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First published online March 27, 2008
Stem Cells Vol. 26 No. 6 June 2008, pp. 1464 -1473
doi:10.1634/stemcells.2007-0808; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

CXCR4+/FLK-1+ Biomarkers Select a Cardiopoietic Lineage from Embryonic Stem Cells

Timothy J. Nelson, Randolph S. Faustino, Anca Chiriac, Ruben Crespo-Diaz, Atta Behfar, Andre Terzic

Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, and Medical Genetics, Mayo Clinic, Rochester, Minnesota, USA

Key Words. Biomarker • Cardiopoiesis • Cardiac differentiation • Network • Transcriptome

Correspondence: Correspondence: Andre Terzic, M.D., Ph.D., Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. Telephone: 507-284-2747; Fax: 507-266-9936; e-mail: terzic.andre{at}mayo.edu

Received on September 23, 2007; accepted for publication on March 14, 2008.

First published online in STEM CELLS EXPRESS  March 27, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi-lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue-specified progenitor cells. From a comprehensive pool of 11,272 quality-filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane-associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over-represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4+ progenitors were sorted from the embryonic stem cell pool into mesoderm-restricted progeny according to co-expression with the early mesoderm marker Flk-1. In contrast to CXCR4–/Flk-1– cells, the CXCR4+/Flk-1+ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha-fetoprotein markers were all depleted. In fact, the CXCR4+/Flk-1+ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4+/Flk-1+ transcriptome shared 97% identity with the CXCR4–/Flk-1– counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4+/Flk-1+ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Stem cells are the focal point for applications in cardiac regenerative medicine [1, 2] and have recently progressed from pre clinical consideration to clinical trials [3]. Initial experience using adult stem cells in patients with myocardial infarction demonstrates that cell transplantation into the heart is feasible and safe, but limited by lack of uniformity in outcome [4–8]. The observed variability in therapeutic efficacy is, in part, attributed to heterogeneity of transplanted adult stem cell populations, which are notable for their unpredictable capacity for cardiac differentiation [9].

Embryonic, rather than adult, stem cell types have consistently demonstrated a robust capacity for cardiogenic differentiation both in vitro and in vivo [10–12]. The inherent aptitude of embryonic stem cells for cardiogenesis ensures that the early-stage embryo establishes a cardiovascular system to meet the obligatory metabolic demands of ongoing organogenesis. This innate cardiogenic potential of embryonic stem cells contributes to de novo re-muscularization in models of ischemic heart disease producing functional improvement with sustained benefit [13–17]. However, the unrestricted mitogenic potential associated with pluripotency hinders therapeutic translation due to risk for uncontrolled growth once stem cells are transplanted outside the native embryonic niche [18–20].

Embryonic development secures predictable differentiation by segregating early progenitor cells programmed to execute cardiac differentiation [21–23]. Multipotent cells, known as mesendoderm, migrate out of the primitive streak towards the primitive ectoderm to form the primary heart field, which evolves into a specialized, mesoderm-derived population of cardiogenic progenitors [24, 25]. In this locale, adjacent endoderm provides critical inductive signals necessary to guide lateral plate mesoderm into the embryonic heart [21, 26]. Selection outside the embryo of a progenitor cell population, analogous to embryonic pre-cardiac mesoderm, would thus offer an approach to separate cardiac progenitors derived through unrestricted differentiation.

Systems-expression profiling, coupled with bioinformatic network analysis at distinct stages of embryonic stem cell differentiation prioritized membrane-associated genes during in vitro cardiac specification. Surface biomarkers, identified through unbiased screens, segregated by primordial stem cell subpopulations harboring a cardiac pedigree characterized by a primitive developmental network that executed functional cardiogenesis. In this way, stage-specific dynamics of a biomarker combination provides a molecular foundation to predict and sort cardiopoietic progenitors from pluripotent stem cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic Stem Cell Cardiopoiesis
Murine embryonic stem cells (GCR8 and R1-derived lines) were maintained in Glasgow's Minimum Essential Medium (BioWhittaker-Cambrex, Walkersville, MD, http://www.cambrex.com) supplemented with pyruvate and L-glutamine (Cellgro, Mediatech, Inc. Herndon, VA, http://www.cellgro.com) non-essential amino acids (Mediatech) β-mercaptoethanol (Sigma-Aldrich, St Louis http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html) 10% fetal calf serum (FCS) (Invitrogen Corporation http://www.invitrogen.com) and leukemia inhibitory factor (LIF) (ESGRO, Chemicon International, Inc, Temecula, CA, http://www.chemicon.com). Stem cells were differentiated into three-layer embryoid bodies using the hanging-drop method in differentiation media supplemented with 20% FCS and TNF-{alpha} (Invitrogen) as described [18, 27–29]. A time course of differentiation starting with undifferentiated embryonic stem cells maintained in LIF was obtained from cell aggregates in suspension at days 3–5 and from plated embryoid bodies at days 7–9. Dual interface Percoll gradient (Invitrogen) was used to enrich sarcomere-rich high density cardiomyocytes [10, 11] from the lower density sarcomere-poor cardiopoietic phenotype from day 7 embryoid bodies. Embryonic stem cell-derived progeny were fixed in 3% paraformaldehyde, permeabilized with 1% Triton X-100, and immunostained with antibodies specific for Mef2c (1:400, Cell Signaling Technologies, Danvers, MA, http://www.cellsignal.com) Nkx 2.5 (1:150, Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) Mesp-1 (1:500, Novusbio, Littleton, CO, http://www.novusbio.com) AFP (1:100, Cell Signaling Technologies, Danvers, MA) and sarcomeric protein {alpha}-actinin (1:1,000, Sigma-Aldrich) along with DAPI staining to visualize individual nuclei [18, 27–29]. Microscopy was performed using an LSM 510 laser scanning confocal microscope (Carl Zeiss Inc., Jena, Germany, http://www.zeiss.com).

