Neurospheres from rat adipose-derived stem cells could be induced into functional Schwann cell-like cells in vitro
- Yongfeng Xu†1,
- Zhengshan Liu†2,
- Lan Liu3,
- Cuiping Zhao1,
- Fu Xiong4,
- Chang Zhou1,
- Yong Li2,
- Yanchang Shan1,
- Funing Peng1 and
- Cheng Zhang1, 2Email author
© Xu et al; licensee BioMed Central Ltd. 2008
Received: 15 July 2007
Accepted: 12 February 2008
Published: 12 February 2008
Schwann cells (SC) which are myelin-forming cells in peripheral nervous system are very useful for the treatment of diseases of peripheral nervous system and central nervous system. However, it is difficult to obtain sufficient large number of SC for clinical use, so alternative cell systems are desired.
Using a procedure similar to the one used for propagation of neural stem cells, we could induce rat adipose-derived stem cells (ADSC) into floating neurospheres. In addition to being able to differentiate into neuronal- and glial-like cells, neurospheres could be induced to differentiate into SC-like cells. SC-like cells were bi- or tri-polar in shape and immunopositive for nestin and SC markers p75, GFAP and S-100, identical to genuine SC. We also found that SC-like cells could induce the differentiation of SH-SY5Y neuroblastoma cells efficiently, perhaps through secretion of soluble substances. We showed further that SC-like cells could form myelin structures with PC12 cell neurites in vitro.
These findings indicated that ADSC could differentiate into SC-like cells in terms of morphology, phenotype and functional capacities. SC-like cells induced from ADSC may be useful for the treatment of neurological diseases.
Schwann cells (SC) play a central role in the regeneration of peripheral nerve, and are essential for peripheral nerve development . It is recognized that SC can provide an option for the treatment of diseases of central nervous system (CNS), such as multiple sclerosis . In CNS, SC transplantation can promote the re-growth of nerve fibres despite unfavorable environment ; SC can remyelinate demyelinated axons of CNS . SC can clear debris by phagocytosis and break down devastated myelin , which can provide an important prerequisite for successful remyelination in demyelinating diseases of CNS . However, it is difficult to obtain sufficient large number of SC for clinical use, so alternative cell systems are desired.
Bone marrow stromal cells (MSCs) can be obtained easily, can be expanded in culture conditions for autologous transplantation, and MSCs can transdifferentiate along a SC lineage in vitro  and in vivo . So, MSCs may be one of alternative cell systems for SC. However, for clinical use, MSCs have presented problems: MSCs procurement procedures are painful and frequently require general or spinal anesthesia and may yield low number of MSCs upon harvest . For these reasons, many researchers begin to investigate alternative sources for MSCs.
Adipose tissue, like bone marrow, is derived from embryonic mesoderm. Cells isolated from adipose tissue, termed adipose-derived stem cells (ADSC), are self-renewal and can differentiate along several mesenchymal tissue lineages, including adipocytes, osteoblasts, myocytes, chondrocytes, endothelial cells and cardiomyocytes [10, 11]. ADSC may also be induced into neurospheres [12, 13] and neuronal-like cells in vitro , and intracerebral transplantation of human ADSC can improve the neurological deficits after cerebral ischemia in rats . Subcutaneous adipose tissue is abundant, readily accessible, and relatively expendable. Liposuction is a common surgical procedure and it is safe, and a large number of cells can be obtained with minimal risk . ADSC may be an ideal alternative cell source for SC. However, it is not known up to now whether ADSC could be induced into SC.
In this study, we found that rat ADSC could be converted into neurospheres, and these neurospheres could be induced into SC-like cells. SC-like cells could induce the differentiation of SH-SY5Y neuroblastoma cells efficiently, and could form myelin structures with neuronal neurites.
Rat ADSC characterization
Conversion of rat ADSC into neurospheres
Neurospheres could be induced to differentiate along a SC lineage
SC-like cells could induce the differentiation of SH-SY5Y cells efficiently
We used SH-SY5Y cells to evaluate whether SC-like cells could secrete soluble factors since genuine SC can induce the differentiation of SH-SY5Y neuroblastoma cells efficiently through production of soluble factors .
