American Society of Hematology

Figure 2

$Figure 2. Native BM MSCs. (A) Flow cytometric sorting of native cells. BM mononuclear cells were prepared and labeled as indicated in Document S1. The sorting gate was determined on the FL2 vs SSC as shown. For SSEA-1, no specific cell subset corresponding to the sorting gate was identified. The percentages (mean ± SEM, n = 7) of BM cells recovered from the total MNCs was (highest to lowest) 1.9% (± 0,4%; CD105), 0.7% (± 0.2%; CD49b), 0.28% (± 0.03%; CD90), 0.17% (± 0.025%; CD130), 0.16% (± 0.035%; CD73), 0.16% (± 0.015%; CD146), 0.15% (± 0.04%; CD200), and 0.014% (± 0.004%; integrin alphaV/beta5). (B) Cell recovery and enrichment in CFU-Fs in the sorted fractions. Red bars indicate cell recovery (percentage of cells recovered in the sorted fraction). Blue bars indicate enrichment in CFU-Fs (cloning efficiency in sorted cells related to that in total mononuclear cells before sorting). Mean values are indicated on top of each bar; error bars represent SEM (n = 4). AV/B5 indicates integrin alphaV/beta5. (C) Protein expression after differentiation. The pattern of protein expression was studied by flow cytometry before differentiation of culture-amplified BM MSCs at passage 1 (continuous red line) and 10 days after induction in osteogenic (continuous green line) and adipogenic (continuous blue line) media (1 representative experiment of 3). Discontinuous lines indicate irrelevant isotype controls. Notice the decrease in expression for CD73, CD105, CD146 and CD200; for CD90 the expression declined only after adipogenic induction; for CD130 there was no decrease. (D) CFU-Fs from CD200+ cells. CD200+ sorted cells (n = 6) were cultured in alpha-MEM plus 10% FCS plus 1 ng/mL bFGF plus supplements as indicated in Document S1. Cultures were screened at days 2, 5, and 10. CFU-Fs were counted at day 10. CFU-Fs could not be grown from CD200− cells. Similar results were obtained for the different sortings using antibodies indicated in panel A. (E) In vitro adipocytic, osteoblastic, and chondrocytic differentiation of CD200+ cells: histochemical markers. P1 confluent layers obtained from CD200+ cells were trypsinized and cells were seeded in differentiation media as indicated in Document S1. Adipocytic differentiation was assessed after 14 days by revealing the presence of cells containing large Nile Red O+ intracytoplasmic vesicles. Osteoblastic differentiation was assessed after 21 days by revealing the presence of von Kossa+ and Alizarin Red+ mineralized areas. Chondrocytic differentiation was assessed after 21 days by revealing the presence in the micropellets of cartilage-specific glycosaminoglycans stained by Safranin O, Toluidine Blue and Alcian Blue. Similar results were obtained for CD146+ cells (data not shown). Experiments were performed in duplicate. Micrographs were acquired with a Leica Microsystems microscope fitted with 10×/0.22 or 20×/0.30 objectives, a Nikon digital camera (DMX1200F; Nikon, Champigny-sur-Marne, France), and Nikon AXT-1 acquisition software (v2.63). (F) In vitro adipocytic, osteoblastic and chondrocytic differentiation of CD200+ cells: molecular markers. RNA was extracted from CD200+ cells cultured in proliferation medium (P) or differentiated into adipocytes (A), osteoblasts (O) and chondrocytes (C). Experiments were performed in parallel on CD200+ and CD146+ cells. RT-PCRs were performed using primers specific for C: transcription factor SOX-9 (SOX9), aggrecan core protein (AGC1), collagen 2, alpha1 chain (COL2A1), collagen 10, alpha1 chain (COL10A1), A: peroxisome proliferator-activated receptor gamma (PPARG), fatty acid-binding protein (FABP4), lipoprotein lipase (LPL), perilipin (PLIN), O: Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALPL), osteopontin (SPP1), collagen 1, alpha1 chain (COL1A1). Housekeeping gene analyzed was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Experiments were performed in duplicate. (G) In vivo ectopic bone formation by CD200+ cells. CD200+ BM MSCs were cultured in proliferation medium (Passage 1) before loading on MBCP ceramic discs that were implanted subcutaneously in nude mice. Mice were killed after 4 weeks. Ceramic discs implanted subcutaneously without cells served as negative controls. (i) Histology picture (Goldner trichrome stain). Bone is stained green; ceramic has a shadowy white appearance. Bar = 50 μm. (ii) Back-scattered electrons mode (BSEM) picture. Mineralized bone is gray with typical osteocyte lacunae, ceramic is white and nonmineralized tissue is black. Bar = 50 μm. Micrograph i was acquired with a Zeiss Axioplan 2 light microscope (Carl Zeiss, Oberkochen, Germany) fitted with a 40× objective, a Kappa OX-40 CDD camera, and Kappa imageBase software (Kappa Opto-electronics, Gleichen, Germany). Micrograph ii was acquired with a scanning microscope with backscattered electron mode (SEM, LEO1450VP, Germany).$

