Molecular Analysis of LEFTY-Expressing Cells in Early Human Embryoid Bodies

Authors


Abstract

Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

Introduction

Most knowledge regarding early human embryonic development comes from the mouse model and from a limited number of sectioned human embryos. The derivation of human embryonic stem cells (HESCs) [1, 2] is now providing a new in vitro approach to study events occurring during early stages of differentiation of the human embryo [3]. HESCs are derived from the inner cell mass (ICM) of preimplantation blastocyst-stage embryos [1, 2]. These are true pluripotent cells that may differentiate in vitro and in vivo into many different cell types. In vitro, when HESCs are grown in suspension, they spontaneously differentiate while forming aggregates named human embryoid bodies (HEBs) [4]. Initially, these HEBs are densely packed cell aggregates surrounded by endoderm-like single cell layer. After a few days, they cavitate and eventually become cystic and accumulate fluid [4]. It has been shown that the mature HEB consists of various types of cells representing the three embryonic germ layers. It is also possible to direct HESC differentiation in vitro by administration of various growth factors to the media [5]. There are multiple pieces of evidence that HESCs may differentiate to various cell types that originate from the different embryonic lineages, namely ectoderm (e.g., neurons and epidermal cells), endoderm (e.g., hepatocytes and pancreatic cells), and mesoderm (e.g., muscle cells, cardiomyocytes, chondrocytes, blood cells, and endothelial cells) [6]. In vivo, engraftment of HESCs into immunodeficient mice yields differentiated tumors called teratomas. These teratomas consist of cells representing the three embryonic germ layers, demonstrating the pluripotency of these cells. Recently, we demonstrated, by performing a large-scale transcriptional analysis of HESCs and their differentiated derivatives (HEBs), that activation of gene expression during HESC differentiation occurs in a stepwise manner. Accordingly, five clusters of genes were recognized, and representatives of these clusters were suggested to correlate to different developmental stages [7]. One such developmentally regulated pathway is the NODAL/LEFTY pathway, which is known to play a major role in mouse gastrulation. NODAL, a member of the set of transforming growth factor-β (TGF-β)-like molecules, is secreted and transduces signals through its receptors ALK4, ALK7, ActRIIa, and ActRIIb to activate SMAD2, SMAD3, SMAD4, and FAST transcription factors. These transcription factors maintain NODAL's own transcription, as well as initiating expression of LEFTY A/B. LEFTY A/B, also one of the transforming growth factor (TGF)-β-like molecules, is secreted and inhibits the signaling of NODAL, possibly by competitive binding to common receptor components [8].

We hypothesized that the formation of differentiated cells from HESCs representing the three embryonic germ layers should be initiated by the formation of progenitor-like cell populations in the early HEB. Here, we demonstrate activation of the NODAL pathway during differentiation of HESCs. We suggest that this pathway is active in early stages of differentiation of HEBs, directing specific cells into the mesoderm lineage.

Materials and Methods

Cell Culture

Human ESCs (H9 and H13) were cultured [4, 5] on a mitomycin C-treated mouse embryonic fibroblast (MEF) feeder layer (obtained from 13.5-day embryos) in 85% Knockout Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, http://www.invitrogen.com), supplemented with 15% Knockout serum replacement (a serum-free formulation) (Gibco), 1 mM glutamine, 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1% nonessential amino acids stock (Gibco), penicillin (50 units/ml), streptomycin (50 μg/ml), ITS ×100 (insulin-transferrin-selenium; Gibco) at a 1:200 dilution, and 4 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). To obtain a feeder-free culture, the cells were plated on 0.1% gelatin (Merck & Co., Whitehouse Station, NY, http://www.merck.com)-coated plates and grown in media conditioned for at least 24 hours by MEFs. In vitro differentiation into embryoid bodies was performed by withdrawal of bFGF from the media and allowing aggregation in Petri dishes. HEBs were collected for RNA analysis after 2, 10, and 30 days of aggregation. For the inhibitor assays, 1 or 10 μM SB-431542 (Tocris Bioscience, Bristol, U.K., http://www.tocris.com) diluted in dimethyl sulfoxide was added to the culture.

Immunostaining

HEBs were immersed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (2 hours, 4°C), embedded in either in paraffin or in O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA, http://www.sakura-americas.com) and sectioned at 6 μm. HESCs were washed once with PBS and fixed with 4% paraformaldehyde. Sections were deparaffinized in xylene and microwave-treated (15 minutes) in 0.01 M citric buffer. Blocking and permeabilization was performed with 2% bovine serum albumin (BSA), 10% low-fat milk, and 0.1% Triton-X in PBS. The sections were incubated for 1 hour with two antibodies: mouse anti-human OCT4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) and goat anti-LEFTY A and B (Santa Cruz Biotechnology Inc.). As secondary antibodies, fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H+L, dilution 1:200; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) and Cy3-conjugated mouse anti-goat IgG (H+L, dilution 1:200; Jackson Immunoresearch).

