Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Prrx1 + stem cells demonstrated a critical role in BMP-2-induced osteogenesis . ( A ) Schematic illustration of a mouse distraction osteogenesis model. ( B ) Genes of osteogenic stem cells sorted by time of peak expression during jaw regeneration.( C ) Expression patterns of Bmpr1a and Prrx1 . ( D ) Co-localization of Prrx1 and BMPR1A in hPDLSCs. ( E ) Schematic diagram of cell migration experiment using a three-channel microfluidic chip. ( F ) After hPDLSCs adhered in the central channel, culture medium containing 200 ng/mL BMP-2 was perfused in the upper channel while the standard culture medium in lower channel; cell migration is observed after 8 h. ( G ) Schematic of semi-open silk sponges subcutaneously implanted with open sides facing either the epithelial or muscular region in Mice. ( H ) Sp7/ALP co-staining was performed to assess osteogenic effects in the localized region on day 7. ( I ) Schematic of silk sponges for dorsal subcutaneous transplantation in Prrx1-cre; R26R tdTomato mice. ( J ) ALP immunofluorescence staining was utilized to trace the osteogenic differentiation of BMP-2-recruited Prrx1 + stem cells. n = 3 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Expressing, Migration, Staining, Transplantation Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Channel structure and BMP-2 induced ectopic recruitment of Prrx1 + stem cells for osteogenesis . ( A ) Schematic illustration of the single-channel PDMS film for dorsal subcutaneous transplantation in rats. ( B ) Evaluation of tissue infiltration and collagen formation within the channel structure using HE staining and Masson's trichrome staining on days 4 and 7. ( C ) Prrx1/ALP co-staining detects the osteogenesis of BMP-2 ectopically recruited Prrx1 + stem cells on day 7. ( D and E ) SP7 (D) and OCN (E) immunofluorescence staining for ectopic osteogenesis within channel structure on day 7. n = 4 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Transplantation Assay, Staining, Immunofluorescence

Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Synergistic osteogenic effects of BMP-2 and channel structure in PDMS cube and subgingival tissue . ( A ) Schematic illustration showing the PDMS cube implanted subcutaneously in rats as a BCS model. ( B ) Evaluation of collagen formation within the PDMS cube via Masson's trichrome staining on day 14. ( C ) Assessment of the sequential induction of stem cell infiltration and osteogenesis by BMP-2 and channel structures through Prrx1/ALP co-staining on day 14. ( D to F ) Whole-sample scan of CD31 staining on day 14 showing overall vascularization within the PDMS cubes (D), along with magnified views of the channel region (E) and the central region (F). ( G ) Schematic illustration of the single-channel PDMS film for subgingival transplantation in rats. ( H ) HE staining and Masson's trichrome staining were used to assess tissue infiltration and collagen formation within channel structures on days 4 and 7. ( I ) Prrx1 and OCN immunofluorescence staining for ectopic osteogenesis within channel structure on day 7. n = 4 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Staining, Transplantation Assay, Immunofluorescence

Journal: Stem Cell Discovery

Article Title: Alkaline phosphatase-positive cells isolated from human hearts have mesenchymal stem cell characteristics

doi: 10.4236/scd.2011.13008

Figure Lengend Snippet: Figure 3. Purification of the ALPL+ population from auri- cle-derived cell cultures. Isotype control, TAD cells (and the ALPL+-enriched population after selection on micromagnetic beads and evalution by flow cytometry, a representative ex- periment (a) Markers for pericytes (b) Markers for endothe- lium (c) MSC-positive Markers (d) and MSC-negative mark- ers (e) Imunnofenotyping was evaluated in TAD (total au- ricle-derived cell population), white bars and in ALPL+ cells (tissue nonspecific alkaline phosphatase positive cell popula- tion), black bars. Data are expressed as medians and standard error (n = 3). Von Willebrand factor (vWF).

Article Snippet: For the selection of tissue nonspecific alkaline phosphatase-positive cells (ALPL+), the total auricle-derived cell population (TAD) was incubated with a biotinylated antibody directed against the human tissue nonspecific alkaline phosphatase (R&D Systems, USA).

