Clinical significance of hyaluronan levels and its pro-osteogenic effect on mesenchymal stromal cells in myelodysplastic syndromes

A total of 82 patients with MDS and 28 healthy donors from our own center between Jun 2011 and March 2014 were investigated in this study. All patients were untreated when they were recruited into this study. MDS was diagnosed in accordance with the minimum diagnostic criteria established by the conference on MDS (Vienna, 2006) [16].

The bone marrow serum and cell culture medium samples were centrifuged at 8000 g for 10 min. The supernatant was used to determine the total HA concentration. The HA levels were determined via radioimmunoassay [17].

Following the isolation by density centrifugation, the BM mononuclear cells (MNC) were seeded at a concentration of 1 × 10⁶ cells/mL and cultured in Human Mesenchymal Stem Cell Growth Medium (Cyagen Biosciences Inc, Guangzhou, China) at 37 °C with 5% CO₂ in a humidified atmosphere as previously described [18]. The supernatant containing non-adherent cells was removed and medium was changed every 3 days. MSC used in all experiments were derived from passages 2–4. To fulfill the criteria of the International Society for Cellular Therapy, MSC were evaluated by flow cytometry for the absence of CD34, CD45 antigens and the presence of CD73, CD90, CD105 and CD166 [19].

RNA from MSC was purified using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. cDNA was prepared using the First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) following the manufacturer’s protocol. PCR was performed on an ABI 7500 real-time PCR machine (Applied Biosystems). The primer sequences of runt related transcription factor 2 (RUNX-2), bone sialoprotein (BSP), alkaline phosphatase (ALP), Type1 collagen (COL-1), osteopontin (OPN), osteocalcin (OCN) and hyaluronan synthase 1/2/3 (HAS-1/2/3) are listed in Table 1. GAPDH served as reference control, and differences in mRNA expression levels were calculated as fold changes by the 2^−△△Ct method.

MSC were seeded at 3 × 10⁴ cells/well in 6-well plate in Human Mesenchymal Stem Cell Osteogenic Differentiation Medium (Cyagen Biosciences Inc, Guangzhou, China), and the medium was replaced every 3 days. After 3 weeks differentiation, cells can be fixed and stained with Alizarin red, visualized using light microscopy. The bound staining was eluted with 10% (wt/vol) cetylpyridinium chloride (sigma), and alizarin red-S in samples was quantified by measuring absorbance at 572 nm.

HA solution was prepared by dissolving HA powder (Mw = 1010 − 1800 KDa; LifeCore, MN, U.S.) in double distilled water and adjusted to working concentration before use. After the HA solution was applied to a 6-well plate surface, the coated substratum was kept to dried at 45 °C for 30 min. We used HA coated surfaces at a concentration of 30 μg/cm².

In total, 82 patients were enrolled in this study. The follow-up cutoff date was defined as the end of June 2015. The median age was 58 years old (range 21–83 years), and the male-to-female ratio was 1.05:1. The patients were classified according to WHO 2016 [20] into MDS with single lineage dysplasia (MDS-SLD, n = 8), MDS with ring sideroblasts (MDS-RS, n = 4), MDS with multilineage dysplasia (MDS-MLD, n = 40), MDS with excess of blasts-1 (MDS-EB-1, n = 17), MDS with excess of blasts-2 (MDS-EB-2, n = 13). According to the IPSS risk category [21], 59 patients classified to higher-risk MDS (Intermediate-2/High, IPSS score ≥ 1.5) and 23 patients classified to lower-risk MDS (Low/Intermediate-1, IPSS score ≤ 1.0). Among the 82 patients, 54 patients had good karyotypes (including 51 normal karyotypes and 3 good abnormal karyotypes: sole 5q-, -y, and 20q-), 15 patients had intermediate risk abnormal karyotypes and 13 patients had poor risk abnormal karyotypes (Table 2).

Firstly we analyzed BM serum levels of HA from 82 MDS patients and 28 normal controls by radioimmunoassay. The mean HA levels of MDS patients was 176.1 μg/L (range from 18.1 to 800.0 μg/L), whereas the mean HA levels of normal controls was 148.2 μg/L (range from 22.3 to 304.2 μg/L). There was no obvious difference between MDS patients and normal controls (p = 0.408; Fig. 1a). According to the IPSS risk category, significantly elevated levels of HA were observed in higher-risk MDS patients compared to normal controls (243.5 ± 45.41 vs. 148.2 ± 15.07, p = 0.037), meanwhile,the levels of HA in higher-risk MDS patients were higher than those of lower-risk MDS patients (243.5 ± 45.41 vs. 149.8 ± 18.52, p = 0.025; Fig. 1a). Then we analyzed the HA levels in different WHO classification of MDS patients, the results showed that the HA levels in patients with MDS-SLD were significantly lower than normal controls (103.1 ± 26.41 vs. 148.2 ± 15.07, p = 0.032). The levels of HA in patients with MDS-EB-2 were higher than those of patients with MDS-SLD (200.6 ± 25.53 vs. 103.1 ± 26.41, p = 0.021) and MDS-RS (200.6 ± 25.53 vs. 93.7 ± 24.7, p = 0.046; Fig. 1b). Within karyotype categories, The HA levels in patients with poor karyotype were higher than normal controls (296.9 ± 74.34 vs. 148.2 ± 15.07, p = 0.01). However, the HA levels in patients with intermediate karyotype were lower than normal controls (88.26 ± 15.01 vs. 148.2 ± 15.07, p = 0.014) and patients with good karyotype (88.26 ± 15.01 vs. 171.4 ± 20.01, p = 0.037). Meanwhile, the patients with poor karyotype exhibited higher HA levels than those of patients with good and intermediate karyotype (296.9 ± 74.34 vs. 171.4 ± 20.01, p = 0.024; 296.9 ± 74.34 vs. 88.26 ± 15.01, p = 0.007, respectively; Fig. 1c). We also found that there was no obvious correlation between HA levels and percentage of BM blast cells (p = 0.45, r = 0.088).