Genomics
Total RNA was extracted at selected developmental stages using a combination of gDNA Eliminator and RNeasy columns (Qiagen, Valencia, CA, http://www1.qiagen.com). cDNA was prepared from total RNA samples using MMLV Reverse Transcriptase (Invitrogen). Samples were subjected to microarray analysis by labeled cRNA hybridization to the mouse genome 430 2.0 GeneChip (Affymetrix, Inc, Santa Clara, CA, http://www.affymetrix.com) [18, 27–29]. Embryonic stem cells in the presence and absence of LIF, cardiopoietic cells and cardiomyocytes were analyzed using cDNA from triplicate biological samples. Real-time polymerase chain reaction (PCR) was performed using a standard TaqMan PCR kit protocol on an Applied Biosystems 7,900HT Sequence Detection System (Applied Biosystems, Foster City, CA). The 50 µl PCR included 3 µl RT product, 25 µl x TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), 19.5 µl RNase-free water and 2.5 µl TaqMan Gene Expression Assays (pre designed, pre-optimized probe and primer sets for each gene of interest (Applied Biosystems). TaqMan Gene Expression Assays contained two unlabeled PCR primers (900 nM each final concentration) and one FAM dye-labeled TaqMan MGB probe (250 nM final concentration). The reactions were incubated in a 96-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All reactions were run in triplicate. The threshold cycle (CT) was defined as the fractional cycle number at which fluorescence passes the fixed threshold. TaqMan CT values were converted into relative fold changes determined using the 2–{Delta}{Delta}CT method, normalized to GAPDH (P/N 435,2662-0506003) expression. Genes representative of each germinal layer were selected for analysis, which included Lhx1 (Mm00521776_m1), Nkx2.5 (Mm00657783_m1), Gata4 (Mm00484689_m1), Myocd (Mm00455051_m1), Oct4 (Mm00658129_gH), Fgf4 (Mm0000438917_m1), Cxcr4 (Mm01292123_m1), Mef2c (Mm01340839_m1), Gsc (Mm00650681_g1), Sox17 (Mm00488363_m1), Sox7 (Mm00776876_m1), Flk1 (Mm00440099_m1), Sox1 (Mm00486299_s1), Sox2 (Mm00488369_s1), Tbx5 (Mm00803521_m1), Myh11 (Mm00443013_m1), and PECAM (Mm00476702_m1). Comparisons between groups were performed by Student's t-tests with 95% confidence intervals.

Gene Expression Profiling
Gene expression changes of microarray data acquired using the GeneChip Scanner 3,000 (Affymetrix) were profiled with the Genespring GX 7.3 analysis software suite (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). Dataset was deposited at the Gene Expression Omnibus website as series GSE6689 [NCBI GEO] . The derived gene list was limited to report transcripts with expression levels above background and then subjected to one-way analysis of variance, using a Benjamini-Hochberg post hoc multiple testing correction for all p < .01. Filtered significant genes were delimited by flag value, excluding those absent in all samples. Gene Ontology Consortium designated membrane-associated transcripts ("integral to membrane") were reproducibly present in all samples. Co-ordinated gene profiles hierarchically assembled into an expression heatmap using gene tree clustering. Genes were selected from membrane-restricted genes according to normalized expression values >1.5-fold. Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways within the derived upregulated cluster (cytokine, adhesion, Jak/STAT) were identified with associated P-values as a measure of similarity between the KEGG-established pathway and the experimentally derived list, with (P) calculated as follows:


Formula

The probability (P) of overlap corresponds to (k) or more genes between (n) and (m) gene lists when randomly sampled from a universal list (u). Comparison among membrane restricted, cytokine clustered and quality filtered lists identified overlapping transcripts, from which upregulated expression profiles were identified. Subcellular location and interrelationships of upregulated, cytokine-clustered genes were recompiled into a format usable by the Institute for Systems Biology Cytoscape 2.2 software (Institute for Systems Biology Cytoscape, Seattle, WA, http://www.cytoscape.org) and expressed as a localized network. Phenotypes associated with identified transcripts were investigated through bioinformatic mining of the Mouse Genome Informatics database (www.informatics.jax.org) to identify impacts of transgenic candidate gene knockouts on cardiac differentiation for ultimate restriction of the transcript pool.

