Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

Abstract

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

FIG. 1

Inhibition of Noto-GFP expression by high Activin A concentration and BMP4. FACS analysis of Noto-GFP expression after 4 days of culture in the presence of (A) 0.1–100 ng/mL Activin A and 10 μM SB431542, (B) 10-1000 ng/mL Nodal, (C) 10 ng/mL BMP4 and 50 ng/mL Noggin, (D) 50 ng/mL Wnt3a and 320 ng/mL Dkk1, and (E) 10-500 ng/mL Fgf2 and 1 μM SU5402. The data represent the mean ± SD of at least 3 independent experiments. See also Supplementary Fig. S1. FACS, fluorescence-activated cell sorting; SD, standard deviation.

FIG. 2

Noto induction requires simultaneous inhibition of BMP, Wnt, and RA signaling. FACS analysis of Noto-GFP expression after 5 days of culture in the presence of different growth factors. The data represent the mean ± SD of at least 3 experiments. (A) Cells were cultured for 3 days in the presence of 1 ng/mL Activin A. During days 4 and 5 the medium was additionally to Activin A supplemented with growth factors and/or their inhibitors: 50 ng/mL Wnt3a, 320 ng/mL Dkk1, 10 ng/mL BMP4, 50 ng/mL Noggin, 0.1 μM RA, and 10 μM AGN193109. (B) Cells were cultured for 3 days in the presence of 1 ng/mL Activin A. During day 4 and 5 the medium was in addition to 1 ng/mL Activin A or 1 ng/mL Activin A plus 100 ng/mL Fgf2, supplemented with 10 μM AGN193109, 320 ng/mL Dkk1, and 50 ng/mL Noggin and the different combinations of them. (C) Schematic view of the final differentiation protocol. (D) FACS analysis of Noto-GFP expression after 5 days of differentiation with or without 1 ng/mL Activin A. After 1 day of differentiation cells were transfected with a Noto-IRES-dTomato plasmid. Ninety-six hours after transfection FACS analysis was performed. Therefore, GFP expression within the dTomato⁺ cell population was measured. See also Supplementary Fig. S2. RA, retinoic acid.

FIG. 3

Characterization of Noto-GFP ⁺ cells. (A) Quantitative reverse transcriptase-polymerase chain reaction analysis of marker expression during differentiation of Noto^Gfp/+ ES cells. The ratio of gene expression against undifferentiated Noto^Gfp/+ ES cells is shown. The data represents the mean ± standard error of the mean of at least 3 independent experiments. (B) Immunocytochemistry of T, Foxa2, Shh (red), and GFP (green) in Noto^Gfp/+ cells was performed at day 5 of the differentiation process. Cell nuclei were stained with DAPI (white). Scale bar = 50 μm. (C) Transverse section of a chicken embryo with the Noto-GFP ⁺ graft lateral to the neural tube. The bracket indicates the expanded Foxa2⁺ region toward the graft. nt, neural tube; nc, notochord; Scale bar = 50 μm. (D) Transmission electron micrograph of Noto-GFP ⁺ cell clusters. Note the dilated mitochondria (M) adjacent to the nucleus (N) and undilated rough endoplasmic reticulum (ER). Scale bars = 20 μm (upper) and 1 μm (lower). See also Supplementary Fig. S3. ES, embryonic stem.

FIG. 4

Proliferation of Noto-GFP⁺ cells. (A) FACS analysis of differentiating Noto-GFP ES cells at day 4 to 6. At the same time points the absolute cell number for each well was counted. (B) Based on % Noto-GFP⁺ and total cell number per well the absolute cell number of Noto-GFP⁺ cells per well was determined. (C) The proliferation of Noto-GFP⁺ cells in the presence or absence of Fgf2 was analyzed by EdU incorporation. Data represents the % EdU⁺GFP⁺ cells within the Noto-GFP⁺ cell population of 3 independent experiments (mean ± standard error of the mean). (D) Immunocytochemistry of EdU (red) and GFP (green). Cell nuclei were stained with DAPI (white). Arrow heads indicate EdU⁺GFP⁺ double-positive cells. Scale bar = 50 μm.

FIG. 5

Maintenance of the Noto-GFP⁺ cell population. Noto^Gfp/+ cells were differentiated using the 2-step differentiation protocol followed by a third step in the presence of 100 ng/mL Fgf2, 100 ng/mL Shh, or 25 ng/mL Wnt or combinations of them. Additionally, culture was continued in step 2 medium. (A) Schematic view of culture procedure. (B) FACS analysis of Noto-GFP expression was performed at day 5 to 8. (C) In the same setup the absolute cell number per well was counted. (D) Based on percentage GFP⁺ and absolute cell number per well, the absolute number of Noto-GFP⁺ cells per well was determined. The data represent the mean ± SD of 3 independent experiments.

FIG. 6

Contribution to the midline of Noto-GFP⁺ cells grafted into chicken embryos. Noto-GFP⁺ cells were labeled with a CellTracker™ Orange and grafted into the medial sector of the Hensen’s node in HH st 4 chicken embryos (A-C). The chicken embryos were further cultured for 20–24 h and stained for GFP (A′–C′) and Foxa2 (A″–C″). (A‴–C‴) represent overlays. (A) Dorsal view of the midline where GFP⁺ cells (bracket) have been laid down within the Foxa2⁺ midline. (B) Transverse section of chicken embryos grafted at HH st 4 showing contribution of GFP⁺ cells to the notochord or (C) showing contribution of grafted cells to the endoderm (arrow heads). All images are optical sections. a, anterior; p, posterior; aip, anterior intestinal portal; nt, neural tube; nc, notochord; *, background; scale bar = 50 μm.