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: Stem Cell Research & Therapy

Article Title: M2 macrophages promote heterotopic ossification through MSX2 binding to LEF1-mediated endothelial-mesenchymal transition

doi: 10.1186/s13287-026-04916-7

Figure Lengend Snippet: M2 macrophages promote osteogenic differentiation of MACECs via MSX2 regulation. A AR staining and B ALP staining of MAOECs cultured in osteogenic differentiation medium for 14 days. Original magnification: ×100. C Protein expression of OPN, OCN, and Runx2 of MAOECs cultured in osteogenic differentiation medium for 14 days detected by western blotting. D mRNA expression of OPN, OCN, and Runx2 of endothelial cells cultured in osteogenic differentiation medium for 14 days detected by qPCR. E Alcian Blue staining of MAOECs cultured in chondrogenic differentiation medium for 14 days. Scale bar: 100 μm. F Protein expression of SP7 and SOX9 of MAOECs cultured in chondrogenic differentiation medium for 14 days detected by western blotting. G mRNA expression of SP7 and SOX9 of MAOECs cultured in chondrogenic differentiation medium for 14 days detected by qPCR. **P < 0.01, ***P < 0.001. All in vitro experiments were repeated three times. Full-length blots are presented in Figure 3 of Original Images for Blots file

Article Snippet: For osteogenic/chondrogenic differentiation, after the above grouping, the MAOECs were further cultured in osteogenic differentiation induction medium (HS-B-001, Hasen, Wuxi, China)/chondrogenic differentiation induction medium (PD-005, Procell, Wuhan, China) with added cytokines.

Techniques: Staining, Cell Culture, Expressing, Western Blot, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Alleviated the Inflammatory Response in Mice Infected with the Influenza Virus A (H1N1)

doi: 10.3390/ijms26188839

Figure Lengend Snippet: Characterization of human umbilical cord mesenchymal stem cells (hUCMSCs) and extracellular vesicles derived from hUCMSCs (hUCMSC-EVs). ( A ) hUCMSCs passed to the 3rd generation grew adherently to the wall and were in good condition, with the morphology of elongated pike/pike shape and the overall distribution of vortex (Scale bar = 200 μm). ( B ) Oil red O staining of hUCMSCs after lipogenic induction, visible with obvious red lipid droplets (Scale bar = 100 μm). ( C ) Alizarin red staining of hUCMSCs after osteogenic induction, visible with obvious red calcium nodules (Scale bar = 100 μm). ( D ) Identification of hUCMSCs surface markers by flow cytometry. ( E ) ZETAVIEW particle potential titration and particle size analyzer for hUCMSC-EVs particle size detection. ( F ) Transmission electron microscopy to observe hUCMSC-EVs morphology (scale bar = 200 nm), the red arrow indicates the hUCMSC-EVs. ( G ) Western blotting to identify the expression of hUCMSC-EVs marker protein.

Article Snippet: When the cell density reached 80–90%, the cells were cultured in adipogenic differentiation induction medium (Dakewe, Beijing, China, #6114531) and osteogenic differentiation induction medium (Dakewe, Beijing, China, #6114541) for 18–21 days.

Techniques: Derivative Assay, Staining, Flow Cytometry, Titration, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Marker

Journal: Cells

Article Title: Inflammatory Modulation of Toll-like Receptors in Periodontal Ligament Stem Cells: Implications for Periodontal Therapy