Flow Sorting
GCR8 and R1-derived embryonic stem cells were placed in 25 µl hanging drops (~2,500 drops per experiment) that initially contained 250 cells each and were cultured for 2 days in 20% FCS (Invitrogen) in the absence of LIF, then allowed to differentiate as large aggregates in suspension for three additional days. Aggregates were washed in phosphate buffered saline (PBS) and dissociated using non-enzymatic dissociation buffer (Invitrogen Corporation, Carlsbad, CA) for 10 minutes at 37°C. Aggregates were triturated using 10 ml pipette until single cell suspension was obtained. Derived cells were spun down at 1,000g for 5 minutes and resuspended in propagation media (7.5% FCS) for 10 minutes to allow cells to recover. Cells (2 x 107) obtained from initial aggregates were collected and immuno-stained for both Flk-1 and CXCR4 expression. Cells were washed with PBS and resuspended in 1 ml PBS which contained goat-CXCR4 antibody (1:150, Abcam, Cambridge, U.K., http://www.abcam.com), placed on ice for 30 minutes incubation, followed by single wash with 10 ml PBS. Secondary anti-goat Alexa 488 (1:500, Molecular Probes, Invitrogen) and phycoerythrin (PE)-conjugated primary antibody for Flk-1 (1:200, BD Biosciences, San Diego, http://www.bdbiosciences.com) were incubated on ice for 30 minutes followed by single 10 ml PBS wash. Cells were isolated using a FACS Aria SE flow cytometer (BD Biosciences). Alexa-488 was excited with a 488 nm argon laser and detected through a 530/30 nm bandpass filter. PE was excited with the 488 nm laser line and detected through a 575/26 bandpass filter. Forward and side scatter parameters were used to gate on viable cell population sorted into subpopulations. Once collected in standard culture media, cells were centrifuged and frozen in liquid nitrogen for RNA isolation and microarray analysis. Alternatively, fresh cells were suspended in propagation media, diluted to 400,000 cells/ml, and 25 µl drops were suspended for 48 hours prior to plating the re-aggregates on 0.1% gelatinized plates with or without visceral endoderm-like cells. Derived from an F9 cell population (American Type Culture Collection) with retinoic acid (1 µM), dbcAMP (0.5 mM) and theophylline (0.5 mM), with phenotype confirmed through comparison with END-2 cells [27, 30], these visceral endoderm-like cells were used to recapitulate the embryonic environment for 4 days of in vitro differentiation to complete the 9-day protocol.

Ontological and Network Analysis
Differentially expressed genes (p < .05) were excluded from subthreshold transcripts using Volcano plot analysis, according to a minimum 1.5-fold change, and ontologically dissected to determine physiological system priority emphasized within changing transcripts. Significant association of sortable cell surface biomarkers was determined, through Ingenuity Pathways Analysis, within each prioritized physiological system. Molecular interactions of expression profiles comprising Cardiovascular Development were examined and formatted for Cytoscape 2.2, which provided an ad hoc network map of integrated up- and down-regulated pro-cardiac candidate genes.

Beating Activity and Calcium Transients
Sorted subpopulations of stem cell-derived progenitor aggregates cocultured with visceral endoderm-like cells were monitored daily. Beating activity was recorded at 20 frames per second with phase contrast microscopy using a Zeiss Observer.Z1 microscope (Zeiss) with ApoTome and live cell imaging system controlled at 37°C. Once beating activity was recorded, cells were loaded with the Ca2+-selective probe fluo-4-acetoxymethyl ester (Molecular Probes) with a final concentration of 5 µM in serum-free cell culture media and incubated for 30 minutes at 37°C before washing in PBS and allowed to recover in standard culture media for 1 hour. Using the temperature controlled live cell imaging system on a Zeiss ApoTome microscope (Zeiss), fluo4-loaded cells were illuminated with a mercury lamp at 400 ± 20 nm and fluorescence was recorded with a AxioCam MRm camera and AxioVision software (Zeiss). Intracellular Ca2+ transients were deconvoluted as a function of time and analyzed with MetaMorph software (Universal Imaging Corporation, West Chester, PA, http://www.moleculardevices.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Systems Bioinformatics Prioritizes a Chemokine Cluster During Cardiopoiesis
Blastocyst-derived embryonic stem cells maintain pluripotency in vitro when treated with leukemia inhibitory factor, and on removal of this mitogen differentiate into embryoid bodies [31]. Within the heterogenous lineages of a differentiating embryoid body, tumor necrosis factor-{alpha} (TNF-{alpha}) expedites cardiac commitment [27]. Derived progenitor cells, referred to as cardiopoietic stem cells, engage cardiogenesis upon nuclear translocation of cardiac transcription factors, and represent an intermediate phenotype preceding sarcomerogenesis during cardiac differentiation [27, 29, 32]. Compared to the parental embryonic stem cell source, genome-wide microarrays identified a distinctive cardiopoietic progenitor transcriptome defined by a spectrum of 11,272 transcripts (Fig. 1A). To screen for biomarkers predictive of lineage-specification, restriction of the cardiopoietic profile to individual transcripts encoding proteins expressed in the cell membrane resolved 736 hierarchically clustered genes (Fig. 1B). Within this candidate surface marker pool, 306 genes associated with plasmalemmal signaling components were significantly increased in expression at the cardiopoietic stage (Fig. 1C). Ontological mining for curated functional families unmasked cytokine/chemokine receptor signaling as the overrepresented functional cluster with highest priority (p= 8.65 x 10–5), followed by cell adhesion (p= 6.46 x 10–3) and JAK/STAT (p= 2.91 x 10–3) clusters. Thus, using an unbiased bioinformatic algorithm, transcriptome analysis during embryonic stem cell differentiation extracted a cytokine/chemokine gene set as the leading cell membrane-associated cluster at the cardiopoietic stage.