In Situ Hybridization and Hematoxylin and Eosin Stainings

In situ hybridization of HEB paraffin sections was performed using the method described by Ma et al. [9]. Antisense and sense probes for LEFTY A were synthesized using the DIG RNA labeling kit (1175025; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Each probe was generated by subcloning polymerase chain reaction (PCR) fragments synthesized using the LEFTY A primers (Table 1) in the pGEM T-Easy vector system (A1360; Promega, Madison, WI, http://www.promega.com). Hematoxylin and eosin stainings were performed using hematoxylin solution Gill no. 3 GHS316 (Sigma-Aldrich) and eosin Y solution, alcoholic, with phloxine HT110316 (Sigma-Aldrich).

Table Table 1.. Primers used
original image

Transfection and 5-Bromo-4-chloro-3-indolyl-β-d-galactoside Staining

Wild-type HESCs were transfected using a calcium-phosphate method as described [10]. The cells were transfected either transiently with the vector hL2ASE-LacZ [11] (kindly provided by H. Hamada, Osaka, Japan) harboring the human LEFTY A (LEFTY 2) asymmetric enhancer (ASE) followed by hsp68 minimal promoter and the LacZ reporter gene or stably with the vector hL2ASE-enhanced green fluorescence protein (EGFP) harboring the human LEFTY A (LEFTY 2) ASE followed by hsp68 minimal promoter regulating an EGFP reporter gene followed by a neomycin-resistant gene. Transfected HESCs were either analyzed for LacZ expression 48 hours after transfection or allowed to aggregate 24 hours following transfection and analyzed for LacZ expression after 2 days of aggregation using a previously described method [12].

Fluorescence-Activated Cell Sorting Analysis

Fluorescence-activated cell sorting (FACS) analysis of LEFTY-EGFP-expressing cells was performed on a FACSCalibur system (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), according to their green fluorescent emission. HEBs were trypsinized and resuspended in PBS. Transfected cells were analyzed for fluorescence intensity and compared with control cells using CellQuest software (Becton Dickinson). FACS analysis for TRA-1-60 expression was performed after trypsinization of the HEBs. The cells were washed with 3% BSA in PBS, incubated with TRA-1-60 antibody (kind gift from Prof. Peter Andrews, Sheffield, U.K.) for 1 hour, incubated with Cy3-conjugated rabbit anti-mouse IgM (Jackson Immunoresearch), and, after washes, analyzed using the FACSCalibur system (Becton Dickinson). Analysis was performed on CellQuest software (Becton Dickinson). Forward and side scatter plots were used to sort TRA-1-60 and LEFTY-EGFP-expressing populations, as well as to exclude dead cells and debris from the histogram analysis.

RNA Extraction and Reverse Transcription-PCR Analysis

RNA was extracted using RNeasy mini/micro kit for total RNA isolation according to the manufacturer's instructions (Qiagen, Hilden, Germany, http://www1.qiagen.com). cDNA was synthesized using random hexamer primers. Amplification was performed on the cDNA using Takara Ex-Taq (Otsu, Japan, http://www.takara.co.jp). PCR conditions included a first step of 3 minutes at 94°C, a second step of 25 to 35 cycles of 30 seconds at 94°C, a 45-second annealing step at 60°C–64°C, 1 minute at 72°C, and a final step of 7 minutes at 72°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to evaluate and compare the quality and quantity of different cDNA samples. Descriptions of the primers and annealing temperatures are given in Table 1. The final products were examined by gel electrophoresis on 2% agarose ethidium bromide-stained gels.

DNA Microarray Analysis

Total RNA was extracted according to the manufacturer's protocol (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) from populations of undifferentiated and differentiated cells derived from HESCs. When extracting RNA from undifferentiated ESCs, the cells were grown for one passage on gelatin-coated plates in conditioned media to avoid contamination of feeder cells. Hybridization to the U133A and B DNA or U95Av2 microarrays, washing, and scanning were performed according to the manufacturer's protocol (Affymetrix), and expression patterns were compared between samples. Signals were normalized by dividing each probe in the average value of the DNA microarray and normalizing to a value of 100, to avoid differences between different DNA microarrays and experiments. U133A and B DNA microarrays were used to compare the global expression of H9 HESCs and 2-, 10-, and 30-day-old HEBs. U95Av2 DNA microarray was used to compare global expression of 2-, 10-, and 30-day-old HEBs to examine the effect of SB-431542 on lineage differentiation, as well as to compare expression of the two cell populations isolated by FACS. Bioinformatics analysis of the results was performed using the Gene Ontology (GO) tool (http://www.geneontology.org), Online Mendelian Inheritance in Man (National Center for Biotechnology Information) database, and the Gene Expression Atlas (Novartis International, Basel, Switzerland, http://www.novartis.com).