SC-like cells could form myelin structures with neuronal neurites
Our results demonstrate that rat ADSC could be converted into neurospheres using a procedure similar to the one used for propagation of genuine neural stem cells. In addition to generating neuronal- and glial-like cells, neurosphere cells from rat ADSC could differentiate into SC-like cells. We showed further that SC-like cells were functional since these cells could secrete soluble factors and form myelin structures with neuronal neurites. Functional properties, especially formation of myelin structures with neuronal neurites, further indicated that SC-like cells from rat ADSC were closely similar to genuine SC.
Primary cultures of adipose tissue are heterogeneous, containing hepatopoietic cells, endothelial cells, smooth muscle cells and pericytes . However, the number of these other cells is small, and the frequency of these other cells will diminish quickly through serial passages . Also, ADSC can differentiate into several mesenchymal tissue lineages, including adipocyte and osteoblast. Rat ADSC within 3–5 passages we used in our experiment were CD29 and CD44 positive, CD31, CD106, CD184, CD34 and CD45 negative, and could undergo osteogenic and adipogenic differentiation. All these characteristics of rat ADSC in our experiments are consistent with previous reports .
Although ADSC and MSCs share many common biological characteristics, the two populations are not identical . Immunocytochemical analysis shows that surface epitope profiles of the two populations are different ; although it is well established that both MSCs and ADSC can undergo chondrogenic differentiation , Kang et al show that ADSC but not MSCs could undergo chondrogenic differentiation under the conditions used in their study; ADSC may have significantly higher neural differentiation capacities than those of MSCs ; the distinctions between the two populations may also extend down to the gene level [10, 12]. Although MSCs can be induced to differentiate along SC lineage , the differences between the two populations mentioned above suggest that whether ADSC could be induced to differentiate along SC lineage needs to be confirmed.
Some recent studies show that neural crest stem cells can be harvested by means of neurosphere method from various seemingly "mesodermal" tissues of adult animals, such as heart  and hair follicular dermal papilla . Kang et al. show that ADSC can be converted into neurospheres , and a preliminary report further suggests that neurosphere cells derived from ADSC may have neural crest-like properties . In our experiment, since neurospheres converted from rat ADSC could differentiate into SC-like cells which belong to peripheral nervous system, these neurospheres should have the characteristics of peripheral nervous system.
Recently, nestin expression has also been observed in myogenic cells, hepatic cells and endothelial cells, which indicates that nestin may not be used as a specific marker for neural stem cells. However, in our experiment, neurosphere cells from rat ADSC can be induced into neuronal- and glial-like cells, which strongly indicates that neurosphere cells derived from rat ADSC have neural stem cell-like properties. Hermann et al suggest that neural stem cell-like cells converted from MSCs are real neural stem cells . The immature neural stem cells would be more suitable for the treatment of neurodegenerative diseases than fully differentiated neural cells, because fully differentiated neurons can not survive detachment and subsequent transplantation procedures .
Neural stem cells from CNS can be maintained in an undifferentiated status by bFGF and EGF [27, 28]. When exposed to RA, neural stem cells will exit from cell cycle and differentiate into nerve cells . In our experiment, neurosphere cells will differentiate in the presence of RA and in the absence of EGF and bFGF.
FSK can elevate the level of intracellular cyclic adenosine monophosphate (cAMP) and cAMP signal may be an intracellular signal during several different stages of SC development. In cultured SC, cAMP elevation can mimick SC responses in the presence of axons during myelination in vivo . In addititon, FSK can enhance the responsiveness of SC to SC mitogens, such as PDGF-BB and glial growth factor . PDGF-BB can induce SC proliferation in the presence of serum and FSK . Heregulin is a subtype of neuregulin-1 and neuregulin-1 is now regarded as the pivotal signal that controls SC at every stage of the lineage . Neuregulin-1 type II, also known as glial growth factor, can induce instructively cultured neural crest cells into SC . In the presence of Heregulin (neuregulin-1 type I), MSCs can be induced into SC-like cells . A mixture of cytokines mentioned above may synergize to induce neurosphere cells into SC-like cells.
SC can produce a number of neurotrophic factors, and a combination of these and other SC-derived soluble factors have been referred to as 'anti-neuroblastoma' agents . Pigment epithelium-derived factor is now regarded as the key factor responsible for SC's ability to induce the differentiation of SH-SY5Y cells . It is likely that SC-like cells from rat ADSC in our experiment produced at least some 'anti-neuroblastoma' agents produced by genuine SC since SC-like cells could induce the differentiation of SH-SY5Y cells efficiently. SC-like cells induced from MSCs can cause neurite growth of dorsal root ganglion neurons in vitro , which supports that SC-like cells from rat ADSC may produce some soluble factors. These SC-derived factors, such as pigment epithelium-derived factor, can promote survival and neurite outgrowth of neurons. SC-like cells from ADSC may be useful for the treatment of diseases in peripheral nervous system (e.g., nerve injuries) and CNS (e.g., multiple sclerosis).
