We evaluated the functional properties of BM stromal

We evaluated the functional properties of BM stromal ephrin receptor isolated from a large number of ON and SCD-ON patients (n=340). Our CFE values for ON patients were consistent with those in the literature (Bernardo et al., 2007; Oreffo et al., 1998), although they were lower than CFE values reported in some publications (Doucet et al., 2005; Kuznetsov et al., 2000). This discrepancy may be due to the different sampling techniques used. In our case, a large volume of BM was collected before buffy coat concentration. We cannot exclude the possibility of a variable degree of peripheral blood contamination, which may explain the low CFE values per 1×106 nucleated marrow cells. Our source of BM collection cannot be compared with cell suspensions obtained after flushing fragments of human trabecular bone, as described in others studies (Doucet et al., 2005; Kuznetsov et al., 2009). Although their numbers are low, the BM stromal cells capable of forming osteoprogenitor colonies (CFU-ALP+) represented more than 85% of the CFE values for ON, N and SCD-ON samples, reflecting their osteogenic potential. We found that the total nuclear cells counts were significantly higher in SCD-ON patients than in the ON group. Among these nuclear cells, the CFE values were also significantly higher in SCD patients than in the age and sex-matched ON group. Consistent with previous reports, we observed that CFE value frequencies were inversely proportional to age for both conditions, with younger patients showing higher CFE counts than older patients (D\'Ippolito et al., 1999; Galotto et al., 1999; Kuznetsov et al., 2009). Although the CFE values were higher in the BM of SCD-ON patients compared with the ON group, we observed a decrease with age. This inverse correlation with age may reflect an increase in the frequency of bone complications, such as delayed fracture healing in elderly individuals (with or without SCD). The high CFE values in SCD patients were first described by Kuznestov et al. for seven pediatric patients (Kuznetsov et al., 2009). Our results confirm this finding in a homogeneous large population of adult patients. SCD is characterized by abnormal hemoglobin, which requires a high turnover of hematopoietic cells in the BM. This disturbance in the BM may explain the higher CFE values in SCD patients. The chronic hematopoietic hyperactivity in the BM enables sickled RBCs to be destroyed and renewed with RBCs to compensate for hemolytic anemia. BM stromal cells reside with hematopoietic stem cells. They cooperate through direct and indirect interactions through the release of cytokines and growth factors, thus forming the hematopoietic niche (Despars & St-Pierre, 2011). The high CFE values in SCD patients may sustain a high rate of hematopoiesis through these various interactions, which maintain bone metabolism and hematopoiesis in the BM cavity. Elevated BM activity is also observed in other hematological disorders, such as in some patients with myeloma (Takahira et al., 1994). However, despite the constitutive activation of the BM in SCD-ON patients, cytometric analysis based on MSCA1+/CD73+/CD90+/CD271+/CD45- expression did not detect BMSCs in the peripheral blood of these patients (data not shown). Because BMSCs do not or rarely circulate (Kuznetsov et al., 2009), the peripheral blood collected during erythrocyte exchange procedures is not a viable source of BMSCs, and the BM remains the main source of primary osteoprogenitor cells. The BM of SCD-ON patients contained more stromal progenitors compared with the ON and N groups. Thus, we investigated whether SCD-ON BMSCs were functionally similar to ON and N BMSCs. Regarding their proliferative capacity, BMSCs from SCD-ON patients adhered to plastic and could be expanded for several passages, similar to BMSCs from ON patients and N donors. The doubling time of each BMSCs population increased similarly with every passage. In addition to adherence to plastic, the expression of specific surface antigens is another criterion for defining BMSCs (Dominici et al., 2006). Cultured BMSCs from both SCD-ON and ON strongly expressed several antigens used to identify BMSCs (≤ 95% CD105, CD90 and CD73) and did not express hematopoietic antigens. We evaluated their osteogenic capacity in vitro and found that these cells produced calcium deposits to a similar extent in response to a classical osteoinduction cocktail. Furthermore, a semi-quantitative assay of mineralization and analysis of osteoblastic gene expression showed no significant differences between SCD-ON and N BMSCs. Several osteoblastic genes were up-regulated during differentiation, although the differences were not significant for ALP and BSP. This result was due to the use of human PL-supplemented culture medium, which has been shown to prime BMSCs cultures to undergo osteoblastic differentiation (Chevallier et al., 2010). We detected a higher level of ALP gene expression from two of six SCD-ON patients, but expression was not significantly upregulated compared with the normal donors; this result mainly reflected the cell heterogeneity between BMSCs from different BM donors in terms of their osteogenic activity. However, in vitro osteogenic differentiation assays cannot predict the in vivo osteogenic activity of BMSCs (Janicki et al., 2011; Mendes et al., 2004). Tissue regeneration is a complex process that requires the migration, adhesion and differentiation of BMSCs. To reach the site of an injury and initiate the healing process, cells must migrate to the target area and adhere. Cell number and cell morphology are directly linked to cell differentiation and bone formation (Mankani et al., 2007; Yang et al., 2012). For this reason, we investigated cellular adhesion and found that both SCD-ON and N BMSCs attached well to a scaffold and were uniformly distributed. Thus, SCD does not affect cellular morphology, adhesion or distribution. We then used an ectopic model to investigate the ability of SCD-ON BMSCs to contribute to bone formation in vivo. We found that the bone-forming capacity after in vivo ectopic implantation was similar for BMSCs obtained from SCD-ON patients and those derived from controls (ON patients and N donors). Disparities between our results and those of other studies are not related to the disease; rather, they are related to donor-to-donor heterogeneity and several other factors, including sampling bias during BM aspiration, age, sex and medication of the BM donor (Janicki et al., 2011; Phinney, 2012). Moreover, in vivo bone formation could lead to extramedullary hematopoiesis in each condition. In conclusion, despite a low number of sample sizes tested, these results suggest that there is no deficiency in the osteogenic potential of SCD patient\'s marrow stromal cell populations.