Results

LEFTY Proteins Are Transiently Expressed During HESC Differentiation

HESCs are isolated from the ICM of blastocyst-stage embryos and may differentiate spontaneously in culture into HEBs. These HEBs consist of multiple differentiated cell types, such as neuronal and cardiac cells [4]. We thus hypothesized that during the differentiation process, intermediate cell types are also formed. These intermediate cell types may be mimicking progenitor cells that appear during normal development. We assumed that genes that are responsible for the events occurring during early development should be transiently expressed upon differentiation of HESCs into HEBs. Indeed, we could demonstrate that during HESC differentiation, several temporal gene expression patterns can be identified. Among them are a cluster of genes that are relatively scarce in the undifferentiated HESCs and in the fully differentiated 30-day-old HEBs but are abundant in the early differentiated stages (2-day-old HEBs) [7]. LEFTY genes, which are involved during mouse gastrulation in mesoderm formation and left-right axis definition, were shown to be a part of this cluster; these genes are transiently expressed during HESC differentiation. To analyze the temporal expression of LEFTY proteins, we followed in vitro differentiation of HESCs from the undifferentiated stage through the early differentiation stage (2- and 5-day-old HEBs), mid-differentiation stage (10-day-old HEBs), mid-late differentiation stage (20-day-old HEBs), and fully differentiated (30-day-old HEBs). Immunohistochemistry analysis showed that LEFTY proteins (stained red) are not expressed by the undifferentiated H9 and H13 HESCs (Fig. 1B, 1D), which do express the undifferentiated cell marker OCT4 (stained red) (Fig. 1A, 1C). Immunohistochemistry analysis of sections of 2-day-old H9 and H13 HEBs with antibodies against human OCT4 (stained green) (Fig. 1E, 1G, 1H, 1J) and LEFTY (stained red) (Fig. 1F, 1G, 1I, 1J) was performed. The segregation of LEFTY (stained red) and OCT4 (stained green) to different cells suggests that in the early HEBs there are cells that have already differentiated and gained markers that determine their fate (i.e., LEFTY-positive cells) and lost markers of the undifferentiated cells (i.e., OCT4).

Figure Figure 1..

LEFTY protein was transiently expressed during human ESC (HESC) differentiation in a discrete cell population separate from the OCT4-expressing cell population. Shown are immunostainings of H9 HESCs (A, B) and H13 HESCs (C, D) stained with anti-LEFTY (B, D) and anti-OCT4 (A, C) antibodies. The presence of LEFTY proteins was not detected in the undifferentiated cells present in HESC culture (B, D), although OCT4 was expressed by the undifferentiated cells (A, C). (E–J): Immunostaining analysis of LEFTY expression in 2-day-old H9 (E–G) and H13 (H–J) human EBs (HEBs). (F, I): LEFTY protein expression (red). (E, H): OCT4 protein expression (green). (G, J): Overlay of LEFTY and OCT4. OCT4 and LEFTY segregated to different cells in the early HEBs. (K–O): Analysis of LEFTY protein expression in HESCs (K), 2-day-old HEB (L), 5-day-old HEB (M), 10-day-old HEB (N), 20-day-old HEB (O), and 30-day-old HEB (P) with an anti-LEFTY (A, B) antibody (red). In the early HEBs (2 and 10 days old), a significant LEFTY protein expression was detected. LEFTY protein expression progressively vanished upon further differentiation in 10- and 20-day-old HEBs and completely disappeared in the mature 30-day-old HEB.

Upon early differentiation, a significant cell population began to express LEFTY proteins, as is made evident by analysis of 2- and 5-day-old HEB paraffin sections (Fig. 1L, 1M). This expression diminished upon further differentiation, as shown in 10- and 20-day-old HEBs sections (Fig. 1N, 1O), and vanished in the matured HEBs (Fig. 1P). Hence, we show that when the undifferentiated cells were allowed to aggregate and form HEBs, a subset of cells began to express LEFTY proteins. This expression diminished throughout differentiation and completely disappeared in the fully differentiated HEBs. We sought to determine the molecular identity of the cells composing the early HEBs.

Molecular Labeling of LEFTY-Positive Cells

To investigate whether the regulation of LEFTY protein expression is at the transcriptional level and to identify cells expressing LEFTY, we transfected HESCs with a vector containing the LacZ reporter gene regulated by the human LEFTY A (LEFTY 2) asymmetric enhancer (hL2ASE) [11]. Transfection of HESCs with the vector resulted in cultures in which only a few differentiated cells expressed the LacZ reporter gene, whereas none of the undifferentiated colonies expressed the LacZ reporter gene (Fig. 2A). Thus, LEFTY expression was activated only in differentiated cells that are naturally present in the HESC culture. However, only 2 days after the HEBs were formed, it was clear that a group of cells began to express LacZ reporter gene (Fig. 2B), demonstrating that the LEFTY A gene is activated in these cells.

