Figure 1.
Characterization of HTR-8/SVneo and TCL-1 cell lines.
Immunocytochemistry of HTR-8/SVneo and TCL-1 cells with the trophoblast differentiation markers CK7, CSH1, CGB, HLA-G, ITGAV/ITGB3 and PHLDA2 (scale bars, 50 µm). Each protein expression is shown as green signals and nuclei are shown as blue signals. TCL-1 cells showed abundant expression of EVT markers, HLA-G and ITGAV/ITGB3. In contrast, HTR-8/SVneo cells showed abundant expression of CGB and PHLDA2 but low expression of HLA-G and ITGAV/ITGB3.
Figure 2.
Isolation of SP and NSP cells from HTR-8/SVneo cell line.
(A) Flow cytometry results of HTR-8/SVneo cells stained with Hoechst 33342 with or without verapamil (Vera). SP cells were obtained from HTR-8/SVneo cells (0.53±0.59% from 4 independent experiments). Verapamil treatment blocked the dye efflux, increased staining and rendered the SP cells undetectable by flow cytometry. The cell fraction gated with a triangle is SP. (B) Both HTR-8/SVneo-SP cells and -NSP cells were reanalyzed by flow cytometry after culture for 3 weeks in HBM. SP and NSP subpopulations were obtained again from the former HTR-8/SVneo-SP cells. In contrast, NSP cells produced only NSP fraction. Similar results were obtained from three independent experiments. (C) Morphologically, the SP cells were small and round in shape, in contrast to NSP cells. The NSP cells were spindle-shaped (scale bars, 50 µm).
Figure 3.
Characterization of SP and NSP cells from HTR-8/SVneo cells.
(A) Immunocytochemistry with the trophoblast differentiation markers, CK7, CSH1, CGB, HLA–G and ITGAV/ITGB3, and PHLDA2, on HTR-8/SVneo-SP and -NSP cells (scale bars, 50 µm). Each protein expression is shown as green signals. (B) Comparison of gene expression between HTR-8/SVneo-SP and -NSP cells by real-time RT-PCR analysis. Fold changes of mRNA level in SP cells against NSP cells on each transcript are shown. The messenger RNA levels were normalized to that of GAPDH and error bars show standard deviations (n = 3). Asterisks indicate statistically significant (*, p<0.05; **, p<0.01). (C) Western blot analysis revealed that HTR-8/SVneo-SP cells expressed ID2 and BMP4 (both mature and precursor) proteins. In contrast, NSP cells failed to express them.
Figure 4.
SP cells had capacity for long-term proliferation and self-renewal.
(A) SP cells derived from HTR-8/SVneo were cultured in HBM, MTSM and HSM for 3 weeks. SP fraction was analyzed by Epics ALTRA (n = 3). Asterisk indicates statistically significant (p<0.05). (B) HTR-8/SVneo-SP cells cultured in HSM were analyzed by Epics ALTRA every week through 4 weeks (n = 3). (C) Cell numbers of HTR-8/SVneo-SP and -NSP cells cultured in HSM were counted for 6 weeks. After 4 weeks, SP cells began to grow, while NSP cells stopped growing after 3 weeks. Data of cell numbers are represented as the mean ± SEM from three independent experiments. (D) Morphologically, the colonies of SP cells cultured in HSM were similar to those cultured on feeder cells. (E) Images of HTR-8/SVneo SP cell colonies cultured on feeder cells immunostained with the trophoblast differentiation markers, CK7, CSH1, CGB, ITGAV/ITGB3, HLA-G and PHLDA2 counterstained with Hoechst 33852. Each protein expression is shown as green signals (scale bars, 50 µm).
Table 1.
List of culture media.
Figure 5.
IL7R and IL1R2 were abundantly expressed in HTR-8/SVneo-SP cells.