doi: 10.3390/cells14060432

Figure Lengend Snippet: ( A ). Crystal violet staining of PDLSCs‘ colonies formed after 12 days and their ( A1 ) microscopic and ( A2 ) macroscopic appearance. ( B – D ). PDLSCs stimulated with osteogenic inductive medium stained with Alizarin Red ( B1 ) and their controls ( B2 ) with the analysis of SPARC ( B3 ), ALP ( B4 ), and RUNX2 ( B5 ) expression. PDLSCs stimulated with adipogenic differentiation medium stained with Oil Red O ( C1 ) and their controls ( C2 ) with the analysis of LPL ( C3 ) and PPARɣ ( C4 ) expression. PDLSCs stimulated with chondrogenic differentiation medium stained with Alcian Blue and Nuclear Fast Red ( D1 ) and their controls ( D2 ) with the analysis of ACAN ( D3 ). (Box-and-whisker plots with medians and quartiles; Wilcoxon signed rank test, statistical significance marked with asterisk, * p < 0.05). ACAN, aggrecan; ALP, alkaline phosphatase; LPL, lipoproteinlipase; PPARɣ, peroxisome proliferator-activated receptor-gamma; RUNX, Runt-related transcription factor-2, SPARC, Osteonectin. ( E ) Flow cytometric analysis of the surface marker expression profile of PDLSCs.

Article Snippet: To evaluate the osteogenic differentiation potential, 2 × 10 4 third passage PDLSCs were plated on 6-well culture plates and cultured in an osteogenic induction medium (PromoCell, Heidelberg, Germany).

Techniques: Staining, Expressing, Whisker Assay, Marker

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: Model of LncMSTRG25 regulation of hBMSC osteogenic differentiation.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques:

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: Differentially expressed genes in hBMSC osteogenetic differentiation. (A) Cluster heat map of differentially expressed lncRNAs between the osteogenic differentiation IL-1β group and control group. P < 0.05 and fold change > 2 applied as criteria for screening genes with marked changes in expression. (B) Statistics of differentially expressed genes; 112 genes were up-regulated and 69 were down-regulated. (C) lncRNA–mRNA GO enrichment analysis showed that the up-regulated genes might be associated with different biological processes, among which the biological process with the highest enrichment is shown. (D) Each GO term is enriched, and the 10 most significant nodes are represented by rectangles, the colors of which represent the significance of enrichment (increasing from yellow to red).

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Control, Expressing

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: hBMSC identification and gene expression in hBMSCs. (A) Flow cytometry analysis of hBMSC surface markers. Antibodies against CD19, CD34, CD44, CD45, CD73, and CD90 were used for flow cytometry. (B) ARS, Oil Red O staining, and Alcian Blue staining results showing the osteogenic, adipogenic, and chondrogenic differentiation abilities of hBMSCs. (C) Bioinformatics prediction of LncMSTRG25 and the predicted mRNA and miRNA targets. (D to F) PCR detection of the relative expression of LncMSTRG25, PAX8, and miR-939-5p on days 0, 3, 5, 7, 10, 14, and 21. (G) FISH results showed that LncMSTRG25 was localized to the cytoplasm. Data are the mean ± SD of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Gene Expression, Flow Cytometry, Staining, Expressing

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: LncMSTRG25 knockdown inhibited osteogenic differentiation of hBMSCs. (A and B) hBMSCs were transfected with siLncMSTRG25-1, siLncMSTRG25-2, and the negative control and induced to differentiate into osteoblasts for 7 d. To evaluate protein expression levels, Western blotting was performed like PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN. (C and D) qPCR was used to detect the relative expression levels of miR-939-5p, LncMSTRG25-1, PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN. (E) hBMSCs were transfected with siLncMSTRG25-1, siLncMSTRG25-2, or a negative control and induced to differentiate into osteoblasts for 14 d. ARS and ALP staining methods were utilized to identify osteoblast differentiation. (F) Immunofluorescence with specific antibodies was used to detect the expression of PAX8, BMP2, and RUNX2 following 7 d of osteoblast differentiation. Data are the mean ± SD of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Knockdown, Transfection, Negative Control, Expressing, Western Blot, Staining, Immunofluorescence

