Journal: Stem cells (Dayton, Ohio)

Article Title: TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

doi: 10.1002/stem.2249

Figure Lengend Snippet: Figure 1. Human adipose stromal cell (hASC) differentiation in various combinations of transforming growth factor-b1 (TGF-b1), growth differentiation factor 5 (GDF5), and dexamethasone (DEX). hASCs derived from patients 1 and 2 were cultivated in the presence of the basic medium enriched with TGF-b1, GDF5, and DEX (one representative patient) for 28 days. (A): Whole pellets were stained with alcian blue. Black arrowheads indicate the peripheral crown of hASC pellets cultivated with TGF-b1, GDF5, and DEX. Scale bar 5 1 mm. (B): Pellets were histologically prepared and sectioned for alcian blue staining. Scale bar 5 100 mm. hASCs derived from patients 2 and 3 were cultivated in the presence of the BM, the BM TGF-b1 DEX, BM GDF5 DEX, and the BM TGF-b11GDF5 DEX for 28 days. (C): The surface stained for alcian blue was quantified in hASCs pellets. (D): The expression of ACAN, COL2A1, CD24, OVOS2, and PAX1 was ana- lyzed by reverse transcriptase quantitative polymerase chain reaction. Black crosses on the x-axis represent a Cq value > 38 cycles. Abbreviations: BM, basic medium; DEX, dexamethasone; GAG, glycosaminoglycan; GDF5, growth differentiation factor 5; TGF-b1, trans- forming growth factor-b1.

Article Snippet: Sections were then incubated overnight at 4 8C with primary antibodies for OVOSTATIN 2 (1:400 dilution, Ab110548, Abcam/Cambridge/United Kingdom/www.abcam.com), PAX1 (1:300 dilution, NBP119767, Novus Biologicals/Littleton/Colorado/www.novusbio. com), P-Smad2 (1:200 dilution, 3101, Cell Signaling Technology/Danvers/Massachusetts/www.cellsignal.com), P-Smad1/5/ 8 (1:200 dilution, 9511, Cell Signaling Technology), and CD24 (1:20 dilution, LS-C87659, LSbio/Seattle/Washington/www. lsbio.com).

Techniques: Derivative Assay, Staining, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

doi: 10.1002/stem.2249

Figure Lengend Snippet: Figure 2. Time course analysis of the chondrogenic and nucleopulpogenic differentiation of human adipose stromal cells (hASCs). hASC pellets from patients 2, 3, and 4 were cultivated in the presence of basic medium, chondrogenic medium, and nucleopulpogenic medium for the times indicated. (A): Pellets were histologically prepared and sectioned for alcian blue staining. Scale bar 5 100 mm. (B): The expression of ACAN, COL2A1, CD24, OVOS2, and PAX1 was analyzed using the Taqman low-density array. Abbreviations: BM, basic medium; CHM, chondrogenic medium; NPM, nucleopulpogenic medium; T0, time of pellet formation.

Article Snippet: Sections were then incubated overnight at 4 8C with primary antibodies for OVOSTATIN 2 (1:400 dilution, Ab110548, Abcam/Cambridge/United Kingdom/www.abcam.com), PAX1 (1:300 dilution, NBP119767, Novus Biologicals/Littleton/Colorado/www.novusbio. com), P-Smad2 (1:200 dilution, 3101, Cell Signaling Technology/Danvers/Massachusetts/www.cellsignal.com), P-Smad1/5/ 8 (1:200 dilution, 9511, Cell Signaling Technology), and CD24 (1:20 dilution, LS-C87659, LSbio/Seattle/Washington/www. lsbio.com).

Techniques: Staining, Expressing, TLDA Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

doi: 10.1002/stem.2249

Figure Lengend Snippet: Figure 3. Reproducibility of the nucleopulpogenic differentiation of human adipose stromal cells (hASCs). hASC pellets from patients 4, 5, 6, and 7 were cultivated in the presence of basic medium (hASC-BM), chondrogenic medium (hASC-CHM), and nucleopulpogenic medium (hASC-NPM) for 28 days. (A): The expression of ACAN, COL2A1, CD24, OVOS2, and PAX1 was analyzed in the three hASC- cultivated conditions and in native human nucleus pulposus cells (represented by a red circle) using the Taqman low-density array. *, p 0.05 versus BM and **, p 0.01 versus BM. Relative expression was represented when Cq value < 38 cycles. (B): The volume of the BM-, CHM-, and NPM-derived hASC pellets was measured. **, p 0.01 versus BM. (C): The cell density was measured in BM-, CHM-, and NPM-derived hASC pellets. *, p 0.05 versus BM. (D): Pellets were histologically prepared, stained with alcian blue, and immuno- stained for aggrecan, type II collagen, CD24, OVOSTATIN 2, and PAX1. Scale bar 5 100 mm. Abbreviations: BM, basic medium; CHM, chon- drogenic medium; hASC, human adipose stromal cell; hNP, human nucleus pulposus; NPM, nucleopulpogenic medium.

