Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: Co-culture with SSCs rescues the function of irradiated osteogenic precursor cells. (A, B) Cell apoptosis was analyzed by flow cytometry with Annexin V-PE/7AAD double staining. (A) Representative flow cytometry plots. (B) Quantitative analysis of the apoptotic rate. (C, D) ALP activity was assessed. (C) Representative ALP staining images (Scale bar: 50 μm). (D) Quantitative analysis of the relative ALP activity. (E, F) Mineralization capacity was evaluated using Alizarin Red S staining. (E) Representative staining images of mineralized nodules (Scale bar: 100 μm). (F) Quantitative analysis of the relative mineralization level. (G, H) Cell migration was determined by a migration assay. (G) Representative images of migrated cells (Scale bar: 50 μm). (H) Quantitative analysis of the relative cell migration level. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01).

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Co-Culture Assay, Irradiation, Flow Cytometry, Double Staining, Activity Assay, Staining, Migration, Two Tailed Test

Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: SSCs exert rescue effects via the Wnt/β-catenin signaling pathway. (A) Representative ALP staining images of cells in each group (Scale bar: 50 μm). (B) Quantitative analysis of ALP activity in each group. (C) Representative Alizarin Red S staining images of cells in each group (Scale bar: 100 μm). (D) Quantitative analysis of Alizarin Red S staining in each group. (E) Relative mRNA expression levels of osteogenic marker genes ( Runx2 , Col1a1 , and OCN ) detected by qRT-PCR. GAPDH was used as an internal reference gene. (F) Representative Western blot images showing the expression levels of RUNX2, COL1A1, OCN, and β-catenin in each group. GAPDH was used as a loading control. (G) Quantitative analysis of Western blot results (gray value ratio of target protein to GAPDH) in each group. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Staining, Activity Assay, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control, Two Tailed Test

Journal: Dose-Response

Article Title: Skeletal Stem Cells Rescue Radiation-Induced Osteogenic Precursor Cell Dysfunction via the Wnt/β-Catenin Signaling Pathway

doi: 10.1177/15593258261440983

Figure Lengend Snippet: SSCs alleviate the radiation-induced bone injury in mice. (A–G) Micro-CT analysis of bone microstructure. (A) Representative micro-CT images of femurs. Quantitative analysis of (B) bone mineral density (BMD), (C) bone volume fraction (BV/TV), (D) trabecular thickness (Tb.Th), (E) trabecular number (Tb.N), (F) connectivity density (Conn.D), and (G) trabecular separation (Tb.Sp) at 2- and 4-weeks post irradiation. (H–K) Histological analysis (Scale bar: 100 μm). (H) H&E staining showing steatosis (arrows) and (I) quantitative analysis of steatotic lesions per field. (J) TRAP staining showing osteoclasts (arrows) and (K) quantitative analysis of osteoclast number per field. (L–O) Immunohistochemical staining of osteogenic markers (Scale bar: 100 μm). (L) Osterix staining and (M) quantitative analysis of Osterix-positive area. (N) β-catenin staining and (O) quantitative analysis of β-catenin-positive area. All experiments were conducted in three groups: Control, irradiation (IR), and IR plus SSC (IR+SSC) at 2- and 4-weeks post-irradiation. All data are presented as mean ± SD, with statistical significance determined by unpaired two-tailed Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: After irradiation and corresponding interventions, cells were cultured in osteogenic induction medium (iXCells Biotechnologies, San Diego, CA, Cat. No. MD-0006) for 7 days.

Techniques: Micro-CT, Irradiation, Staining, Immunohistochemical staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: A Mesenchymal Cell Niche in Skin for Acute Myeloid Leukemia

