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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Shiro Babaa, Toshio Heikea, Katsutsugu Umedaa, Toru Iwasaa, Shinji Kaichia, Yoshimi Hiraumia, Hiraku Doia, Momoko Yoshimotoa, Mito Kanatsu-Shinoharab, Takashi Shinoharab, Tatsutoshi Nakahataa


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      【摘要】7 y) H) M9 F7 d2 c' h0 x0 j
   Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%¨C40% of each cell type differentiated into Flk1  cells. The sorted Flk1  cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1  mGS cells generated more contractile colonies than did the Flk1  ES cells, whereas the Flk1  EG cells generated equivalent numbers as the Flk1  mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0¨C4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1  mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells.* V0 N6 r4 A: l- Z: x% d
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Mouse Germline Somatic stem cells Pluripotent stem cells Embryonic stem cell In vitro differentiation0 T0 c) o1 r8 s5 C$ [4 G  y
   INTRODUCTION  I% M0 Z6 M5 i6 g" H& E; c
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Heart transplantation is one of the most effective therapies for patients who suffer from severe heart failure caused by cardiac infarction or other cardiomyopathies. However, insufficient numbers of matching donor hearts are available . Thus, for all cell types identified to date as candidate transplantable cells, problems with regard to their therapeutic clinical use exist. Moreover, these cell types are difficult to maintain in vitro at the stem/progenitor cell level, which adds an extra impediment to their utility in clinical practice.2 ~0 c4 t* C4 S" y5 w

Recently, it has been proposed that embryonic stem (ES) cells and adult cardiac stem (CS) cells maybe transplantable cell candidates that do not suffer from the problems described in the preceding paragraph. ES cells are known to have multipotent differentiation capacity and are easily maintained in their undifferentiated state .7 p6 H6 [1 }7 \: }( n
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In 2004, we established the multipotent germline stem (mGS) cell line from murine testes and showed that this cell line can differentiate into various lineages, including endodermal, mesodermal, and ectodermal lineages, almost as well as ES cells can . This implies that it will be very important and useful to further examine these somatic stem cells with respect to their mesodermal commitment.

MATERIALS AND METHODS2 `6 {$ W9 [# ~+ g; U: U1 f- V

Cell Culture and Differentiation

mGS cells were maintained as described previously .
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Reverse Transcriptase Polymerase Chain Reaction Analysis, N) `( r0 {1 p/ V

The cDNA product was amplified by polymerase chain reaction (PCR) using primers that target the following genes: Rex1, FGF-5, GATA-4, Flk1, Brachyury, MHC, ¦ÂMHC, ANF, cardiac calsequestrin (Csq), SERCA2, 1c, SCN5A, Kir2.1, Kv4.3, MERG A and MERG B variants, KvLOT1, Nkx2.5, MLC2v, MLC2a, Cx43, and GAPDH. These ion channel primers were referred to in a previous report . The construction of these primers is described in Table 1. All primers were used in the following PCR condition: DNA denaturation at 94¡ãC for 5 minutes followed by 25¨C35 cycles of amplification involving 94¡ãC for 30 seconds, 55¡ãC¨C60¡ãC for 30 seconds and 72¡ãC for 60 seconds, concluded by a final extension step at 72¡ãC for 7 minutes.

Table 1. Polymerase chain reaction primers
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Real-Time Quantitative Reverse Transcriptase PCR Analysis% Q( O% C. j1 J' g

Forward and reverse primers for Rex1 and FGF-5 as well as fluorogenic probes were designed according to PerkinElmer guidelines (Primer Express Software; PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com), and those of Flk1 were described in a previous report . GAPDH primers and probes were purchased from Applied Biosystems (Foster City, CA, http://www.appliedbiosystems.com). Quantitative assessment of mRNA expression was performed using a GAPDH internal standard. The expression of each mRNA was compared with each day 0 mRNA expression.

The primers and probes were as follows: mouse Rex1¡ªAAGCAGGATCGCCTCACTGT and CCGCAAAAAACTGATTCTTGGT with the probe CTGCCTCCAAGTGTTGTCCCCAAATACC; mouse FGF-5¡ªAGAGTGGGCATCGGTTTCC and CTTCGTGGGAGCCATTGACT with the probe CTGCAGATCTACCCGGATGGCA; and mouse Flk1¡ªGTGGTCTTTCGGTGTGTTGCT and TCTCCTACAAAATTCTTCATCAATCTTG with the probe TTCCTTAGGTGCCTCCCCATACCCTG.

