The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

Abstract

Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.

Fig 1. Porcine IVF and PA blastocysts quality identification.

(A-B) Morphology of IVF blastocysts (A) and PA Blastocysts (B) at day 6. (C) Dual differential staining of porcine blastocyst, blue nucleus stained by hoechest33342 are designated as ICM cells, pink nucleus stained by both hoechest33342 and PI are designated as TE cells. (D-F) Immunofluorescence staining of pluripotency and trophoblast stem cells markers of porcine blastocysts. IVF blastocysts are OCT4 (D), CDX2 (E) and SOX2 (F) positive. There were no staining differences between IVF and PA blastocysts (S1 Fig). All scale bars represent 100μm.

Fig 2. Derivation and morphology of porcine trophoblast cell.

(A) Outgrowth of IVF blastocyst after 7 days of culture. (B) Outgrowth of PA blastocyst after 7 days of culture. (C-D) Morphology of pTR cell colonies at different magnification. All scale bars represent 200μm.

Fig 3. Characteristic of the pTR cells.

(A) Immunofluorescence staining exhibited pTR cells (pPATR-5) is SSEA1, CDX2, KRT18 and KRT7 positive. There is no difference between IVF and PA pTR cells (S2 Fig). The scale bar represents 100μm (B) Alkaline phosphatase staining of pTR cells is positive. The scale bar represents 200μm. (C) Oil red staining of pTR cells (pPATR-5) showed the abundant lipid droplets. The scale bar represents 200μm. (D-E) Chromosome analyses of pTR cells. pPATR-5 is tetraploid (chromosome number is 76) showed in D, pIVFTR-7 is diploid (chromosome number is 38) shown in E. The scale bar represents 5μm.

Fig 4. Genes expression analysis of pTR cells.

(A) RT-PCR analysis of trophoblast and pluripotency genes in pTR cells. The expression of TSC and TR markers such as CDX2, TEAD4, ELF5, HAND1, CDH3, GATA3, EST2, MASH2 and PAG was detected in pPATR-5 and pIVFTR-7, and ESRRB was not detected in both pTR cells but in blastocysts. The pluripotentcy marker OCT4 is only expressed in blastocysts. β-ACTIN gene is used as control. (B) Real-time PCR analysis of trophoblast genes in pTR cells. The genes including CDX2, ELF5 and KRT18 are expressed significantly higher in pPATR-5 than in pIVFTR-7. The gene, HAND1 and GCM1 are expressed significantly higher in pIVFTR-7 than in pPATR-5. (C) Real-time PCR results of imprinted genes in pTR cells. Three paternally imprinted genes, IGF2, PEG1 and PEG10, were expressed lower in pPATR-5 than in pIVFTR-7. (D-E) Real-time PCR results of telomerase activity-related genes in pTR cells. TERC and TERF2 had significantly higher expression in pPATR-5 and pIVFTR-7 than in PFF. β-ACTIN is used as an endogenous control for trophoblasts genes in B) and imprinted genes in C), and GAPDH is for genes, TERC and TERF2. PFF: porcine fetal fibroblast; STO: feeder cells; BL, blastocysts. Error bars indicate ± SEM. The * indicates P < 0.05 and the ** indicates P < 0.001. ^a,b indicates a statistically significant difference from any another group (P<0.05).

Fig 5. ROCK inhibitor Y-27632 on the morphology and growth of porcine trophoblast cells.

(A) Dome structure formed after cultured with Y-27632. The scale bar represents 200μm. (B) The difference of pTR cell (pPATR-5) colony area with or without Y27832 for 8 days. (C-F) The growth curve of pTR cells cultured with or without Y-27632. C) The growth of PFF is suppressed by Y-27632, cells are decreased in numbers after culture 4 days. D) pPATR-5 cells that cultured in the present of Y-27632 are significantly improved. E) The mean colony area curve of pPATR-5 shows similar trend with the cell number curve. F) The mean colony area curve of pIVFTR-7. Y-27632 promoted the growth of both pIVFTR cells and pPATR cells.

Fig 6. ROCK inhibitor Y-27632 on the gene expression of porcine trophoblast cells.

(A-B) The real-time PCR analyses of gene expression changes of pTR cells cultured with Y-27632. Most trophoblast genes analyzed are up-regulated in Y-27632 treated cells (pIVFTR-7 A) and pPATR-5 B)). ROCK1 and ROCK2 are down-regulated. (C) Western blot detected proteins changes pTR cells cultured with Y-27632. CDX2 was remarkably increased in Y-27632 treated pIVFTR-7 and pPATR-5 cells. pTR cells cultured without Y-27632 in the same condition are used as control. β-ACTIN serves as an endogenous control. The * indicates P < 0.05 and the **indicates P < 0.001.

Fig 7. Culture pTR-E Cells in Different Medium.

(A) Adherent cells grown in DMEM supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2, ELF5, HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the DMEM/F12/FBS medium for 14 days.