Analyse des sagittalen lumbosakralen Alignments nach Korrekturspondylodese isthmischer Spondylolisthesen

(A) Representative TEM images of c-kit+ cells used for initiating co-cultures after immunomagnetic bead selection. (B) TEM images of 4- and 14-day cultured c-kit+ cells on 500-μm β-TCP scaffolds, 500-μm β-TCP/collagen I/III scaffolds, and 500-μm β-TCP/Matrigel® scaffolds. Collagen I/III gels and Matrigel® alone are shown as controls. C-kit+ progenitor cell morphology (of freshly isolated cells) consisting of visible scattered chromatin, prominent nucleoli, and approximated cell diameter of 8–10 μm is shown as reference

Additional file 2: Figure S1. of An engineered multicomponent bone marrow niche for the recapitulation of hematopoiesis at ectopic transplantation sites

Representative SEM images of 500- or 800-μm β-TCP scaffolds pre-seeded with hMSCs maintained in culture for 3 weeks with or without addition of collagen I/III. Collagen I/III embedded with hMSCs cultured under standard conditions were undifferentiated controls. Collagen I/III gels with embedded hMSCs cultured under osteogenic differentiation conditions were osteogenic controls (xii)

Additional file 3: Figure S2. of An engineered multicomponent bone marrow niche for the recapitulation of hematopoiesis at ectopic transplantation sites

(A) Monitorization of the CD34+CD38− primitive phenotype in human-derived CD34+ progenitors co-cultured with hMSCs for 5 and 12 days in the different β-TCP/matrix hybrids. On the left data presented is a mean ± SD of three independent experiments; on the right, dot plots of one representative experiment are shown. (B) Representative SEM images CD34+ HSPCs co-cultured for 12 days in 800-μm β-TCP scaffolds in the presence of hMSC-containing collagen I/III matrix. CD34+ HSPCs (red arrows) are seen in close contact to hMSCs (white arrows) within the scaffold macropores. β-TCP scaffolds (asterisks) were reinforced with collagen I/III (green arrows)

Additional file 7: Figure S6. of An engineered multicomponent bone marrow niche for the recapitulation of hematopoiesis at ectopic transplantation sites

Representative SEM images of collagen I/III gels pre-seeded with mBMSCs and later seeded with c-kit+-isolated cells. Co-cultures observed were maintained for 4 days (vi) and 14 days (i, iv, v). SEM in cryogenic mode (ii) and dry mode (iii) shows the typically high microporosity rate of collagen I/III

Additional file 1: Table S1. of An engineered multicomponent bone marrow niche for the recapitulation of hematopoiesis at ectopic transplantation sites

Primers used for RT-qPCR

Additional file 4: Figure S3. of An engineered multicomponent bone marrow niche for the recapitulation of hematopoiesis at ectopic transplantation sites

Dot plot representations of the putative LSK (lin−Sca1+c-kit+) population in starting cultures and 4-, 7-, and 14-day cultures of c-kit+-isolated cells in co-culture with mBMSCs on 500- and 800-μm β-TCP scaffolds with or without collagen I/III gels or Matrigel®. Flow cytometry data shown is of one representative experiment

Data_Sheet_1_Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro.PDF

IntroductionVascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.Materials and methods and resultsHuman coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs–a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease–a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP.ConclusionOur direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.</p

Mesenchymal Stem Cells from Rats with Chronic Kidney Disease Exhibit Premature Senescence and Loss of Regenerative Potential

<div><p>Mesenchymal stem cell (MSC) transplantation has the potential for organ repair. Nevertheless, some factors might lessen the regenerative potential of MSCs, e.g. donor age or systemic disease. It is thus important to carefully assess the patient's suitability for autologous MSC transplantation. Here we investigated the effects of chronic kidney disease (CKD) on MSC function. We isolated bone marrow MSCs from remnant kidney rats (RK) with CKD (CKD-RK-MSC) and found signs of premature senescence: spontaneous adipogenesis, reduced proliferation capacity, active senescence-associated-β-galactosidase, accumulation of actin and a modulated secretion profile. The functionality of CKD-RK-MSCs <i>in vivo</i> was tested in rats with acute anti-Thy1.1-nephritis, where healthy MSCs have been shown to be beneficial. Rats received healthy MSCs, CKD-RK-MSC or medium by injection into the left renal artery. Kidneys receiving healthy MSCs exhibited accelerated healing of glomerular lesions, whereas CKD-RK-MSC or medium exerted no benefit. The negative influence of advanced CKD/uremia on MSCs was confirmed in a second model of CKD, adenine nephropathy (AD). MSCs from rats with adenine nephropathy (CKD-AD-MSC) also exhibited cellular modifications and functional deficits <i>in vivo</i>. We conclude that CKD leads to a sustained loss of <i>in vitro</i> and <i>in vivo</i> functionality in MSCs, possibly due to premature cellular senescence. Considering autologous MSC therapy in human renal disease, studies identifying uremia-associated mechanisms that account for altered MSC function are urgently needed.</p></div

Table_1_Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro.XLSX

IntroductionVascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.Materials and methods and resultsHuman coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs–a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease–a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP.ConclusionOur direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.</p

Analysis of renal function and histology on Day 4 and Day 6 of anti-Thy1.1-nephritis.

<p>(A) Experimental design. (B–D) Comparison of rats that had anti-Thy1.1-nephritis and received H-MSC (“Healthy”, n = 7), TG-MSC (“TG”, n = 8), CKDmod-RK-MSC (“CKDmod-RK”, n = 6) or control DMEM (“Medium”, n = 10) injected into the left renal artery on Day 2 after disease induction and were analysed on Day 4. (E) Experimental design. (F–H) Comparison of rats that had anti-Thy1.1-nephritis and received H-MSC (“Healthy”, n = 7), TG-MSC (“TG”, n = 7), CKDmod-RK-MSC (“CKDmod-RK”, n = 6) or control DMEM (“Medium”, n = 9) injected into the left renal artery on Day 2 after disease induction and were analysed on Day 6. * p<0.05; ** p<0.01; *** p<0.001. All data: mean ± SD.</p