Fluorescence-Activated Cell Sorting Analysis and Comparison of the Ability of Flk1  and Flk1¨C Cells to Differentiate into Cardiac and Endothelial Colonies

The cell surface marker antibodies used in these fluorescence-activated cell sorting (FACS) experiments were phycoerythrin-conjugated anti-Flk1 antibody (BD Biosciences), purified anti-E-cadherin antibody (Takara Bio Inc., Otsu, Japan, http://www.takara.co.jp), purified anti-PDGFRa antibody (BD Biosciences) and purified anti-VE-cadherin antibody (BD Biosciences). Allophycocyanin-conjugated anti-rat IgG antibody served as a secondary antibody for unlabeled primary antibody. The cells labeled with these antibodies were analyzed by FACSCalibur (BD Biosciences). The ES, EG, and mGS cell derivatives were divided into Flk1  and Flk1¨C cells by anti-Flk1 labeling followed by sorting with a FACSVantage SE (BD Biosciences). The resulting Flk1  and Flk1¨C cells were cocultured on OP-9 stromal cells in differentiation medium at 500 or 5,000 cells per well in 24-well plates (FALCON). The contractile colonies per well were then counted on an inverted microscope. Dil-acetylated low-density lipoprotein (Dil-Ac-LDL; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) was used to detect of endothelial colonies, and the endothelial colonies per well were counted under a fluorescent microscope. For single-cell culture, Flk1  cells derived from ES, EG, and mGS cells were individually sorted with a FACSVantage SE CloneCyt Plus (BD Biosciences) and cocultured with OP-9 stromal cells at 1 Flk1  cell per well of a 96-well plate (FALCON).
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Immunohistochemistry
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The cells were fixed with 4% paraformaldehyde (PFA) and incubated with following antibodies: anti-myosin light chain 2v (MLC2v; Alexis Biochemicals Inc., Nottingham, U.K., http://www.axxora.com), anti-atrial natriuretic peptide (ANP; Protos Biotech Corporation, New York, http://www.protosantibody.com), anti-tropomyosin (CH1; Sigma-Aldrich), anti-cardiac troponin-I (cTn-I; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), anti-CD45 antibody (Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com) and anti-CD31 antibody (Becton Dickinson). Peroxidase-conjugated donkey anti-mouse IgG, alkaline phosphatase (ALP)-conjugated donkey anti-mouse IgG, ALP-conjugated donkey anti-rabbit IgG, ALP-conjugated donkey anti-goat IgG and peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories Inc. Laboratories, West Grove, PA, http://www.jacksonimmuno.com) served as secondary antibodies. ALP or peroxidase was visualized by using the Alkaline Phosphatase Substrate Kit III or the DAB Substrate Kit (Vector Laboratories Inc.).0 o+ F; S$ y  P. ^) f4 {* V

Electrophysiological Examination

Day 4-sorted Flk1  mGS cells were cocultured with OP-9 stromal cells on MED-probe dishes (Alpha MED Sciences, Torrance, CA, http://www.med64.com) and electrical potentials of contractile colonies derived from these cells were recorded by using the MED64 System (Panasonic multielectrode system; Alpha MED Sciences).$ n# D$ p: e8 t6 B& e

Statistical Analysis

The data were analyzed with the paired two-tailed t test of Microsoft Excel (Microsoft Corp., Redmond, WA). Statistical significance was defined as a p value of less than .05.

