Conceived and designed the experiments: JZ JK. Performed the experiments: JZ Ky FL XL PM. Analyzed the data: FL XL. Contributed reagents/materials/analysis tools: FL XL. Wrote the paper: JZ JG JS JK.
Current address: Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
The authors have declared that no competing interests exist.
Bone marrow stromal cells (BMSCs) are pluripotent and thereby a potential candidate for cell replacement therapy for central nervous system degenerative disorders and traumatic injury. However, the mechanism of their differentiation and effect on neural tissues has not been fully elucidated. This study evaluates the effect of BMSCs on neural cell growth and survival in a retinal ganglion cell (RGCs) model by assessing the effect of changes in the expression of a BMSC-secreted protein, thrombospondin-1 (TSP-1), as a putative mechanistic agent acting on RGCs.
The effect of co-culturing BMSCs and RGCs
Our data suggest that the TSP-1 signaling pathway might have an important role in neural-like differentiation in BMSCs and neurite outgrowth in RGCs. This study provides new insights into the potential reparative mechanisms of neural cell repair.
BMSCs can self-renew,proliferate, and/or differentiate into a variety of cell types, such as cardiomyocytes, rhabdomyocytes, hepatocytes, osteocytes, chondrocytes, tencoytes, adipocytes, smooth muscle cells, and possibly even neural cells
Some researchers, including our group, have reported that BMSCs can differentiate into neural-like cells, with the expression of neural-associated proteins
In order to elucidate the underlying reparative mechanisms, BMSCs and RGCs were co-cultured to evaluate for morphological changes. Additionally, RD-PCR was performed to detect differentially expressed BMSC genes when BMSCs were co-cultured with RGCs versus BMSCs cultured alone. Our data show that BMSCs and RGCs affect each other when co-cultured
Interestingly, we found that TSP-1, an extracellular matrix glycoprotein with a variety of functions and best known for its antiangiogenic properties, was up-regulated in BMSCs co-cultured with RGCs compared to BMSCs cultured alone. TSP-1 is critically important in the formation of neural connections during development
BMSCs were isolated by previously described methods. We utilized a well characterized technique that has been previously used by our lab (21, 22) and multiple others
RGCs were purified by sequential immunopanning to >99.5% purity from Sprague Dawley rats by previously described methods
2×105 BMSCs and RGCs were co-cultured in a transwell system (0.45-uM pore size; BD Bioscience, Bedford, MA) in complete medium. After 5 days, the cover slips were fixed for 7 min in 4% paraformaldehyde, washed 3 times with PBS, and then incubated with 0.1% DAPI for 5 min. Following incubation, the cover slips were washed five times in PBS and mounted in Vectashield mounting medium on glass slides. Under fluorescent microscopy, 10 pictures were randomly taken from one slide in 10× microscopic fields. Cells with an intact nucleus were considered viable. For neurite outgrowth of RGCs, 10 pictures were taken randomly under a regular microscope from one slide. The neurite outgrowth of RGCs was assessed with a camera-lucida projection onto concentric circles. Outgrowth was determined by summing the lengths of all the cells and dividing by the total number of cells in 10× microscopic fields. The percentages of surviving and outgrowth of RGCs were determined on two or three cover slips for each experimental condition and normalized to the control cells examined in a similar manner. The average relative percent of surviving and outgrowth of RGCs from at least three separate experiments for each experimental condition is expressed in the text and figures as the mean+/−S.D.
BMSCs and RGCs were co-cultured in complete medium in the transwell system described above. After 3 days, the total RNA, from the experimental and control conditions, respectively, was isolated with TRIzol Reagent (10296010, Invitrogen, Carlsbad, CA). RD-PCR was performed by previously described methods
The differentially expressed gene fragments or expressed sequence tags (EST) were isolated from the gel, and a second PCR was performed to investigate a band's contents. The PCR products were ligated with pMD-18-T Vector (D101C, Takara, Dalian China), and then sequenced with pMD-18-T sequencing primers. A BLAST search was performed on the EST sequences (
Total RNA was isolated with TRIzol Reagent. RT-PCR was performed using the one-step RT-PCR system (A1250, Promega, Madison, WI). The primer TSP-1 A (sense) is
1×106 BMSCs and RGCs were co-cultured in a transwell system (0.45-uM pore size; BD Bioscience, Bedford, MA) in complete medium. After 24 hours, the medium was replaced with 10 ml of serum-free medium and the cells were incubated for 24 hours. The conditioned medium was collected, concentrated with a Centricon-10 (UFC910024, Billerica, MA), and protein concentrations were determined with the BCA assay (#23250, Pierce, Rockford, IL). Twenty µg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; after electrophoresis the proteins were transferred to a nitrocellulose membrane. Western blot analysis was performed by described methods above.
