标题: Analysis of the Origin and Population Dynamics of Cardiac Progenitor Cells in a [打印本页] 作者: 江边孤钓 时间: 2009-3-5 00:56 标题: Analysis of the Origin and Population Dynamics of Cardiac Progenitor Cells in a
作者：Tao-Sheng Li, Ryo Suzuki, Kazuhiro Ueda, Tomoaki Murata, Kimikazu Hamano作者单位：Department of Surgery and Clinical Science and Institute of Laboratory Animals, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan 1 a# ?' W: U r 4 T' h- x& F! S2 J+ O3 H
2 I; H) K8 H- ]0 P8 R. B/ a
" ^+ m; X7 {( v' u
- ?& Z" M3 w: @8 N1 G9 y( F 2 A1 e* [- Y1 j& H/ B4 d
. g' B6 L4 H( `6 f
% w' q3 j" L; r1 p5 Y / G p! r7 Q* P! I) ^7 ?3 e7 y. D 3 s5 g. |% q& S' u' C : g: p9 n1 Q' V) J# [" A 9 x, ?2 ]* R6 }9 q _" S E( h+ j2 `9 E$ @3 w( w3 B
【摘要】 8 b2 _8 o% k5 Y/ r" R& p7 o' n F Cardiac progenitor (stem) cells have recently been detected in and isolated from the myocardium of neonatal and adult mice, rats, and humans; however, the precise origin and characterization of these cells remain unclear. Using a heterotopic mouse heart transplantation model, we investigated the origin and population dynamics of cardiac progenitor cells. Donor hearts from wild-type C57/BL6 female mice were transplanted into green fluorescent protein (GFP)-transgenic C57/BL6 male mice. The donor hearts were collected 0, 2, 4, 8, and 12 weeks after transplantation. We used quantitative flow cytometry to analyze the number and origin of stem cells in the donor hearts and immunostaining to evaluate the time-related changes in their characteristics. Extracardiac GFP-positive stem cells immigrated into the donor hearts soon after transplantation. Immunostaining revealed that these GFP-positive stem cells in the donor hearts gradually lost expression of the hematopoietic markers of CD45 and CD34 and shifted to express the cardiac-specific transcription factors GATA-4 and NKx2.5. A few of the GFP-positive cells in the donor hearts finally acquired the mature cardiac phenotype in the absence of cell fusion with donor cardiomyocytes. Our discovery provides the first evidence that extracardiac stem cells may be of bone marrow origin, from which they can transform into cardiac progenitor cells in response to myocardial environment cues.% Y, ~4 v+ v/ V6 i. g6 Q8 u
% k3 E! B7 E' w S) F1 L
Disclosure of potential conflicts of interest is found at the end of this article. 0 ?% T' b" @# t3 {& j 【关键词】 Heart Stem cell Bone marrow7 r" M) V" J0 A% i/ A1 c
INTRODUCTION ! [$ g6 W: W2 [ # k# n2 Q& t0 T. x" ~1 _9 `Cardiac progenitor (stem) cells have been found in the adult hearts of humans and other mammals and have demonstrated the distinct characteristics of a cardiac progenitor population. Various cell surface markers, including Sca-1, c-kit, and LIM-homeodomain transcription factor islet-1 ; and there is no convincing evidence that these cardiac progenitor cells are heart-specific endogenous precursors that have been preprogrammed to differentiate into myocytes. , `! b1 L* i; I9 W : f9 T' h3 d- |/ c3 t& t/ XWe used a heterotopic heart transplantation model to test this hypothesis . This model allowed us to monitor the immigration of recipient stem cells (GFP-positive, GFP ) into donor hearts (GFP-negative, GFP¨C). In the donor heart, the replacement of endogenous cardiac progenitor cells by extracardiac stem cells could be measured quantitatively at any time. We were also able to follow the changes over time of the phenotypes and characteristics of extracardiac stem cells in response to myocardial environment cues after immigration into the donor heart. ?' s) F6 S2 r* s; P" v. o+ g6 Z5 c0 s! m
MATERIALS AND METHODS . `1 n; |0 S* J q8 z7 r0 n3 e1 G* H! k
Animals$ X2 w# X3 B8 I, g4 z
' c6 A# P; p# X
GFP-transgenic mice (C57BL6/Tg14) were kindly provided by Masaru Okabe (Genome Research Center, Osaka University, Osaka) and bred in the Animal Center of Yamaguchi University. The wild-type male C57BL/6 mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). All experiments were approved by the Institutional Animal Care and Use Committee of Yamaguchi University. ! q3 w3 V( V M: W6 Z6 K7 R d z9 e2 k6 Q* z# Z" U4 iHeterotopic Heart Transplantation Model 1 J6 G0 Y( V7 g& e6 k4 P' \, I1 @3 P1 Y$ A