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: LncMSTRG25 overexpression promotes osteogenic differentiation of hBMSCs. (A) Fluorescent images showing the transfection efficiency of Lv-LncMSTRG25. (B,C, and F) PCR was conducted to evaluate the relative expression of LncMSTRG25-1, miR-939-5p, PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN. (D and E) Lv-LncMSTRG25 and Lv-NC were transfected into hBMSCs and induced to differentiate into osteoblasts for 7 d. Protein expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN were assessed using Western blotting.(G) Lv-LncMSTRG25 and Lv-NC were transfected into hBMSCs and induced to differentiate into osteoblasts for 14 d. ARS and ALP staining were used to detect osteoblast differentiation. (H) Expression of PAX8, BMP2, and RUNX2 was detected by immunofluorescence staining with specific antibodies after 7 d of osteoblast differentiation. Results are presented as the mean ± SD of 3 independent trials, with significance levels of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Staining, Immunofluorescence

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: Osteogenic effects of miR-939-5p on hBMSCs. (A and B) PCR analysis was conducted to measure the relative expression of miR-939-5p, PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN. (C and D) hBMSCs were transfected with the miR-939-5p agomir, antagomir, or NC, and induced to differentiate into osteoblasts for 7 d. Western blotting was carried out to measure the protein expression of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN. (E) hBMSCs were transfected with miR-939-5p agomir, antagomir, or NC and induced to differentiate into osteoblasts for 14 d. ARS and ALP staining was used to detect osteoblast differentiation. (F) hBMSCs were transfected with specific miRNA for 7 d, and immunofluorescence analysis was performed to detect the expression of PAX8, BMP2, and RUNX2. Results are presented as the mean ± SD of 3 independent trials, with significance levels of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Expressing, Transfection, Western Blot, Staining, Immunofluorescence

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: LncMSTRG25 has miR-939-5p binding targets, and the interaction affects osteogenic differentiation. (A) Using miReap, miRanda, and TargetScan tools, we predicted potential binding sites between LncMSTRG25 and miR-939-5p, and validated these predictions through a dual-luciferase reporter assay. (B) Cotransfection of the miR-939-5p plasmid and LncMSTRG25-WT plasmid significantly decreased luciferase activity, as shown by dual-luciferase reporter gene analysis. (C) FISH data showed that LncMSTRG25 and miR-939-5p were both present in the cytoplasm of hBMSCs. (D and E) Lv-LncMSTRG25, Lv-NC, and miR-939-5p mimics or miRNA negative controls were cotransfected into hBMSCs. PCR analysis was conducted to measure the relative expression of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN following 7 d of osteogenic differentiation induction. (F and G) The protein expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN were determined by Western blotting. (H) Osteogenic differentiation was carried out for 14 d, and ARS and ALP staining was performed to determine the differentiation. (I) Following 7 d of osteogenic differentiation, the expression of PAX8, BMP2, and RUNX2 was analyzed by immunofluorescence staining with specific antibodies. Results are presented as the mean ± SD of 3 independent trials, with significance levels of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Binding Assay, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Activity Assay, Expressing, Western Blot, Staining, Immunofluorescence

Journal: Research

Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis

doi: 10.34133/research.0601

Figure Lengend Snippet: LncMSTRG25–PAX8–miR-939-5p regulatory axis regulates hBMSC osteogenesis. (A) Potential target-binding sites of PAX8 and miR-939-5p were predicted using TargetScan and StarBase, and then validated through a dual-luciferase reporter assay. (B) The results from the dual-luciferase reporter gene assay indicated that cotransfection with the miR-939-5p plasmid and PAX8-WT1 and PAX8-WT2 plasmids caused a significant decrease in luciferase activity. (C to E) RNA RIP experiments were performed on hBMSCs using PAX8 antibody to evaluate the enrichment effect of anti-PAX8 on LncMSTRG25 and miR-939-5p. qPCR was used to detect RNA levels in the immunoprecipitates. The miR-939-5p level in the anti-PAX8 group was higher compared with that in the anti-IgG group, and the level of LncMSTRG25 was significantly increased in the anti-PAX8 group compared with that in the anti-IgG group. The LncMSTRG25 PCR product was subjected to agarose electrophoresis, and in the anti-PAX8 group, the expression level was markedly increased. (F and G) Silver staining and Western blotting results of RNA pull-down samples showed that the biotin-labeled LncMSTRG25-specific probe group significantly enriched PAX8. (H and I) Using Lv-LncMSTRG25, Lv-NC, siPAX8, or an siRNA negative control, osteogenic differentiation was induced for 7 d. Protein expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN were analyzed by Western blotting. (J) To detect the relative expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN, PCR was performed. Osteogenic differentiation continued for 14 d. (K) ARS and ALP staining were used to monitor osteogenic differentiation. (L) Specific antibody immunofluorescence staining was used to detect the expression of PAX8, BMP2, and RUNX2 after 7 d of osteogenic differentiation. Results are presented as the mean ± SD of 3 independent trials, with significance levels of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.