The siRNA sequences used for targeted silencing of TSP-1 expression and the control sequences were as follows: #TSP-1,
BMSCs were transfected with TSP-1 siRNA, and if rescued, they were then transduced with the TSP-1-expressing recombinant adenovirus as previously described. 48 hours after the first siRNA transfection, the cells were washed twice with sterile PBS. Trypsin buffer 0.125% was added to the petri dish and incubated at 37°C for 3 min. Trypsin digestion was inhibited by the addition of complete medium. Then, the BMSCs and RGCs were co-cultured in complete medium in the transwell system described above. After 3 and 5 days, an assessment of RGC survival and neurite outgrowth was performed as described above.
BMSCs were transfected with TSP-1 siRNA, and if rescued, they were then transduced with the TSP-1-expressing recombinant adenovirus as described previously. 48 hours after the first siRNA transfection, the cells were washed twice with sterile PBS. Trypsin buffer 0.125% was added to the petri dish and incubated at 37°C for 3 min. Trypsin digestion was inhibited by the addition of complete medium. The BMSCs and RGCs were then co-cultured in complete medium in the transwell system described above. After 3 days, RGCs were fixed for 7 min in 4% paraformaldehyde (PFA), washed three times in PBS, and blocked for 30 min in 100 uL of a blocking buffer containing 0.1% Triton X-100. After blocking, the cover slips were washed three times in PBS, and 100 uL of mouse anti-synaptophysin (BM0125, Boster Biological Technology, Wuhan China), diluted 1∶100 in antibody buffer, was added to each cover slip. For the control, the cover slips were incubated with PBS. The cover slips were then incubated overnight at 4°C, washed three times in PBS, and incubated for 2 hr with 100 uL of Cy3 conjugated sheep anti-mouse (BA1031, Bios, Beijing China), diluted 1∶50 in antibody buffer. Following incubation, the cover slips were washed five times in PBS and mounted in Vectashield mounting medium on glass slides. The pictures were taken by laser scan confocol microscopy (LSM 510 Meta, Zeiss, Germany). Western blot analysis was performed as described above.
Total RNA was isolated with TRIzol Reagent (10296010, Invitrogen, Carlsbad, CA). One microgram of total RNA was subjected to reverse transcription with the SYBR PrimeScriptTM RT-PCR kit (DRR063S,Takara, Dalian China) following the manufactures protocol. Real-time PCR was employed to measure TSP-1, TSP-2, CD36, CD47, TGF-β, MAP-2, synaptophysin expression using the SYBR Green system (DRR063S,Takara, Dalian, China). The primers are displayed in
TSP-1 | Sense | |
Antisense | ||
TSP-2: | Sense | |
Antisense | ||
CD36 | Sense | |
Antisense | ||
CD47 | Sense | |
Antisense | ||
TGF-β | Sense | |
Antisense | ||
Map-2 | Sense | |
Antisense | ||
Synaptophysin | Sense | |
Antisense |
Data are expressed as means+/−SE. The differences between mean values were evaluated with the two-tailed Student's t-test (for 2 groups) and the analysis of variance (ANOVA, for >2 groups). All calculations and statistical tests were performed by the computer programs Microsoft Excel 2003 (Microsoft, Redmond, WA) or SPSS 11.5 (SPSS, Chicago, IL). p<0.05 was considered significant for all analyses.