Heterotopic intra-abdominal heart transplantation was performed as described previously . Briefly, donor hearts were harvested quickly from wild-type C57/BL6 female mice and placed in ice-cold saline while each recipient GFP-transgenic C57/BL6 male mouse was prepared. We anastomosed the donor aorta and pulmonary artery to the recipient abdominal aorta and inferior vena cava with 10-0 nylon sutures. All procedures took approximately 50 minutes./ v, y6 a' o. h( r
9 k& ~- S& p4 Y p% \/ Q$ OIsolation and Magnetic Enrichment of the Cardiac Progenitor Cells0 U2 a0 `9 \( \& r4 v5 X5 @% A
7 O" ^5 \; d4 I0 w; S
We extracted the donor hearts immediately after transplantation (baseline, n = 4) or 2, 4, 8, and 12 weeks after transplantation (n = 5 at each time point). Each donor heart was perfused immediately with 20 ml of ice-cold phosphate-buffered saline (PBS) through the donor aorta to wash out the circulating blood cells. All of the myocardium from each heart was minced and then incubated in 0.1% collagenase at 37¡ãC for 30 minutes. We collected all of the isolated cells from each donor heart and washed them with PBS. Single cell suspensions in PBS containing 2% fetal calf serum were then filtered through 40-µm BD Falcon Cell Strainers (BD Biosciences, San Diego, http://www.bdbiosciences.com) for flow cytometry analysis.4 G+ \8 E3 q0 v! X/ l g9 \
+ _% |: r, o/ h9 ]
Using magnetic cell sorting (MACS), we also purified the Sca-1- and c-kit-positive cells for analysis. The total population of cardiac cells was isolated and collected from three donor hearts 2, 4, 8, and 12 weeks after transplantation (n = 1 at each time point). The collected cells were incubated with rat monoclonal antibodies against mouse Sca-1 or c-kit at 4¡ãC for 30 minutes. After washing twice with PBS, the cells were incubated with anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) at 4¡ãC for 20 minutes. The Sca-1- or c-kit-positive cells were separated from the total cardiac population by passing a MACS column. Enriched Sca-1- and c-kit-positive stem cells were used for flow cytometry analysis. ' U2 Q$ O7 q3 m0 E/ n, ~: @5 I1 H- t3 X6 p( k
Flow Cytometry Analysis 5 X( t; Z0 B. N- z# j6 h- ]1 f7 d3 k5 t) v" d
All of the isolated cells were stained with phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse Sca-1 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), PE-conjugated rat monoclonal antibody against mouse c-kit (BD Pharmingen), or goat polyclonal antibody against CD133 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) at 4¡ãC for 30 minutes. On the other hand, magnetic enriched Sca-1- and c-kit-positive stem cells were stained with PE-conjugated monoclonal rat anti-mouse antibodies reactive to CD45 and CD34 (all from BD Pharmingen) at 4¡ãC for 30 minutes. Respective isotype controls were used as a negative control. After washing three times with PBS, quantitative flow cytometry analysis was done using a FACScan flow cytometer. We analyzed the acquired data using Cell Quest software (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). $ h* P( b( F$ u# e' ? 0 y, o% @* T/ Y; i( Q$ m& nHistological Analysis0 F P8 ]9 m0 z( V1 L
, M1 \" t/ S) K; `
Donor hearts were extracted 2, 4, 8, and 12 weeks after transplantation (n = 5 at each time point) and perfused with ice-cold PBS as described above. Samples were frozen immediately in liquid nitrogen. We used 5-µm-thick frozen sections for histological analysis. For the cell count, the GFP cells were detected directly under fluorescence microscopy. For photography under confocal microscopy, the GFP cells were enhanced by immunostaining with rabbit anti-GFP antibody (Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com) followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody. We used PE-conjugated monoclonal antibodies against mouse CD4, CD8, CD68, and Gr-1 to examine the infiltration of cells. PE-conjugated monoclonal antibodies against Sca-1, c-kit, and CD133 were used for staining the stem cells, and PE-conjugated monoclonal antibodies against mouse CD45 and CD34 were used as hematopoietic markers. We stained the cardiac transcript factors of GATA-4 and NKx2.5 to identify the cardiac progenitor cells. Cardiac-specific proteins were detected by staining with antibodies against -myosin heavy chain and cardiac troponin T. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Immunostaining was visualized under fluorescence microscopy or under confocal microscopy (Zeiss LSM 510; Carl Zeiss, Jena, Germany, http://www.zeiss.com). & E$ v7 Z @/ _+ n, n& r3 K q8 I8 H3 l9 Z4 g. k8 h! qFluorescence In Situ Hybridization Analysis' G8 J8 f& F+ T+ H# S: U& G
" U3 ?: Q* O# g6 p1 M2 j1 t8 FWe investigated the property of cell fusion with fluorescence in situ hybridization (FISH) analysis using a hybridization DNA probe mixture of Cy3-labeled Y chromosome paint and FITC-labeled X chromosome paint (Star FISH; Cambio, Dry Drayton, U.K., http://www.cambio.co.uk). We also combined immunostaining with FISH analysis to identify whether the myogenic differentiation of GFP extracardiac stem cells was dependent on cell fusion with donor cardiomyocytes. After immunostaining with GFP and -myosin heavy chain, FISH analysis was performed immediately in selected sections using the hybridization DNA probe mixture described above. X and Y chromosomes were localized within the nucleus, but the expression of GFP and myosin was localized in the plasma. Nuclei were counterstained with DAPI. Labeling was confirmed by digital images under confocal microscopy.$ B F7 I6 I- ]8 u( b
4 _$ ]* O% u4 p, j& f; v/ TStatistical Analysis6 E/ O6 \4 z: K* f/ D2 H
2 h8 j% F9 \7 i* `# S" u# N. R
All data are expressed as mean ¡À SD. Statistical analysis was performed by a one-way analysis of variance and the Bonferroni t test or by the 2 test using StatView software (Version 5.0; SAS Institute Inc., Cary, NC, http://www.statview.com). Values of p 2 O- X' |* ^% R9 [" z" j. Y' H% G6 ~+ \7 k- u
RESULTS; }8 p+ y' E7 {! E, ^1 u
0 y$ y8 E( ~! C6 B
Detection of Extracardiac Stem Cells in the Donor Hearts After Transplantation9 A6 @* d* w) Y' l- z% @
5 B1 C" z$ e' P% n m( X' r) gWe extracted the donor hearts for cytological and histological analysis 0 (baseline), 2, 4, 8, and 12 weeks after transplantation. All of the donor hearts were perfused with approximately 20 ml of PBS immediately after extraction to wash out the circulating blood cells. Although a few macrophages and granulocytes were detected, we did not observe obvious lymphocyte infiltration or any other abnormal histological findings related to immunological rejection in these syngeneic donor hearts 2, 4, 8, or 12 weeks after transplantation (Fig. 1). However, GFP cells were detected in the donor hearts 2 weeks after transplantation (Fig. 2A¨C2C), and many of these cells showed positive expression of various stem markers, including Sca-1, c-kit, and CD133 (Fig. 2D, 2F). This indicates that the GFP stem cells of recipient origin immigrated into the donor heart within 2 weeks. These GFP stem cells were observed both as clusters and as single cells (Fig. 2D, 2F). Some of the cardiac stem cell clusters consisted of both GFP and GFP¨C cells (Fig. 2G). These GFP stem cells were distributed uniformly throughout the donor heart with no obvious regional variation.2 c7 v9 P$ o3 h- N# A
; O$ `3 q6 g2 h- j N" [
Figure 1. Histological findings and lymphocyte infiltration in the donor hearts. H&E staining (A) revealed normal myocardium in the donor hearts 2 and 12 weeks after transplantation. Immunostaining analysis of CD4 (B), CD8 (C), CD68 (D), and Gr-1 (E) infiltration revealed no obvious lymphocyte infiltration in the myocardium, although a few CD68 and Gr-1 positive cells (arrows) were detected 2 weeks after transplantation. The numbers of CD4, CD8, CD68, and Gr-1 positive cells are expressed as mean ¡À SD. Abbreviation: GFP, green fluorescent protein. : p0 P y% o; Q6 z3 `, l G$ c8 O7 z8 h3 ]+ e! a' L
