Article Figures & Data
Figures
- Figure 1. Induction of trophoblast stem (TS) cell differentiation is associated with trans-differentiation of a population of TS cells into endothelial phenotype.
(A, B, C) Immunofluorescence staining of TS cells maintained for 6 d in stemness condition and day 6–differentiated cells (TC) using antibodies against endothelial markers, CDH5 (A), PECAM1 (B), and endoglin (C). Nuclei were counterstained with Hoechst. Scale bar represents 20 µm. Images were taken at a magnification of 200×. (D, E, F) Flow cytometric analysis of TS and TC dually stained with trophoblast marker cytokeratin (Ck) and the endothelial specific markers, CDH5 (D), PECAM1 (E), and endoglin (F). (G, H, I) Quantification of the percentage of trophoblast cells showing dual positive staining for Ck and CDH5 (G), PECAM1 (H), and endoglin (I). Data are representative of three biological replicates, and error bars represent SEM. **P < 0.01.
Source data are available for this figure.
Source Data for Figure 1[LSA-2022-01583_SdataF1.pdf]
- Figure S1. Induction of mesenchymal phenotype in human trophoblast cells is associated with increased acquisition of endothelial phenotype.
(A, C) Flow cytometric analysis of JEG3 cells (pre- and post-induction of mesenchymal phenotype) dually stained with trophoblast marker HLAG and the endothelial-specific markers CD144 (A) or CD105 (C). (B, D) Quantification of the percentage of JEG3 cells showing dual positive staining for HLAG and CD144 (B) or CD105 (D). Data are representative of three biological replicates, and error bars represent SEM. *P < 0.5.
Source data are available for this figure.
Source Data for Figure S1[LSA-2022-01583_SdataFS1.pdf]
- Figure S2. Endothelial cell function promoting factors enhances expression of endothelial markers by differentiating trophoblast cells.
(A, C) Flow cytometric analysis of trophoblast cells cultured in the absence or presence of 50 ng/ml VEGF165 and 10 ng/ml bFGF and dually stained with cytokeratin (Ck) and CDH5 maintained in stemness conditions for 6 d (A) or in differentiating condition for 6 d (C). (B) Quantification of the percentage of TS cells showing dual positive staining for Ck and CDH5. (D) Quantification of the percentage of TC cells showing dual positive staining for Ck and CDH5. (E) Flow cytometric analysis of day 6–differentiated trophoblast cells cultured in the absence or presence of 50 ng/ml VEGF165 and 10 ng/ml bFGF and dually stained with cytokeratin (Ck) and ENG. (F) Quantification of the percentage of TC cells showing dual positive staining for Ck and ENG. Data are representative of three biological replicates, and error bars represent SEM. ***P < 0.001; ns, nonsignificant.
Source data are available for this figure.
Source Data for Figure S2[LSA-2022-01583_SdataFS2.pdf]
- Figure 2. Functional transcriptome analysis reveals acquisition of endothelial phenotype by trophoblast cells upon differentiation.
(A) Scatter plot of the real-time PCR–based array of mouse endothelial cell–specific genes demonstrating differential expression patterns of 84 different genes from trophoblast stem cells (TS) and differentiated trophoblast cells (TC) on day 6 of differentiation. Normalization was done using the housekeeping gene that showed least change in an online software provided by SABiosciences. Up-regulated genes are marked by red dots, down-regulated by green, whereas those that remain unaltered are marked by black dots within regression lines. (B) Schematic representation of the functional annotations of the 13 differentially regulated genes.
- Figure 3. Real-time PCR analysis of endothelial cell–specific transcripts that are regulated in trophoblast during differentiation.
(A, B, C) Quantitative real-time PCR (qRT) analysis of the differentially expressed endothelial cell–specific genes in trophoblast stem cells (TS) and differentiated trophoblast cells (TC). qRT–PCR results are grouped based on functional annotations. (A) Angiogenesis-related genes: Cx3cl1,c-kit, Kdr, Plau, Mmp. (B) Cell adhesion molecules: Cdh5, Pecam1, Itgβ3, Col18a1. (C) Apoptosis-related genes: Tnfsf10, Bcl2, Cradd, Casp3. Data are representative of three biological replicates, and error bars represent SEM. *P < 0.5, **P < 0.01.
Source data are available for this figure.
Source Data for Figure 3[LSA-2022-01583_SdataF3.pdf]
- Figure 4. Trophoblast differentiation marks differential expression of endothelial cell signature proteins.