Figure 1
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  		Figure 1. Cytokine gene cluster prioritized from a hierarchically organized transcriptome of embryonic stem cells at the cardiopoietic stage. Differentiating embryonic stem cells at four distinct stages of cardiogenesis, that is, undifferentiated embryonic stem cells developmentally restrained by LIF (ES), released from LIF (ES'), CP and sarcomere-rich CM, were collected and analyzed with microarray gene expression assays. (A): Restricted expression profiling demonstrated dynamic transcriptional changes from ES to CP, delimiting 11,272 genes. Color coding of upregulated, downregulated, and unchanged genes were red, blue, and yellow, respectively. Numbers to the right of the color scale (insert) indicate fold change. (B): Expression heatmap of 736 membrane restricted genes extracted from thresholded CP transcripts were hierarchically clustered, displaying reproducible molecular fingerprints for three biologically distinct samples. (C): Data mining of 306 up-regulated, membrane-associated CP transcripts revealed prominence of a hierarchically arranged cytokine cluster comprised of 11 genes. Inset color scale represents fold change. Abbreviations: CM, cardiomyocyte; CP, cardiopoietic; ES, embryonic stem; ESC, embryonic stem cell; LIF, leukemia inhibitor factor.

 
Deconvoluted Cytokine/Chemokine Cluster Reveals Cardiogenic Linkage with CXCR4
The cytokine/chemokine cluster incorporated 4% of the total 306 upregulated membrane-restricted genes identified at the cardiopoietic stage of differentiation (Fig. 1). Comparison among the GOC roster of genes integral to membrane (2,893 genes), the KEGG pathway designated cytokine/chemokine family (223 genes), and the experimentally determined transcripts in cardiopoietic stem cells (11,272 genes) independently identified candidate genes within the cytokine family (Fig. 2A). Venn diagram resolution of intersecting gene cohorts demarcated 16 overlapping transcripts, verifying the 11 cardiopoietic genes of the identified cytokine cluster, that is, CXCR4, Il11ra1, Ghr, Ltbr, Tnfrsf1a, CX3CL1, Acvr1, Tnfrsf12a, Il10rb, Osmr, and Csf1, with the remaining five neither upregulated nor present (Fig. 2B). Linkage of these 11 cytokine clustered genes to cardiovascular development was interrogated by mining the annotated phenotypes within the Mouse Genome Informatics database. Although 10 genes were associated with non-specific systemic disruptions in organismal growth and cell size (Ghr, Tnfrsf1a, Tnfrsf12a, Acvr1), abnormalities in immunological and hematopoietic systems (Ltbr, Csf1, Osmr, Il10rb), vascular defects in fetal placental development (IL11ra1) and atherosclerotic susceptibility (CX3CL1), cardiac abnormalities were identified in the CXCR4-null mutant [33]. The chemokine receptor, CXCR4, integrated into a non-stochastic network built on cardiogenic TNF-{alpha} and transforming growth factor (TGF)-β signaling pathways (Fig. 2C). Thus, deconvolution of the cytokine/chemokine cluster resolved the CXCR4 chemokine receptor as a candidate biomarker linking progenitor cells to cardiogenesis.


Figure 2
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  		Figure 2. CXCR4 chemokine receptor resolved within the cardiopoietic cytokine cluster. (A): Analysis of overlapping transcripts among the Kyoto Encyclopedia of Genes and Genomes pathway designated cytokine/chemokine family, the GO Consortium classification of genes integral to membrane, and the cardiopoietic transcriptome yielded 16 common genes. (B): Logical set exclusion identified 69% (11/16) of overlapping genes both Venn-restricted and upregulated in the CP stage (colored circles). (C): Analysis of bioinformatically qualified genes (colored circles) integrates into a functional network with relationships curated by the Ingenuity Pathways Knowledge Base. The contextualized cytokine cluster integrates the CXCR4 chemokine receptor (blue circle) that directly couples the extracellular signaling milieu with the intranuclear transcriptional machinery. Abbreviation: CP, cardiopoietic.

 
Pre-Cardiac Induction of CXCR4 Is Unique to the Chemokine Receptor Family
The chemokine family was screened with microarrays that contained chemokine receptor probes to quantify individual gene expression levels during cardiopoiesis. Only CXCR4 expression peaked at the cardiopoietic stage with sustained expression during cardiac maturation (Fig. 3A). Transcripts of three additional chemokine receptor genes, that is, CXCR3, CXCR6, and Gpr27, were present at stable levels during cardiogenesis (Fig. 3A). Confirming this profile, RT-PCR of CXCR4 mRNA was significantly increased as embryonic stem cells transitioned to cardiopoietic progeny (Fig. 3A inset). To trace temporal expression of CXCR4, mRNA expression was quantified using RT-PCR at sequential days of differentiation (Fig. 3B, 3C). At day 0, pluripotent genes (Oct-4, Fgf-4) demonstrated maximal transcript levels that progressively decreased with differentiation (Fig. 3C). Loss of pluripotent markers at day 5 was associated with an upsurge in CXCR4 (Fig. 3C). At day 7, CXCR4 was expressed along with cardiac-specific transcripts (Mef2c, Myocardin and Nkx2.5; Fig. 3C). Initiation of cardiogenic differentiation was confirmed by nuclear translocation of the canonical cardiac transcription factor MEF2C at day 7 and expression of sarcomeric {alpha}-actinin by day 9 (Fig. 3B), which correlated with the onset of spontaneous beating at day 8. Thus, CXCR4 displays a unique expression profile preceding cardiac specification.