Figure Figure 2..

Molecular labeling of LEFTY-positive cells. Human ESCs (HESCs) were transfected with a vector containing the LacZ or enhanced green fluorescence protein (EGFP) reporter gene regulated by human LEFTY asymmetric enhancer (hL2ASE). (A): Shown is a colony of HESCs after transfection with the LacZ vector. Note that LEFTY promoter was active only in the differentiated cell. (B): Shown are 2-day-old human EBs (HEBs) expressing the LacZ reporter gene. (C): Shown are 2-day-old HEBs expressing the vector hL2ASE-EGFP. (D): Fluorescence-activated cell sorting analysis of 2-day-old HEBs either with (green area) or without (red line) the hL2ASE-EGFP vector. Abbreviation: FL, fluorescence.

To expand our observations, we followed up the previous experiment with a stable transfection of HESCs with a vector containing the EGFP reporter gene regulated by the human LEFTY A (LEFTY 2) asymmetric enhancer (hL2ASE) [11]. EGFP was rarely observed in differentiated cells in transfected HESC culture (data not shown); however, in 2-day-old HEBs, a significant cell population expressed the EGFP (Fig. 2C, 2D). FACS analysis indicated that the EGFP-expressing cells contributed approximately 10%–20% of the HEB cell population (Fig. 2D). This percentage of LEFTY-positive cells is supported by our observation when correlating LEFTY in situ hybridization signals with the distribution of cell morphologies in the early HEBs (supplemental online Fig. 1).

LEFTY-EGFP cells were analyzed by FACS. LEFTY-expressing cells (Fig. 3A, green dots) seem to have specific characteristics of size and granularity that set them apart from the cells that are positively stained for TRA-1-60 (Fig. 3B, red dots) (undifferentiated HESC marker) in the early HEBs. These two cell populations were sorted apart, total RNA was extracted from each population, and their global expression patterns were compared using U95Av2 DNA microarray. LEFTY A expression was found to be 12-fold upregulated in the sorted LEFTY-EGFP cell population compared with the TRA-1-60-positive population, were it was absent (Fig. 3C). LEFTY B and CRIPTO (TDGF1) were also upregulated in the LEFTY-EGFP-sorted population. To characterize the LEFTY-EGFP population, we decided to look for cell surface markers that are upregulated in this population compared with the TRA-1-60-positive population. Looking at genes that were at least 10-fold upregulated in the LEFTY-EGFP cell population (288 of 12,625 probes), we could find, using the Gene Ontology annotation for cellular components, six genes that were annotated as localized to the cell membrane (GO annotation 16,020) (Fig. 3C). Among these six genes, the two that were most prominently upregulated in the LEFTY-EGFP population were from the Cadherin family, CADHERIN 2 and CADHERIN 11.

Figure Figure 3..

Isolation and transcriptional characterization of LEFTY-enhanced green fluorescence protein (EGFP) cell population. Analyzing the LEFTY-EGFP cells in the early human EBs according to their size and granularity (forward and side scatter plots) LEFTY-expressing cells (green dots) (A) TRA-1-60 (undifferentiated human ESC marker)-expressing cells (red dots) (B). (C): Histograms of LEFTY A and TDGF1 (CRIPTO) expression in the sorted LEFTY-EGFP (green bars) cell population compared with the TRA-1-60-positive population (red bars), as well as a similar histogram for the six genes that were annotated as localized to the cell membrane (Gene Ontology cellular component annotation) and upregulated in the LEFTY-EGFP population, namely CDH2, CDH11, DEGS1, TDE1, RAP1A, and CNIH. Abbreviations: CDH, cadherin 2; CNIH, cornichon homolog; DEGS1, degenerative spermatocyte homolog 1, lipid desaturase; FSC, forward scatter; RAP1A, member of RAS oncogene family; TDE1, tumor differentially expressed 1.