RESULTS3 V4 W4 S9 G9 B  @

mGS Cells Express Mesodermal and Endothelial Markers  u" S$ y0 J8 o" b" r, x

ES, EG, and mGS cells were maintained on mitomycin C-treated MEFs. Like undifferentiated ES and EG colonies, the undifferentiated mGS colonies are round and their surfaces smooth, and each in situ appearance was almost the same. To differentiate these cell lines into cardiac and endothelial cells, we used the mesodermal differentiation method employing the OP-9 stromal cell coculture system that was reported previously . Thus, although the mesodermal marker expression of mGS cells was slightly retarded compared to that of ES and EG cells, the overall pattern indicates that mGS cells differentiate similarly to ES and EG cells.
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Figure 1. E-cadherin, PDGFRa, Flk1, and VE-cadherin expression by differentiating ES, EG, and mGS cells. ES (A, D, G, J), EG (B, E, H, K), and mGS (C, F, I, L) cells were induced to differentiate on OP-9 stromal cell layers and subjected at day 0, 3, 5, or 6 to fluorescence-activated cell-sorting analysis for E-cadherin (A¨CC), PDGFRa (D¨CF), Flk1 (G¨CI), and VE-cadherin (J¨CL) expression. (M¨CO): Coexpression of Flk1 by day-5 VE-cadherin  cells derived from ES, EG, and mGS cells. Abbreviations: EG, embryonic germ; ES, embryonic stem; mGS, multipotent germline stem.
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Flk1  mGS Cells Differentiate Efficiently into Contractile and Endothelial Colonies7 J2 O5 Y$ \! V4 V* U
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Flk1 has reported to be a marker of progenitor cells that have the potential to differentiate into cardiomyocytes and endothelial cells . The contractile and endothelial colonies first appeared at similar time point for all three cell types, and the Flk1  cells of each cell types differentiated into contractile colonies much more abundantly than did the Flk1¨C cells (Fig. 2D¨C2F). Notably, as shown in Figure 2J, the mGS and EG Flk1  cells both generated significantly more contractile colonies than did the ES Flk1  cells (5,000 Flk1  ES, EG, and mGS cells generated 4.1 ¡À 2.0, 14 ¡À 5.4, and 10 ¡À 7.3 contractile colonies by day 13 after differentiation induction; p
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Figure 2. Differentiation of Flk1  and Flk1¨C cells derived from day-4 differentiating ES, EG, and mGS cells into contractile or endothelial colonies. (A¨CC): ES, EG, and mGS cells were induced to differentiate and on day 4 were sorted into Flk1  and Flk1¨C populations by FACSVantage (BD Biosciences). The sorted populations were at least 99% pure. (D¨CK): The sorted Flk1  and Flk1¨C populations were cultured on OP-9 stromal cells in differentiation medium. The contractile colonies generated by the Flk1  and Flk1¨C cells from ES (D), EG (E), and mGS cells (F) were counted during days 5¨C13 after differentiation induction. The contractile colonies started to appear on day 8 after differentiation induction. The endothelial colonies generated by the Flk1  (black bars) and Flk1¨C (open bars) cells from ES (G), EG (H), and mGS cells (I) were counted during days 5¨C10 after differentiation induction. (J, K): Summary of the number of contractile (J) and endothelial (K) colonies generated by Flk1  cells derived from ES (gray bars), EG (open bars), and mGS cells (black bars). Abbreviations: EG, embryonic germ; ES, embryonic stem; mGS, multipotent germline stem.
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mGS Cells Differentiate into the Mesodermal Lineage As Efficiently As ES and EG Cells: W: `  W4 n& v

Next, we used reverse transcriptase (RT)-PCR analysis to compare the gene expression patterns of mGS, ES, and EG cells before and during their differentiation over the first 4 days. When undifferentiated, all three cells strongly expressed Rex1, which is an undifferentiated cell marker, but did not express the epiblast (primitive ectoderm) marker FGF-5, the mesodermal markers Flk1 and Brachyury, or the mesodermal and cardiac marker GATA-4 (Fig. 3A). The Rex1 expression of all three cell types gradually decreased during their culture in differentiation medium. FGF-5 expression of all three cell types was simultaneously detected by day 2 and maximally by day 3 after differentiation induction. However, ES cells expressed Flk1, Brachyury, and GATA-4 earlier (weak expression on day 2, strong expression thereafter) than did EG and mGS cells (expression detected only from day 3 onwards). Thus, maximal expression of Flk1, Brachyury, and GATA-4 by the three cell types was detected on days 3 or 4 after differentiation induction. The mRNA expression patterns of Rex1, FGF-5, and Flk1 were also confirmed by real-time RT-PCR (Fig. 3B). These results indicate that although mGS cells differentiate into the mesodermal lineage via epiblasts a bit more slowly than do ES cells, the expression of the mesodermal markers Flk1 and Brachyury in the three cell types is maximal at almost the same time.
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Figure 3. Gene expression patterns of differentiating embryonic stem, embryonic germ, and multipotent germline stem cells. ES, EG, and mGS cells were induced to differentiate. (A): On days 0¨C4, reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression of Rex1, FGF-5, Brachyury, Flk1, and GATA-4. (B): Expressions of Rex1, FGF-5, and Flk1 were evaluated by real-time quantitative RT-PCR. (C): The differentiated cells derived from Flk1  ES, EG, and mGS cells were harvested on day 13 and RT-PCR was used to determine the expression of MHC, ¦ÂMHC, ANF, SERCA2, cardiac Csq, Nkx2.5, GATA-4, MLC2a, MLC2v, and Cx43. The controls used consisted of water instead of cellular mRNA (a), mRNA from OP-9 stromal cells (b), and mRNA from mouse ventricle cells (c). Abbreviations: EG, embryonic germ; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mGS, multipotent germline stem.