Figure 3
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  		Figure 3. CXCR4 is the only chemokine receptor family member to be induced prior to cardiac differentiation. (A): Differentiating embryonic stem cells collected at the four distinct stages of cardiogenesis: ES, ES', CP, and CM. Expression levels for all 26 genes within the chemokine receptor families were analyzed using the microarray data from three independent experiments run in triplicate. Five genes (CXCR4, CXCR3, CXCR6, and GPR27) had detectable expression, with CXCR4 demonstrating a fourfold induction in cardiopoietic embryonic stem cells compared to ES cells (* p < .05). Real-time polymerase chain reaction (RT-PCR) confirmed induction of CXCR4 relative to GAPDH expression in cardiopoietic cells compared to undifferentiated embryonic stem cells (Inset). (B): Differentiating embryonic stem cells spanning the cardiopoietic window demonstrated transition from day 5 embryonic stem cell-derived progeny devoid of cardiac Mef2c or {alpha}-actinin to day 7 cardiopoietic stem cells with nuclear Mef2c expression (green), followed by maturation to sarcomere-rich cardiomyocytes expressing {alpha}-actinin (red) at day 9. All nuclei were stained with DAPI (blue). (C): Embryonic stem cells were harvested every 48 h between day 0 and day 9 for mRNA extraction and gene expression analysis using Taqman RT-PCR. Pluripotent markers, Oct4 and FGF4, were decreased by day 3 of differentiation with significant reduction at day 5. Conversely, CXCR4 expression surged by day 5 and remained significantly elevated compared to undifferentiated embryonic stem cells. CXCR4 induction preceded canonical cardiac markers, Myocardin, Mef2c and Nkx2.5 initially expressed at day 7 of differentiation. Abbreviations: CM, cardiomyocyte; CP, cardiopoietic; DAPI, 4',6'-diamidino-2-phenylindole; ES, embryonic stem; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

 
Induction of CXCR4 Coincides with Co-Expression of Mesoderm Restricted Flk-1
Fluorescence activated cell sorting (FACS) quantified CXCR4 protein expression in embryonic stem cells at different days of differentiation (Fig. 4). As predicted by transcriptome analysis (Fig. 3), <5% of cells from three-day-old embryoid bodies expressed CXCR4 protein. By day 5, 50% of total embryoid body-derived cells expressed abundant levels of cell surface CXCR4 protein (Fig. 4A). This induction marked a phenotypic shift from a CXCR4-negative pluripotent stem cell population to a mixture of CXCR4-positive progenitors, indicative of mesendoderm formation [34–36]. Indeed, at day 5 of embryonic stem cell differentiation a marked induction was observed in mesoderm (Goosecoid, Lhx1), as well as endoderm genes (Sox 7, Sox17; Fig. 4B). The early mesoderm specific marker Flk-1, also known as KDR or VEGF-2 receptor [37], demonstrated an mRNA transcription profile similar to CXCR4 (Fig. 4C). Cell sorting according to the additional marker, Flk-1, was thus applied to separate mesoderm-specific progeny within the CXCR4-positive mesendoderm population of embryonic stem cell progeny. The subpopulation that co-expressed both CXCR4 and Flk-1 was <1% at day 3 of differentiation, peaked at ~30% by day 5, and decreased by day 7 (Fig. 5A). To determine pre cardiac mesoderm potential at day 5 of differentiation, pools of CXCR4–/Flk-1–, CXCR4+/Flk-1+, and CXCR4–/Flk-1+ subpopulations were simultaneously collected for gene expression profiling. As quality control to ensure effective sorting, CXCR4+/Flk-1+ cells demonstrated quantitative gene expression differences in CXCR4 and Flk-1 compared to the CXCR4–/Flk-1– subpopulation; CXCR4–/Flk-1+ cells expressed higher levels of Flk-1 compared to CXCR4–/Flk-1– counterparts (Fig. 5B). The pluripotent marker Oct-4, whereas, abundant in CXCR4–/Flk-1– cells, was significantly downregulated in both CXCR4+/Flk-1+ and CXCR4–/Flk-1+ subpopulations (Fig. 5B). Moreover, the mesoderm-specific markers Lhx and Gsc were expressed at significantly elevated levels in CXCR4+/Flk-1+ compared to CXCR4–/Flk-1– cells (Fig. 5C). Furthermore, transcription factors GATA-4 and Tbx5, associated with the embryonic primary heart field, were significantly enriched in CXCR4+/Flk-1+ cells when compared to either CXCR4–/Flk-1– or CXCR4–/Flk-1+ subpopulations, indicating that the pre cardiac mesoderm genotype was segregated with cells expressing the combination of CXCR4 and Flk-1 (Fig. 5C). The cardiogenic profile in CXCR4+/Flk-1+ cells was separated from the phenotypic expression of vascular smooth muscle and endothelium markers, demonstrated by decreased expression of Myh11 and CD31 (PECAM) genes compared to the CXCR4–/Flk-1+ subpopulation (Fig. 5C). Thus, co-expression of CXCR4 and Flk-1 tags a mesoderm-restricted subpopulation of progenitors during differentiation of embryonic stem cells that demonstrates a unique enrichment of genes associated with early embryonic heart development.


Figure 4
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  		Figure 4. CXCR4 cell surface induction coincides with mesoderm specification during in vitro differentiation of embryonic stem cells. (A): Differentiating embryonic stem cells in embryoid bodies at day 3 and 5 were dissociated into single cells and stained for CXCR4 protein expression on living cells. Flow sorting analysis revealed that 3% of total cells expressed detectable levels of CXCR4 at day 3. The number of cells expressing CXCR4 increased to 50% by day 5 in embryoid bodies. (B): Differentiating embryoid bodies were harvested at 48 h intervals and lineage specific gene expression was analyzed with Taqman RT-PCR. Temporal expression dynamics of endoderm genes Sox7 and Sox17 as well as mesoderm genes Gsc and Lhx1 demonstrated peak expression levels between days 5 and 7. (C): Flk-1 (VEGF-receptor 2), a specific marker of primitive mesoderm, also demonstrated an expression profile induced at day 5 that peaked at day 7, coincident with induction kinetics of CXCR4.