Figure 2. Identification of extracardiac GFP stem cells in a donor heart 2 weeks after transplantation. GFP cells were detected by immunostaining with anti-GFP antibody followed by fluorescein isothiocyanate-conjugated secondary antibody (green, ) as clusters and as single cells. Scale bar, 20 µm. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein. - q9 y$ ]; g1 p9 O& I- E: V! k1 q9 `6 [* F5 q' M2 ~
Quantitative Analysis of Cardiac Stem Cells in the Donor Hearts ' \) ?- Z; N# v) s5 ?( E# U/ @ @, _+ m. r. i
To measure the number of endogenous cardiac progenitor cells and extracardiac GFP stem cells, all the cells were isolated from the donor hearts 0, 2, 4, 8, and 12 weeks after transplantation. We collected the isolated heart cells for two-color flow cytometry analysis and measured quantitatively the number of Sca-1-, c-kit-, and CD133-positive stem cells in the donor hearts and divided them into GFP and GFP¨C populations (Fig. 3A). The Sca-1-, c-kit-, and CD133-positive stem cell counts were calculated at approximately 5,000¨C10,000 cells per native heart (baseline) (Fig. 3B). Interestingly, 2 weeks after transplantation, approximately 40%¨C60% of the stem cells in the donor hearts were GFP cells (36.0% ¡À 2.7% for Sca-1, 56.0% ¡À 5.0% for c-kit, and 65.6% ¡À 2.6% for CD133). Thus, approximately half of the cardiac progenitor cells in the donor hearts were renewed by extracardiac stem cells of recipient origin within 2 weeks. The percentage of GFP stem cells in the donor hearts increased further 4 weeks after transplantation (82.2% ¡À 2.6% for Sca-1, 79.5% ¡À 3.9% for c-kit, and 85.8% ¡À 2.8% for CD133; p ?; d( ]" m% C8 h
# m! Q: Q/ [9 U6 M9 L- x! jFigure 3. Quantification and classification of stem cells in the donor hearts using two-color flow cytometry analysis. (A): Representative scatter plot of flow cytometry analysis for GFP (x-axis) and Sca-1, c-kit, or CD133 (y-axis) expression among the total isolated cells from the donor hearts 0 (baseline), 2, 4, 8, and 12 weeks after transplantation. (B): The total number of Sca-1-, c-kit-, and CD133-positive stem cells were counted in each donor heart, and these stem cells were presented by dividing them into GFP and GFP¨C populations. Each column represents a donor heart, and data show the numbers of stem cells expressed as mean ¡À SD for GFP and GFP¨C subpopulations at each time point (* p , u* n# s5 f Z- x
+ o6 e9 p7 L, `# J/ U z+ ?- HCharacterization of Endogenous and Exogenous Cardiac Stem Cells/ g/ f( k D3 Z9 ^; G9 R) g
+ p% ^- m! O( A" W5 t. {% N
To investigate the difference in phenotypes and characteristics between the exogenous GFP stem cells and the endogenous GFP¨C stem cells in the donor hearts, Sca-1- and c-kit-positive cells were purified from the total isolated cells of the donor hearts by magnetic separation (both >90% purity). In accordance with previous reports , we found that the endogenous GFP¨C subpopulation in these enriched Sca-1- and c-kit-positive cells expressed hematopoietic markers of CD45 and CD34 poorly, at less than 2% (Fig. 4). Relatively, the expression of CD45 in the GFP population of these enriched Sca-1- and c-kit-positive cells was approximately 20-fold higher, at almost 30%, than that in the endogenous GFP¨C stem cell population by 2 weeks after transplantation. This suggested a different phenotype between the new exogenous GFP stem cell population and the endogenous GFP¨C stem cell population in the donor hearts. The expression of CD34 in these enriched Sca-1- and c-kit-positive cells was also much higher in the GFP population than in the endogenous GFP¨C population. The relatively extensive expression of hematopoietic markers suggests that these exogenous GFP stem cells in the donor heart originate from the recipient's bone marrow. However, the expression of CD45 in the GFP stem cells tended to decrease over time, stabilizing at about 10% 8 and 12 weeks after transplantation. This suggests that the GFP stem cells gradually lost their expression of hematopoietic markers after immigration into the heart.( S; F$ j. t8 q& w! b# P* u7 K/ ~: P' r( L
