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First published online 31 January 2006
doi: 10.1242/jcs.02788

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TGFß inhibition of yolk-sac-like differentiation of human embryonic stem-cell-derived embryoid bodies illustrates differences between early mouse and human development

Ellen Poon^1,*, Frederic Clermont¹, Meri T. Firpo^2, and Rosemary J. Akhurst^1,

¹ Cancer Research Institute, Comprehensive Cancer Center, University of California San Francisco, Box 0875, 2340 Sutter Street, Room S231, San Francisco, CA 94143, USA
² Department of Obstetrics and Gynaecology, University of California San Francisco, San Francisco, CA 94143, USA

View larger version (57K): [in a new window]   Fig. 1. The organisation of yolk-sac-like structures in D13 human EBs. (A,B) Phase-contrast image of day 13 EB where (B) is a higher magnification of the boxed area within (A) to demonstrate the appearance of vessels (arrows). (C) PECAM1 and ENG RNA expression detected by RT-PCR at different stages of EB differentiation. (D-H) Staining of day 13 EBs with (D) anti-PECAM1, (E) anti-AFP antibodies, (F) DAPI to highlight nuclei, (G,H) both anti-PECAM1 and anti-AFP antibodies together with DAPI to distinguish the two cell layers. (H) is a higher magnification of the boxed area within (G).

View larger version (63K): [in a new window]   Fig. 2. Expression of the TGFß pathway during EB formation. (A) Human ES cells were allowed to spontaneously differentiate to form EBs. The expression of components of the TGFß pathway was examined by RT-PCR. GAPDH primers were used as loading control. (B) The expression of TGFBR2 in undifferentiated ES cells was examined in three independent human ES cell cultures in which the expression of POU5F1 (OCT4) was also verified. A negative control experiment (-) was performed using cDNA from MEFs. The ACVRL1 primers showed some cross-reactivity with mouse Acvrl1.

View larger version (78K): [in a new window]   Fig. 3. Effect of TGFß on EB morphology and expression of endothelial markers. Day 18 EBs treated with 1 ng/ml of TGFß for 18 days. Sections of (A) control and (B) TGFß-treated EBs were examined by H&E staining. Prominent cavities can be seen in both samples. The expression of (C) PECAM1 and (D) KDR was assessed by qRT-PCR, normalized to GAPDH. Results were presented relative to expression in untreated samples.

View larger version (36K): [in a new window]   Fig. 4. Expression of lineage markers in response to TGFß treatment. EBs were treated with different concentrations of TGFß: (A-D) from day 3 to day 8, or (E-F) from day 13 to day 18. Expression was measured by qRT-PCR, normalized to GAPDH. Results were presented relative to expression in untreated samples. The expression of PECAM1 in control (I,K) and treated (L,N) samples was examined by immunofluorescence staining. DAPI-stained nuclei are also shown (J,M). K and N are high power magnifications of the boxed areas within I and L, respectively.

View larger version (12K): [in a new window]   Fig. 5. Expression of hematopoietic markers in response to TGFß treatment. EBs were treated with different concentrations of TGFß from day 13 to day 18. Expression was measured by qRT-PCR, normalized to GAPDH. Results were presented relative to expression in untreated samples

View larger version (36K): [in a new window]   Fig. 6. The effect of TGFß on endoderm. Day 13 EBs were treated with various concentrations of TGFß1 for 5 days. (A) The expression of AFP was assessed by qRT-PCR and normalized to GAPDH. Results are presented relative to expression in untreated samples. (B) AFP protein expression in untreated and TGFß-treated (1 ng/ml) EBs was compared by western blotting. An anti-ß-actin antibody was used as a loading control. The expression of AFP (C,D) in control and treated samples was examined by immunofluorescence staining. DAPI-stained nuclei are also shown (E,F). Day 3 EBs were treated with various concentrations of TGFß1 for 5 days. The RNA level of (G) AFP and (H) TTR and (J) THBD was measured relative to GAPDH. Results are presented relative to expression in untreated samples. (I) Endo A protein expression in untreated and TGFß-treated (1 ng/ml) EBs was compared by western blotting.

View larger version (21K): [in a new window]   Fig. 7. Model for differential responses of mouse and human EBs to TGFß. The schematic shows the alternative lineages leading to the hematopoietic and endothelial lineages of the human and mouse yolk sac. The key species difference is highlighted as a bold connecting arrow to the yolk-sac mesoderm (solid for human, dashed for mouse). TGFß inhibition of hypoblast formation would thus affect descendants of the hypoblast in human but not mouse. Cell types downregulated by TGFß in both species are indicated as solid gray oblongs, those down-regulated by TGFß in human but not in mouse are indicated as hatched gray oblongs.

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