Article Snippet: Sections were then incubated overnight at 4 8C with primary antibodies for OVOSTATIN 2 (1:400 dilution, Ab110548, Abcam/Cambridge/United Kingdom/www.abcam.com), PAX1 (1:300 dilution, NBP119767, Novus Biologicals/Littleton/Colorado/www.novusbio. com), P-Smad2 (1:200 dilution, 3101, Cell Signaling Technology/Danvers/Massachusetts/www.cellsignal.com), P-Smad1/5/ 8 (1:200 dilution, 9511, Cell Signaling Technology), and CD24 (1:20 dilution, LS-C87659, LSbio/Seattle/Washington/www. lsbio.com).

Techniques: Expressing, TLDA Assay, Derivative Assay, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

doi: 10.1002/stem.2249

Figure Lengend Snippet: Figure 5. In vivo biological behavior of human adipose stromal cell (hASC)-derived nucleopulpocytes. hASC pellets from patients 2, 3, and 8 were cultivated in the presence of basic medium, chondrogenic medium, and nucleopulpogenic medium for 21 days before their association with the si-HPMC hydrogel and their transplantation in nude mice subcutis for 6 weeks. (A): The expression of ACAN, COL2A1, CD24, OVOS2, and PAX1 was assessed before transplantation by reverse transcriptase quantitative polymerase chain reaction. Black crosses on the x-axis represent a Cq value > 38 cycles. (B): Explanted samples and a human nucleus pulposus (hNP) were histologi- cally prepared and sectioned for alcian blue and Masson’s trichrome staining. (C): Explanted samples and a hNP were histologically pre- pared and sectioned for type II collagen, OVOSTATIN 2, and PAX1 immunostainings. Scale bar 5 10 mm. Abbreviations: BM, basic medium; CHM, chondrogenic medium; NPM, nucleopulpogenic medium; hNP, human nucleus pulposus.

Article Snippet: Sections were then incubated overnight at 4 8C with primary antibodies for OVOSTATIN 2 (1:400 dilution, Ab110548, Abcam/Cambridge/United Kingdom/www.abcam.com), PAX1 (1:300 dilution, NBP119767, Novus Biologicals/Littleton/Colorado/www.novusbio. com), P-Smad2 (1:200 dilution, 3101, Cell Signaling Technology/Danvers/Massachusetts/www.cellsignal.com), P-Smad1/5/ 8 (1:200 dilution, 9511, Cell Signaling Technology), and CD24 (1:20 dilution, LS-C87659, LSbio/Seattle/Washington/www. lsbio.com).

Techniques: In Vivo, Derivative Assay, Transplantation Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

doi: 10.1002/stem.2249

Figure Lengend Snippet: Figure 7. Role of Smad2/3 and Smad1/5/8 pathways during nucleopulpogenesis of human adipose stromal cells (hASCs). hASCs derived from patients 6, 7, and 9 were cultivated in the presence of basic medium (BM), chondrogenic medium, and nucleopulpogenic medium supplemented with dorsomorphin or SB505124 for 28 days. (A): The expression of ACAN, COL2A1, CD24, OVOS2, and PAX1 was analyzed using the Taqman low-density array. *, p 0.05 versus BM. Relative expression was represented when Cq value < 38 cycles. (B): Pellets were histologically prepared and sectioned for alcian blue and Masson’s trichrome staining. Scale bar 5 100 mm. Black arrowheads indi- cate adipocyte-like cells. Scale bar 5 100 mm. Abbreviations: BM, basic medium; CHM, chondrogenic medium; NPM, nucleopulpogenic medium.

Article Snippet: Sections were then incubated overnight at 4 8C with primary antibodies for OVOSTATIN 2 (1:400 dilution, Ab110548, Abcam/Cambridge/United Kingdom/www.abcam.com), PAX1 (1:300 dilution, NBP119767, Novus Biologicals/Littleton/Colorado/www.novusbio. com), P-Smad2 (1:200 dilution, 3101, Cell Signaling Technology/Danvers/Massachusetts/www.cellsignal.com), P-Smad1/5/ 8 (1:200 dilution, 9511, Cell Signaling Technology), and CD24 (1:20 dilution, LS-C87659, LSbio/Seattle/Washington/www. lsbio.com).

Techniques: Derivative Assay, Expressing, TLDA Assay, Staining

Journal: JOR Spine

Article Title: Harmonization and standardization of nucleus pulposus cell extraction and culture methods

doi: 10.1002/jsp2.1238

Figure Lengend Snippet: qRT‐PCR assays used for quantitative PCR

Article Snippet: PAX1 , , , , , Hs00196352‐m1 , , Hs00196352‐m1.