(A, C, E) Western blot analysis of proteins isolated from TS and TC showing differential expression of (A) angiogenesis-related markers: C-KIT, KDR, PLAU, MMP9, (C) cell adhesion molecules: CDH5, PECAM1, ITGβ3, COL18A1; (E) apoptotic protein, BCL2, CRADD, and caspase-3. (A, B, C, D, E, F) Densitometric analysis of blots from (A, C, E), respectively, using NIH ImageJ software. Normalization was done with GAPDH using three biological replicates. (G, H) ELISA analysis of secreted cytokines TNSF10 and CX3CL1, respectively. Cytokines were normalized to total cellular proteins. Error bars represent SEM. *P < 0.5, **P < 0.01.
Source data are available for this figure.
Source Data for Figure 4[LSA-2022-01583_SdataF4.pdf]
- Figure 5. Acquisition of endothelial markers upon trophoblast differentiation is associated with down-regulation in Hes1.
(A) Western blot analysis for HES1 and endothelial cell–specific proteins CDH5, PECAM1, and ENG using cell lysates from TS and TC. (A, B) Densitometric analysis of the proteins from blots in (A) using NIH ImageJ software after normalization with GAPDH. (C) Quantitative real-time PCR of Hes1 using RNA from TS cells transfected with either scrambled or Hes1 siRNA. (D, E) Quantitative real-time PCR of Cdh5 (D) and endoglin (E) using RNA from TS cells transfected with either scrambled or Hes1 siRNA. (F) Western blot analysis of HES1, CDH5, PECAM1, and ENG using cell lysates from TS cells transfected with either 100 nM scrambled or Hes1 siRNA followed by induction of differentiation till day 2. (F, G) Densitometric analysis of the proteins from (F) using NIH ImageJ software after normalization with GAPDH. Data are representative of three independent biological replicates. Error bars represent SEM. *P < 0.5, **P < 0.01.
Source data are available for this figure.
Source Data for Figure 5[LSA-2022-01583_SdataF5.pdf]
- Figure S3. Expression of HES1 and endothelial markers with progressive days of differentiation.
(A) Western blot analysis for HES1 and endothelial cell–specific proteins CDH5 and PECAM1 using cell lysates from TS and TC grown for 2, 4, and 6 d post-induction of differentiation. (A, B) Densitometric analysis of the proteins from blots in (A) using NIH ImageJ software after normalization with GAPDH. Data are representative of three biological replicates, and error bars represent SEM. ***P < 0.001.
Source data are available for this figure.
Source Data for Figure S3[LSA-2022-01583_SdataFS3.pdf]
- Figure S4. Endothelial marker, CDH5-expressing differentiated trophoblast cells are HES1 negative.
(A) Flow cytometric analysis of TS and TC cells dually stained with HES1 and the endothelial specific marker CDH5. (B) Quantification of the percentage of cells showing single positive staining for CDH5, HES1 and dual positive staining for HES1 and CDH5. Data are representative of three biological replicates, and error bars represent SEM. ***P < 0.001.
Source data are available for this figure.
Source Data for Figure S4[LSA-2022-01583_SdataFS4.pdf]
- Figure S5. Dose-dependent siRNA-mediated down-regulation of Hes1.
Real-time PCR analysis of Hes1 showing down-regulation in Hes1 transcript levels in TS cells using a combination of two pre-validated Silencer Select siRNAs at three different dosages. Scramble siRNAs were used as controls. Data represent mean of three independent biological replicates. Error bar represents SEM. **P < 0.01. A 100 nM dose of total siRNAs (at 50 nM each) was used for further experiments.
Source data are available for this figure.
Source Data for Figure S5[LSA-2022-01583_SdataFS5.pdf]
- Figure S6. Hes1 down-regulation is mandatory for acquisition of endothelial phenotype by trophoblast cells even in the presence of endothelial cell function–promoting factors.
(A, C) Flow cytometric analysis of TS cells transfected with either scrambled or Hes1 siRNA (100 nM) dually stained with trophoblast marker cytokeratin (Ck) and the endothelial specific marker CDH5 (A) and with Ck and ENG (C) in the presence and absence of 50 ng/ml VEGF165 and 10 ng/ml bFGF. (B, D) Quantification of the percentage of cells showing dual positive staining for Ck and CDH5 (B), Ck and ENG (D). Data are representative of three biological replicates, and error bars represent SEM. ***P < 0.001; ns, nonsignificant.