Techniques: Binding Assay, Luciferase, Reporter Assay, Reporter Gene Assay, Cotransfection, Plasmid Preparation, Activity Assay, Electrophoresis, Expressing, Silver Staining, Western Blot, Labeling, Negative Control, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Identification of human cranio-maxillofacial skeletal stem cells for mandibular development

doi: 10.1126/sciadv.ado7852

Figure Lengend Snippet: ( A ) Schematic drawing of the combined scRNA-seq and LCM-seq. ( B ) Representative images showing the areas of CMSSCs1 and MCCs1 collected by LCM from GW9 mandible sections. Scale bars, 100 μm. ( C ) Volcano plot exhibiting DEGs distribution between CMSSCs and MCCs, and the top two DEGs in CMSSCs ( ASPN and OGN ) and MCCs ( COL2A1 and COL9A1 ) labeled, respectively. ( D ) GO analysis of DEGs in MCCs showing the top 10 terms ranked by enrichment score. ( E ) GO analysis of DEGs in CMSSCs showing the top 10 terms ranked by enrichment score. ( F ) Violin plots showing the expression level of COL2A1 , COL9A1 , ASPN , and OGN in the clusters of scRNA-seq data. ( G ) Projection of pseudo–single-cell data processed by LCM-seq data of CMSSCs and MCCs into the scRNA-seq data. ( H ) Venn diagram illustrating the numbers of DEGs in CMSSCs and osteogenic cells and the numbers of intersections. ( I ) UMAP distribution of top five membrane markers among intersection genes. ( J ) Hematoxylin and eosin (H&E) staining of GW9, GW12, GW14, and GW20 mandible sections. Immunofluorescence staining for IFITM5 performed on the areas marked with black dashed lines. Scale bars, 100 μm. ( K ) H&E staining of GW9, GW12, GW14, and GW20 maxilla sections. Immunofluorescence staining for IFITM5 performed on the areas marked with black dashed lines. Scale bars, 100 μm.

Article Snippet: For osteogenic differentiation, the expanded cells from single colony were seeded in 24-well plate with osteogenic differentiation induction medium (Cyagen Biosciences, HUXMX-90021), and medium was changed every 3 days until the calcium nodules were observed under the microscope.

Techniques: Labeling, Expressing, Membrane, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Identification of human cranio-maxillofacial skeletal stem cells for mandibular development

doi: 10.1126/sciadv.ado7852

Figure Lengend Snippet: UMAP atlas of integrated analysis from cellular lineages (common progenitors, osteogenic cells, and chondrogenic cells) of human embryonic mandibles and mesenchymal cells of ( A ) human 8-WPC calvaria, ( B ) human 8-WPC long bone, ( C ) human GW9 vertebra, and ( D ) mouse E10.5 to E14.5 mandibles. CMSSC-like cells were circled with a dashed line and feature plots to the right visualize expression of DLX5 , RUNX2 , and IFITM5 . H&E staining of ( E ) human GW9 calvaria, ( F ) human GW9 long bone, ( G ) human GW9 vertebra, and ( H ) mouse E14.5 and E16.5 mandibles. Immunofluorescence staining of RUNX2 or IFITM5 of the area in the dotted box. Scale bars, 100 μm.