Our research indicated that ADSC could differentiate into SC-like cells in terms of morphology, phenotype and functional capacities. SC-like cells induced from ADSC may be useful for the treatment of neurological diseases.
The local ethics committee approved the animal experimentation protocols and all animal experiments were performed according to Sun Yat-sen university guidelines for animal care. Four- to 8- week-old, male, Sprague-Dawley rats were used for the isolation of rat ADSC. Animals were housed under standard conditions. After sacrifice of the rats, the inguinal fat pad was harvested, and rat ADSC were isolated using a published method . Briefly, the adipose tissue was dissociated mechanically, digested using collagenase type I (Gibco, Carlsbad, CA, USA). The suspension was centrifuged to separate the floating adipocytes from the stromal vascular fraction. Then the cells in the stromal vascular fraction were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). After 24 hours, the non-adherent cells were eliminated by changing the medium. Rat ADSC were passaged for 3–5 times before being used for the experiments.
SH-SY5Y neuroblastoma cell line and PC12 cells (rat pheochromocytoma cell line) were obtained from the American Tissue Type and Culture. SH-SY5Y cells were cultured in DMEM plus 10% FBS in 5%CO2 at 37°C. PC12 cells were cultured in DMEM/F12 (1:1, Gibco) supplemented with 15% horse serum (Gibco) and 2.5% FBS at 37°C in 5% CO2.
Rat ADSC within 3–5 passages after the initial plating of the primary culture were harvested by trypsinization, then the cells were fixed in neutralized 2% paraformaldehyde solution for 30 minutes. The fixed cells were washed twice with PBS and incubated with antibodies to the following antigens: CD31, CD106, CD184, CD34, CD45, CD29 and CD44 (all from Chemicon, Temecula, CA, USA) for 30 minutes. Primary antibodies were directly conjugated with FITC. For isotype control, nonspecific FITC-conjugated IgG was substituted for the primary antibodies . Flow cytometry was performed with a FACscan flow cytometer (Becton Dickinson, San Jose, CA).
Adipogenic and osteogenic differentiation of rat ADSC
Rat ADSC within 3–5 passages were used to verify the multi-potential differentiation capacity. Cells were grown to at least 80% confluence before being cultured in the induction medium. To induce osteogenic differentiation, rat ADSC were cultured for three weeks in DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, 10 mM beta-glycerophosphate. Mineralization of the extracellular matrix was visualized by staining with Alizarin Red S. To induce adipogenic differentiation, rat ADSC were cultured for three weeks in DMEM supplemented with 10% FBS, 0.5 mM isobutyl-methylxanthine (IBMX), 1 μM dexamethasone, 10 μM insulin, 200 μM indomethacin. Adipogenic differentiation was confirmed by staining with Oil-Red O.
Induction of rat ADSC into neurospheres
Rat ADSC within 3–5 passages were induced into neurospheres. In detail, we dissociated rat ADSC (80–90% confluence) with 0.25% trypsin (Gibco) and then plated them on culture flasks at a concentration of 1–2 × 105/cm2 in DMEM/F12 (1:1) supplemented with 20 ng/ml EGF (Peprotech, London, UK), 20 ng/ml bFGF (Peprotech) and B27 (1:50, Gibco) (neurosphere growth medium, NG medium) at 37°C in 5%CO2 [12, 13]. We added fresh NG medium every 3 to 4 days and changed the medium once a week. Neurospheres were passaged every 7 to 10 days by being triturated using a fire-polished Pasteur pipette and being re-plated in fresh medium. We triturated neurospheres and re-plated them in poly-L-lysine (Sigma, St Louis, MO, USA)-coated six-well chamber slides for terminal differentiation experiments.
Terminal differentiation of neurospheres
To induce neurospheres into neural cells, neruospheres from rat ADSC were plated in poly-L-lysine-coated six-well chamber slides and cultured in Neurobasal® medium (Gibco) supplemented with B27 (1:50) for 10 days. During differentiation, 70% of the medium was replaced every 4 days .
