Late stage definitive endodermal differentiation can be defined by Daf1 expression
© The Author(s). 2016
Received: 19 January 2016
Accepted: 23 May 2016
Published: 31 May 2016
Definitive endoderm (DE) gives rise to the respiratory apparatus and digestive tract. Sox17 and Cxcr4 are useful markers of the DE. Previously, we identified a novel DE marker, Decay accelerating factor 1(Daf1/CD55), by identifying DE specific genes from the expression profile of DE derived from mouse embryonic stem cells (ESCs) by microarray analysis, and in situ hybridization of early embryos. Daf1 is expressed in a subpopulation of E-cadherin + Cxcr4+ DE cells. The characteristics of the Daf1-expressing cells during DE differentiation has not been examined.
In this report, we utilized the ESC differentiation system to examine the characteristics of Daf1-expressing DE cells. We found that Daf1 expression could discriminate late DE from early DE. Early DE cells are Daf1-negative (DE-) and late DE cells are Daf1-positive (DE+). We also found that Daf1+ late DE cells show low proliferative and low cell matrix adhesive characteristics. Furthermore, the purified SOX17low early DE cells gave rise to Daf1+ Sox17high late DE cells.
Daf1-expressing late definitive endoderm proliferates slowly and show low adhesive capacity.
The definitive endoderm (DE) gives rise to the gastrointestinal and digestive system. In the mouse embryo, the DE progenitors reside at the posterior region of the epiblast that derived from the inner cell mass . During gastrulation, as the cells ingress through the primitive streak, the epiblast segregates into the three germ layers that form the somatic cell lineages of the ectoderm, mesoderm, and definitive endoderm (DE). DE arises from the Forkhead box A2 (Foxa2)-expressing anterior primitive streak (APS) [2–5] and is then regionalized into the fore-, mid-, and hindgut .
The DE is identified by the expression of SRY (sex-determining region Y)-box 17 (Sox17) [7–9] and chemokine (C-X-C motif) receptor 4 (Cxcr4) [10–12]. Sox17 mutant mouse embryos have a reduced DE, apoptosis of the foregut, and abnormal morphogenesis of the mid- and hindgut . Sox17 is also required for the assembly of the basement membrane, as the Sox17 mutant embryo fails to segregate the DE from the mesoderm . Activin is a frequently used inducer for DE differentiation from pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) [14–16]. When SOX17 is overexpressed, human ESCs spontaneously differentiate into the DE, independent of Activin . In zebrafish embryos, cxcr4a regulates directional migration [11, 18] and DE proliferation during gastrulation . In chick embryos, Cxcr4 is expressed in the DE and angioblasts. Cxcr4 and its ligand Cxcl12 form a reciprocal signaling loop that triggers angioblast migration to the pancreatic endoderm and induces pancreatic development. Inhibition of Cxcr4 suppresses angioblast migration into the pancreatic endoderm region. As a result, the size of the pancreas decreases . Although Cxcr4 is also expressed in the mesodermal cells, it is often used in combination with E-cadherin for purifying ESC-derived DE cells using flow cytometry .
Daf1 is an inhibitor of complementary activation . Daf1 is expressed by immune cells and DE-derived tissue, such as intestine and airway . Using microarray analysis and in situ hybridization, we previously identified Daf1 as a DE cell surface marker based on its expression in ESC-derived and embryonic DE. Daf1 is also expressed in pancreatic progenitor cells [22, 23]. However, the role of Daf1 in the DE is not well understood. In this study, we found that the DE population that expresses Daf1 (Daf1 + DE) has slow cell cycling and low cell-matrix adhesive characteristics. Furthermore, Daf1-negative DE cells (Daf1-DE) turn out to be Foxa2 + Sox17low cells and Daf1-positive DE (Daf1 + DE) cells to be Foxa2 + SOX17high cells. Our results therefore suggest that E-cadherin + Cxcr4 + DE is composed of two populations: Sox17low early DE and Sox17high late DE. Sox17high late DE cells were positive for Daf1, and were slow proliferative and low cell-matrix adhesive cells.