 


Figure 5
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  		Figure 5. Pre-cardiac mesoderm genotype segregates with cells expressing the combination of CXCR4 and Flk1. (A): CXCR4+/Flk-1+ subpopulation distinguished from CXCR4–/Flk-1– and CXCR4–/Flk-1+ counterparts by flow cytometry. Progenitor cells were sorted from embryoid bodies at day 3, 5, and 7 using CXCR4 and Flk-1 antibodies. CXCR4 and Flk-1 cell surface protein expression was largely absent from day 3 cells with less than 3% and 1% of cells expressing each marker within the total population, respectively. Both CXCR4 and Flk-1 biomarkers were induced at day 5, consistent with temporal gene expression profile. The double positive CXCR4+/Flk-1+ subpopulation surged transiently to 32% of total population at day 5. The CXCR4–/Flk-1– (blue), CXCR4+/Flk-1+ (red) and CXCR4–/Flk-1+ (green) subpopulations were collected at day 5 for gene expression analysis (B–C). (B): Both CXCR4 and Flk-1 were highly expressed in CXCR4+/Flk-1+ cells, while CXCR4–/Flk-1+ and CXCR4–/Flk-1– subpopulations expressed, respectively, only Flk-1 at high levels or did not express either marker indicating cell sorting quality. The pluripotency marker Oct-4 was highly expressed in CXCR4–/Flk-1– cells compared to either CXCR4–/Flk-1+ or CXCR4+/Flk-1+ subpopulations. (C): CXCR4+/Flk-1+ was characterized by high expression of mesoderm markers, Lhx and Gsc, and genes associated with the primary heart field, GATA-4 and Tbx5, and with low expression of smooth muscle and vascular endothelial markers, Myh11 and CD31, compared to CXCR4–/Flk-1– or CXCR4–/Flk-1+ cells. Star indicates p < .05 throughout. Abbreviation: AU, arbitrary unit.

 
Divergence between CXCR4+/Flk-1+ and CXCR4–/Flk-1– Transcriptomes Reveals an Early Cardiovascular Developmental Program
A quality-controlled progenitor pool (Fig. 6A) was used to further document characteristic features of a developing cardiac phenotype supported by robust expression of the pre cardiac mesoderm marker Mesp-1 in CXCR4+/Flk-1+ compared to CXCR4–/Flk-1– subpopulations (Fig. 6B). Global transcriptome comparison of the two subpopulations sorted at day 5 of differentiation (Fig. 6A) demonstrated an overall 97% similarity with significant changes in a set of 818 genes (Fig. 6C and inset). This differentially expressed gene set represents 14 distinct physiological systems identified by gene ontology analysis (Fig. 6D). Priority ranking, calculated according to the number of genes contributing to functional clusters established predominance of "Cardiovascular Development" as the most significant represented functional system (Fig. 6D). Significant co-expression of both CXCR4 and Flk-1 was exclusive to the "Cardiovascular Development" classification, as interrogation of all other systems involved either CXCR4 alone or neither of the markers (Fig. 6D). Curated analysis of transcripts specifying "Cardiovascular Development" generated a network integrating CXCR4 and Flk-1 along with 16 other upregulated and nine downregulated genes (Fig. 6E). Cross-referencing with the Mouse Genome Informatics database revealed that the majority of network nodes (14/27) were directly associated with cardiovascular development (Fig. 6E). Thus, the CXCR4+/Flk-1+ subpopulation harbors a defining transcriptome fingerprint characterized by overrepresentation of cardiovascular developmental genes.


Figure 6
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  		Figure 6. Biomarker-sorted embryonic stem cells select progenitors with a pre-cardiac transcriptome. (A): Post-sort analysis of collected CXCR4–/Flk-1– (–/–) and CXCR4+/Flk-1+ (+/+) cells demonstrated >90% purity in respective subpopulations. (B): While CXCR4–/Flk-1– cells demonstrated poor immunocytochemical labeling for Mesp-1 (left), the CXCR4+/Flk-1+ subpopulation expressed high levels of this pre-cardiac mesoderm marker. (C): Volcano plot analysis segregates differentially expressed genes in CXCR4+/Flk-1+ compared to CXCR4–/Flk-1– subpopulations. Blue identifies genes significantly changing ≥1.2-fold, while gray marks all sub-threshold genes. Inset: Proportion of interrogated transcriptome showing no change (97%) and significant change (3%) between the two subpopulations. (D): Left, ontological analysis of circumscribed significantly changing genes yield overrepresented functional families, listed in order of priority. Right, Contribution of CXCR4 and Flk-1 in respective functional systems identifies the combined presence of both markers for "Cardiovascular Development". The P-values are provided with exponential notation. Dashes indicate p > .05. (E): Curated network construction integrating genes involved in "Cardiovascular Development". CXCR4 and Flk-1 are outlined, with red and green node colors indicating up- and down-regulated genes, respectively. Abbreviation: DAPI, 4',6'-diamidino-2-phenylindole.