mGS Cells Differentiate into Mature Cardiac and Endothelial Cells% c* }) _, d* a6 U/ R

We then investigated the expression of mature cardiac cell markers by contractile colonies derived from each cell types. For this purpose, the Flk1  cells were isolated by FACSVantage on day 4 after differentiation induction and were cultured further in differentiation medium, and their mRNA was harvested on day 13 and analyzed by RT-PCR. Regardless of the cell type from which they were derived, all day-13 differentiated cells expressed MHC, ¦ÂMHC, ANF, cardiac Csq, and Sarcomeric Ca2 -ATPase 2 (SERCA2); Nkx2.5, which is a cardiac marker that is detected from an early-stage in the developing heart; GATA-4; the mature ventricular marker MLC2v; myosin light chain 2a (MLC2a), which is a marker of developing cardiomyocytes and atrial cells; and the gap junction marker connexin 43 (Cx43) (Fig. 3C). The expression of these markers was a little weaker in the ES-derived cells than in the EG- or mGS-derived cells. This may reflect the lower potential of ES cells to differentiate into the cardiac lineage that was also revealed by their lesser ability to produce contractile colonies (Fig. 2J).
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We also subjected the day-13 contractile colonies of mGS cells to immunohistochemical analysis to determine their expression of MLC2v; ANP, which is a marker of developing cardiomyocytes and atrial cells; CH1; and cTn-I (Fig. 4A¨C4D). Interestingly, double staining for ANP and MLC2v revealed the presence in the wall of individual ANP  cells located away from the MLC2v  cell mass, although other ANP  cells were in physical contact with the MLC2v  cells (Fig. 4B). We also subjected the day-10 endothelial colonies of mGS cells to immunohistochemical analysis to determine their expression of CD31 and VE-cadherin (Fig. 4E¨C4F). The endothelial sheets showed ample staining of both markers. These results suggest that mGS cells can efficiently differentiate into mature cardiomyocytes and endothelial cells.
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Figure 4. Expression of mature cardiac and endothelial cell markers by contractile and sheet-like colonies derived from multipotent germline stem (mGS) cells. Contractile (A¨CD) and sheet-like colonies (E, F) derived from Flk1  mGS cells were subjected to immunohistochemistry to examine their expression of the mature cardiac cell markers MLC2v (A), ANP (blue) and MLC2v (brown) (B), CH1 (C), and cTn-I (D), or the mature endothelial cell markers CD31 (E) and VE-cadherin (F). (G¨CI): Ability of single Flk1  mGS cells to differentiate into three cardiac, endothelial, and hematopoietic lineages. A single colony generated by a single Flk1  mGS cell that showed the presence of cardiac, endothelial, and hematopoietic cells was subjected to immunohistochemistry. In (G), the colony was stained with the cardiac cell marker MLC2v (blue) and the hematopoietic cell marker CD45 (brown; arrowheads). (H): The colony was analyzed for low-density lipoprotein uptake and an endothelial sheet-like structure. The visual fields of (G) and (H) are the same. (I): The area indicated in (G) by an open square was subjected to higher magnification and stained with the hematopoietic cell marker CD45 (brown). Scale bars = 100 µm.* v1 J$ r$ b+ T& k/ D6 i2 U
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Single Flk1  mGS Cells Can Differentiate into Three Mesodermal Lineages
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It has been reported previously that single Flk1  ES cells can differentiate into cardiomyocytes, endothelial cells, and hematopoietic cells . To test whether mGS cells share the same capacity, we isolated single Flk1  cell derived from ES, EG, and mGS cells (n = 960 for each cell type) on day 4 of the differentiation and plated them individually in 96-well plates on OP-9 stromal cell layers in differentiation medium. The competence of these cells to differentiate into the three mesodermal lineages was then examined on day 13 after differentiation induction. Cardiac colonies were identified on the basis of their contractility; endothelial colonies were identified by their LDL uptake, VE-cadherin staining, and sheet-like structure; and hematopoietic clusters were identified by their in situ cobblestone appearance and immunostaining with anti-CD45 antibody. Each well was subsequently categorized as containing a cardiac colony alone, an endothelial colony alone, a hematopoietic cluster alone, or mixed colonies with two or three lineages (Table 2). The individual Flk1  cells derived from ES, EG, and mGS cells had very closely matching outcomes, indicating equivalent abilities to differentiate into the three mesodermal lineages. Moreover, when we subjected one of the three colonies generated by single Flk1  mGS cells that simultaneously contained cardiac, endothelial, and hematopoietic lineage cells to immunohistochemical analysis, we found that the middle of the colony expressed MLC2v, whereas CD45  hematopoietic cells and LDL  endothelial cells bearing a sheet-like structure were detected around this MLC2v  contractile area (Fig. 4G¨C4I). Thus, single Flk1  mGS cells isolated on day 4 after differentiation induction are as capable of differentiating into all three of the cardiac, endothelial, and hematopoietic lineages as are ES and EG cells.. h. {: k! d1 t* `  n" V6 f/ w6 G& a5 w