Inhibition of NODAL Pathway During HESC Differentiation Specifically Affects Expression of the Mesodermal Markers

NODAL, a member of the set of TGF-β-like molecules, is responsible for the transcriptional activation of LEFTY A/B. The receptors ALK4, ALK7, ActRIIa, and ActRIIb mediate NODAL action by phosphorylation of SMAD2, SMAD3, and SMAD4 and activation of FAST transcription factor [8]. Recently, it has been shown that NODAL signals through ActRIIb and ALK7 [13]. NODAL brings together these two receptors; consequently, type II receptor phosphorylates the type I receptor. This results in phosphorylation of the intracellular transcription factors of the SMAD family. When these transcription factors are phosphorylated, they form complexes, which subsequently accumulate in the nucleus and initiate transcription of target genes (Fig. 4B). We could show by reverse transcription (RT)-PCR that LEFTY A and LEFTY B mRNA levels elevate transiently in 2- and 10-day-old HEBs and diminish thereafter (Fig. 4A). We carried out gene expression profiling of HESCs and their differentiated progenies in 2-, 10-, and 30-day-old HEBs using the Affymetrix GeneChip microarray. We demonstrated that upon HESC differentiation, the NODAL/LEFTY pathway, known to be crucial in the early mouse embryonic development, is activated in a synchronized manner. FAST, which is a regulatory transcription factor, is already present at the HESC stage and is downregulated only in the terminally differentiated 30-day-old HEBs. Transcription of NODAL, LEFTY A, and LEFTY B is transiently upregulated at the early HEB stage and subsequently downregulated when the differentiation progresses. NODAL pathway receptors and cellular mediators are expressed at moderate to high levels throughout HEB differentiation. PITX2, which is known to be expressed at later stages of differentiation, is expressed only in 30-day-old HEB (Fig. 4C). Thus, upon HESC in vitro differentiation, the NODAL pathway is activated in a sequential manner.

Figure Figure 4..

SB-431542 inhibits the NODAL/LEFTY pathway specifically, as well as the mesodermal marker BRACHYURY, during human ESC (HESC) differentiation. (A):LEFTY A and B expression was demonstrated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis in undifferentiated HESCs and 2d, 10d, and 30d human EBs (HEBs). (B): NODAL signaling pathway. NODAL, which is secreted from the cell, initiates a signal transduction pathway via activin receptor type IIB that activates SMAD and FAST to induce NODAL, LEFTY A, LEFTY B, and PITX2 expression. SB-431542, a potent and selective inhibitor of transforming growth factor-β1 receptor-like kinase ALK4/5/7, has been shown to inhibit this pathway. (C): Shown are expression patterns of the NODAL/LEFTY pathway members along the differentiation of HESC (HESCs and 2d, 10d, and 30d HEBs). The expression is given in normalized intensity units as obtained from the U133A and B DNA microarray. (D): SB-431542 specific inhibition is demonstrated in 2d HEBs by the downregulation of LEFTY A, LEFTY B, and NODAL gene expression, whereas genes that are induced in the early HEBs [7] remain to be expressed at similar levels. (E): RT-PCR analysis of 2d HEBs mRNA demonstrated that SB-431542 inhibits expression of NODAL, LEFTY A, LEFTY B, CRIPTO, and the early mesodermal marker BRACHYURY. Abbreviations: d, day-old; DMSO, dimethyl sulfoxide; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Inhibition of the NODAL/LEFTY pathway might reveal its role in HESC differentiation in vitro. We have designed several RNA interference (RNAi) oligos and transfected them to HESCs using the pSUPER system [14]. However, since we were unsuccessful in downregulating the mRNA levels of NODAL by RNAi (supplemental online Fig. 2) and knockout of both alleles of autosomal genes in HESCs is currently not achievable, we decided to use chemical inhibition of the NODAL/LEFTY pathway in order further uncover its function.

Lately, an activin-like kinase (ALK) potent and selective inhibitor, SB-431542, has been developed [15]. This molecule has been shown to effectively inhibit type I receptor ALK7 by acting as a competitive ATP binding site kinase inhibitor [16]. We subjected the HESCs to treatment with SB-431542 at two different concentrations (1 and 10 μM) that have been shown to be effective [16]. Cells were trypsinized and allowed to form HEBs in the presence of the inhibitor, and transcriptional analysis was performed on RNA collected from 2-day-old HEBs. RT-PCR analysis, at nonsaturating conditions, demonstrated that transcriptions of LEFTY A, LEFTY B, and NODAL are inhibited even at the lower concentration of SB-431542 (Fig. 4D). To verify whether the inhibitor effect is specific, we performed an RT-PCR analysis of several genes (Unigene numbers Hs.510523, Hs.71913, Hs. 444459, Hs.127797, Hs. 203963, and Hs.197683, http://www.ncbi.nlm.nih.gov) that are induced in early HEBs, similar to the expression pattern of the LEFTY genes. It was demonstrated that although the LEFTY pathway gene transcription was dramatically inhibited by SB-431542, none of the other genes tested respond to the presence of the inhibitor, and equivalent amount of transcripts were detected without the inhibitor and after its administration in low and high concentrations (Fig. 4D). The NODAL/LEFTY pathway is known to play a major role in mouse gastrulation; moreover, LEFTY and NODAL are recognized as early mesodermal lineage markers. It is also known that when HESCs differentiate in vitro, the HEBs that are formed consist of cells representing the three germ layers. Thus, we examined whether NODAL/LEFTY pathway inhibition in HEBs might affect other mesodermal markers. We showed that SB-431542 not only inhibits transcription of NODAL, LEFTY A, and LEFTY B but also affects the expression of Cripto, a cofactor of NODAL's receptor that is essential for mouse mesodermal development [17], and Brachyury, a well-known mesodermal marker (Fig. 4E).