Techniques: Purification, Derivative Assay, Control, Selection, Flow Cytometry

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of AS-IV on proliferation and osteogenic differentiation of PDLSCs. ( A ) CCK-8 analysis of AS-IV effects on PDLSCs on day 1, 3, and 5. ( B and C ) ALP activity ( B ) and extracellular matrix mineralization ( C ) quantification of PDLSCs affected by AS-IV. ( D ) ALP staining. Scale bar: 500 μm. ( E ) Alizarin Red staining. Scale bar: 500 μm. ( F – H ) qRT-PCR analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV. ( I – L ) Western blot and semi-quantitative analysis of COL-1 (J), ALP(K), and RUNX-2 ( L ) in PDLSCs cultured with AS-IV. 20 μM AS-IV group had a significant promoting effect. Data are presented as mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: CCK-8 Assay, Activity Assay, Staining, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of LY294002 on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and D ) ALP staining( A ) and ALP activity quantification( D ) of PDLSCs affected by LY294002. Scale bar: 500 μm. ( B and E ) Immunofluorescence staining( B ) of COL-1 (green) and DAPI staining (blue) and quantification(E). Scale bar: 50 μm. ( C and F ) Immunofluorescence staining( C ) of eNOS (green) and DAPI staining (blue) and quantification(F). Scale bar: 50 μm. ( G ) NO2- concentration represented the level of NO in cell supernatants. ( H–K ) Western blot and semi-quantitative analysis of COL-1(I), ALP(J), and RUNX-2 ( K ) in PDLSCs cultured with AS-IV and LY294002. ( L–O ) Western blot and semi-quantitative analysis of eNOS(M), iNOS( N ) and p-AKT/AKT ( O ) after addition of AS-IV at different time points. ( P–S ) Western blot and semi-quantitative analysis of eNOS(Q), iNOS( R ) and p-AKT/AKT ( S ) in PDLSCs cultured with AS-IV and LY294002. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Concentration Assay, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of L-NAME on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and B ) ALP staining( A ) and ALP activity quantification( B ) of PDLSCs affected by L-NAME. Scale bar: 500 μm. ( C and D ) Immunofluorescence staining( C ) of COL-1 (green) and DAPI staining (blue) and quantification(D). Scale bar: 50 μm. ( E–H ) Western blot and semi-quantitative analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV and L-NAME. ( I–K ) Western blot and semi-quantitative analysis of eNOS( J ) and p-AKT/AKT ( K ) in PDLSCs cultured with AS-IV and L-NAME. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns: P> 0.05. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Hematoxylin and eosin staining and immunohistochemical staining. ( A ) HE staining of the tension-side periodontal tissues of the control group (A-a) and AS-IV group (A-b) after OTM for 14 days. Scale bar: 50 μm. ( B ) Expression of ALP of the control group (B-a) and AS-IV group (B-b) after OTM for 14 days and quantitative analysis (B-c). Scale bar: 20 μm. ( C ) Expression of COL-1 of the control group (C-a) and AS-IV group (C-b) after OTM for 14 days and quantitative analysis (C-c). Scale bar: 20 μm. ( D ) Expression of eNOS of the control group (D-a) and AS-IV group (D-b) after OTM for 14 days and quantitative analysis (D-c). Scale bar: 20 μm. Data are presented as mean ± SD (n=8 rat/group). * P <0.05, ** P <0.01.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Staining, Immunohistochemical staining, Control, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

doi: 10.1186/s11658-020-00242-9

Figure Lengend Snippet: Effect of W8B2 + CSCs in vitro differentiation on cardiac markers. a Transcript coding for cardiac transcription factors analyzed by TLDA in differentiated vs undifferentiated W8B2 + CSCs. b Determination by immunostaining of protein expression profile of GATA4, MEF2C and NKX 2.5 ( b1 ) and troponin T, a-actinin and b-MHC (b2) after in vitro differentiation of W8B2 + CSCs. Nuclei are represented in blue with TOPRO staining. Scale bar 100 μm. c Transcript coding for cardiac contractile structures analyzed by TLDA in differentiated vs undifferentiated W8B2 + CSCs. d Transcript coding for natriuretic peptides [atrial (ANP) and brain (BNP)] analyzed by TLDA in differentiated vs undifferentiated W8B2 + CSCs. The graphs illustrate the gene expression variations (expressed in log 2) of the differentiated cells versus the undifferentiated cells. Values between 1 and − 1 (horizontal red lines) indicate that gene expression does not vary (this corresponds to variations of + 2 times and − 2 times compared to undifferentiated cells). Each bar represents the mean value ± S.E.M. (standard error of the mean). (N = 4 patients)

Article Snippet: After magnetic cell sorting, confluent cells were labeled with W8B2 PE-conjugated antibody and released using a FACS Aria Flow cytometer and sorter using MSCA-1 (W8B2) antibody coupled to phycoerythrin (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: In Vitro, Immunostaining, Expressing, Staining, Gene Expression

Journal: Cellular & Molecular Biology Letters

Article Title: In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

doi: 10.1186/s11658-020-00242-9

Figure Lengend Snippet: Evolution of spontaneous calcium activity parameters during W8B2 + CSC differentiation. a Types of spontaneous calcium activity recorded at the beginning of the differentiation process [day 2 after differentiation (1)] and at the end of the differentiation process [day 28 after differentiation (2)]. The graphs illustrate variations in [Ca 2+ ] i represented by ΔF/F0 ratio, where F0 represents the basal fluorescence emitted by the GCaMP, and ΔF the difference between basal and actual fluorescence (F) at a given time point. b Bar graphs (1), (2), (3), (4) represent the evolution of the frequency, the peak amplitude, the duration and TTP (time to peak) of calcium oscillations, respectively, recorded during the 4 weeks of W8B2 + CSC differentiation. (N = 6 patients, n: represents the number of cells analyzed, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01, ***p < 0.001 vs. week 1; one-way ANOVA followed by a Newman-Keuls test