According to the BM serum levels of HA, all the patients were divided to two groups: high HA (> 100 μg/L) and low HA (0 to 100 μg/L). As shown in Fig. 1d, the median survival of patients with high HA levels was 18 months and the median survival of patients with low HA levels did not reach. The median survival between the two groups was statistically different (HR: 2.149, 95% CI 1.125 to 4.105, p = 0.021). However, no significant impact was seen on time to AML progression (HR: 2.372, 95% CI 0.724 to 7.772, p = 0.154; Fig. 1e).

We next measured the HA levels in culture medium supernatants from MNC by radioimmunoassay. However, there was no difference between MDS patients and normal controls (Fig. 2a). We also examined HA levels in culture medium supernatants from MSC, we found that the HA levels were elevated in culture medium supernatants from MDS-MSC compared to normal controls (1121 ± 29.99 vs. 969.1 ± 54.12, p = 0.013), especially in higher-risk MDS-MSC (1159 ± 42.64 vs. 969.1 ± 54.12, p = 0.022; Fig. 2b). Furthermore, we determined the mRNA expression of HAS-1/2/3 in MDS-MNC. The results showed that the expression of HAS-1 in MDS-MNC was higher than normal controls (p = 0.027), and lower-risk MDS-MNC also exhibited significant higher HAS-1 expression than normal controls (p = 0.011; Fig. 2c). The expression of HAS-2 was up-regulated in higher-risk MDS-MNC compared to normal controls (p = 0.004) and lower-risk MDS-MNC (p = 0.002; Fig. 2d). No significant difference in HAS-3 expression was observed between the MNC of normal controls and MDS patients (Fig. 2e). We also investigated the mRNA expression of HAS-1/2/3 in MDS-MSC. HAS-2 mRNA expression was 2.3-fold greater in higher-risk MDS-MSC compared with normal MSC (p = 0.003), meanwhile, HAS-2 mRNA expression was significantly increased in higher-risk MDS-MSC compared to lower-risk MDS-MSC (p = 0.019; Fig. 2g). However, there was no difference between any groups in HAS-1 or HAS-3 (Fig. 2f, h).

In a next step we selected ten patients with high BM serum levels of HA and ten patients with low BM serum levels of HA,then investigate the osteogenic differentiation potential of MSC between two groups. After 21 days of osteogenic induction differentiation, relative calcium production by MSC from MDS patients with low BM serum levels of HA was reduced compared to MDS patients with high BM serum levels of HA (p = 0.022; Fig. 3a, b). In addition, mRNA expression of genes associated with osteogenic differentiation in MSC was investigated, the results showed that OPN mRNA expression was significantly increased in MSC from patients with high BM serum levels of HA (p = 0.010). However, the mRNA expression of the other genes (RUNX-2, BSP, ALP, COL-1, OCN) investigated were no obvious difference between two groups (Fig. 3c).

Finally, to further study the involvement of HA in the regulation of osteogenic differentiation of MSC, we performed an osteogenic induction culture of MSC with low BM serum HA levels on surface with and without HA-coating, and evaluated the mineralization at day 7, 14, and 21, respectively. As shown in Fig. 4a, mineralization was evident from day 14, which was confirmed by alizarin red S staining. Compared with normal controls, the relative calcium production increased in MSC with HA-coated surface at day 14 and day 21 (p = 0.013, p = 0.002, respectively; Fig. 4b). Next we investigated the expression of genes associated with osteogenic differentiation in MSC following osteogenic induction at day 0, 7, 14, and 21. MSC with HA-coated demonstrated higher ALP gene expression compared with normal controls at each of the time points (day 7, p = 0.002; day 14, p = 0.003; day 21, p = 0.008, respectively). The results also showed that the expression levels of COL-1 was increased in MSC with HA-coated at day 7 compared to normal controls (p = 0.046). Moreover, MSC with HA-coated showed the higher OPN expression than normal controls at day 21 (p = 0.002; Fig. 4c).