We expanded our analysis of the effect of SB-431542 on NODAL/LEFTY pathway inhibition to later stages of HESC differentiation and 2-, 10-, and 30-day-old HEBs, and we performed a large-scale DNA microarray analysis (using a U95Av2 microarray). The most significantly inhibited gene (approximately 100-fold downregulation) was LEFTY A, which was absent in cells exposed to the inhibitor (supplemental online Fig. 3) (Fig. 5). Examining the effect of the inhibitor at the different time points, overall downregulation of mesoderm-related genes, such as CRIPTO (TDGF1), and upregulation of ectoderm-related genes were detected (supplemental online Fig. 3). Moreover, we could identify, among the mesoderm- and ectoderm-related genes that were affected by the NODAL/LEFTY pathway inhibition, genes with different temporal expression patterns. These genes could be related to early, mid-, and late mesoderm or ectoderm differentiation within the HEBs. For example, LEFTY A, vascular endothelial growth factor C (VEGFC), and INTEGRIN α4 (representing early, mid-, and late mesodermal differentiation, respectively) were downregulated, and Distal-less homeobox 5 (DLX5), PERIPHERIN, and ENOLASE 2 (representing early, mid-, and late ectodermal differentiation, respectively) were upregulated (Fig. 5).

Figure Figure 5..

NODAL pathway inhibitor inhibited mesoderm-related genes and promoted ectoderm-related genes. Shown are expression patterns of three mesoderm-related (left panels) and three ectoderm-related (right panels) genes possessing early (LEFTY A and DLX5), mid- (VEGFC and PERIPHEIN), and late (INTEGRIN α4 and ENOLASE 2) expression patterns during normal (blue line) HEB differentiation (2d, 10d, and 30d HEBs). In the presence of the NODAL pathway inhibitor (red line), mesoderm-related genes were shown to be downregulated and ectoderm-related genes were shown to be upregulated in response to the inhibitor. Abbreviations: d, day-old; DLX5, distal-less homeobox; VEGFC, vascular endothelial growth factor C.

Discussion

The formation of the whole intact embryo is dependent upon precise implementation of a developmental program. The developmental program is based on a strict temporal, spatial, and sequential gene expression that allows the formation of various specialized cells from the undifferentiated cells in the blastula. The generation of specialized differentiated cells from undifferentiated pluripotent cells is assumed to begin by differentiation into intermediate cell types (stem cells or progenitor cells). These intermediate cells have a more committed fate than their undifferentiated ancestors and will presumably appear at the beginning of the differentiation process.

Our aim was to identify and isolate progenitors for different embryonic lineages in humans. We thus envisioned that the in vitro system to some extent would recapitulate the early developmental processes occurring in vivo. Since, in humans, the in vivo analysis of early differentiation processes is extremely limited, initially we had to rely on the mouse model as a reference. It is known that in the mouse, NODAL signaling acts upon gastrulation to specify progenitors for mesoderm and endoderm. For example, it has been shown that in the mouse, Nodal mutants do not form a primitive streak, which is normally formed by mesendoderm progenitors [18]. In addition, the level of Nodal signaling affects the formation of mesendoderm in the mouse [19]. Nodal expression initiates a signal transduction cascade that induces its own expression, as well as that of Lefty 1, Lefty 2, and Pitx2, at later stages of embryonic development. In this study, we followed this family of genes, which is known to play a major role in mouse mesoderm differentiation. We could indeed demonstrate that genes that are developmentally regulated in vivo are also temporally expressed upon HESC differentiation. LEFTY A and B seem to serve a similar function during HESC differentiation. Still, LEFTY A appears to hold a more central role in mesoderm differentiation [20]. In our experiments, the induction of LEFTY A during HEB differentiation was more significant than that of LEFTY B. Moreover, upon inhibition of the NODAL pathway, the effect on downregulation of LEFTY A was more pronounced than that of LEFTY B.