To induce neurospheres into SC-like cells, we triturated neurospheres and re-plated them in poly-L-lysine-coated six-well chamber slides at a density of 2.0–2.5 × 105 cells/cm2. We cultured the cells in NG medium for 6 to 8 hours first, then we removed the NG medium, and washed the cells twice with phosphate buffered saline (PBS). Then the cells were induced to differentiate for 48 hours in DMEM supplemented with 10% FBS, 0.5 μM RA (Sigma), 5 μM FSK (Alexis, Lausen, Switzerland), 10 ng/ml PDGF-BB (Peprotech) and 200 ng/ml Heregulin-beta1 (Peprotech) (SC differentiation medium). In some experiments, the SC differentiation medium was replaced with DMEM plus 10% FBS 48 hours after differentiation.
CM was collected from SC-like cells. SC-like cells were grown to 80% confluence. We aspirated the medium and rinsed the cells twice with 5 ml of PBS. We then aspirated the rinse medium and added 4 ml of DMEM supplemented with 2% FBS. The cells were cultured at 37°C in 5% CO2. Forty-eight hours later, we harvested and centrifuged the medium (1000 g, 5 minutes), and collected the supernatant as SC-like cell-CM .
Assessment of the differentiation of SH-SY5Y cells
We dissociated SH-SY5Y cells, and seeded 1 ml of cell suspensions containing 1.25 × 104 cells/ml in each well of 24-well plates coated with poly-L-lysine. Twenty-four hours later, we washed SH-SY5Y cells twice with PBS and cultured the cells for 3 days in the following medium: 1) DMEM with 2% FBS (control group); 2) SC-like cell-CM (SC-like cell-CM group). A cell whose neurite length was longer than 50 μm was regarded as differentiated . We used antibody against beta-tubulin III protein (Chemicon) to confirm the neuronal differentiation of SH-SY5Y cells.
We detected the expression of each antigen for 2 to 4 times in independent experiments. We fixed the cells with 4% paraformaldehyde, blocked the cells with normal goat serum. Then anti-nestin (mouse monoclonal, 1:40), anti-beta-tubulin III (mouse monoclonal, 1:100), anti-glial filament acidic protein (GFAP; mouse monoclonal, 1:400), anti-S-100 (mouse monoclonal, 1:100), anti-p75 nerve growth factor receptor (p75; mouse monoclonal, 1:222) (all from Chemicon) and anti-fibronectin (mouse monoclonal, 1:250; Neomarkers, Fremont, CA, USA) were added. The primary antibodies were incubated overnight at 4°C. We used Cy3-conjugated goat anti-mouse antibody (Chemicon) as secondary antibody which was incubated at room temperature for 1 hour. Then we used DAPI (Sigma) to label the nuclei. Primary antibodies were omitted for control. We examined the cells with a fluorescence microscope (Olympus DP70, Japan).
In vitro myelination assay
Before being used for the co-culture experiment, PC12 cells were cultured in DMEM plus 10% FBS for a few passages until most of PC12 cells stretched out noticeable processes . PC12 cells cultured in DMEM plus 10% FBS for a few passages were dissociated and re-plated at a density of 500 cells/cm2 in poly-L-lysine coated culture dishes in DMEM plus 10% FBS. After 12–24 hours, the medium was removed from PC12 cells, and 500 dissociated rat ADSC or 500 SC-like cells from rat ADSC were seeded into each dish, respectively. PC12/SC-like cells and PC12/rat ADSC were cultured in DMEM plus 10% FBS for 14 days, and the medium was changed every 2–3 days . After 14 days, the cocultures were fixed in 2% glutaraldehyde in sodium cacodylate buffer at 4°C. Following treatment with 1% osmium tetroxide and 1% uranyl acetate, samples were embedded in epon. Ultra-thin sections (50–70 nm) were cut and mounted on Formvar-coated slot grids, and stained for 20 s in 1:1 supersaturated uranyl acetate in acetone followed by staining in 0.2% lead citrate. For examination a CM10 transmission electron microscope (Philips, Netherlands) was used. Electron microscopy was performed at electron microscopy center of Sun Yat-sen university.
We photographed ten random fields per marker. We counted the number of positively stained cells and the total cell number as indicated by DAPI nuclear labeling, respectively. Data are expressed as means ± S.D for all samples. We used SPSS 11.0 to analyze the data. Statistical comparisons were made by student t test. We set statistical significance at p < 0.05 for all the tests performed.
adipose-derived stem cells
bone marrow stromal cells
central nervous system
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