Source data are available for this figure.
Source Data for Figure S6[LSA-2022-01583_SdataFS6.pdf]
- Figure 6. Hes1 binds to transcriptionally active promoter sites of endothelial marker genes in trophoblast stem cells.
(A, C, E) Schematic representation of the putative HES1-binding sites on promoter/enhancer region about 5 kb upstream to the transcription start site of the endothelial marker genes Cdh5 (A), Pecam1 (C), and Eng (E). (B, D, F) Chromatin immunoprecipitation assay with either HES1 or RNA polymerase II antibody using genomic DNA from TS cells followed by PCR analysis. BS, binding site.
Source data are available for this figure.
Source Data for Figure 6[LSA-2022-01583_SdataF6.pdf]
- Figure 7. Differentiated trophoblast cell induces apoptotic death of endothelial cells through activation of extrinsic apoptotic pathway.
(A) Apoptotic death induced by increasing dose of TRAIL in endothelial cells (MS1) and differentiated trophoblast cells (TC) assessed using annexin V–PI–based flow cytometric analysis. Corresponding untreated cells were kept as controls. (A, B, C) Quantitative analysis of percentage of from (A) in MS1 cells and (C) TC has been shown in bar graphs. Data are representative of three independent biological replicates. Error bar represents SEM. ***P < 0.001; ns, nonsignificant. (D, E) Annexin V–PI–based flow cytometric analysis to assess apoptotic death of endothelial cells (MS1) co-cultured either in the absence or presence of differentiated trophoblast cells for 48 h (D) or 72 h (E). Quantitative analysis of percentage of cells undergoing apoptotic death has been shown in adjacent bar graphs. (F) Western blot analysis of the TNFSF10 (TRAIL) agonistic receptor DR4 using cell lysate from MS1 co-cultured either in the absence (control) or presence of differentiated trophoblast cells and cell lysate from co-cultured trophoblast cells (TC). (F, G) Densitometric analysis of the proteins from (F) using NIH ImageJ software after normalization with GAPDH. Data are representative of three independent biological replicates. (H, J) Western blot analysis of proteins from extrinsic (H) and intrinsic (J) apoptotic pathway proteins using lysates from MS1 cells co-cultured in the absence or presence of TC. (G, I, K) Densitometric analysis of the proteins from (G, I), respectively, using NIH ImageJ software after normalization with GAPDH. Data are representative of three independent biological replicates. Error bars represent SEM. **P < 0.01; ns, nonsignificant.
Source data are available for this figure.
Source Data for Figure 7[LSA-2022-01583_SdataF7.pdf]
Tables
- Table 1.
Differentiation-induced up-regulation of endothelial cell function genes in trophoblast cells.
Sl No. Gene symbol Accession no. TS cell Ct average Diff. cell Ct average Fold change Gene description 1 Tnfsf10 NM_009425 30.76 23.84 261.99 Tumor necrosis factor (ligand) superfamily, member 10 2 Cdh5 NM_009868 24.29 21.37 16.38 Cadherin 5 3 Pecam1 NM_008816 24.87 22.49 7.89 Platelet/endothelial cell adhesion molecule 1 4 Kit NM_021099 24.72 23.98 3.63 Kit oncogene 5 Cx3Cl1 NM_009142 25.02 23.88 2.75 Chemokine (C-X3-C motif) ligand 1 6 Itgβ3 NM_016780 22.25 21.92 2.72 Integrin β 3 - Table 2.
Differentiation-induced down-regulation of endothelial cell function genes in trophoblast cells.
Sl No. Gene symbol Accession no. TS cell Ct average Diff. cell Ct average Fold change Gene description 1 Col18a1 NM_009929 22.75 29.44 −47.58 Collagen, type XVIII, alpha 1 2 Mmp9 NM_013599 16.54 23.2 −46.53 Matrix metallopeptidase 9 3 Kdr NM_010612 19.82 26.17 −37.65 Kinase insert domain protein receptor 4 Bcl2 NM_009741 21.62 26.42 −12.86 B-cell leukemia/lymphoma 2 5 Cradd NM_009950 22.4 26.78 −9.6 CASP2 and RIPK1 domain containing adaptor with death domain 6 Plau NM_008873 25.78 29.98 −8.51 Plasminogen activator, urokinase 7 Casp3 NM_009810 22.08 25.75 −5.86 Caspase-3
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