Table 2. Single-cell culture: Potential of single Flk1  cells derived from ES, EG, or mGS cells to differentiate into cardiac, endothelial, and/or hematopoietic cells
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Differentiated mGS Cells Have Electrical Potentials and Sodium, Potassium, and Calcium Channels

Finally, we measured the electrical potentials of contractile colonies derived from Flk1  mGS cells. The contractile colonies had action potentials as assessed by the MED64 system (Fig. 5A). Moreover, as assessed by RT-PCR, these cells expressed the L-type calcium channel (1c), sodium channel (SCN5A), inward rectifier (Kir2.1), transient outward channel (Kv4.3), delayed rectifier IKr (MERG A and MERG B variants), and IKs (KvLOT1). These results confirm the cardiomyogenesis of mGS cells in vitro.9 {. ~6 O) M8 n9 Z6 E' v5 Z2 W
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Figure 5. Contractile colonies derived from mGS cells have action potentials and sodium, potassium, and calcium channels. (A): Electrical activities were measured from contractile colonies. (B): The differentiated cells derived from Flk1  ES, EG, and mGS cells were harvested on day 13, and RT-PCR was used to determine the expression of 1c, SCN5A, Kir2.1, Kv4.3, MERG A and MERG B variants, and KvLOT1. The controls used consisted of water instead of cellular mRNA (a), mRNA from OP-9 stromal cells (b), and mRNA from mouse ventricle cells (c). Abbreviations: EG, embryonic germ; ES, embryonic stem; mGS, multipotent germline stem.8 [/ d. A, Z4 `" E% w" u1 x
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DISCUSSION
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There have been many reports of heart cell transplantation using various cardiomyopathy models. The candidate for transplantable cell sources identified to date include fetal cardiomyocytes, adult skeletal muscle, smooth muscle, bone marrow, adult CS cells, and ES cells. Moreover, clinical trials assessing the efficiency of performing transplants on patients with severe heart failure with skeletal muscle or bone marrow cells have been reported . However, all cell sources are associated with various impediments to their use in the clinical, and thus it remains unclear which candidate is best for cell transplantation therapy. In this article, we revealed that mGS cells are more capable of differentiation into cardiomyocytes than are ES cells, and that they generate endothelial cells as efficiently as do ES and EG cells.