Article Snippet: For osteogenic differentiation, the expanded cells from single colony were seeded in 24-well plate with osteogenic differentiation induction medium (Cyagen Biosciences, HUXMX-90021), and medium was changed every 3 days until the calcium nodules were observed under the microscope.

Techniques: Expressing, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Identification of human cranio-maxillofacial skeletal stem cells for mandibular development

doi: 10.1126/sciadv.ado7852

Figure Lengend Snippet: ( A ) Experimental flowchart of IFITM5 + cells isolation and characterization in vitro. ( B ) Flow cytometry gating strategies for sorting IFITM5 + cells ( n = 3 embryos). ( C ) Flow cytometry plots showing serial colony formation from a single IFITM5 + cell ( n = 3 clones). ( D ) Representative crystal violet staining of fibroblast colony-forming unit (CFU-F) colonies from IFITM5 − and IFITM5 + cells. ( E ) Numbers and mean diameters of CFU-F colonies ( n = 3 embryos). ( F , H , and J ) Representative alizarin red, alcian blue, and oil red O stainings after in vitro differentiation of clonally expanded IFITM5 − and IFITM5 + cells. ( G , I , and K ) qPCR analyses of osteogenic, chondrogenic, and adipogenic marker genes and quantification of alizarin red in clonally expanded IFITM5 − and IFITM5 + cells after in vitro differentiation ( n = 3 embryos). ( L ) Workflow of IFITM5 + cell characterization in vivo. ( M ) Bright-field images and H&E staining of subcapsular xenografts of IFITM5 − and IFITM5 + cells ( n = 5). ( N , P , and Q ) Immunofluorescence staining of COLII, COLI, OPN, and Stem101. ( O ) Micro–computed tomography (CT) three-dimensional (3D) reconstruction images (top) and coronal images (bottom) of mandibular defect repair after GelMA, IFITM5 − , and IFITM5 + cell transplantation. ( R ) Quantification of bone formation parameters at defect region ( n = 5). ( S ) H&E staining (bone defect edge demarcated by black dashed lines) and Masson staining in coronal sections of GelMA, IFITM5 − , and IFITM5 + cell groups. ( T ) Immunohistochemical staining for human nucleoli, RUNX2, and OPN in areas marked by white dashed lines in Masson staining images. Scale bars, 100 μm in all figures. [(E), (G), (I), and (K)] ** P < 0.01 and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (R) * P < 0.05, ** P < 0.01, and *** P < 0.001 versus GelMA; ### P < 0.001 versus IFITM5 − cells determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. SSC-A, side scatter-area; OD, optical density.

Article Snippet: For osteogenic differentiation, the expanded cells from single colony were seeded in 24-well plate with osteogenic differentiation induction medium (Cyagen Biosciences, HUXMX-90021), and medium was changed every 3 days until the calcium nodules were observed under the microscope.

Techniques: Isolation, In Vitro, Flow Cytometry, Clone Assay, Staining, Marker, In Vivo, Immunofluorescence, Micro-CT, Transplantation Assay, Immunohistochemical staining, Two Tailed Test

Journal: Science Advances

Article Title: Identification of human cranio-maxillofacial skeletal stem cells for mandibular development

