Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro

Animals

Immunization of perlecan to Japanese white rabbits and isolation of MSCs from Wistar rats (both animals from Japan Laboratory Animals, Tokyo, Japan) were performed in accordance with the ethics committee of Hokkaido University (Japan).

Purification of perlecan

Perlecan was purified from Bovine kidneys (kindly gifted from Hokkaido Research Organization) as described by Kanwar et al. (1984) with some modifications. Frozen kidneys (stored at −20°C) were minced, washed with running cold water, and extracted in 4 mol/L guanidine chloride. The extract was then precipitated by an equal volume of 95% ethanol containing 2% potassium acetate. The ethanol precipitant was sequentially washed with water, 0.5 mol/L NaCl, 4 mol/L urea, and further extracted in a solution containing both 4 mol/L urea and 0.5 mol/L NaCl. The extract was subjected to anion-exchange chromatography. Fractions rich in perlecan were adjusted to 1.33 g/mL of density by adding cesium chloride, and fractionated into five fractions by centrifugation at 100 000 × g for 48 h. The undermost fraction was again precipitated by ethanol. The precipitant was finally dissolved in PBS (pH 7.4), dialyzed against PBS, and used as purified perlecan for culturing MSCs. In order to remove HS chains, prior to ethanol precipitation, purified perlecan was treated with heparitinase I (SEIKAGAKU, Tokyo, Japan), 20 mU for 1 mg glycosaminoglycan (GAG), at 37°C for 1 h.

Purity of perlecan was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The bands observed by Coomassie Brilliant Blue-staining were all detected by Western blotting using monoclonal antibody against bovine perlecan domain I or V (both were from Bio Porto Diagnostics, Gentofte, Denmark). Protein and GAG contents in the perlecan solution were determined by the Lowry method (Lowry et al. 1951) and carbazole reaction method of Bitter and Muir (Bitter & Muir 1962), respectively. GAG/protein ratio was determined as 0.156.

Preparation of antiserum against bovine perlecan

A Japanese white rabbit was anesthetized by sodium pentobarbital (Dainippon Sumitomo Pharma, Osaka, Japan), followed by immunization with the purified bovine kidney perlecan previously emulsified with an equal volume of Freund complete adjuvant (BD Biosciences, Franklin Lakes, NJ, USA). Six weeks later, antiserum against perlecan (Anti-Pln) was collected. Non-immune serum was also collected from the same rabbit before immunization.

Specific reactivity of Anti-Pln was confirmed by enzyme-linked immunosorbent assay, Western blotting and immunostaining of tissue sections. Anti-Pln had no cross-reactivity with laminin, type IV collagen or fibronectin. In addition, similar to commercial antibodies, Anti-Pln could also recognize perlecan bands during Western blotting.

Culture of MSCs

MSCs were isolated from bone marrow of rat femurs as described by Nakamura et al. (2009) with some modifications. Male Wistar rats of 5 weeks old were euthanized by overdose of sodium pentobarbital, and their femurs were cleaned. The tip of each femur was removed, and the marrow was harvested by flushing the bone cavities with growth medium, alpha-modified minimum essential medium (α-MEM; Life Technologies, Carlsbad, CA, USA) containing 15% fetal bovine serum (FBS) (Cell Culture Technologies, Gravesano, Switzerland), 63.3 μg/mL of penicillin G potassium (Nacalai Tesque, Kyoto, Japan), and 100 μg/mL of streptomycin sulfate (Nacalai). After culturing the harvested marrow for a day, the non-adherent cells were removed by rinsing with sphosphate buffered saline (PBS). Adherent cells were expanded in growth medium.

Adipogenic and osteogenic differentiation was induced as described by Tan et al. (2011) and Nakamura et al. (2009) with some modifications. The MSCs were cultured in adipogenic differentiation medium (ADM) and osteogenic differentiation medium (ODM). Dulbecco's modified Eagle's medium (DMEM)-high glucose (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS, 27 μg/mL L-ascorbic acid phosphate magnesium salt n-hydrate (Wako, Osaka, Japan), 10 μg/mL insulin (Sigma), 1 mmol/L octanoate (Wako), 500 μmol/L 3-isobutyl-1-methylxanthin (IBMX; Nacalai) and 250 nmol/L dexamethasone (Nacalai) was used as ADM; on the other hand, α-MEM containing 15% FBS, 82 μg/mL L-ascorbic acid phosphate magnesium salt n-hydrate, 10 mmol/L β-glycerophosphate (Nacalai) was used as ODM. The media were exchanged at every 3 days' interval.