ES cells are isolated from the inner cell mass of mouse blastocysts, whereas EG cells are isolated from embryonic germline cells. Both of these unique cells can proliferate in culture in the undifferentiated state over a prolonged period, and differentiate into every tissue type in the body. These undifferentiated cells are round and smooth, and express the undifferentiated cell marker Rex1. Like the ES and EG cells, undifferentiated mGS cells also strongly express Rex1 and show a decrease in this marker over time after differentiation induction. In murine embryogenesis, the three ectodermal, mesodermal, and endodermal lineages are all generated from epiblasts with totipotency during gastrulation . Because mGS cells are established from testes, we suppose that this mechanism would induce the mGS developmental acceleration especially into mesodermal lineages.1 u# `, {: y4 X1 w$ L
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Flk1 has been reported to be an early mesodermal marker and plays a significant role in not only hematopoietic and endothelial differentiation but also in cardiac differentiation . We show here that Flk1  mGS and EG cells also generate cardiomyocytes and endothelial cells significantly more efficiently than do the Flk1¨C cells. Thus, in terms of their in vitro differentiation, the potential of mGS cells to differentiate into cardiomyocytes and endothelial cells is at least as great as that of ES cells. Although we will, of course, have to determine whether this potential is repeated in the in vivo situation, these results indicate that the mGS cell is likely to be at least as suitable as the ES cells as a candidate cell source for cell transplantation into patients with heart failure.# H3 t& y. d6 Z; u! b, d8 ~

It has been reported previously that single Flk1  ES cells have the potential to differentiate into three mesodermal lineages as cardiomyocytes, endothelial cells, and hematopoietic cells .

Another major problem of cell transplantation is an immunological problem. It is not possible to avoid this problem in the clinical transplantation use of ES cells. In contrast, mGS-like cells would eliminate many problems associated with transplantation, such as rejection and graft-versus-host disease, at least for male heart failure patients. However, for this to be feasible, it will be necessary to efficiently collect mGS-like cells from the testes of these adult patients as somatic multipotent stem cells. Furthermore, when we establish the system to produce ES-like cells such as mGS cells from somatic cells, these cells will be suitable for transplantation in clinical setting in the near future regardless of the sex of the patient.

In conclusion, mGS cells, like ES cells, have a satisfactory potential to differentiate into mature cardiomyocytes and endothelial cells in vitro. Moreover, this cell source is suitable for cell transplantation at will because undifferentiated mGS cells can grow abundantly in vitro. In addition, pluripotency of spermatogonial stem cells from adult mouse testis has been reported recently . Thus, although adult CS and ES cells are suitable transplantation candidates because of their strong potential to differentiate into cardiomyocytes and endothelial cells, mGS cells are also highly attractive candidates.& G0 ]8 w  o* \) S
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. |* {9 Q* \+ l2 t
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R. Pedersen has acted as a consultant to and has a financial interest in Stemnian, LLC (Pittsburgh, PA).
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ACKNOWLEDGMENTS

This study was supported by the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (03-2) and Research of Japan, and by a Grant-in-Aid for Creative Scientific Research (13GS0009). We thank Dr. Yasuhisa Matsui for providing the EG cell line and Dr. Hiroaki Kodama for providing the OP-9 stromal cell line.0 ^8 ]# A* ?6 F' O3 X
      【参考文献】8 }  ^8 ]- X/ d3 f  [$ ^

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Etoch SW, Koenig SC, Laureano MA et al. Results after partial left ventriculectomy versus heart transplantation for idiopathic cardiomyopathy. J Thorac Cardiovasc Surg 1999;117:952¨C959.

Reinecke H, Zhang M, Bartosek T et al. Survival, integration, and differentiation of cardiomyocyte grafts: A study in normal and injured rat hearts. Circulation 1999;100:193¨C202.

Yoo KJ, Li RK, Weisel RD et al. Heart cell transplantation improves heart function in dilated cardiomyopathic hamsters. Circulation 2000;102:III204¨CIII209.

Li RK, Jia ZQ, Weisel RD et al. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999;31:513¨C522.
# \" _6 U$ P6 V( Q# Y) [9 r
Borenstein N, Bruneval P, Hekmati M et al. Noncultured, autologous, skeletal muscle cells can successfully engraft into ovine myocardium. Circulation 2003;107:3088¨C3092.

Tambara K, Sakakibara Y, Sakaguchi G et al. Transplanted skeletal myoblasts can fully replace the infarcted myocardium when they survive in the host in large numbers. Circulation 2003;108 (suppl 1):II259¨CII263.