doi: 10.1101/2022.05.20.491183

Figure Lengend Snippet: ( A ) A representative FACS profile showing FACS sorting/analysis of the Ebf2 + and Ebf2 − cells in dorsal skin. The cells were first gated within non-hematopoietic (CD45 − TER119 − ) and non-endothelial (CD31 − ) live (PI − ) stromal cells. Then, these cells lacking expression of CD44 were further analyzed for their expression of SCA1, PDGFRa/CD140a (PαS) and CD51. The numbers in the panel are the mean frequencies. ( B-C ) The Ebf2 + cell frequency within total PI − ( B ) or PI − CD45 − TER119 − CD31 − stromal cells ( C ) in dorsal skin. ( D ) The fractions of PαS cells within the Ebf2 + and Ebf2 − stromal cells. Each dot in B-D represents data from a single mouse in 3-6 experiments with the horizontal line as a mean value. ( E ) CFU-Fs in the Ebf2 + and Ebf2 − stromal cells. ( F ) CFU-Fs were exclusively found in the Ebf2 + SCA1 + cell fraction. Data in E-F are from 3 independent experiments and each dot represents replicate assays from 2-3 mice in each experiment. The horizontal line represents mean value. Wilcoxon matched-signed pair rank test was used for statistical analysis. (G-J) Single-cell analysis of CFU-Fs and lineage differentiation from the FACS-sorted Ebf2 + ( G-H ) and Ebf2 − PαS ( I-J ) stromal cells. The CFU-F frequencies ( G, I ) were determined by limiting dilution at a density of 1, 2, 5, 10 cells per well in a 96-well plate and the frequency of the single cells with bi-lineage plasticity ( H, J ) were assessed by multilineage differentiation potential of single CFU-Fs derived from the cells. ( K ) Representative images of the osteogenic and adipogenic differentiation from single CFU-F clones derived from Ebf2 + and Ebf2 − PαS cells. ( L ) Population-doubling time (PDT) of randomly selected CFU-Fs derived from single Ebf2 + and Ebf2 − PαS cells. Each line represents the growth kinetics of a single clone. See also in Figure S1.

Article Snippet: For osteogenic differentiation, cells were cultured with complete osteogenic medium mixed by human/mouse StemXVivo osteogenic / adipogenic base medium (CCM007, R&D Systems) and mouse StemXVivo ostegenic supplement (CCM009; R&D Systems) under normoxic condition for 14-21 days.

Techniques: Expressing, Single-cell Analysis, Derivative Assay, Clone Assay

Journal: Molecular medicine reports

Article Title: MicroRNA‑125b suppresses the proliferation and osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells.

doi: 10.3892/mmr.2014.2024

Figure Lengend Snippet: Figure 1. Comparison of the proliferation and osteogenic differen tiation of hBMSCs isolated from osteoporotic patients and normal subjects. (A) Growth and viability of hBMSCs were determined by the MTT assay after cells were cultured for 2, 4, 6, 8 and 10 days; (B) hBMSCs at passage 3 were cultured in osteogenic medium for 14 days, followed by staining and assessment of ALP activity. Data were analyzed using the Student's t‑tests; (C) quantitative polymerase chain reaction analysis of miR‑125b expression in hBMSCs. miR‑125b expression levels were increased in hBMSCs isolated from elderly patients. Data were subjected to Student's t‑tests. Error bars represent the mean ± standard deviation of three independent experiments. *P<0.05. hBMSCs, human bone marrow‑derived mesenchymal stem cells; ALP, alkaline phosphatase; miR, micro RNA; OD, optical density.

Article Snippet: On every third day, the medium was replaced with osteogenic differentiation medium (10% fetal bovine serum, Hyclone; 100 nM dexamethasone, 45 mM L-ascorbic acid and 10 mM β-glycerophosphate; Sigma, St. Louis, MO, USA).

Techniques: Comparison, Isolation, MTT Assay, Cell Culture, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: Molecular medicine reports

Article Title: MicroRNA‑125b suppresses the proliferation and osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells.

doi: 10.3892/mmr.2014.2024

Figure Lengend Snippet: Figure 3. (A) Overexpression of miR‑125b suppressed the osteogenic differentiation of hBMSCs. Osteoblast differentiation of hBMSCs was induced by osteogenic differentiation medium and hBMSCs were collected 14 days after osteogenic induction. qPCR analysis measured the expression of Runx‑2, ALP, OC and COL1α1 in hBMSCs. qPCR data were subjected to Student's t‑tests. Error bars represent the mean ± standard deviation of three independent experi ments. *P<0.05. (B) ALP staining was performed after 14 days of culture to detect ALP activity and Alizarin Red staining was performed after 21 days to evaluate mineralized bone matrix formation. (C) ALP activity of hBMSCs was determined 14 days after osteogenic induction by p‑Nitrophenyl Phosphate Liquid Substrate System and absorbance was measured at 405 nm. Data were subjected to Student's t‑test. *P<0.05. hBMSCs, human bone marrow‑derived mesenchymal stem cells; qPCR, quantitative polymerase chain reaction; Runx‑2, Runt‑related transcription factor‑2; ALP, alkaline phosphatase; OC, osteo calcin; COL1α1, collagen type I α1; NC, negative control; miR, micro RNA; OD, optical density.

Article Snippet: On every third day, the medium was replaced with osteogenic differentiation medium (10% fetal bovine serum, Hyclone; 100 nM dexamethasone, 45 mM L-ascorbic acid and 10 mM β-glycerophosphate; Sigma, St. Louis, MO, USA).

Techniques: Over Expression, Expressing, Standard Deviation, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control