doi: 10.1126/sciadv.ado7852

Figure Lengend Snippet: ( A ) Flow cytometry gating strategies for sorting CADM1 + cells ( n = 2 embryos). ( B ) Representative crystal violet staining of CFU-F colonies from CADM1 + and IFITM5 + cells. Scale bars, 100 μm. ( C ) Numbers and mean diameters of the CFU-F colonies ( n = 2 embryos). ( D ) Representative alizarin red staining after osteogenic differentiation of CADM1 + and IFITM5 + cells. Scale bars, 100 μm. ( E ) Quantification of alizarin red staining in CADM1 + and IFITM5 + cells after in vitro differentiation ( n = 2 embryos). ( F ) qPCR analyses of osteogenic marker genes in vitro differentiation ( n = 2 embryos). ( G ) Micro-CT coronal images (top) and 3D reconstruction images (bottom) of mouse mandibular defect repair after transplantation of CADM1 + and IFITM5 + cells. ( H ) Quantification of bone formation parameters at defect region ( n = 5). ( I ) Representative images of H&E staining (bone defect edge demarcated by black dashed lines) in coronal sections of GelMA, CADM1 + , and IFITM5 + cell groups. Scale bar, 100 μm. [(C), (E), and (F)] * P < 0.05, ** P < 0.01, and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (H) ** P < 0.01 and *** P < 0.001 versus GelMA; # P < 0.05 and ### P < 0.001 versus CADM1 + cells determined by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: For osteogenic differentiation, the expanded cells from single colony were seeded in 24-well plate with osteogenic differentiation induction medium (Cyagen Biosciences, HUXMX-90021), and medium was changed every 3 days until the calcium nodules were observed under the microscope.

Techniques: Flow Cytometry, Staining, In Vitro, Marker, Micro-CT, Transplantation Assay, Two Tailed Test

Journal: Science Advances

Article Title: Identification of human cranio-maxillofacial skeletal stem cells for mandibular development

doi: 10.1126/sciadv.ado7852

Figure Lengend Snippet: ( A ) Schematic diagram of characterization IFITM5 + cells in vivo and in vitro. ( B ) H&E and immunofluorescence staining of IFITM5 and RUNX2 in mandible fragments aged 19, 32, and 49 (white boxes indicating magnified regions). y, years. ( C ) Flow analysis with MSC-specific surface markers and morphology of BMSCs ( n = 3). ( D ) Gating scheme for sorting Zombie − IFITM5 + BMSCs ( n = 3 patients). ( E ) Crystal violet staining of CFU-F colonies from IFITM5 − and IFITM5 + BMSCs. ( F ) Numbers and mean diameters of CFU-F colonies ( n = 3 patients). ( G , J , and L ) Alizarin red, alcian blue, and oil red O stainings after in vitro differentiation. ( H and M ) Quantification of alizarin red and oil red O stainings. ( I , K , and N ) qPCR analyses of osteogenic, chondrogenic, and adipogenic marker genes ( n = 3 patients). ( O ) Bright-field images and H&E staining of subcapsular xenografts of IFITM5 − and IFITM5 + BMSCs ( n = 5). ( P , Q , and R ) Immunofluorescence staining images of COLII, COLI, OPN, and Stem101. ( S ) Micro-CT 3D reconstruction images and coronal images of mandibular defect after GelMA, IFITM5 − , and IFITM5 + BMSC transplantation. ( T ) Quantification of bone formation parameters at defect region ( n = 5). ( U ) H&E staining (bone defect edge demarcated by black dashed lines) and Masson staining in coronal sections of GelMA, IFITM5 − , and IFITM5 + BMSC groups. ( V ) Immunohistochemical staining for Human nucleoli, RUNX2, and OPN in areas marked by white dashed lines in Masson staining images. Scale bars, 100 μm in all figures. [(F), (H), (I), (K), (M), and (N)] ** P < 0.01 and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (T) ** P < 0.01 and *** P < 0.001 versus GelMA; ## P < 0.01 and ### P < 0.001 versus IFITM5 − cells determined by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: For osteogenic differentiation, the expanded cells from single colony were seeded in 24-well plate with osteogenic differentiation induction medium (Cyagen Biosciences, HUXMX-90021), and medium was changed every 3 days until the calcium nodules were observed under the microscope.

Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Marker, Micro-CT, Transplantation Assay, Immunohistochemical staining, Two Tailed Test