(A) IL7R and IL1R2 mRNA expressions between HTR-8/SVneo-SP and -NSP cells were compared by real-time RT-PCR. The mRNA levels were normalized to those of GAPDH and error bars show standard deviations (n = 3). Asterisk indicates statistically significant (p<0.01). (B) Western blot analysis of IL7R, IL1R2 and CXCR7 proteins in HTR-8/SVneo-SP and -NSP cells. SP cells exclusively expressed IL7R and IL1R2, while NSP exclusively expressed CXCR7. (C) The protein expression of IL7R, IL1R2 or CXCR7 was analyzed by immunocytochemistry. All images counterstained with Hoechst 33852. Each protein expression is shown as green signals (scale bars, 50 µm). (D) IL7R and IL1R2 expressions in HTR-8/SVneo-SP cells were analyzed by immunocytochemistry. All images were counterstained with Hoechst 33852. IL7R and IL1R2 expressions are shown as red and green signals respectively (scale bars, 50 µm).
Table 2.
Representative genes, whose expressions were upregulated in HTR-8/SVneo-SP, compared to –NSP cells.
Table 3.
Representative genes, whose expressions were downregulated in HTR-8/SVneo-SP, compared to –NSP cells.
Figure 6.
SP and IL1R2/IL7R double-positive cells differentiated into multiple trophoblast cell lineages.
(A) Immunocytochemistry images of IL7R, IL1R2, CK7, CSH1, CGB and HLA−G expression in SP cells sorted from HTR-8/SVneo parent cells. Each protein expression is shown as green signals (scale bars, 50 µm). The cells were assayed after culture for 2 weeks in HSM or HBM. (B) Flow cytometry results of IL1R2-positive cells after being sorted by MACS. SP cells were obtained from IL1R2-positive cells after culture for 2 weeks (4.78±1.45% from 3 independent experiments). (C) Induction of trophoblast differentiation of IL7R/IL1R2 double-positive HTR-8/SVneo cells confirmed by differentiation marker, CSH1 and CGB expression. IL7R and IL1R2 expressions are shown as red signals; CSH1 and CGB expressions are shown as green signals (scale bars, 50 µm). The cells marked with an arrow show positive for CSH1 or CGB and negative for IL1R2 or IL7R expression.
Table 4.
Percentages of HTR-8/SVneo-SP cells positive for each marker expression after 2 weeks culture in HSM or HBM.
Figure 7.
Isolation of SP cells from human first trimester vCTB.
(A) Immunocytochemistry images of CGB, CK7, CSH1, HLA−G, CD9 and HLA1 expression in primary vCTB cells isolated by Percoll gradient centrifugation. Each protein expression is shown as red signals (scale bars, 50 µm). The isolated cells were plated on chamber slides with HBM and stained the next day. (B) The SP cells represent 0.12±0.28% (n = 22) of the total primary vCTB. Co-incubation of the cells with verapamil erased the SP cell fraction. (C) The expression of CD105, CD146 and VIM in SP cells from primary vCTB cells was analyzed by immunocytochemistry. All images show counterstaining with Hoechst 33852. Each protein expression is shown as red signals (scale bars, 50 µm). (D) Immunocytochemistry images of IL7R and IL1R2 expression in SP and NSP cells counterstained with Hoechst 33852. IL7R and IL1R2 expressions are shown as red signals (scale bars, 50 µm). (E) The expression of CGB, CSH1 HLA−G, CK7, PHLDA2 and EOMES in IL7R/IL1R2 double-positive cells was assessed by immunocytochemistry. These expressions are shown as green signals (scale bars, 50 µm). (F) Induction of differentiation in IL7R/IL1R2 positive cells was determined by IL7R, IL1R2, CSH1 and CGB expression. IL7R/IL1R2 positive cells from primary human vCTB were analyzed after being cultured in HBM for 2 weeks. IL7R and IL1R2 expressions are shown as red signals; CSH1 and CGB expressions are shown as green signals (scale bars, 50 µm). The cell marked with an arrow show positive for CGB and negative for IL7R expression. In contrast, the cell marked with an arrowhead was positive for IL7R and negative for CGB expression. The outlines of the cells are indicated by broken lines.