) H. ^8 L/ t+ T" J+ XFigure 4. Characterization of cardiac stem cells using two-color flow cytometry analysis. Representative scatter plot of flow cytometry analysis for CD45 and CD34 expression in both the enriched Sca-1- (A) and c-kit-positive (B) stem cells from the donor hearts 4 weeks after transplantation (upper panel). Quantitative data showed distinct differences in the expression of CD45 and CD34 between the exogenous GFP stem cell population and the endogenous GFP¨C stem cell population (lower panel). The percentage expression of CD45 and CD34 in these exogenous GFP stem cells decreased over time after transplantation. Abbreviation: GFP, green fluorescent protein./ W& a7 d- H. ]4 t
. F& ~+ v4 I* K$ `Histological Analysis of the Phenotype and Differentiation of Extracardiac Stem Cells 1 p" v" b, R2 ^8 o, ]: P: W$ c& ^0 \ B/ A
We performed histological experiments to clarify whether these bone-marrow-derived GFP stem cells acquired the characteristics of cardiac progenitors and differentiated into mature cardiomyocytes. Unexpectedly, expression of the myogenic-specific transcription factors GATA-4 and Nkx2.5 in these GFP cells was almost negative 2 weeks after transplantation (Table 1). However, expression of GATA-4 or Nkx2.5 in the GFP cells was observed in the donor hearts 4 weeks after transplantation (Fig. 5A, 5B, and Table 1), suggesting that some of the GFP stem cells completed their phenotype transformation into cardiac progenitors within 4 weeks but not within 2 weeks. The expression of GATA-4 and Nkx2.5 in the GFP stem cells increased significantly over time (p * y' Z8 i2 d5 N0 P " Y* j" r% d& d/ t# wTable 1. The expression of hematopoietic markers, cardiac transcript factors, and cardiac-specific proteins in green fluorescent protein (GFP) cells within donor hearts after transplantation ! f0 P/ Z' M: h$ o7 C* z6 ~& Q0 P/ j+ [8 z/ t
Figure 5. Transformation of cardiac progenitor cells and cardiomyogenic differentiation from extracardiac GFP stem cells. (A): One of three distributed GFP cells (green) showing positive expression of GATA-4 (red dot, double arrows) in a donor heart 4 weeks after transplantation and one GFP¨C cell also showing positive expression of GATA-4 (red dot, single arrow). (B): One Nkx2.5-positive cell (red dot, double arrows) was detected within a GFP cell cluster (green) in a donor heart 8 weeks after transplantation. (C): A single rod-shaped GFP cell (green, arrowhead) was stained positively for -myosin heavy chain (red) in a donor heart 12 weeks after transplantation, whereas multiple adjacent GFP cells were negative. (D): GFP cardiomyocytes were also identified occasionally by immunostaining for troponin T (red) and GFP (green) in a donor heart 12 weeks after transplantation. Scale bar, 20 µm. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein. $ o8 \, u/ a& F$ i: u) l. a* o& ?7 X' a8 }) I
Our histological findings strongly support the data on the phenotypes and characteristics of purified Sca-1- and c-kit-positive stem cells based on flow cytometry analysis, revealing that approximately 30% of the GFP cells expressed CD45 2 weeks after transplantation. The expression of the hematopoietic markers CD45 and CD34 decreased significantly thereafter (p 7 q# ~/ l+ d3 A r' M0 o6 _9 {- A
3 N$ j" D( ?. m' I5 R# L
The differentiation of these bone-marrow-derived GFP cells into mature cardiomyocytes was also determined by expression of the cardiac-specific proteins -myosin heavy chain and cardiac troponin T. These GFP cells did not express -myosin heavy chain or cardiac troponin T 2 or 4 weeks after transplantation (Table 1); however, a sparse subset of GFP cells, accounting for less than 0.5%, that expressed -myosin heavy chain or troponin T was detected 8 and 12 weeks after transplantation (Fig. 5C, 5D, and Table 1), suggesting the potential cardiomyogenic differentiation of these exogenous GFP cells.5 c2 b* Y9 D0 j* h. N
1 M% E6 U% S; O; V. z7 C) P