Perlecan or heparitinase-I treated perlecan (Pln ΔHS) was added to the media to achieve a final protein concentration of 5 μg/mL. Sodium heparin (Nacalai) solution, which contains uronic acid equivalent to the purified perlecan solution was also added. Perlecan-blocking was performed by adding 1/500 volume of Anti-Pln to the media every day, whereas non-immune serum was added as control. Alternatively, in order to know the effective period of perlecan on MSC differentiation, MSCs were cultured in ADM and ODM for 9 days in which Anti-Pln was added at 0–3, 3–6, 6–9 or 0–9 days. Non-immune serum was either added for the entire culture period, or for the remaining period of Anti-Pln treatment.

Analysis of MSC differentiation

MSCs were induced to differentiate into adipocytes and osteoblasts on 96-well cell culture plates (Thermo Fisher Scientific, Waltham, MA, USA). They were fixed in 10% formalin for 10 min, and stained with Oil Red O and Arizarin Red S. Microscopic images were obtained by using EVOS xl core AMEX-1200 (Advanced Microscopy Group, Bothell, WA, USA). To evaluate the proportion of stained area, microscopic images were obtained from central regions of each well at ×4 magnification, which covered roughly a 50% area of each well, and was analyzed by using ImageJ (downloaded from the National Institute of Health, Bethesda, MD, USA, available at http://rsb.info.nih.gov/ij).

Immunofluorescence

The formalin-fixed cells were rinsed with PBS, and then reacted with primary antibodies, anti-mouse laminin and osteocalcin antisera (both from LSL, Tokyo, Japan), and Anti-Pln. After counterstaining with fluorescein isthiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG) (MP Biomedicals, Solon, OH, USA) and 4′,6-diamidino-2-phenylindole (DAPI; DOJINDO LABORATORIES, Kumamoto, Japan), fluorescent images were obtained under inverted fluorescence microscope, EVOS fl AMF-4302 (Advanced Microscopy Group).

RT-PCR

MSCs cultured in ADM were lysed in Isogen (Wako), and total RNA was isolated according to the manufacturer's instruction. The first strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA by using oligo d(T) 18 primer and reverse transcriptase (Moloney murine leukemia virus, ribolunlease (RNase) H+ ; Wako). For PCR amplification, Fast Start Taq DNA Polymerase (Roche Diagnostics, Basel, Switzerland) was used according to the manufacturer's instructions. The specific primers were designed as follows: 5′-TATGCTGTTATGGGTGAAAC-3′ and 5′-TGGTAATTTCTTGTGAAGTGCTC-3′ for peroxisome proliferator-activated receptor γ2 (PPARγ2); 5′-GGTGAAGGTCGGTGTCAACGGATT-3′ and 5′-GATGCCAAAGTTGTCATGGATGACC-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The resulting PCR products were electrophoresed on 2% agarose gels, followed by staining with 0.5 μg/mL ethidium bromide (Life Technologies). The intensity of bands were evaluated by ImageJ.

Alkaline phosphatase (ALP) activity

ALP activity of MSCs cultured in ODM was measured by Lab Assay ALP (Wako). Cells in 96-well plates were rinsed with distilled water, reacted with substrate buffer at 37°C for 5 min, and enzyme activity was measured at an absorbance of 405 nm using Ultrospec visible plate reader II 96 (GE Healthcare Japan, Tokyo, Japan).

Statistical analysis

Data were shown as mean ± SD. Multiple comparison was performed for each data by Tukey's highly significant difference (HSD) test. P < 0.05 was considered as significant.

Examination of MSC differentiation and perlecan expression

The MSCs cultured in ADM were stained with Oil Red O to examine the progress of adipogenic differentiation by observing lipid accumulation (Fig. 1A). Perlecan and laminin expression was also evaluated by immunostaining and is shown in Figure 1A. A small amount of lipid droplets was observed in several cells at day 3. As culture in ADM proceeded, the number of cells containing lipid droplets and the amount of lipid droplets in each adipocyte derived from MSCs increased. Many cells at 9 days of culture in ADM appeared as spherical shapes, a typical morphology of adipocytes, and were surrounded by BM components, perlecan and laminin. A spherical staining pattern was observed for laminin at day 6, but not for perlecan.