After BMSCs were co-cultured with RGCs, changes in BMSC morphology were observed after the first day (
A, The morphological changes of BMSCs over 7 days (10×); B, The bar graph demonstrates that the percentage of neural-like BMSCs increases when co-cultured with RGCs. p≤0.05 at all time points. (10 pictures were taken randomly under a regular microscope from culturing cells at different time points. The average relative percent of neural-like BMSCs come from at least three separate experiments).
Phase contrast photographs ((
A, RGC neurite formation was enhanced after 5 days of co-culturing with BMSCs (10×). B, Synaptophysin, MAP-2 and TGF-β expression were up-regulated in RGCs co-cultured with BMSCs as determined by real-time RT-PCR (p = 0.031, p = 0.023, p = 0.015, respectively). C, RGC survival was increased when co-cultured with BMSCs (p = 0.025) (d) The mean outgrowth of neurites in RGCs co-cultured with BMSCs increased (p = 0.03). *p-values≤0.05. (10 pictures were taken randomly under a regular microscope from culturing cells at different time points. The average relative percent of surviving and outgrowth of RGCs come from at least three separate experiments).
To assay mRNA expression profiles which might account for the observed morphological changes when BMSCs are co-cultured with RGCs, RD-PCR was performed with BMSCs cultured alone and with RGCs. The RD-PCR products were separated by eletrophoresis on 5% polyacrylamide gel and stained with a silver solution (
A, 5% polyacrylamide gel eletrophoresis demonstrates two dense bands representing upregulated genes in BMSCs co-cultured with RGC (white arrowheads). B, RT-PCR using total RNA isolated from BMSCs on day three demonstrated increased TSP-1 mRNA expression compared to controls. C, BMSC TSP-1 protein expression was increased as compared to the controls as demonstrated by Western blot. β-actin was used as a loading control in B and C. D, TSP-1, TSP-2 and TSP-1 receptors (CD36, CD47) expression were determined by real-time RT-PCR. E, Western analysis for TSP-1 in co-culture medium. *p-values≤0.05. (All data come from at least three separate experiments).
TSP-1 is an extracellular matrix protein critically important in the formation of neural connections during development. TSP-1 is expressed at high levels in the early postnatal rat cortex and superior colliculus, during the period of intense synaptogenesis, but its expression is reduced in the adult. We sought to determine whether TSP-1 secreted by BMSCs affects neural cell survival and neurite outgrowth. In order to answer this question, siRNA interference was performed to silence TSP-1 expression. Transfection of BMSCs with siRNA oligos using a cationic lipid might affect expression of other proteins and reduce experimental reliability. Thus, we also used controls to overcome siRNA silencing of TSP-1 by transducing BMSCs with a recombinant adenovirus with a CMV-promoter containing TSP-1 cDNA (pAd/CMV-Myc-TSP-1). We used a two-time tandem RNA transfection approach at an interval of 1 day, then transduced BMSCs with the recombinant adenovirus (pAd/CMV-Myc-TSP-1) expressing TSP-1, a fusion protein, and Myc-TSP-1, coincident with the second siRNA transfection. The cells were harvested on the third day after the first transfection. This protocol produced a marked reduction in TSP-1 expression. However, TSP-1 expression was rescued by pAd/CMV-Myc-TSP-1 transduction (
(The experiments were repeated at least three times).
Some BMSCs were transfected with siRNA oligos and infected with a recombinant adenovirus (pAd/CMV-Myc-TSP-1) expressing TSP-1 before co-culturing them with RGCs as described above.
48 hours after the first siRNA transfection, BMSCs and RGCs were co-cultured in a transwell system. A, Neural-like morphological changes (10×) in BMSCs was increased at 3 and 5 days in controls (1). This effect was diminished when TSP-1 was silenced with siRNA (2), but restored in BMSCs infected with a TSP-1-expressing adenovirus (3). B, Bar graph shows the mean and SE of the percentage of neural-like cells in the control, silenced, and rescued BMSCs (p = 0.011, p = 0.022, p = 0.028, respectively). *p-values≤0.05. (10 pictures were taken randomly under a regular microscope from culturing cells at different time points. The average relative percent of neural-like BMSCs come from at least three separate experiments).
