Systemic Dysfunction of Osteoblast Differentiation in Adipose-Derived Stem Cells from Patients with Multiple Myeloma

Abstract

Keywords: multiple myeloma; bone disease; osteogenesis; adipogenesis; adipose-derived stem cells; bone marrow; senescence; Dickkopf-related protein 1; systemic disease.

Figure 1

MM-ASC have normal morphology, proliferation capacity, and immunophenotype. (A) Morphology of the different stem cell populations. HD-ASC (a and b) and MM-ASC (c and d) were visualized at 2× (a and c) or 10× (b and d) magnification, using standard light microscopy. (B) Proliferative capacity of HD-ASC (n = 6) and MM-ASC (n = 11). Left: Mean cumulative expansion rate between P1 and P3. The number of viable cells (Trypan blue staining) was determined at the end of each passage (at confluence) and the cumulative expansion was calculated as the ratio of the total number of cells collected at the end of the passage to the total number of cells plated. Right: Mean doubling time calculated for each passage as follows: Doubling time = (ΔT × ln2)/(ln (Nn) – ln (N0)), where Nn is the number of cells at confluence and N0 is the number of cells seeded. Results are expressed as the mean ± SEM; * p < 0.05, using an unpaired t-test with Welch’s correction. (C) Immunophenotypes of HD-ASC (n = 6) and MM-ASCs (n = 11) at passage 2. The percentage of positive cells (%) (left) and the mean fluorescence intensity in arbitrary units (AU) (right) are indicated for each hematopoietic marker. Results are expressed as the mean ± SEM, * p < 0.05, MM-ASC vs. HD-ASC using unpaired t-test with Welch’s correction. AU: Arbitrary units.

Figure 2

Adipocyte differentiation is functional in MM patients. HD- and MM-ASC were differentiated into adipocytes for 7 or 14 days. (A) The cells were stained with Oil-red-O to visualize lipid droplets after 7 or 14 days of differentiation and representative micrographs and scans are shown (left). Staining was quantified at 520 nm and normalized to the protein content (right). (B) Whole cell lysates were extracted on day 0, 7, or 14 of differentiation and analyzed by immunoblotting. Representative immunoblots of PPARγ, C/EBPα, and tubulin (loading control) are shown in the left panel and Western blot quantifications in the two right panels. AU: arbitrary units. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. day 0. NS, not significant. HD-ASC vs. MM-MSC.

Figure 3

Osteoblast differentiation is altered in MM patients. ASC were differentiated into osteoblasts for 14 days. (A) The cells were stained with Alizarin Red to visualize calcium deposition and representative micrographs and scans are shown (left). Staining was quantified at 560 nm and normalized to the protein content. Alkaline phosphatase (ALP) activity was measured and representative micrographs and scans are shown (right). ** p < 0.01, *** p < 0.001, MM-ASCs vs. control cells (HD-ASC). (B) Whole cell lysates were extracted on day 0 or 14 of differentiation and analyzed by immunoblotting. Representative immunoblots of osteocalcin, RUNX2, DKK1, and tubulin (loading control) are shown in the upper panel and Western blot quantifications in the two lower panels. * p < 0.05, ** p < 0.01, *** p < 0.001. AU: Arbitrary units.

Figure 4

Senescence in MM-ASC versus HD-ASC. (A) SA β-galactosidase activity was assessed according to the ratio of pH 6- to pH 4-positive staining after 14 days of differentiation (left). Representative micrographs of β-galactosidase positive cells are shown (right). (B) Whole cell lysates were extracted from ASC on day 0 or 14 of differentiation and analyzed by immunoblotting. Representative immunoblots of the cell cycle arrest markers p21, p16, tubulin (loading control) are shown in the left panel and Western blot quantifications in the two right panels. * p < 0.05, ** p < 0.01, *** p < 0.001. AU: Arbitrary units.

Figure 5

Comparison of ASC and MSC derived from the same MM patients. (A) MM-ASC and MM-MSC from the same patients were differentiated into adipocytes for 7 or 14 days. The cells were stained with Oil-red-O to visualize lipid droplets and representative micrographs and scans are shown (left). Whole cell lysates were extracted on day 0, 7, or 14 of differentiation and analyzed by immunoblotting. Representative immunoblots of PPARγ, C/EBPα, and tubulin (loading control) are shown (left). (B) MM-ASC (blue) and MM-MSC (pink) were differentiated into osteoblasts for 14 days. The cells were stained with Alizarin Red to visualize calcium deposition and ALP activity was assessed. ns, not significant (left). Whole cell lysates were extracted and analyzed by immunoblotting. Representative immunoblots of osteocalcin, RUNX2, and tubulin (loading control) are shown (right). (C) MM-ASC (blue) and MM-MSC (pink) were differentiated into adipocytes or osteoblasts for 14 days as indicated. SA-β-galactosidase activity was assessed according to the ratio of pH 6- to pH 4-positive staining after 14 days of differentiation. * p < 0.05, adipocyte vs. osteoblast. ns, not significant (left). Whole cell lysates were extracted from MM-ASC and MM-MSC on day 0 or 14 of adipocyte or osteoblast differentiation and analyzed by immunoblotting. Representative immunoblots of cell cycle inhibitors p21, p16, and tubulin (loading control) are shown (right).

Figure 6

DKK1 inhibition rescues RUNX2 expression. (A) MM-ASC were cultured without or with an anti-DKK1 monoclonal antibody (DKK1-Ab) or IgG as control. Whole cell lysates were extracted and analyzed by immunoblotting. Representative immunoblots of RUNX2, DKK1, p16, p21, and tubulin (loading control) are shown. (B) The reverse experiment was performed by adding either recombinant DKK1 protein or conditioned medium (CM) from MM-ASC cultures to HD-ASC cultures, incubated or not with anti-DKK1. Immunoblots are shown.