Scorsin M, Hagege A, Vilquin JT et al. Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function. J Thorac Cardiovasc Surg 2000;119:1169¨C1175., n: Q! A( F& l3 G2 P0 p1 d

Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430¨C436.0 q, ]' k$ \1 N" y* P6 y: |0 e' {: m
* b: _0 I  |7 u% N1 x: K: m) Y$ s8 K
Agbulut O, Menot ML, Li Z et al. Temporal patterns of bone marrow cell differentiation following transplantation in doxorubicin-induced cardiomyopathy. Cardiovasc Res 2003;58:451¨C459.! {9 Z3 ~/ ^. m: v7 b+ U6 J1 C! X5 _

Murry CE, Soonpaa MH, Reinecke H et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664¨C668.9 }4 I$ `3 Y4 l6 z

Balsam LB, Wagers AJ, Christensen JL et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668¨C673.+ Y& i* i7 l( X- `% X, o" {
( t. u% ^; z/ E
Hescheler J, Fleischmann BK, Lentini S et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc Res 1997;36:149¨C162.+ b% S0 F1 a2 \5 w6 s. Y& i7 W# ?

Hidaka K, Lee JK, Kim HS et al. Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells. Faseb J 2003;17:740¨C742.& \7 ?; @, X$ r; L
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Fijnvandraat AC, van Ginneken AC, de Boer PA et al. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube. Cardiovasc Res 2003;58:399¨C409.
  I2 X5 K$ v" P" X+ F/ Z5 {: f: T
Min JY, Yang Y, Converso KL et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 2002;92:288¨C296.: z  z2 G5 F7 j7 b( q0 P
: m) b0 h; w2 D: f) B
Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763¨C776.! q% S* V) u- K) H( l: ]8 w

Kanatsu-Shinohara M, Inoue K, Lee J et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004;119:1001¨C1012.; p4 v7 V+ {! |* C/ q& @

Guan K, Nayernia K, Maier LS et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006;440:1199¨C1203.
6 U6 W( |2 d; s6 ]% e! u
Kanatsu-Shinohara M, Shinohara T. The germ of pluripotency. Nat Biotechnol 2006;24:663¨C664.- o1 G7 Z# h, w& y% P& A
5 {8 p1 H/ R2 z. o
Ogawa M, Kizumoto M, Nishikawa S et al. Expression of alpha4-integrin defines the earliest precursor of hematopoietic cell lineage diverged from endothelial cells. Blood 1999;93:1168¨C1177.2 Z1 m9 w7 w* [' q
' ]. S( V' r( l% L- v0 U* B
van der Heyden MA, van Kempen MJ, Tsuji Y et al. P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level. Cardiovasc Res 2003;58:410¨C422.; j, n' j6 x3 q2 K2 C. i

Redaelli CA, Semela D, Carrick FE et al. Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice. J Hepatol 2004;40:305¨C312.

Nishikawa SI, Nishikawa S, Hirashima M et al. Progressive lineage analysis by cell sorting and culture identifies FLK1 VE-cadherin  cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747¨C1757.
" [. v+ H$ o2 t' v: {4 E$ h
Tada S, Era T, Furusawa C et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 2005;132:4363¨C4374.7 V# Y: T5 R# p, O% H* g

Iida M, Heike T, Yoshimoto M et al. Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation. Faseb J 2005;19:371¨C378.9 v& U: n& Q0 `' p( @

Umeda K, Heike T, Yoshimoto M et al. Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro. Development 2004;131:1869¨C1879.

Menasch¨¦ P, Hagege AA, Scorsin M et al. Myoblast transplantation for heart failure. Lancet 2001;357:279¨C280.
# ^0 I$ x' Y" h# j
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294¨C2302.0 N8 C3 s( i( J: T" g# ^& \. |- P$ h

Svajger A, Levak-Svajger B, Kostovic-Knezevic L et al. Morphogenetic behaviour of the rat embryonic ectoderm as a renal homograft. J Embryol Exp Morphol 1981;65 (suppl):243¨C267.8 x; _8 `% l) R% ~2 l# L; u

Kanatsu M, Nishikawa SI. In vitro analysis of epiblast tissue potency for hematopoietic cell differentiation. Development 1996;122:823¨C830.4 T: y5 y2 x& j  ]# S0 I
  V; u# a, g7 |5 Y- [5 [
Barton SC, Ferguson-Smith AC, Fundele R et al. Influence of paternally imprinted genes on development. Development 1991;113:679¨C687., C% Q# w- Y, Y; k; I/ ?

Swijnenburg RJ, Tanaka M, Vogel H et al. Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 2005;112:I166¨C172.

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