand the characteristics of rat ADSC. However, to our best knowledge, there have been only two published papers on rat ADSC, but neither reported the characterization of these cells (Yang et al., 2004, 2005). In the present study, we for the first time showed that rat ADSC possessed characteristics similar to human ADSC in that (1) both express the widely recognized stem cell marker STRO-1 (Zuk et al., 2002; Strem et al., 2005a), (2) both express hematopoietic cell marker CD34 in early but not late passages (Mitchell et al., 2006), and (3) both are capable of differentiation into various tissue types in vitro. Based on the identification of these stem-cell characteristics, we proceeded to focus on ADSC neural differentiation.
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Fig. 3 Differentiation of rat adipose tissue-derived stromal cells (ADSC) into adipocytes, smooth muscle cells, and neurons. ADSC were induced to differentiate into these three cell types as described in Materials and methods. Oil Red O staining was used to identify adipocytes with red-stained fats in the cytoplasm (upper right panel). Immunofluorescence staining with anti-smooth muscle a-actin and anti-calponin antibodies was performed to identify smooth
muscle cells (green fluorescence, middle panels). Nuclear staining with 4 0 ,6-diamidino-2-phenylindole (blue fluorescence) was done to reveal cellular presence. For neurogenesis following induction with neural induction medium, cell morphological changes were visualized by phase contrast microscopy (lower panels). Their expression of neuronal markers is presented in Fig. 4.
Experimental protocols previously used to induce ADSC neural differentiation were largely adopted from earlier studies on BMSC neural differentiation. These protocols generally relied on exposing cells to retinoic acid (RA) and cytokine cocktails (Sanchez-Ramos et al., 2000; Kim et al., 2002; Case et al., 2005), butylated hydroxyanisole, di-methyl-sulfoxide (DMSO) (Woodbury et al., 2000; Safford et al., 2002; Woodbury et al., 2002; Zuk et al., 2002; Yaghoobi et al., 2005; Guilak et al., 2006), and agents that elevate intracellular cAMP levels (Deng et al., 2001; Ashjian et al., 2003; Suon et al., 2004; Fujimura et al., 2005). While the choice of some of these agents was rationalized in a limited number of studies, there has been a lack of explanation for why it was necessary to use multiple compounds in the induction cocktail. As such, in the present
study we tested each of the three components in the induction cocktail used by Ashjian et al. (2003) and by Fujimura et al. (2005). The results showed that while insulin and INDO were moderately effective, IBMX alone was sufficient for full induction. The inductive effect of IBMX on BMSC neural differentiation was first reported by Deng et al. (2001), and it was believed that IBMX induced neural differentiation by raising intracellular cAMP levels. This interpretation was supported by a later study in which both IBMX and another cAMP-elevating agent, forskolin, were shown to be highly effective in inducing BMSC neural differentiation (Suon et al., 2004). Recently, Kim et al. (2005) further reported that forskolin induces BMSC neural differentiation via activation of the ERK/ MAPK signaling pathway. Specifically they demon-
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Fig. 4 Detection of neuronal markers in neural induction medium (NIM)-treated adipose tissue-derived stromal cells (ADSC). Passage 4 ADSC were incubated in control medium (left panels) or NIM (right panels) for 5 hr and then stained for neuronal markers
S100, NF70, and nestin. 4 0 ,6-diamidino-2-phenylindole staining (data not shown), which overlapped with the fluorescein isothiocyanate staining, indicates all induced cells expressed these markers.