Detection of Cell Fusion by FISH Analysis 1 j" n& w# S6 V1 K& f 1 g( s9 Z0 s) B" O! F) ZThe property of cell fusion between the donor heart cells and exogenous GFP stem cells was identified by FISH and immunostaining analysis (Fig. 6). Compared with a previous study on infarcted hearts , we observed cell fusion as a very rare event in the uninjured heart after transplantation (0.136%, 0.202%, 0.215%, and 0.167% at 2, 4, 8, and 12 weeks, respectively; Fig. 6A). This might be attributable to the different methods of analysis and experimental models in the two studies. Furthermore, we detected one X and one Y chromosome uniformly in a total of 15 GFP cells that expressed -myosin heavy chain (Fig. 6B)., ?; t) Y: x( Q1 w6 |- E, z8 H
+ G; I3 A' d& w( c q1 a! l/ ~
Figure 6. Identification of cell fusion by fluorescence in situ hybridization analysis. (A): Cell fusion was observed as a rare event (arrow) in the donor heart (approximately 0.1%¨C0.2%). (B): A representative GFP cell that expressed -myosin heavy chain in a donor heart was identified distinctly to have one X and one Y chromosome 12 weeks after transplantation. Scale bar, 20 µm. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein.2 v: L! _% C: I5 |: J5 A
$ l9 f" d/ A- {: R2 L' TDISCUSSION 5 F; u3 l7 r( J8 Z " N. z$ `6 d+ D& ^Our findings in this heterotopic heart transplantation model provided evidence that extracardiac stem cells, possibly of bone marrow origin, immigrate into the donor heart and then change their phenotype gradually into cardiac progenitor cells by shifting to express the cardiac-specific transcription factors. Furthermore, the number of extracardiac stem cells increased, whereas the number of endogenous cardiac progenitor cells decreased over time in the donor hearts. These findings indicate that extracardiac stem cells contributed to the renewal of cardiac progenitor cells.2 W L% k# C- F9 N/ N
. w* F7 x* ~$ m' c }
Although the well established bone marrow transplantation model is generally used to identify bone marrow origin , we selected a heterotopic heart transplantation model to identify the origin of cardiac stem cells in this study. Because time is needed to reconstruct a new hematopoietic system after bone marrow transplantation, it is impossible to observe precisely the time-related changes in cardiac stem cells by using the bone marrow transplantation model. Moreover, the instability of the chimerisms of the bone marrow transplantation model would affect the quantitative analysis of cardiac stem cells. Naturally, there are several shortcomings of the donor heart model, including its abnormal physiologic condition. The most critical limitation of this donor heart model is that all cells in the recipient were GFP . Thus, the precise origin of these exogenous GFP stem cells in the donor heart could not be identified, although the relatively extensive expression of hematopoietic markers in these GFP cardiac stem cells at an early phase strongly supports the belief that the exogenous GFP stem cells in the donor heart are hematopoietic stem cells, possibly of bone marrow origin.# \# ]3 w( o/ G
3 B4 V: y9 `1 T( T8 T. KThe reason for the progressive decrease in endogenous GFP¨C cardiac stem cells remains unknown. It was reported recently that the proliferation of endogenous cardiac progenitor cells is largely responsible for maintaining the homeostasis of the stem cell pool in hearts under physiologic conditions . However, we found that less than 1% of these cardiac progenitor cells expressed Ki-67 positivity, indicating a relatively low proliferating potency of cardiac stem cells in the normal heart under physiological condition (data not shown). The progressive decrease in the number of endogenous GFP¨C cardiac progenitor cells might be due to the poor proliferating activity. Therefore, the self-proliferation potency of endogenous cardiac progenitor cells may be inadequate, and some extracardiac stem cells will enter the heart to maintain homeostasis of the stem cell pool in the heart. ?