Native BM MSCs. (A) Flow cytometric sorting of native cells. BM mononuclear cells were prepared and labeled as indicated in Document S1. The sorting gate was determined on the FL2 vs SSC as shown. For SSEA-1, no specific cell subset corresponding to the sorting gate was identified. The percentages (mean ± SEM, n = 7) of BM cells recovered from the total MNCs was (highest to lowest) 1.9% (± 0,4%; CD105), 0.7% (± 0.2%; CD49b), 0.28% (± 0.03%; CD90), 0.17% (± 0.025%; CD130), 0.16% (± 0.035%; CD73), 0.16% (± 0.015%; CD146), 0.15% (± 0.04%; CD200), and 0.014% (± 0.004%; integrin alphaV/beta5). (B) Cell recovery and enrichment in CFU-Fs in the sorted fractions. Red bars indicate cell recovery (percentage of cells recovered in the sorted fraction). Blue bars indicate enrichment in CFU-Fs (cloning efficiency in sorted cells related to that in total mononuclear cells before sorting). Mean values are indicated on top of each bar; error bars represent SEM (n = 4). AV/B5 indicates integrin alphaV/beta5. (C) Protein expression after differentiation. The pattern of protein expression was studied by flow cytometry before differentiation of culture-amplified BM MSCs at passage 1 (continuous red line) and 10 days after induction in osteogenic (continuous green line) and adipogenic (continuous blue line) media (1 representative experiment of 3). Discontinuous lines indicate irrelevant isotype controls. Notice the decrease in expression for CD73, CD105, CD146 and CD200; for CD90 the expression declined only after adipogenic induction; for CD130 there was no decrease. (D) CFU-Fs from CD200⁺ cells. CD200⁺ sorted cells (n = 6) were cultured in alpha-MEM plus 10% FCS plus 1 ng/mL bFGF plus supplements as indicated in Document S1. Cultures were screened at days 2, 5, and 10. CFU-Fs were counted at day 10. CFU-Fs could not be grown from CD200⁻ cells. Similar results were obtained for the different sortings using antibodies indicated in panel A. (E) In vitro adipocytic, osteoblastic, and chondrocytic differentiation of CD200⁺ cells: histochemical markers. P1 confluent layers obtained from CD200⁺ cells were trypsinized and cells were seeded in differentiation media as indicated in Document S1. Adipocytic differentiation was assessed after 14 days by revealing the presence of cells containing large Nile Red O⁺ intracytoplasmic vesicles. Osteoblastic differentiation was assessed after 21 days by revealing the presence of von Kossa⁺ and Alizarin Red⁺ mineralized areas. Chondrocytic differentiation was assessed after 21 days by revealing the presence in the micropellets of cartilage-specific glycosaminoglycans stained by Safranin O, Toluidine Blue and Alcian Blue. Similar results were obtained for CD146⁺ cells (data not shown). Experiments were performed in duplicate. Micrographs were acquired with a Leica Microsystems microscope fitted with 10×/0.22 or 20×/0.30 objectives, a Nikon digital camera (DMX1200F; Nikon, Champigny-sur-Marne, France), and Nikon AXT-1 acquisition software (v2.63). (F) In vitro adipocytic, osteoblastic and chondrocytic differentiation of CD200⁺ cells: molecular markers. RNA was extracted from CD200⁺ cells cultured in proliferation medium (P) or differentiated into adipocytes (A), osteoblasts (O) and chondrocytes (C). Experiments were performed in parallel on CD200⁺ and CD146⁺ cells. RT-PCRs were performed using primers specific for C: transcription factor SOX-9 (SOX9), aggrecan core protein (AGC1), collagen 2, alpha1 chain (COL2A1), collagen 10, alpha1 chain (COL10A1), A: peroxisome proliferator-activated receptor gamma (PPARG), fatty acid-binding protein (FABP4), lipoprotein lipase (LPL), perilipin (PLIN), O: Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALPL), osteopontin (SPP1), collagen 1, alpha1 chain (COL1A1). Housekeeping gene analyzed was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Experiments were performed in duplicate. (G) In vivo ectopic bone formation by CD200⁺ cells. CD200⁺ BM MSCs were cultured in proliferation medium (Passage 1) before loading on MBCP ceramic discs that were implanted subcutaneously in nude mice. Mice were killed after 4 weeks. Ceramic discs implanted subcutaneously without cells served as negative controls. (i) Histology picture (Goldner trichrome stain). Bone is stained green; ceramic has a shadowy white appearance. Bar = 50 μm. (ii) Back-scattered electrons mode (BSEM) picture. Mineralized bone is gray with typical osteocyte lacunae, ceramic is white and nonmineralized tissue is black. Bar = 50 μm. Micrograph i was acquired with a Zeiss Axioplan 2 light microscope (Carl Zeiss, Oberkochen, Germany) fitted with a 40× objective, a Kappa OX-40 CDD camera, and Kappa imageBase software (Kappa Opto-electronics, Gleichen, Germany). Micrograph ii was acquired with a scanning microscope with backscattered electron mode (SEM, LEO1450VP, Germany).

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