Loss of osteogenic but not adipogenic potential in senescent MSC is p53 dependent.

<p>(<b>A</b>) MSC derived from p53 knockout mice (MSC-p53KO) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later stained for the expression of SAβ-gal activity. (<b>B</b>) The proliferation capacity of MSC-p53KO was determined using a CFU assay one week post-exposure or not to IR. (<b>C</b>) One week post exposure or not to IR, MSC-p53KO were placed in adipogenic differentiation media for 7 to 14 days. Representative photographs showing lipid accumulation stained with Oil Red O is shown. Scale bar: 200µm. (<b>D</b>) Quantification of lipid accumulation has determined by the extraction of Oil Red O staining and detection by spectrophotometry. (<b>E</b>) Expression of PPARγ was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO cultured in adipogenic differentiation media. (<b>F</b>) One week post exposure or not to IR, MSC-p53KO were placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown. (<b>G</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S staining and detection by spectrophotometry. (<b>H</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO populations placed in osteogenic differentiation media. Mean ± standard error of at least 3 individual experiments is shown; *: <i>p</i> value < 0.05.</p

Senescence of multipotent and committed stromal lineages following exposure to IR.

<p>(<b>A</b>) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (<b>B</b>) Quantification of the proportion of SAβ-gal positive cells in each population. (<b>C</b>) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (<b>D</b>) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.</p

IR-induced senescent MSC failed to generate bone in vivo.

<p>(<b>A</b>) Schematic of the experiment. Control or IR-induced senescent MSC were mixed with HA/TCP particles along with collagen and injected subcutaneously to the flank of mice. 10 weeks post injection, implants were retrieved from the animals, embedded in plastic, sectioned and stained with Goldner’s trichrome to detect bone formation. (B) Representative images from n= 6 implants per group showing mineralization (Goldner’s trichrome in green) from control or IR-induced senescent MSC. Implants were counterstained with hematoxylin eosin. Scale bar: 300µm.</p

Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.

<p>(<b>A</b>) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (<b>B</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (<b>C</b> and <b>D</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: <i>p</i> value < 0.05.</p

Proliferation of spleen and bone marrow osteoclast precursors.

<p>Spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates at 78×10³ cells/well. At each time point, cells were incubated with BrdU during 3 hours. Incorporated BrdU was measured at 370 nm with a 492 nm reference wavelenght, using a colorimetric immunoassay. White circles: WT cells, black squares: Tg-hDAP12 cells. Results are means ± SE. <i>p</i><0,001 (***);<i>p</i><0,01 (**).</p

Osteoclastogenesis from bone marrow and spleen cells cultures from 3-month-old Tg-hDAP12 and WT female mice.

<p>(A) Osteoclastogenesis from mononuclear bone marrow cells cultured for five days in the presence of M-CSF and RANKL; cells were seeded at 12×10³ cells/well in triplicate in 96-well plates; Scale bar 200 µm. (B and C) Osteoclastogenesis from mononuclear spleen cells cultured in the presence of the two cytokines. Cells were seeded at 78×10³ cells/well in triplicate in 96-well plates (B) Kinetic studies of osteoclastogenesis over 8 days. Scale bar 100 µm. Cells were fixed in paraformaldehyde and stained for TRAP activity at each indicated time point of the kinetic. Arrows show osteoclast precursors on day 2 (D2) in cultures of WT and Tg-hDAP12 splenocytes. On day 4 (D4) of WT cultures, the images show two different microscopic fields, with osteoclasts formed in foci of proliferating precursors (image on the left). On day 6 (D6) of Tg-hDAP12 cultures, the images show also two different microscopic fields with dead osteoclasts (*in the image on the right). On day 8 (D8) of Tg-hDAP12 cultures the stars (*) also show lysed osteoclasts. One experiment is shown, representative of six independent experiments. (C) Quantifications of TRAP-positive multinucleated cells present in spleen cell cultures at each time point of the kinetic were done on two experiments. Results are mean ± SE.</p

Ability of Tg-hDAP12 Bone Marrow-Derived Osteoblasts to Promote Osteoclastogenesis.

