Journal: NPJ Regenerative Medicine

Article Title: Personalized medicine for reconstruction of critical-size bone defects – a translational approach with customizable vascularized bone tissue

doi: 10.1038/s41536-021-00158-8

Figure Lengend Snippet: a EPC after uptake of acetylated LDL-DiI (red) and lectin staining (green). Counterstaining with DAPI (blue). Scale bar 50 µm. b CD34 immunofluorescence staining (green) of EPC. Counterstaining with DAPI (blue). Scale bar 50 µm. c Sprouting of EPC spheroids after 48 h incubation in a fibrin gel. Scale bar 50 µm. d mRNA expression of EPC compared to MSC (set to 1). e Oil Red O staining of MSC after adipogenic differentiation. Scale bar 100 µm. f Alcian Blue staining of MSC after chondrogenic differentiation. Scale bar 200 µm. g Alizarin Red staining of MSC after osteogenic differentiation. Scale bar 200 µm.

Article Snippet: For the adipogenic differentiation, MSC were cultivated in a differentiation medium consisting of MSC adipogenic differentiation basal medium, supplemented with FCS, glutamine, insulin, 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone, rosiglitazone, and penicillin-streptomycin; for chondrogenic differentiation MSC were cultivated in MSC chondrogenic differentiation basal medium, supplemented with FCS, dexamethasone, ascorbate, sodium selenite (ITS), proline, TGF-β3, and penicillin-streptomycin; for osteogenic differentiation MSC were cultivated in MSC osteogenic differentiation basal medium, supplemented with FCS, glutamine, ascorbate, β-glycerophosphate, dexamethasone, and penicillin-streptomycin according to the manufacturer’s recommendation (all from Pelobiotech GmbH, Planegg, Germany).

Techniques: Staining, Immunofluorescence, Incubation, Expressing

Journal: Scientific Reports

Article Title: Prep1 prevents premature adipogenesis of mesenchymal progenitors

doi: 10.1038/s41598-017-15828-1

Figure Lengend Snippet: Bone marrow derived Mesenchymal Stromal Cells (MSC) from Prep1 hypomorphic mice exhibit enhanced adipogenic differentiation potential. ( A ) Immunobloting analysis of Prep1 protein in MSCs at different times of adipogenic differentiation. Days after the induction of differentiation are shown. β-actin Ab was used as a loading control. Full-length blots are presented in Supplementary Fig. . ( B ) Oil-red O staining of MSCs from wild-type (wt) or Prep1 hypomorphic ( Prep1 i/i ) mice bone marrow 7 or 14 days after addition of the differentiation cocktail. Quantification of Oil-red O staining by absorbance at 490 nm is shown. ( C ) qRT-PCR analysis of Prep1 , Ppar γ, Adipoq and Glut4 expression in MSCs from wild-type (wt) or Prep1 hypomorphic ( Prep1 i / i ) mice before (uninduced, UN) and 3 days after (Dif (3d)) induction of adipogenic differentiation. Each graph is representative of 3 independent experiments. Level of significance is indicated as follows *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: MCSs were seeded at p2 at 2 × 10 4 cells/cm2 and at 80% confluence the Complete Medium was replaced with Adipogenic Induction Medium (MesenCult Basal Medium supplemented with 20% AdipogenicStimulatory Supplement (StemCell Technologies) and 1% Pen-Strept (Life Technologies) we followed the manufacturer’s recommendations.

Techniques: Derivative Assay, Western Blot, Control, Staining, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Prep1 prevents premature adipogenesis of mesenchymal progenitors

doi: 10.1038/s41598-017-15828-1

Figure Lengend Snippet: Prep1 down-regulation in 3T3-L1 cells results in an increased tendency towards adipogenic differentiation. ( A ) Oil-red O staining of 3T3-L1 control (C) and Prep1 down-regulated (P) cells 6 days after induction of adipogenic differentiation. ( B ) Quantification of Oil-red O staining by absorbance at 490 nm. UN - undifferentiated (n = 4); Dif (6d) - after 6 days of differentiation (n = 4), *p < 0.01. ( C ) qRT-PCR analysis of Pparγ1 and Pparγ2 RNA transcripts in 3T3-L1 cells before (−2), 2 (2) and 4 days (4) after adipogenic differentiation. Each graph is representative of 3 independent experiments +/− std. deviation. *p < 0.01. ( D ) Clustering heatmaps of gene expression changes assayed by RNA-seq from P and C cells before differentiation (−2 days) and control cells 1 day after differentiation (1 day), where n = 704 represents the number of the genes that changed their expression. The logarithm of ratios for each normalized RPKM is shown. Gene ontology (GO) terms of 3T3-L1 genes enriched by both differentiation of control cells (from day −2 to day +1) and by Prep1 down-regulation at day −2 are shown.

Article Snippet: MCSs were seeded at p2 at 2 × 10 4 cells/cm2 and at 80% confluence the Complete Medium was replaced with Adipogenic Induction Medium (MesenCult Basal Medium supplemented with 20% AdipogenicStimulatory Supplement (StemCell Technologies) and 1% Pen-Strept (Life Technologies) we followed the manufacturer’s recommendations.

Techniques: Staining, Control, Quantitative RT-PCR, Gene Expression, RNA Sequencing, Expressing

Journal: Scientific Reports

Article Title: Prep1 prevents premature adipogenesis of mesenchymal progenitors

doi: 10.1038/s41598-017-15828-1

Figure Lengend Snippet: Prep1 down-regulation affects the expression and phosphorylation of key genes and proteins involved in adipogenic differentiation. ( A ) Immunoblotting analysis of Pparγ, C/EBPα and Vinculin (for loading control) in total cell lysates from C and P cells 3 days after induction of differentiation. For the induction Ins, Dex and IBMX as single agents in the presence of FCS or in various combinations, as indicated, were used. Full-length blots are presented in Supplementary Fig. . ( B ) Immunoblotting analysis of cell extracts from Prep1 down-regulated (P) and control cells (C) at various time points after induction using anti-phospho-Irs1 (Tyr941) Ab. Dex, Ins and IBMX in the presence of FCS were used to induce differentiation. The arrow shows the size of the phosphorylated form (pIrs1). Vinculin Ab was used to assess loading. Full-length blots are presented in Supplementary Fig. . ( C ) Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-phospho-Akt (Thr308) Ab. Total Akt and Vinculin Abs were used as controls. Full-length blots are presented in Supplementary Fig. . qRT-PCR analyses of Srebf1 ( D ), Klf4 ( F ), Klf5 ( G ) and Cebpd ( I ) expression in P (solid line) and C (dashed line) cells at different time points after induction. Relative gene expression was normalized to the levels of Gapdh expression. Immunoblotting of cell extracts from P and C cells at various time points after differentiation induction using anti-Pref1 ( E ) and anti-Klf5 ( H ) Abs. Vinculin Ab was used as loading control. Full-length blots are presented in Supplementary Figs and , respectively.

Article Snippet: MCSs were seeded at p2 at 2 × 10 4 cells/cm2 and at 80% confluence the Complete Medium was replaced with Adipogenic Induction Medium (MesenCult Basal Medium supplemented with 20% AdipogenicStimulatory Supplement (StemCell Technologies) and 1% Pen-Strept (Life Technologies) we followed the manufacturer’s recommendations.

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR, Gene Expression