QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 13 March 2008
doi: 10.1242/dev.011767

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pearson, S. Articles by Kouskoff, V. Search for Related Content PubMed PubMed Citation Articles by Pearson, S. Articles by Kouskoff, V. Social Bookmarking                         What's this?

The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF

Cancer Research UK, Paterson Institute for Cancer Research, Manchester University, Wilmslow Road, M20 4BX, Manchester, UK.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Transition from ES cells to epiblast-like cells. (A) Flk1 and GFP expression analysis by flow cytometry on mouse day-3 embryoid bodies (EBs) grown in serum-free (left) or serum-supplemented (right) culture. (B) GFP-Bry ES cells were differentiated in serum-free media supplemented with serum or with the indicated combination of factors added at the start of the culture (B, Bmp4; A, activin A; F, bFGF; V, VEGF). At day 5, EB-derived cells were tested for the presence of hematopoietic progenitors in a secondary replating assay. Primitive, primitive erythrocytes; definitive, all definitive colonies (macrophage, macrophage/erythrocyte, mixed colonies and granulo-macrophage colonies). (C) Outline of the experimental design employed in subsequent experiments. (D) Gene expression analysis in ES cells and day 1, 2 or 3 serum-free EB-derived cells by RT-PCR. β-actin expression levels were used for cDNA quantity control. cDNA from day-4 EBs grown in serum was used as positive control for Sox17, brachyury, Gata4; cDNA from day-6 EBs grown in neuronal condition (Li et al., 1998) was used as positive control for Sox1 and Wnt1 PCR. (E) ES cells grown in serum-free media rapidly lose their ability to form EBs. ES cells and day 1, 2 or 3 EB-derived cells were replated in semi-solid conditions allowing the formation and quantification of EBs. *A statistically significant difference (P<0.05) compared with ES cell control. All data shown are representative of at least three experiments. For B and E, data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Bmp4 induces mesodermal specification. (A) GFP-Bry ES cells were differentiated in serum-free media either with serum or with various concentrations of Bmp4 added at the start of the culture (day 0). At the indicated time point in days, the percentage of live cells positive for GFP was determined by flow cytometry. (B) Gene expression analysis performed on ES cells and day 1.5, 2.5 or 3.5 serum-free plus Bmp4 EB-derived cells by RT-PCR. β-actin expression levels were used for cDNA quantity control. (C) Cells derived from day-3.5 EBs were assessed for their relative percentage of GFP-Bry and Flk1 expression. (D) Cells derived from day-3.5 EBs were assessed for their ability to form blast and VSM (vascular smooth muscle) colonies upon secondary replating. Colonies were scored 4 days after replating. *A statistically significant difference (P<0.05) compared with the serum control. Data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m. (E) Tie2 and CD45 expression levels were analyzed on pools of replated colonies derived from day-3.5 EBs stimulated with Bmp4. (F) Morphology of VSM colonies in secondary replating at day 4 of culture (20x magnification). Individual colonies were expanded on glass coverslips for 8 days and stained for the expression of CD31 (red) and smooth muscle actin (green); nuclei are stained in blue with DAPI (20x magnification). (G) Individual VSM colonies were cultured for 4 days in Matrigel plugs (10x magnification). All data shown are representative of at least three experiments.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Activin A and bFGF induce hemangioblast specification. (A) VEGF, activin A or bFGF were added at day 2.5 of serum-free culture supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested at day 3 and assessed for their ability to form blast colonies by secondary replating. (B) activin A and bFGF were added at day 2.5 of serum-free culture supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested 3, 6 or 12 hours after the induction with activin A and bFGF and assessed for their ability to form blast colonies by secondary replating. *A statistically significant difference (P<0.05) compared with Bmp4-induced cultures. (C) Tie2, CD45, CD31 and VE-cadherin expression levels were analyzed on pools of blast colonies. Top panel presents CD45 and Tie2 staining; the level of CD31 and VE-cadherin expression was then assessed on CD45+Tie2- (middle) and CD45-Tie2+ (bottom) cells. (Da-c) Individual blast colonies (examples a, b and c) were tested for their potential to generate endothelium tubule-like structures in Matrigel plugs. Colony in c is stained with CD31 antibody. 10x magnification, bright field. (E) At 3, 6 and 12 hours after activin A and bFGF stimulation, cells were tested for their relative expression of GFP-Bry and Flk1 by flow cytometry. (F) EBs were grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4 and then activin A and bFGF were added. At days 4, 5 and 6 of differentiation, EB-derived cells were tested for the presence of hematopoietic progenitors by colony-forming ability in secondary replating assay. Primitive, primitive erythrocytes; definitive, all definitive colonies (see Fig. 1). Primitive colonies were scored 5 days after replating, definitive colonies after 8 days. **A statistically significant difference (P<0.05) compared with the serum control. All data shown are representative of at least three experiments. For A, B and D, data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m.

