Table 1.
GD patients characteristics.
Figure 1.
GBA activity measurement in blood and BM cells.
GBA activity was measured in blood and BM cell subtypes of GD patients (GD) and healthy donors (Ct). Results are expressed as an index defined by the ratio between median fluorescence intensity (MFI) of cells incubated with PFB-FDGlu alone and MFI of cells incubated with CBE and PFB-FDGlu (blood: n = 10 for GD and controls; BM: n = 8 for GD and 6 for controls).
Figure 2.
(A) Relative number of CD34+ cells was measured in GD (n = 7) and controls (n = 10) BM samples according to ISHAGE Guidelines [34]. (B) CD34+ cells clonogenic potential. Granulocyte–macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and megakaryocyte colony-forming units (CFU-Mk) were scored at day 14 by direct visualization using an inverted microscope (n = 8 and 10 for GD patients and controls, respectively). (C) BM-CD34+ cells ex-vivo expansion. Total nucleated cell (TNCs) number was determined after 6 and 12 days culture (n = 6 and 10 for GD and controls respectively). Results are presented as TNCs fold expansion. (D) Primitive hematopoiesis was evaluated by LTC-IC assay. GD patients and controls CD34+ cells were cultured on healthy donors MSCs during a period of 5 weeks. All experiments were performed in triplicate (n = 4 and 8 for GD patients and controls, respectively). (E) Four- to 8-week-old NSG mice were transplanted by retro-orbital injection with 1.105 BM CD34+ cells from GD patients or controls. The presence of human CD45+ cells was assessed by flow cytometry 7 weeks later (n = 5 mice engrafted with CD34+ cells from 2 healthy donors and 4 mice engrafted with CD34+ cells from 2 GD patients).
Figure 3.
(A) Histograms represents CFU-F quantification per 105 mononuclear cells (left panel) and mean cell number per CFU-F (right panel) obtained after culture of control (Ct) or GD BM samples (n = 10 and 7 for GD patients and controls respectively). (B) MSCs growth kinetics: cumulative population doubling (CPD) are shown for Ct and GD-MSCs (n = 5 and 10 respectively). (C) Cell cycle analysis was performed by flow cytometry on Ct and GD-MSCs. The percentage of cells in each phase of the cell cycle was determined by gating G0/G1, S and G2/M cells (n = 8 and 6 respectively). (D) Representative FSC/SSC flow cytometry profile of Ct and GD-MSCs. (E) Ct and GD-MSCs were stained with anti-alpha-tubulin (red) and with Hoechst for nuclear staining (blue). Typical non-round nuclei (angled panels) and large nuclei (arrows) were observed in GD-MSCs. Scale bars represent 50 µm. A and C: * indicates P<0.05.
Figure 4.
Osteoblastic and osteoclastic balance in GD.
MSCs osteogenic differentiation: (A) Box plots represent ALP activity at D0 and after 14 days of differentiation for Ct and GD. Results are expressed as nmol/min/mg protein (n = 10). (B) Osteopontin levels were measured by ELISA at D0 and D14 after differentiation in culture supernatants of Ct and GD-MSCs (n = 10). (C) Relative expression of osteogenic transcription factor RUNX2 measured by RTqPCR at D0 and D14 after differentiation (n = 10). (D) Resorption activity of CD14+-derived osteoclasts cultured on biocoat bone matrix in osteoclastic media: Quantification of resorption potential of monocytes-derived osteoclasts from Ct or GD after 14 days of differentiation or from Ct-osteoclasts in the presence of Ct or GD treated MSCs conditioned media (CM) (n = 6).
Figure 5.
MSCs' soluble factors secretion.
Interleukin-6 (IL-6), IL-8, Monocytes chemotactic protein-1 (MCP-1), Dickkopf-related protein-1 (DKK-1), prostaglandin-E2 (PGE2), and stromal cell-derived factor-1 (SDF-1) levels were measured in supernatants obtained from controls and GD-MSCs (n = 8). Levels were normalized to the number of cells.