strated that blockade of ERK phosphorylation with a MAPK inhibitor abrogated cAMP-mediated morphological changes and induction of neuronal proteins. While there have been numerous reports on the in vitro neural differentiation of BMSC and ADSC, few if any have demonstrated or discussed whether the associated morphological and gene expression changes are the unique properties of these particular cell types. As such, we included VSMC in this study, and for the first time, we showed that a portion of VSMC could also undergo neuron-like morphological changes and express neuronal protein NF70. In addition, we showed that these changes were the results of IBMX treatment. Ashjian et al. (2003) reported that neural differentiation of human ADSC began 3 days after induction and peaked at the 9th or 10th day. However, using the same induction medium (NIM) and protocol, we observed rapid morphological changes in our rat ADSC in 1 hr. Fujimura et al. (2005) also noted rapid induction of neural differentiation in NIM-treated mouse ADSC. Zuk et al. (2002), using a different induction protocol, also observed rapid neural differentiation in human ADSC. Similar observations of rapid induction of neural differentiation in BMSC following various treat-
ments have also been reported (Woodbury et al., 2000; Kim et al., 2002, 2005; Suon et al., 2004; Yaghoobi et al., 2005). Indeed, the rapidity of the morphological transformation and expression of neuronal proteins in BMSC following treatment with DMSO had raised doubts about the authenticity of the observed neural differentiation. In two consecutive papers, Lu et al. (2004) and Neuhuber et al. (2004) contended that the neuron-like morphological changes in the DMSO-treated BMSC were due to disruption of the cytoskeleton rather than true differentiation. They also argued that the increased immunohistochemical staining of neuronal proteins was due to an increase of antigen levels per unit area, as opposed to an overall increase of protein concentrations. Although the above-mentioned concerns were addressed specifically to DMSO-treated BMSC, they could possibly be generalized to include studies using other inducing agents and other cell types (such as ADSC). Indeed, two recent studies that used nonDMSO inducing agents have responded to these concerns. Kim et al. (2005) reported that forskolin-induced BMSC neural differentiation was accompanied by increases of neuronal proteins NSE and NF200 as deter-
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Fig. 5 Effects of neural induction medium (NIM) components and their solvents on adipose tissue-derived stromal cells (ADSC). Photographs in the right panel are ADSC treated with isobutylmethylxanthine (IBMX), indomethacin (INDO), or insulin. Those in the left are ADSC treated with the respective solvents for IBMX, INDO, and insulin. The concentration for each compound and solvent was identical to that in NIM. All cells were stained with Hematoxylin–Eosin.
mined by western blotting (instead of immunohistochemistry). More importantly, they demonstrated that
Fig. 6 Detection of NF70 and CNPase expression in isobutylmethylxanthine (IBMX)-treated adipose tissue-derived stromal cells. Cells were incubated in control (left panel) or IBMX-containing medium (right panel) for 5 hr and then stained for neuronal marker NF70 and oligodendrocyte marker CNPase. 4 0 ,6-diamidino-2-phenylindole staining (data not shown), which overlapped with the fluorescein isothiocyanate staining, indicates all induced cells expressed these markers.
Fig. 7 Neural induction medium (NIM)-induced morphological changes and NF70 expression in vascular smooth muscle cells (VSMC). VSMC were first verified for smMHC expression (A, Cells stained with anti-smMHC antibody; B, Cells stained with 4 0 ,6-diamidino-2-phenylindole (DAPI)). VSMC were then incubated in control medium (C, E, G) or NIM (D, F, H) for 5 hr and then stained with HE (C, D), anti-NF70 antibody (E, F), or DAPI (G, H; DAPI images merged with E, F).
blockade of ERK phosphorylation with a MAPK inhibitor abrogated cAMP-mediated morphological changes and induction of neuronal proteins. In another study Fujimura et al. (2005) used electron microscopic analysis to show that NIM-induced ADSC, but not control cells, possessed microtubules in the soma and processes, thus providing strong evidence that the neurally induced cells had neuronal structures. While in vitro neural differentiation of ADSC is still controversial, a promising in vivo study has been reported by Kang et al. (2003). In this study human ADSC injected into the lateral ventricle of rat brain were able to migrate to multiple sites in the brain, and approximately 2% of the injected cells remained in the brain at 5–14 days after injection. In addition, the authors stated that the cells persisted in the migration sites for at least 30 days after injection. When ADSC were injected into the brain of a rat model of ischemic brain injury, more cells were found to migrate into the injured
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area of the brain cortex. Furthermore, approximately 4% and 9% of the injected cells were found to express neural markers MAP2 and GFAP, respectively. More importantly, ADSC transplantation in the injured brain resulted in significant recovery of motor and somatosensory behavior, suggesting its usefulness in clinical applications. We are interested in ADSC for its potential to treat degenerative diseases such as erectile dysfunction and stress urinary incontinence. Therefore, in addition to this in vitro study, we have examined ADSC’s differentiation potential in vivo by injecting them into various urological organs in rats. The results showed that ADSC were able to differentiate into various tissue types including nerves (manuscript in preparation), thus supporting the above-mentioned study by Kang et al. that ADSC are indeed capable of neural differentiation. Acknowledgments This work was supported by grants from the California Urology Foundation, the Rock Foundation, and the National Institutes of Health. We would like to thank Dr. ChiaCheng Chang of Michigan State University for providing the adipocyte-inducing medium.
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