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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00970

Journal of Cell Science 117, 1269-1280 (2004)
Published by The Company of Biologists 2004
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Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin

Martin F. Pera1,*, Jessica Andrade1, Souheir Houssami1, Benjamin Reubinoff1,{ddagger}, Alan Trounson1, Edouard G. Stanley1, Dorien Ward-van Oostwaard{ddagger} and Christine Mummery2,{ddagger}

1 Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia
2 The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands



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  		Fig. 1. Spontaneous or BMP-2-induced extra-embryonic differentiation of human ES cells. (A,B) Phase contrast morphology of control (A) or BMP-2 (B, 25 ng/ml) cells 7 days after treatment. A shows typical stem cell morphology. (C) Spontaneously differentiating ES cell colony. (D) Hematoxylin and eosin stained section of cells similar to those shown in C after removal from culture dish. (E,F) Phase contrast (E) and indirect immunofluorescence (F) image of BMP-2-treated cells stained with antibody to cytokeratins 8, 18 and 19. (G,H) Phase contrast (G) or indirect immunofluorescence (H) micrographs of BMP-2-treated cells stained with antibody to laminin. (I) Double-label staining of BMP-2-treated cells stained with GCTM-2 recognizing a stem cell surface proteoglycan (red) and SPARC (green). (J,K) Phase contrast (J) or indirect immunofluorescence (K) micrographs of a cystic structure and cells at its base stained with antiserum to alphafetoprotein. Wall of cyst (at left) and cells at base (at right) are stained. Bars, A-C, 50 µM; D, 10 µM; E-K, 20 µM.

 


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  		Fig. 2. Flow cytometric analysis of GCTM-2 stem cell surface proteoglycan staining in control cells and cells treated for 5 days with 25 ng/ml BMP-2. Left panels show side scatter versus forward scatter; middle panels show histograms of cell counts versus fluorescence intensity of cells stained with isotype matched control; right panels show histograms of cell counts versus fluorescence intensity of cells stained with antibody GCTM-2 against stem cell surface proteoglycan.

 


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  		Fig. 3. Feeder cell antagonism of BMP action. (A) The effect of feeder cell density on the response of human ES cells to BMP-2. ES cells were plated onto feeder cells prepared at a density of either 6.6x104/cm2 (high density) or 1.3x104/cm2 (low density) and were grown for 5 days with or without treatment with 50 ng/ml BMP-2. The wells were then fixed and stained with monoclonal antibody GCTM-2 followed by detection with anti-mouse immunoglobulin conjugated to alkaline phosphatase; red staining against blue counterstain indicates activity. (B) RT-PCR analysis for transcripts of three BMP antagonists in ES cells, differentiating cultures of ES cells and mouse embryo fibroblasts. Gremlin transcripts are strongly expressed in mouse embryo fibroblasts.

 


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  		Fig. 4. Gene expression in spontaneously differentiating and BMP-2-treated ES cells, spontaneously differentiating and BMP-2- or retinoic acid-treated EC cells, and noggin-treated ES cells. (A) RT-PCR for transcripts for stem cell markers (Oct-4, Cripto and FoxD3) or markers of extra-embryonic endoderm differentiation (alphafetoprotein (AFP), HNF3-{alpha}, HNF-4, GATA-4, GATA-6, transferring (TRF), vitronectin (Vn) and SPARC) and beta actin in control ES cells (C2, HES-2, and C3, HES-3) or cells treated with BMP-2 (B2, HES-2 treated with BMP; B3, HES-3 treated with BMP) at 25 ng/ml for 5 days. Positive control for ES cell markers, human EC cell line GCT 27X-1 and for extra-embryonic endoderm markers yolk sac carcinoma cell line GCT 72. (B) RTPCR for BMP-2 and BMPR1-A, BMPR-2, ß-actin and activin receptor ß in undifferentiated control and spontaneously differentiating ES cell cultures. Controls are on the right, and differentiated cells are on the left, for each PCR pair shown. (C) RNA blotting analysis for BMP-2 and glyceraldehyde-3 phosphate dehydrogenase transcripts in human EC cells at 0, 12, 24, 48, 96 hours and 7 days after plating in the absence of a feeder cell layer (controls) with or without treatment with BMP-2 or retinoic acid. (D) Immunoblot analysis for BMP-2 in spontaneously differentiating ES cells. Tracks from left to right show 10 ng recombinant BMP-2, 25 ng recombinant BMP-2, black bars indicating the position of 19, 24 and 36 kDa marker standards, cell lysate. (E-G) Phase contrast (E) image of differentiating ES cell colony showing staining by indirect immunofluourescence of BMP-2 (F) and GCTM-2 (G). Bar in E-G, 50 µM.

