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Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation

Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto city, Kyoto, Japan

¹Correspondence: Department of Pediatrics, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho 54, Sakyo-ku, Kyoto city, Kyoto, Japan 606-8507. E-mail: heike{at}kuhp.kyoto-u.ac.jp

	ABSTRACT

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We evaluated the expression of the FLK1, one of the lateral mesoderm early markers where cardiogenesis occurs, to characterize and isolate cardiac stem/progenitor cells from ES cells. Dissociated cells from embryoid bodies (EBs) on day 3, 4, or 5 were collected into two subpopulations with or without FLK1 expression and coculture on OP9 stromal cells was continued to examine whether contracting colonies came out or not. FLK1⁺ cells from EBs at days 3 and 4 formed spontaneous contracting colonies more efficiently than FLK1^– cells on the same days, but not at day 5. Most contracting cardiac colonies derived from FLK1⁺cells mainly on day 4 were detected on endothelial cells along with hematopoietic cells. Further characterization of cells with these capabilities into three lineages revealed the FLK1⁺ CD31^–VE-cadherin^– phenotype. Our findings indicate that FLK1⁺cells, especially FLK1⁺ CD31^–VE-cadherin^– cells, could act as cardiohemangioblasts to form cardiac cells as well as endothelial cells and hematopoietic cells.—Iida, M., Heike, T., Yoshimoto, M., Baba, S., Doi, H., Nakahat, T. Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation.

Key Words: embryoid bodies • ES cells • cardiomyocyte differentiation • blastocysts

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
CURRENT THERAPEUTIC MODALITIES to treat end-stage cardiac failure include pharmacological therapy and mechanical left ventricular assist devices, but their applications are limited because they are inadequate. Cardiac transplantation is the ultimate treatment of choice for end-stage cardiac disease but is hampered by the limited availability of donor organs, complications of immunosuppressive therapy, and long-term failure of grafted organs (1 2 3 4 5) . Thus, the development of alternative therapeutic strategies to overcome intractable cardiac disease remains an important therapeutic goal.

Embryonic stem (ES) cells are continuously growing stem cell lines of embryonic origin first isolated from the inner cell mass of mouse blastocysts. These unique cells are characterized by their capacity to proliferate in undifferentiated state for a prolonged period in culture and by their ability to differentiate into every tissue type in the body. It is anticipated that ES cells might become feasible sources to repair or regenerate ischemic or damaged tissues, including the heart. Investigation of murine ES cells has reviewed important insights into the early steps of development in the mammalian heart, including patterns of gene expression, myofibrillogenesis, ion channel development and function, receptor development, and calcium handling (6) . ES cells can differentiate into various derivatives of three primary germ layers, including cardiomyocytes as well as neuronal cells and hematopieitic cells simultaneously in vitro, leading to the difficulty of isolating tissue-specific cells. This difficulty in preparing pure populations of cardiac lineage has hampered dissection of the mechanism underlying cardiac lineage differentiation. Therefore, development of the privileged characterization system into cardiac lineage is indispensable for further investigation and clinical application.

During differentiation into hematopoietic or endothelial cells from ES cells in vitro, characterization of stem/progenitor cells into these lineages was done using flow cytometer based on cell surface molecules (7 8 9) . FLK1, a receptor for vascular endothelial growth factor, is a marker for lateral plate mesoderm and the earliest differentiation marker for endothelial cells and blood cells (10) . Nishikawa et al. have elucidated that FLK1 could be induced during differentiation of ES cells using an EB system or 2-dimensional culture on plates coated with type IV collagen and characterized hemagioblasts, whose derivatives could form blood cells and vessels (9 , 11) . In a cardiac development system, cell sorting approaches have been done based on the expression of transcription factors. Generally, expression of transcription factor genes precedes that of structural genes. Several transcription factors are expressed in the heart primordium, where they play a pivotal role during heart development by controlling the expression of many cardiac muscle-specific genes (12) . Among these, Nkx2.5 is known to be expressed most strongly in cardiomyocytes of the heart tube during early cardiac development (13) . Hidaka et al. generated an ES cell line in which a GFP reporter gene had been knocked into one of the Nkx2.5 loci and isolated the cells, which differentiated into cardiomyocytes during in vitro culture (14) . However, Nkx2.5 expression cannot be evaluated by flow cytometer based on its expression on cell surface; therefore, this methodology encounters difficulties when applying the characterization of cardiac stem/progenitor cells from the ES cells line without a genetical marker insertion under Nkx2.5 gene promoter before use.