Daf1 + DE are slowly proliferating cells
To identify the differences between Daf1 + DE and Daf1-DE cells, we purified Daf1+/-DE cells and compared their properties. Real time PCR analysis of the sorted Daf1+/-DE cells confirmed Daf1 expression is enriched in Daf1 + DE (Fig. 1b, c). Expressions of an M phase marker, phosphorylated histone H3 (pH3) or Proliferating Cell Nuclear Antigen (PCNA) that marks proliferating cells at every phase of the cell except G0 were enriched in Daf1-DE than in Daf1 + DE cells, revealed by western blot analysis (Fig. 1d). We analyzed the cell cycle phases and found that Daf1 + DE cells showed a longer Go/G1 phase and shorter S, M/G2 phase compared to Daf1-DE cells (Fig. 1e, Additional file 2). This suggested that cell proliferation was decreased in Daf1 + DE.
Daf1 + DE are low adhesive cells
Daf1 is a marker of late stage DE
Daf1+/-DE cells can give rise to pancreatic and intestinal fates
Previously, we identified that Daf1 is expressed in the DE. Here, we identified Daf1 as a late DE marker. Daf1 is an inhibitor of complement activation . Daf1 is expressed in the kidney, spleen, testis, intestine, and bronchi of the adult mouse . Daf1 deficiency is reported in autoimmune hemolytic anemia patients . In Daf1 knockout mice, IFN-γ expression increases, resulting in enhanced T cell response autoimmunity . However, gastrointestinal-tract develops normally in Daf1 knockout mice. Here, we examined the detailed expression patterns during DE differentiation using ESCs.
DE cells are defined as E-cadherin+/Cxcr4+ cells . However, both E-cadherin and Cxcr4 are also expressed in the primitive streak [34, 35]. Therefore, the use of E-cadherin+/Cxcr4+ as a marker to define the DE cells is confined to a limited time window. Moreover, E-cadherin+/Cxcr4+ DE cells are a heterogeneous population. Here we used Daf1 to characterize a subpopulation of E-cadherin+/Cxcr4+ DE cells. We revealed that both Daf1-DE cells represent early DE and Daf1 + DE represent late DE. Daf1-DE and Daf1 + DE cells can give rise to the pancreatic and intestinal lineages. Daf1 + DE formed small colonies, due to their less proliferative and low adhesive characteristics than that of Daf1-DE cells (Fig. 5). A slight decrease in S, M/G2 phase and increase in G0/G1 phase in Daf1 + DE cells might reflect their property as more differentiated cells. Daf1 + DE cells seem to differentiate as efficiently into Pdx1-expressing cells, but not as efficiently into Cdx2-expressing cells, compared to Daf1-DE cells. This might due to a partial loss in differentiation potency of Daf1+ DE cells into the intestinal fate. We previously reported that regional-specific endodermal fates are determined sequentially in the order of stomach, intestine and pancreas, in the chick embryos . It is possible that Daf1 + DE gradually lose potency to differentiate into intestinal lineages, but retains differentiation potency into pancreatic lineages, compared to that of Daf1-DE.
Cell-matrix adhesion is also necessary for cell differentiation [37, 38]. Integrin expression promotes DE differentiation from human pluripotent stem cells . Integrin is a receptor of extracellular matrix expressed in the cells, which enables binding of the cells to the extracellular matrix. Itgα5 and ItgαV are DE-specific Integrins. Knockdown of either Itgα5 or ItgαV inhibits DE differentiation . Both Daf1- and Daf1 + DE cells express Itgα5 and ItgαV. We found that the expressions of Itgα1, Itgα3, and Itgα8 decreased in Daf1 + DE cells. Itgα1 is an attachment molecule of the DE . Itgα3 is expressed in the DE and expression decreases in the Foxa2 null mouse embryo . Itgα8 null mice have abnormal lung morphogenesis . These Daf1-DE specific integrins could regulate DE differentiation and modulate their behavior. The integrins are known to show distinct ligand binding specificities among the superfamily members. Itgα1β1, Itgα3β1 bind specifically to laminin and Itgα8β1 binds specifically to fibronectin . The lowered expression of Itgα1, Itgα3 might explain the decreased adhesion of Daf1 + DE cells to Matrigel, which composed mainly of laminin. The decrease in adhesion to matrix might reflect the developmental transition from early to late DE.