Similarly,
48 hours after the first siRNA transfection, BMSCs and RGCs were co-cultured in a transwell system. A, After 5 days, RGC survival was not diminished by co-culturing with BMSCs with TSP-1 silencing, but the degree of RGC neurite outgrowth was lessened (3). B, Bar graph shows the mean and SE of RGC neurite outgrowth in within (10×) microscopic fields after 5 days of co-culturing. RGC neurite outgrowth was enhanced by co-culturing with BMSCs (2), which was diminished with TSP-1 silencing (3) but restored in TSP-1-rescued co-cultures (4) (p = 0.038). *p-values≤0.05. (10 pictures were taken randomly under a regular microscope from culturing cells at different time points. The average relative percent of outgrowth of RGCs come from at least three separate experiments).
TSP-1 helps to promote normal CNS synaptogenesis in vivo and vitro
BMSCs were transfected with siRNA oligos and infected with a recombinant adenovirus (pAd/CMV-Myc-TSP-1) expressing TSP-1 before co-culturing as described above. A, After 3 days, immunohistochemical analysis of RGCs for synaptophysin (red dots) shows more synaptic puncta when co-culturing with BMSCs compared to RGCs alone (2 & 4), but fewer when TSP-1 was silenced by siRNA (3). B, Western blot shows synaptophysin in RGCs is up-regulated by TSP-1; (2), which was diminished by TSP-1 silencing (3) and restored by TSP-1-expressing adenoviral rescue (4). C, After 3 days, synaptophysin, MAP-2 and TGF-β expression were determined by real-time RT-PCR (p = 0.019, p = 0.020, p = 0.017, respectively). *p-values≤0.05. (The experiments were repeated at least three times).
BMSC differentiation into neural cells is a widely debated phenomenon
Various factors secreted by RGCs might induce BMSC neural-like differentiation. For example, Mizobe and our group have reported that Thy-1.1, NF and MAP-2 expressed by RGCs accelerates neural-like differentiation in BMSCs
Furthermore, BMSCs significantly increased the propensity of RGCs to form neurites with branching growth cones and yielded an increased survival compared to controls. RGC survival increased by approximately 1.7 fold and more than 70% had intact neurites after 5 days of co-culturing with BMSCs, indicating that factors secreted by BMSCs may improve RGC survival and neurite outgrowth. And real-time RT-PCR data showed that synaptophysin, MAP-2 and TGF-β is up-regulated in RGCs co-cultured with BMSCs.
Therefore, BMSCs and RGCs seem to affect each other when co-cultured in vitro through increased expression of various proteins in both cell types. Likewise, the specific interactions leading to BMSC neural-like differentiation probably involve complex interactions with multiple proteins and/or signaling pathways that will need further elaboration.
Why is TSP-1 up-regulated in BMSCs co-cultured with RGCs? TSP-1 is secreted by a wide variety of epithelial and mesenchymal cells, in patterns that mirror developmental changes in the embryo and response to injury in the adult, and is critically important in the formation of neural connections during development
Thrombospondin family includes five members (TSP-1, TSP-2, TSP-3, TSP-4, and TSP-5)
Thrombospondins are a family of multimeric, calcium-binding extracellular glycoproteins. The first identified thrombospondin, TSP-1, was discovered as a stored protein in a-granules that are released upon platelet activation. Initial studies of the function of TSP-1, therefore, focused on its role in platelet aggregation and fibrin clot formation, and also its effect on vascular endothelial and smooth muscle cells
How does TSP-1 induce RGC neurite outgrowth and synaptogenesis? Real-time RT-PCR data showed that TGF-β, activated by TSP-1, is up-regulated in RGCs co-cultured with BMSCs. TGF-β is a multifunctional cytokine with anti-inflammatory, reparative and neuroprotective functions
In conclusion, we have shown that BMSCs improve RGC survival and neurite outgrowth. Meanwhile, the RGC-conditioned medium induces neural-like differentiation in BMSCs. During differentiation, TSP-1 in BMSCs is up-regulated after co-culturing with RGCs, and TSP-1 is probably involved in RGC neurite outgrowth and synaptogenesis. This study provides new insights into BMSC-mediated reparative mechanisms that might one day be translated into novel treatments for neurological disorders.