The heart originates from the splanchnic mesoderm. During development, two primordia of epithelial cells and precardiac mesoderm are observed at paired regions of anterior lateral mesoderm that eventually fuse in the midline of the body, leading to formation of a tubular heart. Cells from the lateral mesodermal region are known to express a FLK1 gene differentially with the potential to differentiate into hematopoietic and endothelial cells (15 , 16) . In this paper, we evaluated the differentiation potential of cells with FLK1 expression derived from EB cells and elucidated that these cells could differentiate into cardiomyocytes as well as hematopoietic or endothelial cells.

	METHODS

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Cell lines
EB5 ES cell line and its derivative, the GACT4 ES cell line, produced by stable transfection of EGFP gene driven by ubiquitous CAG promoter were kindly provided by Dr. Ogawa. The self-renewal activity and differentiation potential could not be distinguished between these lines. The continuous expression of GFP gene during differentiation was confirmed by flow cytometry (data not shown). The cells were maintained on gelatin-coated tissue culture dishes in GMEM containing 10% knockout-SR (Gibco, Grand Island, NY, USA), 1%FCS, 5 x 10^–5 M² mercaptoethanol (2ME), nonessential amino acids, 5000 units/mL leukemia inhibitory factor, Puromycin, and Brastcidine. The OP9 stromal cell line, kindly provided by Dr. Kodama, was cultured as described previously (17 , 18) .

Differentiation of ES cells
ES cells were differentiated as described previously (15) . Briefly, single-cell dispersions of undifferentiated ES cells were suspended in -MEM supplemented with 10% FCS and 5 x 10^–5 M 2ME at a concentration of 2 x 10⁴ cells on bacteriological grade 100 mm Petri dishes. On days 3–5 after differentiation, formed EBs were subjected to additional experiments.

FACS analysis and in vitro cultures of sorted cell populations
ES cells were harvested with a cell dissociation buffer (Gibco), followed by dissociation. Dissociated single cells were stained with phycoerythrin (PE) -conjugated anti-FLK1 antibody (PharMingen, San Diego, CA, USA) and PECAM-1 (anti-CD31) (PharMingen). Monoclonal antibodies AVAS12 (anti-FLK1) (8) and VECD (anti-VE-cadherin) (19) were purified from hybridoma cultured supernatants on a protein G-Sepharose column (Pharmacia, Piscataway, NJ, USA) and labeled with allophycocyanin. These antibodies had not influenced the differentiation potential of ES cells (7 8 9 , 11) . Sorted FLK1 positive and negative cells were cocultured with OP9 stromal cells at a cell density of 2 x 10³/well in 24-well plate culture dishes. FACS sorting systems in this study did not damage ES cell differentiation as in previous reports (9 , 11) . These experiments were repeated 10 times and quantified the differentiation capacity.

Immunohistochemical staining
For immunological staining of the contracting cardiac colonies derived from FLK1⁺and FLK1^–cells on OP9 stromal cells, cultured cells were fixed in situ by 4% paraformaldehyde in phosphate1-buffered saline (for 10 PBS) min at 4°C. After washing with PBS, 2% skim milk in PBS was incubated as a blocking solution for 1 h at room temperature. The fixed dishes were incubated with MF20, monoclonal antibody against myosin heavy chain (Hybridoma bank) and anti-CD45 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) overnight at 4°C, followed by incubation with alkaline phosphatase (ALP)-conjugated antimouse IgG (H⁺L) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 1 h at room temperature. After each step, the cultured cells were washed three times with PBS containing 0.05% Tween 20 (Wako Chemical, Kyoto, Japan). Cells were visualized using 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) substrate (Roche Molecular Biochemicals, Basel, Switzerland). Endogenous ALP activity was blocked by 2 mmol/L levamisole (Sigma, St. Louis, MO, USA) before incubation with MF20. Anti-CD45 antibody was peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories Inc.) and visualized using DAB Substrate Kit (Vector Laboratories, Inc., Burlingame, CA, USA).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from FLK1⁺ and FLK1^–cells dissociated before and after coculture with OP9 cells by acid-phenol extraction using Trizol Reagent (Gibco). Reverse transcription was performed with 1 µg of total RNA treated with RNase-free DNaseI (Gibco) as a template and oligo (dT) as a primer. The cDNA was amplified by PCR using the following primers: brachyury-specific primers 5'-TGCTGCCTGTGAGTCATAAC-3' and 5'-TCCAGGTGCTATATATTGCC-3', which should give a 947 bp product; goosecoid-specific primers 5'-GCACCATCTTCACCGATGAG-3' and 5'-AGGAGGATCGCTTCTGTCGT-3', which should give a 179 bp product; Nkx-2.5-specific primers 5'-GTGGGTCTCAATGCCTATG-3' and 5'-CTCTTTCCCTACCAGGCTC-3', which should give a 233 bp product. PCR conditions for brachyury and goosecoid were performed by denaturing the DNA at 94°C for 5 min, followed by 30 cycles of amplification: 94°C for 30 s, 60°C for 30 s, 72°C for 60 s, and a final extension step at 72°C for 7 min. For Nkx2.5, PCR reactions were performed by denaturing the DNA at 94°C for 5 min, followed by 30 cycles of amplification: 94°C for 30 s, 60°C for 30 s, 72°C for 60 s, and a final extension step at 72°C for 7 min. To normalize the amount of template, 18S rRNA was used as an internalcontrol according to the manufacturer’s instruction (QuantumRNA Internal Standards Kit, Ambion, Austin, TX, USA). Amplified fragments were separated on a 2.0% agarose gel and visualized with ethidium bromide staining.

	RESULTS

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Differential progression into contracting cardiomyocytes between FLK1⁺ and FLK1^–cells from EBs
ES cells do not express FLK1 gene in an undifferentiated state. However, differentiation in EB culture induces various kinds of differentiated markers, including FLK1. We first evaluated the temporal switch of FLK1 gene expression during EB culture on days 3–5 sequentially. As demonstrated in Fig. 1 A, FLK1⁺ cells could be detected during this culture although barely detected in undifferentiated state at day 0. Day 4 was when the highest percentage of FLK1⁺ cells could be detected. FLK1⁺ cells or FLK1^– cells generated from EBs on days 3–5 were sorted, respectively, followed by coculture on OP9 stromal cells (Fig. 1B ). Expression of brachyury gene could be detected in FLK1⁺ cells but not in FLK1^– cells (data not shown). FLK1⁺ cells and FLK1^– cells generated from EBs on day 3 differentiated into contracting cardiac colonies efficiently after a further 8-day culture on OP9 cells (day 3+7), though differentiation from FLK1⁺cells preceded by 1 day that of FLK1^–cells. Mouse fibroblast cell line 3T3 could not support this differentiation at all (data not shown). Similarly, both populations from EBs on day 4 differentiated into contracting cardiac colonies; differentia-tion from FLK1⁺cells preceded by 1 day that of FLK1^–cells. In contrast, FLK1⁺cells and FLK1^– cells from EBs on day 5 failed to form contracting cardiac colonies. This experiment was repeated 10 times with the same tendency, describing a representative result. These results imply that both populations of FLK1⁺ and FLK1^–cells on day 3 or 4 had the capability of differentiation into contracting cardiac cells, with a higher tendency in FLK1⁺ cells.

View larger version (61K): [in this window] [in a new window]   Figure 1. A) ES cells were differentiated as described (17) . On days 3–5 after differentiation, EBs formed were stained with phycoerythrin (PE)-conjugated anti-FLK1 antibody. Stained cells were sorted using a FACS Vantage cell sorter. Sorted FLK1 positive and negative cells were cocultured with OP9 stromal cells at a cell density of 2 x 103/well in 24-well plate culture dishes. Contracting cardiac colonies after 7-day culture on OP9 cells (day 3+7, day 4+7, and day 5+7) differentiated from FLK1+ cells and FLK1– cells generated from EBs on days 3–5. B) Total RNA was isolated from FLK1+ cells and FLK1– cells were generated from EBs on day 3, 4, and 5. By acid-phenol extraction using Trizol Reagent (Gibco), reverse transcription was performed with 1 µg of total RNA treated with RNase-free DNaseI (Gibco) as a template and oligo (dT) as a primer. PCR amplification was performed by RT-PCR analysis for cardiac muscle-specific gene and early mesoderm markers after sorting. Amplified fragments were separated on a 2.0% agarose gel and visualized with ethidium bromide staining.

Cardiac muscle-specific gene expression during differentiation into cardiomyocyte
We examined the expression of cardiac muscle-specific or mesoderm-specific gene during differentiation. RT-PCR analysis was performed to determine the expression of cardiac muscle-specific gene Nkx2.5 (20 , 21) and expression of early mesoderm markers goosecoid and brachyury (22 , 23) (Fig. 1B ). It was confirmed that OP9 cells did not express early mesoderm markers (goosecoid and brachyury) (data not shown). Cardiac muscle-specific Nkx2.5 gene was already detected in FLK1⁺ cells from dissociated EBs on days 3–5, with the highest expression on day 4. In FLK1^– cells, Nkx2.5 gene expression could be faintly or barely confirmed. In contrast, the highest expression of the early mesoderm markers goosecoid and brachyury was confirmed in both populations from EBs at day 3, but decreased gradually on days 4 and 5. The results imply that each population of cells from EBs on days 3–5 has different properties as to developmental stage, leading to the distinct differentiation capability.

Simultaneous generation of endothelial cells and hematopoietic cells during Flk1⁺ cell differentiation into cardiomyocytes
FLK1⁺ cells or FLK1^– cells generated from EBs on days 3 and 4 differentiated into contracting cardiac colonies after coculture on OP9 cells. During differentiation of FLK1⁺ cells from EBs at day 4, endothelial sheet formations were observed along with contracting cardiac colonies for the most part, some of which were accompanied with blood cells simultaneously (Fig. 2 A-b, e, B). These observations were not identified in FLK1^– cells from day 4. On day 3, a part of contracting colonies derived from FLK1⁺ population was accompanied with endothelial cells and hematopoietic cells but not from the FLK1^– population (Fig. 2A-a, d ). FLK1⁺cells and FLK1^– cells from EBs on day 5 did not differentiate into contracting cardiac colonies, although FLK1⁺cells preserved the capability to differentiate into cobble stone-like hematopoetic clusters and endothelial sheets, which were not identified in FLK1^– cultures (Fig. 2A-c, f ). These observations imply that FLK1⁺cells or FLK1^– cells had different differentiation potential depending on the harvesting date during EB culture, and only FLK1⁺cells exhibited the phenotype of cardiohemangioblasts.

View larger version (119K): [in this window] [in a new window]   Figure 2. A) Differentiation of FLK1+ cells from EBs on days 3 (a), 4 (b), and 5 (c) at day 7 after sorting, respectively; day 3+7, day 4+7, and day 5+7 and FLK1– cells from EBs on day 3 (d), 4 (e), and 5 (f). FLK1+ and FLK1–cells from EBs on days 3 and 4 were differentiated into contracting cardiac colonies (arrows) (a, b, d, e). Asterisks indicate endothelial cells under contracting cardiac colony-derived FLK1+ cells. B) Expression of sarcomic myosin on day 4+7. Contracting cardiac colonies (arrows) derived from FLK1+ cells from EBs on day 4 were stained with antibodies to sarcomic myosin (MF20) on OP9 cells.

Conversion of FLK1^– cells into FLK1⁺ cells during coculture on OP9 cells
Although FLK1^–cells from EBs on day 4 did not express the Nkx2.5 gene, they could differentiate into contracting cardiac muscle cells with a 1-day delay compared with FLK1⁺ cells. Nkx2.5 gene is a crucial transcription factor for heart development, so Nkx2.5 gene expression was evaluated during the differentiation into contracting cardiac cells of these FLK1^–cells sequentially (Fig. 3 ). As shown in Fig. 3 , expression of the Nkx2.5 gene was confirmed in a 1-day delayed manner in FLK1^–cells vs. FLK1⁺ cells, which corresponded to the delay of phenotypical change. To investigate the role of FLK1 in this system further, we examined the phenotypical conversion of FLK1^–cells into FLK1⁺ cells during coculture on OP9 cells using ES cells with the GFP gene driven under CAG promoter. After 1-day coculture of FLK1^–cells from EBs at day 4, cells were harvested according to GFP expression and resorted according to the FLK1 expression into two populations with or without FLK1 expression. Surprisingly, nearly 0.1% of FLK1^– cells changed their phenotype into FLK1⁺ cells. These two populations continued to be cultured on OP9 cells as described above (Fig. 4 A). Resorted FLK1⁺ cells formed contracting colonies more efficiently than resorted FLK1^– cells (Fig. 4B ). In addition, contracting colonies derived from resorted FLK1⁺cells began to be detected 1 or 2 days earlier than FLK1^– cells. These results suggest that FLK1^– cells might differentiate into contracting cardiac cells after converting into FLK1⁺ cells predominantly.

View larger version (27K): [in this window] [in a new window]   Figure 3. RT-PCR analysis for cardiac muscle-specific gene on sorted FLK1+ and FLK1– cells generated from EBs on day 4. Total RNA was isolated from FLK1+ and FLK1–cells dissociated after coculture with OP9 cells by acid-phenol extraction using Trizol Reagent (Gibco). Reverse transcription was performed with 1 µg of total RNA treated with RNase-free DNaseI (Gibco) as a template and with oligo (dT) as a primer, then PCR amplification was performed

View larger version (18K): [in this window] [in a new window]   Figure 4. A) Schema of our FACS sorting system for cardiac differentiation. We sorted FLK1+ cells and FLK1– cells from EBs on day 4, recultured 2000 cells/well of 24-well OP9 stromal cell-coated plates, resorted FLK1–cells on day 4+1, and recultured them on OP9 stromal cells in the same way. B) Resorted FLK1+ cells and FLK1– cells were differentiated into contracting cardiac colonies.

Further characterization of cardiac stem/progenitor cells in FLK1⁺cells
We further classified FLK1⁺cells from EBs on day 3, 4, or 5 according to the expression of endothelial markers CD31 and VE-cadherin and sorted them into the following four populations: cells expressing both FLK1 and CD31 (F⁺31⁺), cells expressing FLK1 without expression of CD31 (F⁺31^–), cells expressing both FLK1 and VE-cadherin (F⁺V⁺), and cells expressing FLK1 without expression of VE-cadherin (F⁺V^–), respectively (Fig. 5 A). As shown in Fig. 5B , F⁺31^– or F⁺V^– cells from EBs on days 3 and 4 differentiated predominantly into contracting cardiac cells. Another experiment of single-cell culture with F⁺31^– V^– characteristics demonstrated the differentiation into three lineages of cardiac cells as well as endothelial and hematopoietic cells, confirmed by immunohistochemical analysis (Fig. 6 A–C). These results support the notion that cardiac progenitor cells can be identified as F⁺31^–V^–cells.

View larger version (28K): [in this window] [in a new window]   Figure 5. A) Dissociated single cells were stained with PE-conjugated anti-FLK1 antibody (PharMingen) and PECAM-1 (anti-CD31) (PharMingen). FACS analyses of FLK1, CD31, and VE-cadherin expression during EB development. B) EBs on days 3–5 were sorted for FLK1+CD31+, FLK1+CD31–, FLK1+VE-cadherin+, and FLK1+VE-cadherin– cells. The contracting colonies were counted on days 3+7, 4+7, and 5+7.

View larger version (82K): [in this window] [in a new window]   Figure 6. A) FLK1+CD31–VE-cadherin– single cells were cocultured on 96-well OP9 stromal cells-coated plates. B) Expression of the endothelial cells were characterized using acetylated low density lipoprotein (DiI-Ac-LDL) (Molecular Probes). C) Single-cell culture stained with antibodies to sarcomic myosin (blue) and CD45 (PharMingen) (brown).

	DISCUSSION

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In this paper, we have characterized the cardiac stem/progenitor cells from ES cells using the FACS sorting systems. Cardiac cell differentiation from ES cells could be easily induced in culture in vitro, and many extensive studies have been done from the viewpoint of a functional or physiological standpoint up to now (24) . However, this conventional differentiation system comprises various kinds of differentiated tissues other than cardiac tissue; therefore, precise evaluation of cardiomyocyte differentiation has been prevented. In this study, we evaluated the expression of the FLK1 gene, an early marker of lateral mesoderm where cardiogenesis occurs (25) , to characterize and isolate cardiac stem/progenitor cells from ES cell. We demonstrated that FLK1, especially FLK1⁺CD31^–VE-cadherin^–, was a feasible marker for detecting cardiac stem/progenitor cells.

Several transcription factors are expressed in the heart primordium, where they play a pivotal role during heart development by controlling the expression of many cardiac muscle-specific genes (12) . Among these, Nkx2.5 is known to be expressed most strongly in cardiomyocytes of the heart tube during early cardiac development (13) and is recognized as an early marker for cardiac cell differentiation (20 , 21) . In our experiments, Nkx2.5 gene expression was always detected before that of contracting characteristics, implying that the commitment to cardiac differentiation could be concluded by the acquisition of Nkx2.5 gene expression. Cells with Nkx2.5 gene expression were recognized cardiac stem/progenitor cells. Our PCR experiments suggested that close correlation between expression of the Nkx2.5 gene and that of FLK1 was confirmed, implying that FLK1 expression might become a feasible marker for detecting cardiac stem/progenitor cells. This interpretation could also be supported by the observation that FLK1^– cells differentiated into contracting muscle cells 1 or 2 days later than FLK1⁺ cells, synchronous with Nkx2.5 gene expression. In contrast, FLK1⁺ cells from EBs on day 5 could not differentiate into contracting cardiac muscle cells, implying FLK1 itself is not the sole decisive marker for cardiac stem/progenitor cells and that cells with FLK1 expression are composed of heterogeneous populations from the viewpoint of development. This discrepancy might be explained by the difference in expression patterns of the brachyury or goosecoid gene. FLK1⁺ cells from EBs on day 3 or 4 expressed brachyury, or goosecoid gene, but not on day 5, reflecting the immaturity of FLK1⁺ cells from EBs on day 3 or 4. These fluctuated expressions of Nkx2.5, brachyury, and goosecoid genes during these 3 days suggested that FLK1⁺ cells might be different qualitatively during this period. Taken together, FLK1⁺ cells from EBs on day 3 or 4 might be regarded as cardiac stem/progenitor cells. It suggested that FLK1⁺ cells had quite different characters during differentiation on days 3, 4, and 5; however, until now FLK1 expression was attained only during mesodermal differentiation. Furthermore, these FLK1 characters showed that endothelial and hematopoietic cells had not enhanced development of the cardiomyocyte.

Using the in vitro ES differentiation model system, G. Keller et al. had shown that ES cells differentiated for 2.5–3.5 days contained a unique cell population, the blast colony-forming cell (BL-CFC) (26 , 27) . BL-CFCs form blast colonies in response to vascular endothelial growth factor (VEGF), a ligand for the receptor tyrosine kinase FLK1 (28) , suggesting that these cells express FLK1 gene. These observations are consistent with the interpretation that a fraction of the FLK1⁺ cells would represent the hemangioblasts (29) . Our findings demonstrated that contracting cardiac colonies derived from FLK1⁺ cells at day 4 appeared on endothelial sheets, sometimes with blood cells, supporting the idea that FLK1⁺ cells on day 4 contained three populations: cardiac progenitor cells, endothelial progenitor cells, and hematopoietic progenitor cells. We analyzed, sorted, and recultured FLK1⁺ cells on day 3, 4, or 5 under further classification using VE-cadherin or CD31. Our studies demonstrated that more FLK1⁺CD31^– or FLK1⁺ VE-cadherin^– cells on day 4 differentiated into contracting colonies than FLK1⁺CD31⁺ cells and FLK1⁺VE-cadherin⁺ cells, which predominantly differentiated into endothelial cells, hematopoitic cells. However, these observations could not clarify whether only one kind of single cardiohemangioblasts exists with differential capability into three lineages or whether this population is still heterogeneous, with differential potential only into one or two lineages. Additional experiments are needed to resolve this question.

In our experiments, FLK1^– cells from EBs on day 3 or 4 could differentiate into contracting cardiac cells. This contradicted our idea that FLK1 could be a good marker to isolate cardiac stem/progenitor cells from ES cells. However, precise examination of FLK1 expression on FLK1^– cells 1 day after plating on OP9 cells revealed that conversion of the FLK1 gene in nearly one-third of FLK1^– cells of cells could be observed from negative to positive. Replating of FLK1⁺ cells or FLK1^– cells on OP9 cells (derived from FLK1^– cells at day 4) revealed that FLK1⁺ cells differentiated into contracting cardiac muscle cells predominantly. On the other hand, when we plated sorted cells on fibroblast cells, gelatin-coated plates, and collagen type IV-coated plates, the contracting cardiac cells either did not show or were only a few. These results imply that FLK1^– cells with the ability to differentiate into cardiac cells change their phenotype from negative to positive before differentiating into contracting cardiac cells predominantly, leaving the possibility that FLK1^– cells could still differentiate into cardiac cells directly.

In conclusion, the findings in this study demonstrate that FLK1 plays a significant role not only in hematopoietic and endothelial differentiation, but also in cardiac differentiation, and that cardiohemangioblasts populations contribute somewhat to the formation of contracting cardiac muscle cells. The development of isolation and culture conditions described here in which ES cells depend on cardiohemangioblasts to initiate cardiac differentiation will greatly facilitate further analysis of cardiac stem/progenitor cell specification and cell therapy.

	ACKNOWLEDGMENTS

 
This study was supported by a Health and Labor Science Research Grant, Research on Human Genome, Tissue Engineering Food Biotechnology, Ministry of Health, Labor and Welfare, a Grant-in Aid for Science Research on Priority Areas 122150-67, and a Grant-in-Aid for Creative Scientific Research 13GS-0009.

Received for publication May 3, 2004. Accepted for publication November 1, 2004.

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TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

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