Techniques: Amplification

Journal: JOR Spine

Article Title: Harmonization and standardization of nucleus pulposus cell extraction and culture methods

doi: 10.1002/jsp2.1238

Figure Lengend Snippet: Cells were isolated from human NP tissue using different methods. Human NP tissue was digested with 64 U/ml Collagenase type II for either 4 or 16 h. (A) Number of live cells per gram of tissue. (B–F) Gene expression for ACAN, COL2A, PAX1, KRT18, and COL1A1 in cells immediately following extraction of NP cells from Human NP tissue for 4 or 16 h 64 U/ml collagenase type II. a Wilcoxon matched pairs signed rank test utilized to investigate influence of extraction duration. * P < 0.05.

Article Snippet: PAX1 , , , , , Hs00196352‐m1 , , Hs00196352‐m1.

Techniques: Isolation, Gene Expression, Extraction

Journal: JOR Spine

Article Title: Harmonization and standardization of nucleus pulposus cell extraction and culture methods

doi: 10.1002/jsp2.1238

Figure Lengend Snippet: Re‐differentiation of dog NP cells in reduced FCS media: Histology & IHC: Representative histological pictures of H&E‐stained alginate beads (A) and immunohistochemical results for ACAN, COL1A1, COL2A1, and PAX1 after redifferentiation in six different conditions were (three different expansion media and two different redifferentiation media): (1) E 10% R FCS , (2) E 10% R noFCS , (3) E 5%+FGF R FCS , (4) E 5%+FGF R noFCS , (5) E 5% R FCS , and (6) E 5% R noFCS . n = 3 dogs.

Article Snippet: PAX1 , , , , , Hs00196352‐m1 , , Hs00196352‐m1.

Techniques: Staining, Immunohistochemical staining

Journal: JOR Spine

Article Title: Harmonization and standardization of nucleus pulposus cell extraction and culture methods

doi: 10.1002/jsp2.1238

Figure Lengend Snippet: Re‐differentiation of human NP cells in FCS replacement media. After resuspending human NP cells ( n = 3) into alginate beads 2 weeks in a 3D environment. During the 2‐week re‐differentiation period alginate beads ( n = 24 per patient) were cultured in 1 of 3 different media to test the effect of FCS and alternatives on the re‐differentiation process. The basal composition of each media treatment was identical (low glucose DMEM), with the addition of (1) 10% (w/v) FCS, (2) 1% (w/v) ITS‐X, or (3) 1% (w/v) ITS‐X, 40 μg/ml l ‐proline. Human NP cells extracted from 3 individual patients were used in each separate experiment. Alginate beads were cultured for 2 weeks at 5% O 2 to allow human NP cells to re‐differentiate into an in vivo like phenotype. Beads were harvested after 2 weeks, dissolved and cells were taken for (A) DNA and (B) glycosaminoglycan (GAG) content (normalized to DNA content) analysis, and gene expression analysis of (C) KRT18, (D) KRT19, (E) PAX1, (F) ACAN, (G) COL2A1, (H) MMP3, (I) ADAMTS4, and (J) IL1B. One‐way ANOVA with Tukey's post hoc tests were performed to investigate statistical differences. No statistically significant differences were observed between the three different media conditions.

Article Snippet: PAX1 , , , , , Hs00196352‐m1 , , Hs00196352‐m1.

Techniques: Cell Culture, In Vivo, Gene Expression

Journal: Nature Communications

Article Title: Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids

doi: 10.1038/s41467-024-46181-3

Figure Lengend Snippet: a Identification of previously unidentified genes that might have critical roles in the parathyroids by integrating expression, enhancer and GCM2 -binding data. b GCM2 binding and chromatin states (top) for PAX1 . Y -axis of GCM2 ChIP-seq: normalized read densities. The scale of the value is indicated in parentheses. The arrow indicates the transcription starting site and transcriptional direction. Chromatin states for the PAX1 locus in the parathyroid and other tissues from the Roadmap Epigenomics (middle). Expression levels in the parathyroid and other tissues from the GTEx project (bottom). Orange: parathyroid, Gray: other tissue types from the GTEx project. n = 8, biologically independent samples for parathyroids. The box represents the interquartile range (IQR) divided by the median, and Tukey whiskers extend to a maximum of 1.5 × IQR beyond the box. The colors in the box indicate chromatin states. c Same as in b but for PTHLH that is expressed in many tissue types. Broad and strong enhancers are restricted to the parathyroid. d Same as in b but for DNAH11 .

Article Snippet: Chromatin states for the PAX1 locus in the parathyroid and other tissues from the Roadmap Epigenomics (middle).

Techniques: Expressing, Binding Assay, ChIP-sequencing