In contrast, MSCs cultured in ODM were stained with Arizarin Red S to examine the progress of osteogenic differentiation by observing calcium deposition (Fig. 1B). Perlecan and osteocalcin expression were also evaluated by immunostaining and are shown in Figure 1B. Faint Arizarin Red S staining until day 6 indicates poor calcium deposition, whereas abundant staining was observed at day 9 (Fig. 1B). During culture in ODM, under the phase-contrast microscope the round-shaped cells were observed on the layer of fibroblast-like cells. The number of the rounded cells increased as culture in ODM proceeded, and these cells developed nodules at day 9. Osteocalcin as well as perlecan was intensely stained in the rounded cells at days 3 and 6. At day 9, intense staining for perlecan and osteocalcin was also observed in several cells within the nodules. Similar to perlecan and osteocalcin, intense staining for laminin was detected in the rounded cells at days 3 and 6, and in the cells within nodules at day 9, whereas relatively weak staining on fibroblast-like cells were also observed at all time points (data not shown).

The effect of extrinsic perlecan on adipogenic and osteogenic differentiation of MSCs

The immunofluorescence study suggested perlecan expression is associated with adipogenic and osteogenic differentiation of MSCs. To assess the effect of perlecan on MSC differentiation, MSCs were induced to differentiate into adipocytes and osteoblasts in the presence of extrinsic perlecan, and compared to cells cultured in the presence of PBS, heparin or Pln ΔHS. Figure 2A shows the weak Oil Red O-staining of lipids in the cells cultured in ADM containing perlecan (Fig. 2A). When the proportion of Oil Red O-stained area was quantified using image-analyzing software, the suppressive effect of perlecan on adipogenic differentiation was significant as compared to PBS (Fig. 2B). Pln ΔHS also showed suppressive effect on adipogenic differentiation, and it was more pronounced than that of perlecan. However, heparin did not show any suppressive effect. Contrary to cells cultured in ADM, cells in ODM showed increased calcium deposition in the presence of perlecan (Fig. 2C), and this promotive effect of perlecan on osteogenic differentiation was significant compared to PBS (Fig. 2D). Neither heparin nor Pln ΔHS showed significant effect on osteogenic differentiation.

MSCs were also cultured on a perlecan-coated 96-well plate, but the immobilized perlecan affected neither adipogenic nor osteogenic differentiation of MSCs (data not shown).

The effect of perlecan blocking on adipogenic and osteogenic differentiation of MSCs

To examine whether intrinsic perlecan secreted in medium also shows the effects observed by extrinsic perlecan, MSCs were induced to differentiate in the presence of Anti-Pln and were compared to control cells cultured with non-immune serum (Fig. 3). Increased lipid accumulation in MSCs cultured in ADM containing Anti-Pln was observed by Oil Red O-staining (Fig. 3A). The lipid accumulation was significantly higher when compared with the control (Fig. 3B). Conversely, calcium deposition in cells induced to osteogenic differentiation was reduced by Anti-Pln (Fig. 3C) and was significantly different from the control.

These perlecan-blocked cells were subjected to immunological staining for perlecan, but the distribution pattern of perlecan was observed not to be altered from control cells (data not shown).

The effective period of perlecan blocking on adipogenic and osteogenic differentiation of MSCs

In order to know when perlecan affects differentiation of MSCs, Anti-Pln was added to differentiation media in distinct culture periods either at days 0–3, 3–6, 6–9 or 0–9 (Fig. 4). Perlecan blocking of cells cultured in ADM at days 0–3 and 3–6 showed slight increase of lipid accumulation as compared to control cells, which was cultured in ADM with non-immune serum the during entire period (Fig. 4A). Perlecan blocking applied at days 6–9 showed increased lipid accumulation in cells, and the level of lipid was the same even when perlecan of cells was blocked for the entire period (day 0–9). However, PPARγ2 gene expression was not altered by perlecan blocking (Fig. 4B). Perlecan blocking of cells cultured in ODM at days 3–6 but not 0–3 and 6–9 suppressed calcium deposition in cells, and the level of suppression achieved by perlecan blocking at days 3–6 was unchanged from that achieved by perlecan blocking for the entire period (days 0–9) (Fig. 4C). ALP activities of cells at days 6 and 9 were insignificantly suppressed by perlecan blocking (P = 0.133 and P = 0.114) as compared to control (Fig. 4D).