Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: ( A ) Isolation of MSCs from vertebral bone marrow aspirate by plastic adherence. MSCs were double labeled with PKH26 and PKH67 and labeling was confirmed by flow cytometry. ( B) IVDs with endplates were isolated from bovine tails. Adjacent IVDs were randomly assigned to: time point zero ctrl (T0), day 5 untreated control (ctrl) and day 5 treated disc by MSC homing (treated). After 5 days, the IVDs were digested overnight and the cells were analyzed by flow cytometry. MSCs were excluded by gating, and disc cells were either processed for gene expression analysis (PCR) or analyzed for expression of Tie2, DAPI, Annexin V, or Ki-67 (FACS). ( C) Human IVD tissue was isolated during surgery or from organ donors. Tissue from one donor was divided into halves. One half was treated by MSC homing (treated), the second half was used as untreated control (ctrl). After 5 days, a tissue piece from both groups was embedded in cryocompound and snap frozen for histological analysis (Ki-67). From the remaining tissue, cells were isolated by overnight digestion and analyzed by flow cytometry. MSCs were excluded and disc cells were analyzed for expression of Tie2, DAPI, Annexin V or Ki-67 (FACS). IVD indicates intervertebral disc; MSCs, mesenchymal stem cells; PCR, polymerase chain reaction.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques: Isolation, Labeling, Flow Cytometry, Expressing, Polymerase Chain Reaction

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: List of Antibodies Used for Flow Cytometry Analysis and IHC

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques: Flow Cytometry, Cytometry

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: List of Bovine Genes Analyzed by Real-time RT-PCR

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques:

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: ( A) bovine IVD (bIVD) treatment by MSCs in spatial separation (0.22 μm porous membrane). ( B-C) No changes in Tie2 expression (0.8- ± 0.5-fold) and fraction of dead cells (1.0- ± 0.3-fold) relative to donor corresponding ctrl disc was found at day 5 (n = 5 bovine tails, 2 MSC donors). IVD indicates intervertebral disc; MSCs, mesenchymal stem cells.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques: Expressing

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: ( A) Fraction of Tie2 + bovine IVD (bIVD) cells at day 0 (T0: 3.8% ± 3.4%) and day 5 (ctrl, treated) (n = 4 bovine tails, hMSC#1). ( B) Individual fold change (relative to donor corresponding ctrl) of data points represented in Fig. 3A. ( C) MSC homing significantly enhanced the Tie2 + progenitor cell population at day 5 (ctrl: 1.6% ± 1.9%, treated: 3.0% ± 3.0%) (n = 13 bovine tails, 5 MSC donors). Red dots refer to data presented in Fig 3A, B. ( D) Individual fold change (relative to donor corresponding ctrl) of data points represented in Fig. 3C. ( E) Fraction of dead bIVD cells at day 0 (T0: 5.3% ± 3.9%) and day 5 (ctrl, treated) (n = 4 bovine tails, hMSC#1). ( F) Individual fold change (relative to donor corresponding ctrl) of data points represented in Fig. 3E. ( G) MSC homing significantly reduced the fraction of dead bIVD cells at day 5 (ctrl: 17.2% ± 7.3%, treated: 13.8% ± 6.0%) (n = 13 bovine tails, 5 MSC donors). ( H) Individual fold change (relative to donor corresponding ctrl) of data points represented in Fig. 3G. IVD indicates intervertebral disc; MSCs, mesenchymal stem cells.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques:

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: ( A) Fraction of apoptotic bovine IVD (bIVD) cells at day 0 (T0: 2.9 ± 0.4%) and day 5 (ctrl, treated) (n = 4 bovine tails, hMSC#1). ( B) A slight not significant difference in the fraction of apoptotic cells was found at day 5 (ctrl: 12.7% ± 3.3%, treated: 10.5% ± 4.2%) (n = 5 bovine tails, 2 MSC donors). ( C) The Bax/Bcl2 gene expression ratio was decreased in five and increased in two of seven MSC-treated IVDs. ( D) The gene expression of Tie2 was positively correlated with the expression of Bcl2 ( P = 0.042, R 2 = 0.96) (n = 7 bovine tails, hMSC#6). IVD indicates intervertebral disc; MSCs, mesenchymal stem cells.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques: Expressing

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: An enhanced Tie2 + disc progenitor cell population and a lower fraction of DAPI + disc cells were found in seven of 10 analyzed human IVDs (hIVD) after MSC treatment, although changes were not significant (n = 10, MSC#7,8,9). ( A) Healthy (green) Tie2: 3.8- ± 2.9-fold; DAPI: 0.8- ± 0.2-fold. ( B) Trauma (red) Tie2: 2.2- ± 1.9-fold; DAPI: 0.7- ± 0.6-fold. ( C) Degenerative (blue) Tie2: 2.0- ± 1.9-fold; DAPI: 1.1- ± 0.6-fold.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques:

Journal: Spine

Article Title: Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response

doi: 10.1097/BRS.0000000000003150

Figure Lengend Snippet: ( A) MSC homing nonsignificantly enhanced the fraction of Ki-67 + disc cells (healthy (green) n = 2: 1.2- ± 0.6-fold; trauma (red) n = 2: 1.7- ± 0.2-fold; degenerative (blue) n = 2: 5.6- ± 3.1-fold) in five of six human IVDs. Table: lists fold changes (relative to ctrl disc) of Ki-67, DAPI, Annexin V and Tie2 expression of individual human IVDs. Owing to limited amounts of human tissue, some analyses could not be performed for all donors (−). IHC staining for Ki-67 confirmed results by flow cytometry (healthy n = 3: 1.2- ± 0.6-fold) (scale bar = 50 μm). A higher fraction of proliferating disc cells (indicated by circles) was found in all human IVDs with an enhanced fraction of the Tie2 + disc progenitor cell population. IHC indicates immunohistochemistry.