 
CXCR4+/Flk-1+ Progeny Execute Functional Cardiogenesis
The physiological significance of the primitive cardiac developmental network intrinsic to the CXCR4 +/Flk-1+ progeny was examined through comparative differentiation with CXCR4–/Flk-1– cells. Allowed to complete differentiation in monolayers following cell sorting, CXCR4+/Flk-1+ progeny formed mesenchymal-like cellular structures containing cytoplasmic cardiac {alpha}-actinin and nuclear localized cardiac transcription factors, Mef2c and Nkx2.5 (Fig. 7A, Fig. 7B). In contrast, CXCR4–/Flk-1– counterparts were devoid of the cardiogenic phenotype (Fig. 7A, Fig. 7B), and demonstrated alternative lineage-specification characterized by differential expression of the endoderm-specific marker alpha-fetoprotein absent from CXCR4+/Flk-1+ progeny (Fig. 7C). Confirming distinct cardiac-lineage specification, RT-PCR data validated the disparate genetic programs of the CXCR4+/Flk-1+ progeny after 9 days of differentiation with robust expression of vital genes encoding for cardiac transcription factors (Mef2C, GATA-4, Myocardin, and Nkx2.5), without significant presence of ectodermal/neuroectoderm (Sox1 and Sox2) and endodermal (Sox7 and Sox17) lineage-specific markers (Fig. 7D). In contrast, the CXCR4–/Flk-1– progeny, while depleted of cardiac markers, displayed high levels of pluripotent genes (Oct 4, Fgf4) typical of the primordial germ layer and genes (Sox1, Sox2) of the neuroectoderm lineage (Fig. 7D). To monitor cardiac maturation and acquisition of functional cardiogenic traits, the mesoderm-derived subpopulation of CXCR4+/Flk-1+ sorted progenitors, now devoid of endoderm components, was co-cultured on visceral endoderm-like cells to recapitulate embryonic endoderm environment (Fig. 7E). CXCR4+/Flk-1+ progeny differentiated as layers of aggregated tissue containing beating areas, in contrast to CXCR4–/Flk-1– progeny limited to quiescent sphere-like structures that persisted beyond an extended 21-day differentiation (Fig. 7E inset). Beating activity (Fig. 7F) was recorded in ~40% of CXCR4+/Flk-1+ clusters (see also Supplemental Movie). Calcium transients, absent from CXCR4–/Flk-1– progeny, were detected in CXCR4+/Flk-1+ aggregates with initiation of beating (Fig. 7G), indicating operational excitation-contraction coupling. Thus, dual expression of CXCR4/Flk-1 biomarkers separate pluripotent, neuroectoderm and/or endoderm phenotypes from a cardiopoietic lineage.


Figure 7
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  		Figure 7. CXCR4+/Flk-1+ cells differentiate into functional cardiac tissue. (A–C): Cells sorted from day-5 old embryoid bodies were differentiated in a monolayer for 4 days. In contrast to CXCR4–/Flk-1– progeny (left panels), CXCR4+/Flk-1+ counterparts (right panels) demonstrated positive expression of cardiac {alpha}-actinin (red; A–B) with nuclear localization of cardiac transcription factors Mef2c (green; (A) and Nkx2.5 (green; (B) consistent with cardiac phenotype. Conversely, CXCR4–/Flk-1– (left panel) in contrast to CXCR4+/Flk-1+ (right panel) progeny expressed the endoderm marker {alpha}-fetoprotein AFP (green; C). Cell nuclei were counterstained with DAPI (blue; A–C). Scale bar, 20 µm (A–C). (D): Gene expression analysis using unsorted cells as baseline (100% expression) revealed CXCR4–/Flk– cells enriched for pluripotency (Oct4, Fgf4), neuroectoderm and ectoderm (Sox1, Sox2). In contrast, CXCR4+/Flk+ cells were enriched for markers of cardiac differentiation with Mef2c expression increased by ≥1200% and Nkx2.5, GATA-4, and myocardin increased by ≥300%. (E–G): Spheroids from CXCR4–/Flk– cells, morphologically consistent with less differentiated progeny, were quiescent (E, inset; n= 35), in contrast to CXCR4+/Flk+ cells that formed expansive mesenchymal-like aggregation (E; n= 17) and developed prominent beating areas (~40%) with sustained contractile activity (F). CXCR4+/Flk+ aggregates loaded with Fluo 4-AM demonstrated rhythmic intracellular calcium transients deconvoluted from line scans (G). Abbreviations: AFP, alpha-fetoprotein; AU, arbitrary unit; DAPI, 4',6'-diamidino-2-phenylindole.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cell-based de novo cardiogenesis has shown promise in cardiac regeneration [10, 13, 15–17]. However, clinical translation requires tissue-specific progenitors that balance proliferative potential with safety of a predefined cardiac fate [25, 27]. Lineage-specification of embryonic stem cells has been recently achieved through guided induction using exogenous cardiogenic factors [27] or transgenic overexpression of cardiac promoter-driven report construct [38]. As demonstrated here, cell surface biomarkers CXCR4 and Flk-1 enabled an alternative approach selecting cardiac progenitors from an unrestricted pool of pluripotent embryonic stem cells. In this way, selection of lineage-restricted progenitors containing endogenous cardiac developmental networks was achieved without pharmacologic or genetic manipulation.

Transcriptome analysis efficiently maps the genome involved in tissue-specific differentiation [32, 39–41], and was applied here to predict genes that represent spatiotemporal biomarkers of progenitor cells. Utilizing stem cell populations at discrete stages of differentiation, interrogation of stage-specific transcriptomes [27, 32] identified and prioritized expression of cardiopoietic cell surface markers. By sorting subpopulations according to CXCR4/Flk-1 and applying network analysis, refined bioinformatic recursion reduced transcriptional noise to resolve a pro-cardiogenic network that predicted ensuing cardiac differentiation. Transcriptome-wide dissection of stage-specific subpopulations thereby provided an unbiased algorithm to uncover biomarker candidates that forecast cardiac lineage from embryonic stem cells.