 


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  		Fig. 5. Smad1 phosphorylation induced by BMP addition to hES cells. Undifferentiated hES cells were deprived of fetal calf serum for 4 hours then used as controls (A-D) or treated with BMP2 (50 ng/ml) for 1.5 hours (E-H). After fixing and permeabilization, cells were stained with GCTM2 (green, A and E) and anti-PSmad1 (red, F). Comparison of B and F and the overlays of control and treated cells in C and G indicate that BMP treatment leads to nuclear translocation of Smad1. (I) Nuclear fluorescent intensity quantified as pixel density in ten serial z-sections in a confocal laser scanning microscope.

 


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  		Fig. 6. Effects of noggin on human ES cells. (A) Area of differentiation in ES cell colony; (B) noggin-treated cells. (C) Cells from a colony similar to B after replating onto fresh feeder cell layer. Bar, 50 µm. (D) Proportion of GCTM-2-positive cells in human ES cultures after 5 days of growth under control conditions or in the presence of 200 ng/ml noggin. (E) RT-PCR analysis of gene expression in human ES cultures after 5 days of growth under control conditions or in the presence of 200 ng/ml noggin.

 


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  		Fig. 7. Differentiation of noggin cells into neural precursors. (A) Phase contrast micrograph of control cells after transfer to neural progenitor medium; (B) phase contrast appearance of noggin-treated cells after transfer to neural progenitor medium; (C) phase contrast appearance of noggin-treated cells following transfer to neural progenitor medium and attachment to culture surface; (D) same field as C stained with antibody to nestin. (E) Graph showing proportion of control and noggin-treated cells forming nestin-positive colonies after 5 days of growth on a feeder cell layer in the absence or presence of 200 ng/ml noggin followed by 1 week of growth in neural progenitor medium. Bars, A-D, 50 µM.

 


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  		Fig. 8. Neural derivatives of noggin-treated cells. (A) Outgrowth of cells from a sphere similar to that shown in 7B; (B,C) staining of outgrowth similar to that shown in A with antibody to 200 kDa neurofilament protein (B, phase contrast; C, indirect immunfluorescence); (D,E) staining of outgrowth similar to that shown in A with antibody to Map 2 a,b (D, phase contrast; E, indirect immunofluorescence); (F,G) cells similar to those shown in Fig. 6C following transfer to monolayer culture in the presence of serum without feeder cell support (F, phase contrast; G, indirect immunofluorescence for glial fibrillary acidic protein). Bar shown in B for A-E, 50 µM.

 


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  		Fig. 9. Early differentiation events in human ES cell cultures. ES cells undergo spontaneous differentiation in a BMP-dependent fashion to extra-embryonic tissues; the choice of extra-embryonic endoderm or trophoblast (Xu et al., 2002bGo) depends on environmental factors. Extra-embryonic tissues can produce factors that either drive stem cell renewal or various differentiation pathways. Gremlin produced in feeder cells partially offsets extra-embryonic differentiation locally; addition of noggin to medium completely inhibits this differentiation and allows the formation of neural progenitor through a default mechanism. Lack of extra-embryonic endoderm in noggin-treated cultures results in a loss of signals for stem cell renewal and differentiation into other cell lineages.

 

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© The Company of Biologists Ltd 2004