Article Snippet: Bovine Tie2 , bs-1300R , Bioss , 450–500/1124 , Alexa F488 , 1 st : 1 in 50 2 nd : 1 in 200.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry

Journal:

Article Title: Angiopoietin-1 and Angiopoietin-2 Activate Trophoblast Tie-2 to Promote Growth and Migration during Placental Development

doi:

Figure Lengend Snippet: Effect of Ang-1 on trophoblast migration. Migration of ED77 was observed with a modified Boyden chamber. Cells (2 × 105) were seeded in the upper compartment in 0.5% FCS. A: Increasing concentrations of Ang-1 were placed in the lower compartment. B: Ang-1 was premixed with recombinant Tie-2-FC for 30 minutes before its addition to the lower compartment. C: The effect of Ang-2 on trophoblast migration was assessed in a fashion similar to that of the analysis of Ang-1 in B. Cells that migrated across the polycarbonate filters in response to the vehicle alone or to Ang-1/Ang-2 were counted after a 6-hour incubation. Chemotaxis in response to FCS (20%) (black column) was used as a positive control. Results are expressed as the mean (± SEM) ED77 counted per 10 fields (×200) in representative experiments performed in duplicate and quadruplicate, respectively. Five similar experiments were performed on different cell passages with similar results.

Article Snippet: A polyclonal rabbit anti-Tie-2 antibody (sc-324) was purchased from Autogen Bioclear (Stevenage, UK).

Techniques: Migration, Modification, Recombinant, Incubation, Chemotaxis Assay, Positive Control

Journal:

Article Title: Angiopoietin-1 and Angiopoietin-2 Activate Trophoblast Tie-2 to Promote Growth and Migration during Placental Development

doi:

Figure Lengend Snippet: Quantification of Tie-2 mRNA gestational human placenta. A: Identification of a 265-bp protected mRNA fragment for Tie-2 by RNase protection assay, using total placental RNAs from first (FT) (n = 4), second (ST) (n = 5), and third (TT) (n = 5) trimester, as well as term (n = 6). The abundance of mRNA for 28S rRNA is also shown for each sample for comparison of RNA amounts. B: Graphical representation of Tie-2 mRNA levels. The ratio of Tie-2 to 28S rRNA was calculated from the protected band intensities as assessed by laser densitometric analysis. Levels of Tie-2 increased with increasing gestation.

Article Snippet: A polyclonal rabbit anti-Tie-2 antibody (sc-324) was purchased from Autogen Bioclear (Stevenage, UK).

Techniques: Rnase Protection Assay

Journal:

Article Title: Angiopoietin-1 and Angiopoietin-2 Activate Trophoblast Tie-2 to Promote Growth and Migration during Placental Development

doi:

Figure Lengend Snippet: Immunohistochemical localization of Tie-2 receptor in the human placenta. Serial sections were incubated with a rabbit polyclonal anti-Tie-2 antibody (1:100). A and B: Intense immunostaining staining for Tie-2 receptor is demonstrated in the cytotrophoblast-syncytiotrophoblast bilayer of placental villi of term placenta. C: Intense immunostaining for Tie-2 is demonstrated in the cytotrophoblast and syncytiotrophoblast of first-trimester placenta. D: The endothelial cells of the placental blood vessels also display strong tie-2 immunostaining. The control is negative, with nonimmune antibody. Original magnification: A and B, ×100; C and D, ×400.

Article Snippet: A polyclonal rabbit anti-Tie-2 antibody (sc-324) was purchased from Autogen Bioclear (Stevenage, UK).

Techniques: Immunohistochemical staining, Incubation, Immunostaining, Staining

Journal:

Article Title: Angiopoietin-1 and Angiopoietin-2 Activate Trophoblast Tie-2 to Promote Growth and Migration during Placental Development

doi:

Figure Lengend Snippet: Identification of Tie-2 in a human first-trimester trophoblast cell line. A: Proteins were extracted from serum-starved HUVECs and trophoblast cells (ED27) and immunoprecipitated with anti-Tie-2 antibody (1 μg/100 μl) overnight at 4°C before Western blotting with anti-angiopoietin 1 (1:2000). Both endothelial (E) and trophoblast (T) extracts show a band of 208 kd corresponding to the Tie-2 receptor plus Angiopoietin 1. B: Cells were prepared and immunoprecipitated as in A before Western blotting with an anti-VEGF antibody (1:1000). No specific bands were observed in either endothelial or trophoblast cell extracts. The data shown are representative of three independent experiments.

Article Snippet: A polyclonal rabbit anti-Tie-2 antibody (sc-324) was purchased from Autogen Bioclear (Stevenage, UK).

Techniques: Immunoprecipitation, Western Blot