& b5 j7 H0 z; I & [6 _( {+ l% @: x0 l, ]In this experimental study, we observed rare cardiomyogenic differentiation from GFP stem cells, which may be attributed to the fact that we used uninjured donor hearts. As progenitor (stem) cells are considered to differentiate and mature in response to injury , the lack of injury and cardiac stress in the donor heart model will reduce the requirement for myocyte renewal. It will be of great interest to identify if the induction of heart injury and cardiac stress can increase these GFP cardiac progenitor cells to differentiate into mature cardiomyocytes for cardiac repair. 1 h6 j6 ^# O2 K- X, v ) H* ~' U% ^* v1 }Previous studies have found the cardiomyogenic differentiation of bone-marrow-derived stem cells through cell fusion . Thus, we tried to determine if the cardiomyogenic differentiation of GFP stem cells also occurred through cell fusion. Although we analyzed only a limited number of cells, we detected one Y chromosome and one X chromosome uniformly in the GFP cells that expressed -myosin heavy chain. Furthermore, if GFP stem cells fused with host cardiomyocytes, then GFP cardiomyocytes should also be found in the donor heart soon after transplantation. In fact, none of the GFP cells expressed -myosin heavy chain or troponin T in the donor hearts in the early phase, 2 or 4 weeks after transplantation. These findings suggest that extracardiac stem cells differentiated into cardiac progenitors, and finally into myocytes, independent of cell fusion. 3 V$ `! n) w- O# r5 c) B( X; _( g$ {1 X d. y M
Our data provided strong evidence that extracardiac stem cells, possibly of bone marrow origin, acquire the characteristics of cardiac progenitor cells spontaneously within approximately 4 weeks, differentiating occasionally into mature cardiomyocytes within approximately 8 weeks. We noted expression of the hematopoietic markers, cardiac-specific transcription factors, and cardiac-specific proteins of these GFP cells as a clear pattern at specific times after transplantation in the uninjured donor mouse heart. Although we were unable to determine whether this process would be accelerated in an injured heart, we think that the development of extracardiac stem cells into cardiac progenitor cells, and finally into mature cardiomyocytes, would involve a slow step-by-step process in response to myocardial microenvironment cues. According to our data, a slow step-by-step process, being approximately 8 weeks in mice, would be necessary for the differentiation of extracardiac stem cells into mature cardiomyocytes. The relatively slow process of cardiomyogenic differentiation from hematopoietic stem cells might provide a valid explanation for the conflicting results that intramyocardial implanted bone marrow stem cells did not differentiate into mature cardiomyocytes . Further studies are necessary to identify the specific factors and cell signal pathways for regulating the complex processes involved in the transformation of hematopoietic stem cells into cardiac progenitor cells and their subsequent differentiation into cardiomyocytes.0 v% q% T! N4 T0 S7 j. F
8 r5 c |' K4 ]7 |
In summary, extracardiac stem cells may be of bone marrow origin, from which they can immigrate into the heart and then change into cardiac progenitor cells by shifting to express the cardiac-specific transcription factors, finally differentiating into mature cardiomyocytes in the absence of cell fusion. The results of this study provide the first evidence that extracardiac stem cells can transform into cardiac progenitor cells to maintain the homeostasis of the stem cell pool in the heart.- L1 M( _. [8 k* V9 [
1 _* U% [* e2 B2 i& n* @* X& vDISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST & l. ]: Q6 @/ _ & b1 N3 }* i" a3 c# OThe authors indicate no potential conflicts of interest.. w, m- M" o0 W
# ~7 K3 B; e( q9 H& O C. vACKNOWLEDGMENTS t! l1 b. L( d/ f; r" I4 [6 }3 q
% X1 R- \5 k2 c% D4 q( D/ V/ ^( WWe thank Mako Ohshima for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. / c, s, c! t4 L 【参考文献】+ P4 ]( w. ^, Y8 l
3 j2 c" J/ ~; W, P& p) `7 M$ `( c0 t/ b1 a1 f
Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763¨C776.8 F" |, e' U5 R' e& q( a5 X
' H( F6 i: H! b& L" G
Oh H, Bradfute SB, Gallardo TD et al. Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 2003;100:12313¨C12318.: f7 f' J% n6 P) H) K' m
6 {& O- Q+ N$ L
Laugwitz KL, Moretti A, Lam J et al. Postnatal isl1 cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433:647¨C653." j1 m A7 y2 v5 s: A! @+ [) J
! l' e4 _0 A- ~' Z4 A
Matsuura K, Nagai T, Nishigaki N et al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem 2004;279:11384¨C11391.% M( m9 M$ }! C& ~& y" e# ^# g
& A, B* P A+ z
Messina E, De Angelis L, Frati G et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res 2004;95:911¨C921. 0 @8 O7 }/ S+ f7 | 0 a) ~) o/ j( w4 f; dPfister O, Mouquet F, Jain M et al. CD31- but not CD31 cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52¨C61. / M: u( G# Q; O! R 4 E1 j$ M0 `) F0 w) T8 u7 n. v5 g# D; J# eLaflamme MA, Myerson D, Saffitz JE et al. Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circ Res 2002;90:634¨C640.: x6 A+ f) Q0 Y J5 j1 Z7 S
, ^* I$ v& g w4 H9 P0 r2 K
Quaini F, Urbanek K, Beltrami AP et al. Chimerism of the transplanted heart. N Engl J Med 2002;346:5¨C15. # @7 u8 @/ o5 T1 ` f# `0 n5 }$ J1 h8 B. U% i
Muller P, Pfeiffer P, Koglin J et al. Cardiomyocytes of noncardiac origin in myocardial biopsies of human transplanted hearts. Circulation 2002;106:31¨C35. 5 \3 N, M- q% b" |3 f" K, C5 r
Deb A, Wang S, Skelding KA et al. Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients. Circulation 2003;107:1247¨C1249.* p1 X8 @/ O# Q' C$ Y
" z& n+ x, {' p6 H& j
Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697¨C705. ( I, d; F7 \7 V9 `' l* v" h' M0 g. {2 b6 {7 V. \
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41¨C49. 9 n. P" P0 @" w/ ` G: O' I- f& j- t1 u7 v3 c! B
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701¨C705.8 K2 y( I/ |4 v# {" R+ o& Q! C# Y
& p6 A' W% I1 Z+ W4 WJackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395¨C1402. 9 _8 \! H" [ Q/ U5 o6 a, o2 U0 Z& F# C) p6 y. } s
Li TS, Hayashi M, Ito H et al. Regeneration of infarcted myocardium by intramyocardial implantation of ex vivo transforming growth factor-beta-preprogrammed bone marrow stem cells. Circulation 2005;111:2438¨C2445.- i. ]2 G- M+ q; k% l1 P
( o* ~: j' L7 ]* z
Hamano K, Rawsthorne MA, Bushell AR et al. Evidence that the continued presence of the organ graft and not peripheral donor microchimerism is essential for maintenance of tolerance to alloantigen in vivo in anti-CD4 treated recipients. Transplantation 1996;62:856¨C860.! R4 n1 L9 b0 S2 x+ ^5 n7 ~3 x
* g, u6 I. ]3 r, h% ]2 nOkabe M, Ikawa M, Kominami K et al. ¡®Green mice' as a source of ubiquitous green cells. FEBS Lett 1997;407:313¨C319.4 [! T/ L- Q( y* U0 o
/ s4 b( x8 w2 w% [& G% u0 n
Nygren JM, Jovinge S, Breitbach M et al. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med 2004;10:487¨C493. $ |; A! n3 W/ ]$ M6 S1 M. ]3 O1 X: C, @$ s; R% `
Balsam LB, Wagers AJ, Christensen JL et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischemic myocardium. Nature 2004;428:668¨C675./ Q0 Z( q- m% A" V. j8 n; q. i
- i0 W& {9 ?; j5 C, y2 U
Murry CE, Soonpaa MH, Reinecke H et al. Hematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664¨C668.. t: F) F3 D1 e6 Q9 p
3 a% H6 f0 {6 a$ e m- U+ \5 _+ \
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256¨C2259.- g G& O' J4 n' f( n
) N+ s# K1 T2 I% T4 ]0 H. q6 wMouquet F, Pfister O, Jain M et al. Restoration of cardiac progenitor cells after myocardial infarction by self-proliferation and selective homing of bone marrow-derived stem cells. Circ Res 2005;97:1090¨C1092." l! e/ r/ T b& W( c! i1 ?2 K/ S
# N- Q: E: |5 |0 U) B9 ~LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589¨C601. ; O( R3 A' L; m$ K " U! F* t. E/ n! Z, j& ITerada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542¨C545.8 D4 Y" b3 v6 H5 @+ J3 x: w5 r( R
/ ]2 h* W; d$ h$ U" K7 \Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545¨C548.作者: falconli 时间: 2009-3-20 08:20