It has recently been suggested [21, 22] that LEFTY A and LEFTY B are expressed at the RNA level in the undifferentiated cells. In this study, we also identified LEFTY A and LEFTY B expression by RT-PCR and by DNA microarray in culture of HESCs. However, when we carefully analyzed LEFTY A and B protein by immunostaining, it became clear that in the HESC culture, LEFTY proteins were exclusively expressed by the differentiated cells that are naturally present in the culture. However, soon after the cells were allowed to differentiate in vitro, LEFTY protein expression was upregulated (2- and 5-day-old HEBs); it diminished gradually as differentiation proceeded (10–20-day-old HEBs), as has also been shown for the mRNA transcripts [7, 23], until expression completely disappeared in the matured HEBs. Furthermore, molecular labeling (LacZ or EGFP reporter genes) of LEFTY A-expressing cells indicated that only the differentiated cells derived from HESCs expressed the LEFTY A gene. Hence, we investigated LEFTY expression by various methodologies, quantitative RT-PCR (Fig. 4A), DNA microarray (Fig. 4C), LEFTY protein expression in HESCs and HEBs of two different cell lines (Fig. 1E–1J) and by using LEFTY enhancer to drive a reporter gene expression. We show that LEFTY protein is not expressed by undifferentiated HESCs, although as these cells differentiate they begin to express LEFTY protein. This is also supported by LEFTY mRNA levels at the different time points. Thus, we hypothesize that the differentiated cells in the HESC culture are responsible for the LEFTY transcripts revealed by reverse transcription of RNA from a mixed cell population (undifferentiated and differentiated cells).

The identification and isolation of intermediate progenitor cells is extremely important; it will enable the in vitro developmental study of human embryonic development where early embryos are inaccessible. It will also allow isolation and study of these progenitor-like cells, which are thought to retain the self-renewal characteristic but are more committed. When isolated, these cells will be of great value for transplantation medicine since they are more committed than HESCs. As a result, the derivation of fully differentiated cells from the same lineage will be less problematic, and since the cells are no longer ESCs, they are not predisposed to tumor formation.

Only 2 days after HEB formation, the level of LEFTY mRNA and protein was upregulated dramatically. Therefore, we presumed that the activation of NODAL signaling pathway upon HESC differentiation might be responsible for the acquisition of mesodermal cell fate in the early HEBs (2 days old). We assumed that the acquisition of cell fates would be performed gradually, as known from in vivo studies; therefore, at the beginning of differentiation, stem cell-like or progenitor-like cells would appear transiently. The observation that components from the NODAL/LEFTY pathway are activated sequentially upon HESC differentiation encouraged us to think that NODAL expression followed by upregulation of LEFTY A and LEFTY B might represent the induction of mesoderm and endoderm intermediate cells in the early HEB. Indeed, it was recently suggested by two groups [24, 25] that Activin/Nodal in conjunction with the fibroblast growth factor pathway is responsible for maintaining HESC pluripotency through SMAD2/3 activation. However, when the effect of LEFTY was examined, either by addition of high doses of recombinant LEFTY protein to the media or by LEFTY transgene overexpression, no significant effect on HESC pluripotency was detected [25]. In light of these studies and our results, we speculate that when HEBs are formed, in the first couple of days of HESCs aggregation, high local concentrations of NODAL protein are generated within these cell aggregates. These local high NODAL concentrations induce transcription of LEFTY by SMAD2/3 activation, which in turn inhibits NODAL function and transcription; as a result, the initial steps of differentiation begin.

When we analyzed cell morphology and protein expression of 2-day-old HEBs, it was evident that the early HEB was indeed not homogenous either morphologically or molecularly. Thus, when HESCs were allowed to aggregate, the cells quite rapidly acquired different markers and distinct morphology. Immunostaining analysis indicated that the early HEB still consisted of some undifferentiated cells expressing OCT4, but it also contained LEFTY-expressing cells. We could see that there was practically no overlap between the expression of these markers and that they segregated to different cells. We could also correlate cell morphology and marker expression, as the in situ hybridization signal for LEFTY A colocalized to a differentiated cell morphology. Furthermore, our FACS results and cell distribution analysis demonstrate that LEFTY-expressing cells contributed approximately 10%–20% to the HEB volume. We anticipate that this percentage reflects the actual amount of LEFTY-expressing cells, although the immunostainings suggested a somewhat higher amount. It should be noted that the immunostainings against LEFTY proteins recognize not only intracellular LEFTY proteins but also the secreted fraction of these proteins and therefore overestimate the abundance of the cells expressing LEFTY protein. This supports our previous assumption that the NODAL/LEFTY pathway might be involved in at least the formation of one intermediate cell type in the early HEB.