Identification of CXCR4 as a marker of cardiac progenitors is in line with the temporal gene expression profile of this chemokine receptor gene in embryogenesis [36, 42], which coincides with initiation of lineage-specific migration during gastrulation [24]. The significance of the CXCR4 receptor along with the sole ligand, SDF-1, during embryonic heart development is underscored by ventricular septum defects in homozygous null mutants for both CXCR4 and SDF-1 [33, 43]. The CXCR4/SDF-1 axis also provides a critical signal for cell homing and retention in the post-natal animal [44]. In fact, defective stem cell migration leading to myelokathexis was the first human deficiency attributed to CXCR4 mutations [45, 46]. Myelokathexis may also be associated with congenital cardiac malformation [47], recapitulating the cardiac developmental defects of CXCR4/SDF-1 knockout models [33, 43]. Conversely, overexpression of CXCR4 or SDF-1 in stem cells offers therapeutic advantages for heart repair [48, 49].

Furthermore, the observed association of CXCR4 with the cardiopoeitic stem cell phenotype correlated with the expression dynamics of tri-lineage differentiation [35, 50]. Individual markers are however commonly associated with multiple lineages during stem cell differentiation, and singularly may not be sufficient to distinguish lineage specification [51, 52]. In this regard, the overlapping expression kinetics of CXCR4 and Flk-1 provided a dual biomarker approach that identified a pro-cardiac population. Spatiotemporal expression patterns of CXCR4 and Flk-1 were consistent with pre-cardiac mesoderm in the developing embryo [36, 38, 53] in which Flk-1 expression is limited to mesoderm derivatives [54] and excludes CXCR4-expressing endodermal progenitors. Simultaneous co-expression for CXCR4/Flk-1 preceded expression of conventional cardiac genes, including Myocardin, Mef2C, and Nkx2.5, identifying this biomarker pair as an early predictor of cardiac fate. The cardiovascular potential of Flk-1 selected progenitors has recently been reported [38, 53]. Here, added cardiac specificity is demonstrated by segregating the Flk-1 population according to CXCR4 expression to obtain further enriched cardiopoietic progenitors containing genes, such as Mesp-1, GATA-4, and Tbx5, associated with pre-cardiac mesoderm and the primary heart field.

Indeed, isolated pre-cardiac mesoderm cells, according to CXCR4/Flk-1 co-expression, revealed distinctive genetic features of a significant cardiac developmental potential, absent in the CXCR4–/Flk-1– counterpart population. Upregulated pro-cardiac genes that include BMP-2 [55], Myc [56], Hand2 [57, 58], and GATA-6 [59] integrated into an early cardiac network, supported by downregulation of molecules that modulate differentiation (Id2) [60], antagonize TGF-β signaling (KLF2) [61, 62], or participate in oncogenesis (ERBB3) [63]. In this way, a primitive network, exposed by tandem CXCR4/Flk-1 dependent-selection, demonstrated a significant emerging property of pre-cardiac lineage specification with concomitant pluripotential deactivation. Mapped pro-cardiac frameworks in endoderm-directed embryonic stem cells [27, 64] have recently established the premise for such network evolution conferring an ontological repertoire switch in cardiopoietic precursors [32]. Recapitulation of simultaneous loss of pluripotency with cardiac specification is executed here in a discrete, pro-cardiac population procured from an embryonic stem cell source through strategic dual CXCR4+/Flk-1+ biomarker selection. The physiological significance of cardiopoietic network engagement by the CXCR4+/Flk-1+ subpopulation was validated through successful cardiac phenotype maturation with development of associated beating activity and calcium transients, hallmarks of proper cardiac differentiation [11, 13, 65], in contrast to CXCR4–/Flk-1– cells that were deprived of cardiogenic capacity. Thus, CXCR4+/Flk-1+ biomarkers identify progenitor cells programmed by pro-cardiogenic genes.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
A central aim in regenerative medicine is to balance stemness versus tissue-commitment in order to apply effective yet safe approaches for cell therapy. Therefore, selecting a cardiac progenitor cell over erratic multi-lineage embryonic stem cell populations would achieve a critical milestone for advancing cardiac repair applications. This study identifies stem cell surface biomarkers, CXCR4/Flk-1, that predict cardiopoiesis. The CXCR4+/Flk-1+ sorted population demonstrated cardiac linage propensity, away from pluripotency, neuroectoderm, or endoderm specification. While the CXCR4+/Flk-1+ transcriptome shared 97% identity with the CXCR4–/Flk-1– counterpart, the 3% differentially expressed gene set predominantly encoded cardiovascular developmental functions indicative of pre-cardiac mesoderm and the primary heart field yielding functional heart muscle tissue. This biomarker combination enables thus targeted selection of a cardiopoietic lineage to generate stage-specific, tissue-predetermined progenitors from a pool of pluripotent parental stem cells.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank James E. Tarara, Mayo Clinic Flow Cytometry/Optical Morphology Resource for his expert guidance. We also thank Lois A. Rowe for her technical assistance performing cell culture and FACS analysis, and the technical support with gene array technology by the Mayo Advanced Genomics Technology Center core. This work was supported by grants from the National Institutes of Health, Marriott Heart Disease Research Program, Marriott Foundation, Ted Nash Long Life Foundation, Ralph Wilson Medical Research Foundation, and Asper Foundation. T.J.N. and A.B. are supported by Mayo Clinic Clinician-Investigator Program, and A.C. and R.J.D.C. by Mayo Graduate School.


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 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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