View larger version (34K): [in this window] [in a new window]   Fig. 4. VEGF induces the formation of committed blood progenitors. (A) EBs grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4 were stimulated with activin A and bFGF or with activin A, bFGF and VEGF and assayed at days 4, 5 and 6 for the presence of hematopoietic progenitors by colony-forming ability in secondary replating assay. *A statistically significant difference (P<0.05) compared with the serum control at the same time point. Data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m. (B) CD41 and CD34 expression was determined by flow cytometric analysis of cells derived from day-6 EBs stimulated as indicated. (C) CD45 and Mac1 expression was determined by flow cytometric analysis of cells derived from EBs stimulated with Bmp4 followed by activin A, bFGF and VEGF. Data are shown for EBs harvested at days 5, 6 and 7. All data shown are representative of at least three experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Hematopoietic and endothelium precursors are defined by CD41 and CD34 relative expression. (A) Cells expressing CD41 and CD34 were sorted from day-5 EBs grown in serum-free conditions supplemented with Bmp4, followed by activin A, bFGF and VEGF at day 2.5. Each fraction was tested for the presence of hematopoietic progenitors by colony-forming ability in secondary replating assay. (B) May-Grunwald Giemsa staining of CD41+CD34- sorted cells; arrow indicates more-mature cells (40x magnification). (C) Day-5 serum-supplemented EBs derived from wild-type ES cells or ES cells carrying a truncated human (h) CD4 cDNA knocked into the Runx1 locus, were stained for CD41 and hCD4 coexpression. (D) The CD41 and CD34 fractions were tested for their ability to form VSM (vascular smooth muscle) colonies upon secondary replating. Data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m. All data shown are representative of at least three experiments. PS, presort.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Molecular program at the onset of hemangioblast specification. (A) ES cells were induced to differentiate in serum-free culture conditions with Bmp4 for 6 days (lanes B), Bmp4 followed by activin A and bFGF at day 2.5 (lanes BAF) or Bmp4 followed by activin A, bFGF and VEGF at day 2.5 (lanes BAFV). Day 0, 1 and 2: Bmp stimulation only for the three conditions. Equal amounts of RNA from ES cells or from EB-derived cells were reverse-transcribed and analyzed by RT-PCR for the expression of the indicated genes. (B,C) EBs grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4 were further stimulated with either activin A and bFGF (AF) or with activin A, bFGF and VEGF (AFV) and harvested 1, 2, 3, 6 or 24 hours after stimulation for gene expression analysis by real-time PCR. cDNA derived from EBs grown in serum condition for 4.5 days were used as positive control (C+). All real-time PCR data are presented as mRNA expression level relative to that of β-actin. *A statistically significant difference (P<0.05) compared with Bmp4-stimulated control at 0 hour. Data are mean±s.e.m. of triplicate values from three independent experiments. (D) Schematic representation of the differentiation steps leading to the formation of hematopoietic progenitors. All data shown are representative of at least three experiments.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter