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Prospective identification of cardiac progenitors by a novel single cell-based cardiomyocyte induction

Jun K. Yamashita^*,,1, Makoto Takano, Mina Hiraoka-Kanie^*,, Chikashi Shimazu^*, Yan Peishi^*, Kentoku Yanagi^*, Akiko Nakano^*, Emi Inoue^*, Fumiyo Kita^* and Shin-Ichi Nishikawa^||

^* Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;
PRESTO, Japan Science and Technology Agency, Saitama, Japan;
Department of Physiology, Jichi Medical School, Tochigi prefecture, Japan;
Department of Tissue Engineering and Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan; and
^|| Laboratory for Stem Cell Biology, Center for Developmental Biology, RIKEN, Kobe, Japan

¹Correspondence: Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 Japan. E-mail: juny{at}frontier.kyoto-u.ac.jp

SPECIFIC AIMS

The goal of the present study is the elucidation of cardiomyocyte differentiation mechanisms and exploration of novel cardiac regeneration strategies. We have developed a novel in vitro cardiomyocyte differentiation system using embryonic stem (ES) cells that enables reproduction of cellular process of cardiomyocyte differentiation in vitro and the dissection of the cellular and molecular mechanisms of cardiomyocyte differentiation at the single cell level.

PRINCIPAL FINDINGS

1. Induction and purification of cardiomyocytes from Flk1⁺ mesoderm cells on 2-D culture
Earlier we established an ES cell differentiation system for blood vessels in a 2-dimensional (2-D) culture devoid of embryoid body (EB) formation that reproduces early vascular development from progenitor cells expressing Flk1, a vascular endothelial growth factor receptor. In this study, we induced cardiomyocytes from Flk1⁺ mesoderm cells on 2-D culture. Flk1⁺ cells were induced and purified using an ES cell line carrying -myosin heavy chain promoter-driven GFP gene. Undifferentiated ES cells (E-cadherin⁺) maintained with leukemia inhibitory factor (LIF) were plated onto type IV collagen-coated dishes and cultured in the absence of LIF to induce differentiation. After 96 to 108 h of differentiation, induced Flk1⁺ (E-cadherin^–) cells were sorted and purified by FACS, then recultured (Fig. 1 A). When Flk1⁺ cells were plated onto OP9 stroma cells, which were established from the calvaria of op/op mice, spontaneously beating cells begin to be observed from day 4 or 5 of Flk1⁺ cell culture (Flk-d4, 5) (Supplemental movie 1). Beating cell colonies then grew large and made some remodeling to show cavity-like or network-like structure formation during 1 or 2 wk culture of Flk1⁺ cells (Supplemental movies 2, 3).

View larger version (55K): [in this window] [in a new window]   Figure 1. Identification of cardiac progenitor potentials. A) FACS analyses of Flk1+ cell culture at Flk-d2. PKH-negative populations (ES cell-derived cells) were analyzed. Purified quadrant populations by Flk1 and various markers (CXCR4, c-kit, sca-1, CD44, and CD90) were plated on OP9 and examined cardiomyocytes appearance at Flk-d6 by immunostaining for cTnT. B) Enriched cardiogenic potential in Flk1+/CXCR4+ population. cTnT staining (dark gray). Scale bar: 400 µm. C) Quantitative evaluation of cardiomyocyte induction by fluorescent intensity of cTnT staining (n=9, *P<0.05, **P<0.01). D) RT-PCR analysis for differentiation markers during cardiomyocyte induction. ES: undifferentiated ES cells, Flk1+: purified Flk1+ cells at Flk-d0, FCV: purified Flk1+/CXCR4+/VE-cadherin– cells at Flk-d2, GFP+: purified GFP+ cardiomyocytes.

During the differentiation, bright GFP-positive beating cells were observed at almost the same timing as the appearance of beating cells (Supplemental movie 4). At day 6 of Flk1⁺ cell culture (Flk-d6), 10–18% of ES-derived cells were GFP⁺ cardiomyocytes, which was >2- or 3-fold higher in induction efficiency than that of the EB method. Purified GFP⁺ cardiomyocytes could be recultured on type I collagen gel and could form synchronously contracting cell sheets in the absence of stroma cells (Supplemental movie 5).

2. Characterization of induced cardiomyocytes
Induced spontaneously beating colonies were positive for various cardiomyocyte markers such as cardiac troponin-T (cTnT), ventricular myosin, atrial natriuretic peptide, Nkx2.5, GATA4, and connexin43. Action potential in recultured GFP⁺ cells showed the existence of cells with pacemaker potential and spontaneous beating at various rates, as well as ventricular type cells lacking pacemaker potential that contracted only by electrical stimulation, indicating that cardiomyocytes are induced from Flk1⁺ cells as a mixture of various cardiac cell types (Supplemental movie 6).

3. Role of OP9 stroma cells in cardiomyocyte induction
Cardiomyocytes appeared only when Flk1⁺ cells were cultured on viable OP9 cells. OP9 cells were then prestained with PKH fluorescent dye and Flk1⁺ cells were plated on prestained OP9 cells. FACS analysis at Flk-d6 revealed that PKH⁺ OP9 and induced GFP⁺ cardiomyocytes were largely mutually exclusive, indicating that the main mechanism of cardiomyocyte differentiation on OP9 is fusion-independent.

4. Cardiomyocyte induction from a single progenitor cell
Single Flk1⁺ cells plated onto OP9 cells successfully differentiated into cardiomyocytes (Supplemental movie 7). Double immunostaining with anti-CD31 and anti-cTnT antibody at Flk-d6 demonstrated that three types of colonies appeared from single Flk1⁺ cells: endothelial cell only (EC), cardiomyocyte only (CC), and mixtures of both (Mix). The majority of colonies were either EC (40%) or Mix (43%) colonies; CC colonies (16%) were the minority. These results indicate that Flk1⁺ cells possess the progenitor potential to give rise to cardiomyocytes as well as endothelial cells at the single cell level.

5. Prospective identification of cardiac progenitor cell populations
At Flk-d2, we harvested and recultured the cells on OP9 after subdivision by FACS using various molecular markers (Fig. 1A ). Though reported cardiac stem or progenitor markers, c-kit and sca-1, and mesenchymal stem cell markers such as CD44, CD90, and others were examined together with Flk1 (Fig. 1A ), they failed to prescribe subpopulations from which cardiomyocytes were specifically induced. Cardiomyocytes appeared from CXCR4-expressing cells but not from CXCR4^– cells. We sorted four different cell populations subdivided by Flk1 and CXCR4 expressions and recultured the cells on OP9 for 4 days. cTnT staining demonstrated that cardiomyocyte differentiation potential was highly enriched in Flk1⁺/CXCR4⁺ population that occupied only several percentages of total cells at Flk-d2 (Fig. 1A, B ). Evaluation by fluorescent intensities of cTnT staining showed that 15-fold more cardiomyocytes appeared from Flk1⁺/CXCR4⁺ population than with Flk1^–/CXCR4^– cells (Fig. 1C ). RT-PCR analysis during cardiomyocyte differentiation further demonstrated the cellular process of cardiomyocyte differentiation (Fig. 1D ). An undifferentiated ES cell marker, Oct3/4, was observed only in undifferentiated ES cells. Flk1 and CXCR4 were expressed in Flk1⁺ cells at Flk-d0 and Flk1⁺/CXCR4⁺ but VE-cadherin^– cells at Flk-d2 (FCV cells), but not in GFP⁺ cardiomyocytes. GATA4 was observed from Flk1⁺ cells at Flk-d0 to GFP⁺ cardiomyocytes. Nkx2.5 was first detected in FCV cells. Mature cardiomyocyte markers such as MHC, myosin light chain (MLC) 2v and MLC2a were observed only in GFP⁺ cardiomyocytes. These results indicate that this differentiation system can represent the sequential process of cardiac development at the cellular level, and FCV cells at Flk-d2 are postulated as cardiac progenitor cells.

6. Cardiac progenitor potential at single cell level
Single FCV cells showed more cardiac-specific differentiation potentials than Flk1⁺ cells. CC colonies became dominant (64%), and colonies, including cardiomyocytes (Mix and CC colonies) reached 80% of total colonies (Mix 16%, EC 20%). FCV cells thus have been prospectively demonstrated to possess specified cardiac progenitor potential at the single cell level.

7. Role of Noggin and wnt inhibitors in cardiomyocyte induction
Addition of a BMP inhibitor, noggin, to Flk1⁺ cell culture on OP9 strongly inhibited cardiomyocyte differentiation (Fig. 2 A, B). Cardiomyocyte population decreased to 10% of control (Fig. 2G ). Surprisingly, FACS analysis for FCV cells demonstrated that noggin treatment resulted in almost a total disappearance of FCV cardiac progenitor population (Fig. 2H, I ). These results suggest that noggin suppressed cardiomyocyte differentiation by inhibiting the induction of cardiac progenitors from mesoderm cells.

View larger version (74K): [in this window] [in a new window]   Figure 2. Role of Noggin and wnt inhibitors in cardiomyocyte induction. A–F) Immunostaining of Flk1+ cell culture on OP9 at Flk-d6 for cTnT (brown). A) Control. B) Noggin (300 ng/mL) treatment. C) Dkk-1 (0.1 ng/mL). D) Frizzled-8/Fc (200 ng/mL). E) wnt3a (5 ng/mL). F) wnt3a (1.5 ng/mL) and Frizzled-8/Fc (500 ng/mL). Scale bars: 400 µm. G) Quantitative evaluation of cardiomyocyte induction by fluorescent intensity of cTnT staining. (n=9, *P<0.05, **P<0.01 vs. control). H) FACS analysis of Flk1+ cell culture on OP9 at Flk-d2. I) Noggin-treated cells. Flk1+/CXCR4+ population (FCV cells; arrow) observed in control almost completely disappeared.

On the other hand, Wnt inhibitors Dkk-1 and Frizzled-8/Fc chimera induced an 1.5-fold increase of cardiomyocyte differentiation from Flk1⁺ cells (Fig. 2C, D, G ). On the contrary, wnt3a strongly suppressed cardiomyocyte induction from Flk1⁺ cells (Fig. 2E, G ). The inhibitory effect of wnt3a on cardiomyocyte induction was almost completely reversed by simultaneous administration of Dkk1 or Frizzled-8/Fc (Fig. 2F, G ). These results indicate that wnt signaling exerts an inhibitory effect on cardiomyocyte differentiation from Flk1⁺ mesoderm cells.

CONCLUSIONS AND SIGNIFICANCE

Here we have described a novel cardiomyocyte induction method that is amenable to trace the differentiation process at the cellular level. This achievement enabled prospective identification of cardiac progenitor potentials that is impossible in the conventional EB method. Indeed, we identified a novel cardiac progenitor population, FCV cells.

In our culture system, OP9 cells may play a role similar role to that of the endoderm in cardiomyocyte induction. This in vitro differentiation system should reproduce embryonic cardiomyocyte differentiation process; pluripotent cells -> mesoderm cells -> cardiac progenitors -> cardiomyocytes.

Though knockout studies are able to identify an essential molecule in normal development, it is difficult to identify stem/progenitor cells and trace their differentiation process. In our ES cell system, it is possible to systematically induce cardiovascular cells from common progenitor cells and constructively reproduce their differentiation pathway to form the cardiovascular system (Fig. 3 ). This novel approach would provide novel insights on developmental biology, especially for cell differentiation.

View larger version (41K): [in this window] [in a new window]   Figure 3. Systemic differentiation of cardiovascular cells in ES cell system in vitro. ES cell-derived Flk1+ cells represent lateral plate mesoderm in the embryo and common progenitors for cardiovascular cells. Flk1+ cells give rise to endothelial cells and mural cells on type IV collagen-coated dishes by VEGF and PDGF-BB, respectively (dotted lines). Flk1+ cells can differentiate into cardiomyocytes as well as endothelial cells on OP9 stroma cells (solid lines). Noggin plays an inhibitory role in cardiac progenitor cells (FCV cells) and cardiomyocyte differentiation. Wnts and wnt inhibitors influence the differentiation potential of FCV cells and cardiomyocyte induction from Flk1+ cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3540fje;

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