<p>Four conditions of co-culture were tested: WT bone-marrow-derived osteoblasts co-cultured with either WT or Tg-hDAP12 spleen cells (respectively WTOC/WTOB, TgOC/WTOB) and Tg-hDAP12 bone marrow-derived osteoblasts co-cultured with either WT or Tg-hDAP12 spleen cells (respectively WTOC/TgOB and TgOC/TgOB). 35×10³bone marrow-derived osteoblasts of either 3-month-old WT or Tg-hDAP12 female mice were co-cultured with 12×10⁵ cells, isolated from the spleen of 3-month-old WT or Tg-hDAP12 mice. After 6 days of co-culture in presence of ascorbic acid and 1α,25-dihydroxyvitamin D3 at 10⁻⁸ M, osteoclasts with nuclei ≥3 were counted after fixation and TRAP staining. Results are expressed as mean values of three independent experiments ± SE. ***<i>p</i><0.001.</p

RANKL dose-dependent response of spleen and bone marrow osteoclast precursors.

<p>Bone marrow and spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates. 12,5×10³ cells/well for bone marrow cells and 78×10³per well for spleen cells. Bone marrow and spleen cells were grown in presence of M-CSF and increasing concentrations of RANKL either 20, 30, 50 and 100 ng/ml for bone marrow cells (A) or 20, 30, 40, 50 and 100 ng/ml for spleen cells (B). After 4 days in culture for bone marrow cells or 6 days in culture for spleen cells, TRAP-positive osteoclasts with ≥3 nuclei were counted. White bars:WT cells; Grey bars:Tg-hDAP12 cells. Results are expressed as mean number of osteoclasts with nuclei ≥3 present on the total surface of each of the three wells ± SE. In (B), ***indicated <i>p</i><0.001.</p

Characterization of monocytic precursors in the spleen of 3-month-old Tg-hDAP12 female

<p><b>mice.</b> (A) FACS analyses of myeloid B22O⁻CD11b⁺ cells in the spleen of Tg-hDAP12 mice by comparison with WT mice (four WT and four Tg-hDAP12 animals). The total number of B220⁻CD11b⁺ cells is expressed as a percentage of total nucleated splenocytes. Results are means ± SE. >0.1. (B) One representative FACS dot plot showing the myeloid B220⁻CD11b⁺ population in a black oval and the B220+CD11b–lymphoid population (dashed black oval). (C) Left, Number of CFU-GM colonies obtained after seeding of 2×10⁵ WT or Tg-hDAP12 spleen cells of 3-month-old female mice in Methocult 3534® containing SCF, IL-3 and IL-6 (see Materials and Methods). Results of one representative out of two experiments. Results are means of the number of colonies in the three wells ± SE. *: <i>p</i><0.01. Right, Photographs of one typical colony obtained after 7 days in culture (left picture) and of colony-derived cells after Wright-Giemsa staining. (right picture). The cells have the typical morphology of macrophages. (D) number of CFU-M colonies counted 7 days after seeding WT or Tg-hDAP12 bone marrow or spleen cells in methocult supplemented with 30 ng/ml M-CSF. One experiment performed. Grey bars: Tg-hDAP12 mice; white bars: WT mice.</p

Osteoblastogenesis and osteoclastogenic activities of osteoblasts in bone marrow cell cultures from 3-month-old Tg-hDAP12 female mice.

<p>Osteoblasts were classically obtained after treatment of bone marrow cells with ß sodium-glycerophosphate (10 mM) and ascorbic acid (50 µg/ml) for 7 days. Total RNA was extracted at different times of culture: day 3, day 7 and day 14. (A) Gene expression of osteoblast-associated markers: alkaline phosphatase (ALP), osteocalcin (OCN), measured by RT-PCR at the indicated times of culture. Gene expression was normalized to the L32, house-keeping gene values. Results are means of two independent experiments ± SE. (B) Mineralized colonies obtained from osteoblasts generated from bone marrow. Colonies doubly-stained with alkaline phosphatase and von Kossa appear as black dots. Results are plotted as the mean number of nodules ±SE of three wells for 3-month-old WT and Tg-hDAP12 female mice and were representative of three independent experiments. (C) Expression of human DAP12 transgene in 7-day-old bone marrow-derived osteoblasts using RT-PCR. 1: WT osteoblasts; 2: Tg-hDAP12 osteoblasts. 3: hDAP12 expression in human monocyte-derived osteoclasts used as control; 4: RT-PCR without template. White stars indicate the 373 bp PCR fragment corresponding to hDAP12. One experiment is shown, representative of three independent experiments. Grey bars: Tg-hDAP12 mice; white bars: WT mice.</p