We found that Daf1-DE could turn into Daf1 + DE cells. Sox17 expression was higher in Daf1 + DE than in Daf1-DE cells. Furthermore, expression of a primitive streak marker, Brachyury , was higher in Daf1-DE cells (SO unpublished). Therefore, E-cadherin+/Cxcr4+ DE could be a mixed population of both primitive streak and DE cells.
Our data indicate that DE can be divided into two stages: early and late DEs. Early DE consists of E-cadherin + Cxcr4 + Daf1-Foxa2 + Sox17low cells that show higher proliferative activity and higher cell-matrix adhesive capacity. Later on, these DE cells differentiate into E-cadherin + Cxcr4 + Daf1+ Foxa2 + Sox17high late DE cells that show a decreased proliferation and low cell-matrix adhesion capacity.
Our findings would contribute to the understandings of the differentiation of the primitive streak and DE during gastrulation.
ESC cell lines (R1, SK7 Pdx1/GFP)  or a mouse Nanog iPS cell line (20D-17) , were maintained on a feeder layer of mouse embryonic fibroblasts (MEFs) in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with human recombinant LIF (1:1000, Wako), 10 % fetal bovine serum (FBS, Hyclone), 2 mM l-glutamine (l-Gln, Nacalai Tesque), 100 mM non-essential amino acids (NEAA, Invitrogen), 50 U/mL penicillin, 50 mg/ml streptomycin (PS, Nacalai Tesque), and 100 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich) in 5 % CO2.
Differentiation of ESCs
DE differentiation: ESCs or iPSCs (104 cells/ml) were seeded onto mitomycin C (Sigma)-treated M15 feeders, and cultured in the presence of 10 ng/ml Activin (R&D systems) in DMEM containing 10 % FBS, 2 mM l-Gln, 100 mM NEAA, PS, and 100 μM 2-ME. Pancreatic differentiation: Sorted DE cells were seeded onto MEF feeders and cultured with DE differentiation medium supplemented with 5 ng/ml FGF2 (Peprotech). Intestinal differentiation: Sorted DE cells were seeded onto MEF feeders and cultured with DMEM (2000 mg/ml glucose) and 5 μM bromoindirubin-3′-oxime (BIO) (Wako), 10 μM N-[(3, 5-diflurophenyl) acetyl]- l-alanyl-2-phenylglycine-1, 1-dimethylethyl ester (DAPT) (Peptide), 10 % Knockout Serum Replacement (KSR)(In vitrogen), 2 mM l-Gln, 100 mM NEAA, PS, and 100 μM 2-ME.
For immunocytochemical analysis, goat anti-Sox17 antibody (1:100, R&D systems), rabbit anti-Hnf3b/Foxa2 (1:200, Millipore), mouse anti-Cdx2 (1:500, BioGenex) and rabbit anti-GFP (1:1000, MBL) were used. For flow cytometric analysis, rat anti-E-cadherin (1:500, TaKaRa), biotin anti-Cxcr4 (1:500, BD Biosciences), PE anti-CD55/Daf1 (1:100, BD Biosciences), PE/Cy7 Streptavidin (1:500, Biolegend) antibodies were used. E-cadherin antibody was labeled by Allophycocyanin Labelling Kit-SH2 (DOJINDO). For Western blot analysis, mouse anti-α-tubulin (1:2000, 12G10, Developmental Studies Hybridoma Bank), mouse anti-phospho-Histone H3 (Ser10) antibody (1:500, Millipore), rabbit anti-Sox17 antibody (1:100, Sigma-Aldrich) and mouse anti-PCNA (1:500, Oncogene, NA03-200UG) were used.