Activation of the NODAL/LEFTY cascade could indicate that differentiation of HESCs can serve as a model for early development. Hence, the use of different growth factors that were already shown to affect HESC differentiation fate [5] or selective inhibitors such as SB-431542 could be a valuable tool for directing HESC differentiation and consequently isolating and selecting specific cell populations. It is also possible to isolate cells according to their morphology, since one can assume that cells acquiring different fates will have different morphologies. Another option is to isolate specific cells by genetic labeling, as shown for LEFTY-EGFP cells. Although LEFTY-positive cells could not grow well after sorting, comparing the transcriptome of the LEFTY-positive and TRA-1-60-positive cell populations, we could see that LEFTY transcript was indeed absent from the TRA-1-60 population, and it was present at high levels in the LEFTY-positive population. Moreover, another mesoderm lineage-related marker, CRIPTO (TDGF1), was also expressed at a high level in the LEFTY-positive population and was absent from the TRA-1-60 population. Analyzing the cell surface molecules distinguishing the LEFTY-positive population from the TRA-1-60 population, we could identify few genes. The two most prominent genes belong to the cadherin family, which are thought to play an important role in development through maintenance of cell-cell adhesion. CADHERIN 2 is known to be required during chicken embryo gastrulation, mediating cell adhesion events, and is one of the earliest proteins to be asymmetrically expressed during embryogenesis [26]. Moreover, blocking its activity affected Pitx2 expression but not Nodal or Lefty. Therefore, in the chicken embryo, Cadherin 2 and Nodal/Lefty seem to play a parallel role during gastrulation. CADHERIN 11 is expressed by progenitor cells of osteoblasts, and its expression was shown to be upregulated during osteoblastic differentiation. Therefore, a function in bone cell differentiation was suggested for CADHERIN 11 [27]. Hence, the LEFTY-positive cell population seems to express another mesoderm-related marker, CRIPTO, as well as cell surface markers that are typical for a cell population existing during early stages of embryogenesis and that seem to be associated with the mesoderm lineage.

We further addressed the NODAL/LEFTY pathway role during HESC differentiation by taking the approach of loss-of-function experiments. Inhibition of the NODAL pathway chemically via administration of SB-431542, which selectively prevents the phosphorylation of SMAD3 in this cascade, resulted in a decrease of mesodermal markers such as LEFTY A and LEFTY B (which are related to the same pathway) to negligible amounts. Likewise, BRACHYURY, an unrelated early mesodermal marker, was also significantly downregulated as an effect of the inhibitor administration. Furthermore, an overall inhibition of mesoderm-related genes and promotion of ectoderm-related genes was detected in response to exposure to the inhibitor along differentiation. We could also identify markers affected by the inhibitor that possess different expression patterns related to early, mid-, and late HEB differentiation. LEFTY A as an early marker, VEGFC, a stimulator of endothelial cell growth, as a middle marker, and INTEGRIN α4, which is expressed by blood cells and involved in cell-cell and extracellular matrix adhesion, as a late marker of HEB mesoderm differentiation were shown to be dramatically inhibited by NODAL pathway inhibitor. The opposite is true for the ectodermal markers; DLX5, which is mainly expressed by the fetal brain, PERIPHERIN, which is mainly expressed by the dorsal root ganglia, and ENOLASE 2, which is expressed at the nervous system, as early, mid-, and late HEB ectodermal markers were shown to be dramatically promoted by NODAL pathway inhibitor. Therefore, we suggest here that the NODAL/LEFTY pathway is functional and active in HESCs and takes part in the generation of an intermediate mesoderm-related cell population from the undifferentiated HESCs.

This study insinuates that HESC differentiation in vitro is an extremely important tool for understanding early human development and cellular differentiation. HESC differentiation may elucidate initial processes taking place at the beginning of embryonic development, such as lineage determination and differentiation. Moreover, the existence of a pathway activated sequentially upon differentiation suggests that more pathways might be identified and serve as a helpful tool for isolation of progenitor-like cells to understand early differentiation processes and subsequently for transplantation medicine.

Conclusion

In this study, we demonstrated that during early differentiation of HESCs into HEBs, LEFTY is expressed in a subset of cells. LEFTY expression is regulated by NODAL pathway, and its inhibition affects the mesodermal fate of the cells. We speculate that in early HEBs, several progenitor-like cells appear; these cells should be useful in the study of early human development and as a tool in regenerative medicine.

Disclosures

The authors indicate no potential conflicts of interest.

Acknowledgements

We thank Prof. Joseph Itskovitz-Eldor at the Rambam Medical Center for kindly providing us with the human ESCs as collaboration. We are indebted to Prof. Hiroshi Hamada for kindly supplying hL2LacZ vector. We are grateful to Adi Ben-Nun from the Geographical Information System Department at the Hebrew University for the HEB area analysis. We thank Dr. Rachel Eiges and Dr. Danny Kitsberg for critically reading the manuscript. This research was partially supported by funds from the Ministry of Science and Technology, Israel; the Herbert Cohn Chair (N.B.) of the Israel Science Foundation; and by an NIH grant.

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