FGF4-dependent stem cells derived from rat blastocysts differentiate along the trophoblast lineage

Abstract

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.

Fig. 1. Derivation and characterization of stem cell populations from rat blastocysts

A) A colony of tightly packed cells characteristic of the stem cell state (Stem) and a colony of cells possessing larger nuclei following 8-days of differentiation (Dif). Differentiation was induced by culturing cells in the absence of FGF4, heparin, and REF conditioned medium. Scale bars, 250 μm. B) Germ layer lineage analysis. RT-PCR analysis for lineage markers in the rat blastocyst-derived stem cells in the stem cell state (S) and following 15 days of differentiation. Lineage markers: Cdx2 (trophoblast - stem cell), Prl3b1 (trophoblast - differentiated), T (mesoderm), Pou5f1 (epiblast), Gata6 (endoderm), and Hnf4 (endoderm). RNA isolated from yolk sac and embryos were used as controls for the extraembryonic endoderm and embryonic gene markers, respectively. Gapdh was used as an internal control.

Fig. 2. Stem cell gene marker analysis in rat TS cells

Rat TS cells were cultured under stem cell (S) or differentiation conditions for the indicated days, harvested, and RNA extracted. A) qRT-PCR was used to estimate transcript levels for genes known to be expressed in the stem cell state of TS cells: Cdx2, Eomes, Id1, Id2, Esrrb, Elf5, Bmp4, and Fgfr2. Transcript levels were normalized to 18S RNA. Ratios of tested gene C_T value to 18S C_T value (18S C_T value: 13.63 ± 0.027) in the stem cell state: Cdx2, 1.39; Eomes, 1.54; Id1, 1.41; Id2, 1.78; Esrrb, 1.54; Elf5, 1.81; Bmp4, 2.32; Fgfr2, 1.54. Bars represent means ± the standard error of the mean. Differentiation expression values significantly different from the stem cell level of expression are indicated with an asterisk (*P<0.05). B) Sox2 expression in TS cells. Mouse and rat TS cells were cultured under stem cell or differentiation conditions (d6), harvested, and RNA extracted. RT-PCR was used to estimate Sox2 transcript levels. Gapdh was used as an internal control. E14 mouse ES cells and rat ES-like cells were used as positive controls for Sox2 expression. The positive controls express Oct4, Nanog, and Sox2 but do not express Cdx2.

Fig. 3. Rat TS cell differentiation: morphology and endoreduplication

Rat TS cells were cultured under stem cell conditions (d0; A, D) and differentiation conditions for 2 days (E), 4 days (B, F), 6 days (G), and 8 days (C, H). A–C) The cells were fixed, stained with rhodamine phalloidin, and counterstained with DAPI. Arrowheads show the presence of differentiated cells with trophoblast giant cell morphologies. D–H) DNA contents of the cells were determined by flow cytometry following propidium iodide staining. Scale bar, 100 μm.

Fig. 4. Rat TS cell differentiation: gene expression

A) Rat TS cells were cultured under stem cell or differentiation conditions for the indicated days, harvested, and RNA extracted. qRT-PCR was used to estimate transcript levels for Prl3b1, Prl5a1, Prl2a1, Prl7b1, Tpbpa, Gcm1, Gjb3, Cdh5, Adm, and Hand1. Transcript levels were normalized to 18S RNA. Ratios of tested gene C_T value to 18S C_T value (18S C_T value: 13.63 ± 0.027) at peak expression: Prl3b1, 1.37; Prl5a1, 1.66; Prl2a1, 1.11; Prl7b1, 2.22; Tpbpa, 1.84; Gcm1, 2.47; Gjb3, 1.42; Cdh5, 1.68; Adm, 1.53; Hand1, 1.27. Bars represent means ± the standard error of the mean. Differentiation expression values significantly different from the stem cell level of expression are indicated with an asterisk (*P<0.05).

Fig. 5. Ascl2 gene expression in TS cells

A) Mouse TS cells; B) Rat TS cells; C) Normalized mouse and rat TS cell expression of Ascl2 mRNA. Mouse and rat TS cells were cultured under stem cell or differentiation conditions for the indicated days, harvested, and RNA extracted. qRT-PCR was used to estimate Ascl2 mRNA levels. Transcript levels were normalized to 18S RNA. Ratios of Ascl2 C_T values to 18S C_T values (mouse TS cell 18S C_T value: 13.67 ± 0.058; rat TS cell 18S C_T value: 13.63 ± 0.027) at peak expression in mouse TS cells: 1.67; and rat TS cells: 1.89. Bars represent means ± the standard error of the mean. Differentiation expression values significantly different from the stem cell level of expression are indicated with an asterisk (*P<0.05).

Fig. 6. Steroidogenic capacity of rat TS cells

A) Schematic representation of the steroidogenic pathway. B) Rat TS cells were cultured under stem cell or differentiation conditions for the indicated days, harvested, and RNA extracted. qRT-PCR was used to estimate transcript levels for Cyp11a1, Hsd3b6, Cyp17a1, and Hsd17b2. Transcript levels were normalized to 18S RNA. Ratios of tested gene C_T value to 18S C_T value (18S C_T value: 13.63 ± 0.027) at peak expression: Cyp11a1, 1.29; Hsd3b6, 1.41; Cyp17a1, 1.51; Hsd17b2, 1.41. C) Measurements of progesterone and androstenedione production by stem and differentiating rat TS cells. Steroids were measured in medium conditioned by the rat TS cells using radioimmunoassay. Bars represent means ± the standard error of the mean. Differentiation values significantly different from the stem cell level are indicated with an asterisk (*P<0.05).

Fig. 7. Stability of the rat TS cell phenotype

A) Cdx2, Eomes, Prl3b1, and Tpbpa transcript levels measured by RT-PCR in stem cell and differentiated states from early (passage 9) and late (passage 45) passage rat TS cells. Gapdh was used as an internal control. B) CDX2, EOMES, PRL3B1, and TPBPA protein levels measured by western blotting in stem cell and differentiated states from early (passage 9) and late (passage 45) passage rat TS cells. α-Tubulin was used as an internal control. C) CDX2, EOMES, and TPBPA protein expression assessed by immunocytochemistry in stem cell and differentiated states from early (passage 9) and late (passage 45) passage rat TS cells. Scale bar, 250 μm.