Cells were fixed with 4 % paraformaldehyde (PFA) (Nacalai Tesque) for 5 min. After fixation, cells were permeabilized with 0.1 % TritonX (Nacalai Tesque) for 10 min. Then, cells were blocked with Blocking One (Nacalai Tesque) and stained with antibodies.
Flow cytometry analysis
Cells were dissociated with Cell Dissociation Buffer (Invitrogen) and stained with the appropriate antibodies. The stained cells were recovered using FACS Aria II (BD Biosciences). Data were recorded using the BD FACS Diva Software program (BD Biosciences) and analyzed using the FlowJo program (Tree Star).
Western blot analysis
Cells were homogenized in SDS sample buffer (62.5 mM Tris–HCl, 10 % glycerol, 2 % SDS, pH 6.8). After centrifugation, the supernatants were collected and used as total protein extracts. Total proteins were subjected to 8 % SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P Transfer Membrane, Millipore). The membranes were incubated with antibodies listed above. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG (1:20000, CST) was used as the secondary antibody. Chemiluminescence signals were detected with ECL Plus Western Blotting Detection Reagents (GE Healthcare, Japan).
RNA was extracted from the cells using the RNeasy Micro-Kit (QIAGEN) and then 1 μg of RNA was reverse transcribed using ReverTra Ace (TOYOBO), ribonuclease inhibitor, recombinant (TOYOBO), and Oligo dT primers (TOYOBO). Primer sequences are shown in Additional file 3.
Cell cycle analysis
Cells were dissociated with Cell Dissociation Buffer (Gibco). Dissociated cells were washed with PBS and treated with Vybrant DyeCycle Violet Stain (Life Technologies) for 30 min at 37 °C. Cells were analyzed by FACS Canto (BD Biosciences).
Cell- matrix adhesion analysis
The sorted cells were plated onto matrigel-precoated dishes with serum free medium for 90 min. The attached cells were fixed with 4 % PFA for 5 min, then stained with DAPI (1:2000, Roche). Cell counts were performed as previously described .
For the apoptosis assay, caspase-3/7 activity was measured using CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen Life Technologies Co., Carlsbad, CA, USA) according to the manufacturer’s protocol.
2-ME, β-mercaptoethanol; APS, anterior primitive streak; Cxcr4, chemokine (C-X-C motif) receptor 4; Daf1/CD55, decay accelerating factor 1; Daf1-DE, Daf1-negative DE cells; DAPI, 2-(4-amidinophenyl)-1H -indole-6-carboxamidine; DE, definitive endoderm; DMEM, Dulbecco’s modified eagle medium; ESC, embryonic stem cells; FBS, fetal bovine serum; FoxA2, Forkhead box A2; GFP, green fluorescent protein; HRP, horseradish peroxidase; iPSC, induced pluripotent stem cell, Daf1 + DE, Daf1-positive DE cells; KSR, knockout serum replacement; l-Gln, l-glutamine; MEF, mouse embryonic fibroblasts; NEAA, non-essential amino acids; PCNA, proliferating cell nuclear antigen; PFA, paraformaldehyde; PH3, phosphorylated histone H3; PS, penicillin & streptomycin; RT-PCR, reverse transcription-polymerase chain reaction; Sox17, sex-determining region Y-box 17
We thank Dr. T. Seki (Kumamoto University) for maintenance of FACS Aria II. This work was supported by Grants-in-Aid (to S.K, 26253059 and 26670384) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, and in part by Health and Labor Sciences Research Grants “Research on Regulatory Science of Pharmaceuticals and Medical Devices” from the Ministry of Health, Labour and Welfare, Japan. S.O. is a research resident of Japan Agency for Medical Research and Development. This work was also supported in part by the Takeda Science Foundation and by the Program for Leading Graduate Schools “HIGO” in Kumamoto University from MEXT. S.K. is a member of HIGO Program, MEXT, Japan.
SO conceived, designed experiments and acquired, analyzed, interpreted data and drafted manuscript. HO, MM, and NS acquired and analyzed data. SK provided conceptual input, wrote the manuscript, and obtained funding. All authors read and approved the final manuscript.
The authors declared that they have no competing interests.
Ethics approval and consent to participate
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