Article Snippet: After magnetic cell sorting, confluent cells were labeled with W8B2 PE-conjugated antibody and released using a FACS Aria Flow cytometer and sorter using MSCA-1 (W8B2) antibody coupled to phycoerythrin (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

doi: 10.1186/s11658-020-00242-9

Figure Lengend Snippet: Effect of W8B2 + CSC in vitro differentiation on calcium channels, ion exchangers and contributors to calcium homeostasis. Transcript coding for players in the process of calcium homeostasis analyzed by TLDA in differentiated vs undifferentiated W8B2 + CSCs. Graphs illustrate gene expression variations (in log 2) of differentiated cells versus undifferentiated cells from N = 4 patients. Horizontal red lines correspond to 1 and − 1 values

Article Snippet: After magnetic cell sorting, confluent cells were labeled with W8B2 PE-conjugated antibody and released using a FACS Aria Flow cytometer and sorter using MSCA-1 (W8B2) antibody coupled to phycoerythrin (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: In Vitro, Gene Expression

Journal: Cellular & Molecular Biology Letters

Article Title: In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

doi: 10.1186/s11658-020-00242-9

Figure Lengend Snippet: Role of L-type calcium channel and NCX exchanger activity in W8B2 + CSCs after 28 days of in vitro differentiation. a Effect of 5 µM nifedipine (L-type CaV inhibitor) and c 3 µM SEA-0400 (NCX exchanger inhibitor) on spontaneous calcium activity recorded in differentiated CSCs. Graphs illustrate variations in [Ca 2+ ] i as represented by changes in the ΔF/F0 ratio. ΔF = F − F0 (F: fluorescence emitted by CaMP at a given time; F0: basal fluorescence emitted by GCaMP). Bar graphs (1), (2), (3) represent the evolution of the peak amplitude, the duration and frequency of calcium oscillations, respectively, recorded from differentiated W8B2 + CSCs after treatment with nifedipine (5 µM) ( b ) and SEA (3 µM) ( d ). (N = 3 patients, n: represents the number of events (oscillations) recorded, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; t-test paired followed by a Wilcoxon test

Article Snippet: After magnetic cell sorting, confluent cells were labeled with W8B2 PE-conjugated antibody and released using a FACS Aria Flow cytometer and sorter using MSCA-1 (W8B2) antibody coupled to phycoerythrin (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, In Vitro, Fluorescence, Control

Journal: Cellular & Molecular Biology Letters

Article Title: In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

doi: 10.1186/s11658-020-00242-9

Figure Lengend Snippet: Role of SERCA and IP3 receptors on spontaneous calcium activity recorded in W8B2 + CSCs after 28 days of in vitro differentiation. a Effect of 5 µM xestospongin C (IP3 receptor inhibitor) and c 1 µM thapsigargin (SERCA inhibitor) on spontaneous calcium activity recorded in differentiated CSCs. Graphs illustrate variations in [Ca 2+ ] i represented by changes in the ΔF/F0 ratio. ΔF = F − F0 (F: fluorescence emitted by GCaMP at a given time; F0: basal fluorescence emitted by GCaMP). Bar graphs (1), (2), (3) represent the evolution of the peak amplitude, the duration and the frequency of calcium oscillations, respectively, recorded from differentiated W8B2 + CSCs after treatment with xestospongin C (5 µM) ( b ) and thapsigargin (1 µM) ( d ). (N = 3 patients, n: represents the number of events (oscillations) recorded, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01 vs. control; t-test paired followed by Wilcoxon test

Article Snippet: After magnetic cell sorting, confluent cells were labeled with W8B2 PE-conjugated antibody and released using a FACS Aria Flow cytometer and sorter using MSCA-1 (W8B2) antibody coupled to phycoerythrin (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

doi: 10.1101/2023.02.12.528214

Figure Lengend Snippet: Representative immunohistochemistry staining of hypertrophic chondrocyte markers ALPL and SOX9 in 12-month-old WT and Col2a1- Bmal1 KO mice. Arrows highlight expression of ALPL and SOX9 in the peripheral edge of the heterotopic ossification region in 12-month-old Col2a1- Bmal1 KO lumbar intervertebral discs. ALPL and SOX9-positive cells are prolific throughout this region, particularly towards the peripheral edges. N=3.

Article Snippet: Primary antibodies for ALPL (R&D Systems, AF2910), SOX9 (Cell Signalling) were diluted at 1:1000 and 1:500, respectively, in blocking buffer.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: bioRxiv

Article Title: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

doi: 10.1101/2023.02.12.528214

Figure Lengend Snippet: A) ALPL and SOX9-positive cells were prolific throughout this region, particularly towards the peripheral edges. Cells showed a rounded morphology. B) x40 magnification regions highlighted in A).

Article Snippet: Primary antibodies for ALPL (R&D Systems, AF2910), SOX9 (Cell Signalling) were diluted at 1:1000 and 1:500, respectively, in blocking buffer.

Techniques: