The Role of the Sca-1 /CD31C Cardiac Progenitor Cell Population in Postinfarctio - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Xiaohong Wang, Qingsong Hu, Yasuhiro Nakamura, Joseph Lee, Ge Zhang, Arthur H.L. From, Jianyi Zhang作者单位：Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
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      【摘要】
   Cardiac stem cell-like populations exist in adult hearts, and their roles in cardiac repair remain to be defined. Sca-1 is an important surface marker for cardiac and other somatic stem cells. We hypothesized that heart-derived Sca-1 /CD31¨C cells may play a role in myocardial infarction-induced cardiac repair/remodeling. Mouse heart-derived Sca-1 /CD31¨C cells cultured in vitro could be induced to express both endothelial cell and cardiomyocyte markers. Immunofluorescence staining and fluorescence-activated cell sorting analysis indicated that endogenous Sca-1 /CD31¨C cells were significantly increased in the mouse heart 7 days after myocardial infarction (MI). Western blotting confirmed elevated Sca-1 protein expression in myocardium 7 days after MI. Transplantation of Sca-1 /CD31¨C cells into the acutely infarcted mouse heart attenuated the functional decline and adverse structural remodeling initiated by MI as evidenced by an increased left ventricular (LV) ejection fraction, a decreased LV end-diastolic dimension, a decreased LV end-systolic dimension, a significant increase of myocardial neovascularization, and modest cardiomyocyte regeneration. Attenuation of LV remodeling was accompanied by remarkably improved myocardial bioenergetic characteristics. The beneficial effects of cell transplantation appear to primarily depend on paracrine effects of the transplanted cells on new vessel formation and native cardiomyocyte function. Sca-1 /CD31¨C cells may hold therapeutic possibilities with regard to the treatment of ischemic heart disease.
      【关键词】 Cardiac Sca- /CDC cells Cardiac remodeling Cardiac bioenergetics Myocardial infarction; T" Z+ _4 e: o. t1 t  x9 z
   INTRODUCTION

Postinfarction left ventricular (LV) remodeling is a complicated healing process that involves many molecular signaling pathways and cell types .+ W7 k5 g" ^+ ^4 F; x: w2 B( R

Stem cell antigen-1 (Sca-1) is a member of the ly-6 family, which was first reported as one of the cell surface markers of hematopoietic stem cells . Taken together, these findings indicate that somatic stem cells bearing Sca-1 have potential for clinical use in skeletal muscle and cardiac repair. Thus, further elucidation of the potential role(s) of Sca-1  cells in cardiac disease models is pertinent and might yield strategies for using these cells therapeutically.

In this report, we confirm that Sca-1 /CD31¨C cells isolated from the myocardium are progenitor cells possessing both endothelial cell and cardiomyogenic differentiation potential . Importantly, we report a significant expansion of Sca-1 /CD31¨C cell numbers in the peri-infarction and infarct zones in a murine model of MI and show that transplantation of Sca-1 /CD31¨C cells into the myocardial peri-infarction zone at the time of coronary ligation significantly limits the structural and functional consequences of MI-induced LV remodeling; most strikingly, remodeling-associated degradation of myocardial energetic characteristics are markedly attenuated.

MATERIALS AND METHODS5 F: P9 {0 w' t2 U; L* h- X9 K

Wild-type mice (BALB/c, 8¨C10 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The University of Minnesota Animal Care Committee approved all procedures and protocols. The investigation conformed to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Research.5 k/ L9 K8 X( V, e& a1 e

Sca-1 /CD31¨C Cell Isolation and Fluorescence-Activated Cell Sorting Analysis

Suspensions of cardiac cells or skeletal muscle depleted of myocytes were prepared as follows. Briefly, minced cardiac or skeletal muscle tissue was digested with 10 mg/ml collagenase type B (Roche Diagnostics, Basel Switzerland, http://www.roche-applied-science.com), 2.4 U/ml dispase II (Roche Diagnostics), and 2.5 mM CaCl2 at 37¡ãC for 20 minutes, followed by filtration with a Netwell filter (74-µm pore size; Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com). The cell suspension was centrifuged at 1,500g for 5 minutes and resuspended in phosphate-buffered saline (PBS) containing 3% fetal bovine serum (FBS).0 T3 r# X$ r( u3 H

To determine the fractional content of Sca-1 /CD31¨C cells in myocyte-depleted preparations of normal and post-MI cardiac muscle, as well as in normal skeletal muscle, aliquots containing 106 cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-Sca-1 antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com), PE-conjugated anti-CD31 antibody (BD Biosciences) and APC-conjugated CD45 antibody, or control IgG for 20 minutes at 4¡ãC. Samples were measured using a FACS Calibur cytometer (BD Biosciences), and the data were then analyzed using CellQuest software.7 C/ [. b) U, X& x5 ~, a
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To isolate Sca-1 /CD31¨C and Sca-1¨C/CD31¨C cells from whole heart cardiomyocyte depleted cell suspensions, the suspension was incubated for 20 minutes at 4¡ãC with FITC-conjugated anti-Sca-1 antibody (BD Biosciences) and PE-conjugated anti-CD31 antibody (BD Biosciences), washed in PBS supplemented with 3% FBS, and then incubated with anti-PE microbeads for 20 minutes at 4¡ãC and passed through a magnetic cell sorting (MACS) column in a Miltenyl magnet to separate CD31  and CD31¨C cells. The CD31¨C cells were incubated with anti-FITC microbeads for 20 minutes at 4¡ãC and then separated through the MACS column into Sca-1 /CD31¨C cell and Sca-1¨C/CD31¨C cell populations. The magnetic sorting was repeated to increase the purity of Sca-1 /CD31¨C cell preparations. Subsequent fluorescence-activated cell sorting (FACS) analyses and immunofluorescent staining indicated that fractional Sca-1 /CD31¨C cell content of the purified suspension was approximately 90%.
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Matrigel Tubule Formation Assay( `- |+ u6 J+ V; j. F
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Four-well Lab-Tek chamber slides were coated with growth factor-reduced Matrigel (50 µl/cm2; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) according to the manufacturer¡¯s instructions. Five x 105 cells were plated and incubated at 37¡ãC. Network formation was observed using a phase-contrast microscope, and networks were stained with alkaline phosphatase according to the manufacturer¡¯s instructions (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com)." I1 K& U* `* J9 s" ]

In Vitro Differentiation of Sca-1 /CD31¨C Cells

Sca-1 /CD31¨C and Sca-1¨C/CD31¨C cell isolates were cultured on 1% fibronectin-coated dishes with Iscove¡¯s modified Dulbecco¡¯s medium (IMDM) supplemented with 20% FBS, 100 µg/ml penicillin, and 250 µg/ml streptomycin at 37¡ãC in humidified air containing 5% CO2. To induce endothelial cell differentiation, Sca-1 /CD31¨C cells were plated at 2 x 104 cells per cm2 on fibronectin-coated surfaces in a four-well chamber slide or six-well plate and treated with 10 ng of vascular endothelial growth factor (VEGF) in IMDM supplemented with 1% FBS for 14 days. Endothelial cell makers were determined by immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR). To induce cardiomyocyte differentiation, Sca-1 /CD31¨C cells were plated at 2 x 104 cells per cm2 on fibronectin-coated surfaces and cultured in the basal medium containing 10 ng/ml Wnt antagonist Dickkoff-1 (DDK-1), 0.75% dimethyl sulfoxide (DMSO), 10 ng/ml bone morphogenetic protein (BMP) 2, 100 ng/ml fibroblast growth factor 4 (FGF4), and 10 ng/ml FGF8 in combination (with the addition of 10 µM 5'-azacytidine for the initial 3 days); total time in culture was 14 days. Immunohistochemical and immunofluorescent staining were used to determine the extent of differentiation to cardiomyocytes. Groups of Sca-1¨C/CD31¨C cells cultured under the same conditions in the absence of induction factors served as controls.6 J% q! G9 l# d5 y6 i2 P* U% @' L. W

Coculture of Sca-1 /CD31¨C Cells with Rat Neonatal Cardiomyocytes; p' R! K* Z. [3 P! n3 _
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Primary cultures of neonatal cardiomyocytes were prepared by enzymatic digestion of ventricles obtained from 2-day-old Sprague-Dawley rats as previously described . Coculture experiments were set up in fibronectin-coated chamber slides at a total density of 2 x 104 (1:1 ratio; cardiomyocytes/Sca-1 /CD31¨C cells) in IMDM supplemented with 20% FBS, 100 µg/ml penicillin, and 250 µg/ml streptomycin at 37¡ãC in humid air with 5% CO2. Before coculture, Sca-1 /CD31¨C cells were infected with 100 plaque-forming units per cell nuclear LacZ adenovirus. After infection, cells were extensively washed before being cocultured with rat neonatal cardiomyocytes. After 3 days, cultures were fixed with 2% paraformaldehyde and subjected to ß-galactosidase and immunofluorescence stainings.6 A& `* _2 Q7 A0 z; L
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Immunostaining& D6 k( j5 R- [+ m5 i

Sca-1 /CD31¨C cells and cell cocultures were prepared on chamber slides. Staining for endothelial cell markers included von Willebrand factor (vWF) (BD Biosciences), caveolin-1 (BD Biosciences), and CD31 (BD Biosciences). Staining for cardiac differentiation markers included troponin T (Labvision, Fremont, CA, http://www.labvision.com), GATA-4 (Santa Cruz Biotechnology), and Homeobox protein NKx2.5 (NKx2.5) (Santa Cruz Biotechnology). Visualization was achieved using conjugated secondary antibodies and nuclear staining with 4,6-diamidino-2-phenylindol dihydrochloride (DAPI). All studies were performed in triplicate using samples from different culture preparations. Control staining was performed without primary antibody.3 R, g  i  S$ V  Y3 |: e

RT-PCR Analysis
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Total RNA was isolated using RNeasy columns (Qiagen, Hilden, Germany, http://www1.qiagen.com) with RNase-free DNase treatment. One µg of total RNA was used for reverse transcription reactions using oligo(dT)18 as a primer. Primers for amplification were as follows. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5'-ACCACAGTCCATGCCATCAC-3', antisense, 5'-TCCACCACCCTGTTGCTGTA-3'; Flt1:sense, 5'-CGCGCCTCAGATCACTTGGTTC-3', antisense, 5'-TCCGGCAGGTGGGTGATTTCTTA-3'; CD31: sense, 5'-CCCGGTGGATGAAGTTGTGAT-3', antisense, 5'-CATGTTCTGGGGGTCTTTATTTTG-3'; vWF: sense, 5-CCCCCAGAGCTGTGAAGAAAAGA-3', antisense, 5'-GGCTCGGGGGTATCCTCAACAT-3'. The PCR programs were as follows: Flt1, 40 cycles; CD31, 35 cycles; GAPDH, 25 cycles, annealing at 60¡ãC; and vWF, 30 cycles, annealing at 65¡ãC.  |1 Y) s5 F# |3 O+ a9 g
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Animal Surgery and Cell Transplantation" j; ^& [$ K: d) M

Adult female BALB/c mice aged 10¨C12 weeks were employed for this study. They were housed in trios or quartets with food and tap water ad libitum. Myocardial infarction was induced by left coronary artery (LAD) ligation as previously described . Briefly, mice were anesthetized by intraperitoneal injections of sodium pentobarbital (35 mg/kg) and lidocaine hydrochloride (10 mg/kg), instrumented with a standard limb lead II electrocardiogram (ECG), intubated, and mechanically ventilated using a small-animal respirator (Harvard Apparatus). Under a stereomicroscope, the heart was accessed via left thoracotomy, and the LAD was ligated with a 9-0 surgical suture to produce myocardial infarction and ischemia. Intramyocardial injections of heart-derived Sca-1 /CD31¨C cells in saline, Sca-1¨C/CD31¨C cells in saline, or saline alone were administered at five sites in the peri-infarct zone immediately following LAD ligation (total number of cells injected = 1 x 106).2 W0 F" r6 A7 Z; K
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Echocardiography9 m8 Y: h% ~$ ?4 x
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Echocardiography was performed 1, 2, and 3 weeks after myocardial infarction using an echocardiographic system equipped with a 15.6 MHz phased-array transducer (SONOS 5500; Philips Medical System, Best, The Netherlands). Mice were lightly anesthetized using ketamine HCl (25 mg/kg i.p.) and xylazine (10 mg/kg i.p.), and chest fur was removed using a depilatory cream. Two-dimensional echocardiographic images and M-mode traces were taken from the parasternal short-axis view at the level of papillary muscles. To evaluate LV structural changes, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured. Left ventricular ejection fraction (LVEF) was calculated as an index of systolic function.6 W2 s9 ^3 ~* q8 n
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Open-Chest 31P Nuclear Magnetic Resonance Spectroscopy

31P nuclear magnetic resonance spectroscopy (31P MRS) was conducted as previously described, with some modifications . Briefly, cardiac MRS was performed at 4.7 Tesla using a custom-built, 1H/31P-double-tuned, 10-mm diameter nuclear magnetic resonance surface coil. Mice were anesthetized using a bolus intraperitoneal injection of sodium pentobarbital (35 mg/kg), intubated, and ventilated at 100 breaths per minute with a tidal volume of 0.5 ml. A small Bovie cautery (Medical Resource USA, San Antonio, TX, http://store.mediaresourceusa.com) was used to remove the anterior ribcage and expose the beating heart. The mice were then placed prone onto the coil assembly with the heart centered on the coil axis and inserted into the magnet bore. A thin plastic film was placed between the heart and the coil to support the weight of the heart and maintain mediastinal moisture.

The 1H signal of water was used for positioning and shimming the mouse heart. Nuclear magnetic resonance (NMR) signal acquisition occurred during mid-diastole via gating (SA Instruments Inc., Stony Brook, NY) with a two-lead ECG probe system. The 31P transmitter frequency offset was placed between phosphocreatine and -ATP resonances. 31P NMR spectrum was acquired over a 6,000 Hz spectral width using a 1,000- ms adiabatic half-passage radiofrequency pulse with a repetition time of 6 seconds and 256 free inductive decay averages. Spectra were corrected for 90% phosphocreatine saturation. Phosphocreatine ( ratios were calculated from the integrals of PCr and -ATP resonances.
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Western Blotting& F" j4 L4 U3 k

SDS-polyacrylamide gel electrophoresis and Western blotting were carried out as previously described . The membranes were reprobed with a mouse monoclonal GAPDH antibody to verify equal loading., Q* a  d0 [9 E/ \- M
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Engrafted Cells Number and Differentiation Status' u: j3 I' `: l% C7 x( Q
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Mouse hearts that had received LacZ transduced Sca-1 /CD31¨C or Sca-1¨C/CD31¨C cells were fixed with 2% paraformaldehyde and subjected to ß-galactosidase staining. Hearts were photographed, embedded into Tissue-Tek OCT (Fisher Scientific), transversely sectioned into 8-µm slices using a cryostat, and stained for vWF, phospholamban (Labvision), -myosin heavy chain (Abcam, Cambridge, MA, http://www.abcam.com), and N-cadherin (Novus Biologicals) antibodies. The sections were visualized using fluorescence-labeled secondary antibodies (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). The total number of cell nuclei per high power (20x) were identified by DAPI (Sigma-Aldrich) staining. The engraftment cell rate was determined by counting DAPI and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) DAPI double-stained nuclei in every 10th serial section of the whole heart.  s8 n, j" I/ h9 [

Analysis of Neovascularization) O1 e' b+ X% P. ?" @9 o

Capillaries were counted at a magnification of 20x using an Olympus microscope (Olympus BX51/BX52). Border zones around the infarct site were examined for the number of vWF-stained capillaries. The quality of the computer analysis of capillary numbers (NIH Image J program, http://rsb.info.nih.gov/ij) was checked against manual counting. The capillaries were counted in blinded fashion on 50 sections (two fields per section, five sections per heart, n = 5 for each group) in the peri-infarct zone.
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Statistical Analysis' I! s0 }& K9 }) \+ Q
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Comparisons between two groups were analyzed using Student¡¯s t test (p

Comparisons among three groups were analyzed using one-way analysis of variance followed by Student-Newman-Keuls post hoc tests (p ) z9 y1 ]8 [5 c7 z1 D, m2 Z
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RESULTS
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In Vitro Studies2 Z' u& Z: g& f2 P% U" ?- y6 g

Sca-1  /CD31¨C Cells Demonstrated Endothelial and Cardiomyogenic Differentiation. To assess the endothelial differentiation potential of both cardiac- and skeletal muscle-derived Sca-1 /CD31¨C cell populations, aliquots of both were cultured on Matrigel. By day 3, Sca-1 /CD31¨C cells from both cardiac and skeletal muscle gave rise to numerous microtubular structures. In contrast, no microtubule formation was seen following culture of Sca-1¨C/CD31¨C cells (data not shown). Alkaline phosphatase staining was positive in 50% of the microtubules derived from heart Sca-1 /CD31¨C cells (Fig. 1A) and in 40% of Sca-1 /CD31¨C cells derived from skeletal muscle (not shown). In addition, positive caveolin-1 staining was present in microtubules derived from both cardiac and skeletal muscle-derived Sca-1 /CD31¨C cells (Fig. 1A).

Figure 1. The heart-derived Sca-1 /CD31¨C cell population possesses endothelial cell differentiation capacity. (A): Left, Sca-1 /CD31¨C cells were cultured in growth factor-reduced Matrigel and stained for alkaline phosphatase (purple). Right, H-derived and S-derived Sca-1 /CD31¨C cells were cultured in growth factor-reduced Matrigel; immunofluorescence staining for caveolin-1 (red) was positive in both H and S. (B): Immunofluorescence staining showed heart-derived Sca-1 /CD31¨C cells expressed endothelial cell markers, including caveolin-1 (red), CD31(red), and vWF (red), after being induced by 10 ng/ml VEGF for 14 days. (C): Expression of CD31, Flt1, and vWF mRNA was induced in heart-derived Sca-1 /CD31¨C cells by 14 days of culture with VEGF. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H, heart; S, skeletal muscle; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.3 R( \% j3 c7 ]. u& u, o
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Cardiac-derived Sca-1 /CD31¨C cells were induced by VEGF (10 ng/ml medium) for 14 days. Immunofluorescence staining revealed significant increases in CD31, caveolin-1, and vWF protein expression in Sca-1 /CD31¨C cells after induction by VEGF (Fig. 1B). RT-PCR confirmed CD31, Flt1, and vWF mRNA expression in Sca-1 /CD31¨C cells induced by VEGF (Fig. 1C), suggesting that the Sca-1 /CD31¨C cell population can differentiate into endothelial cells in vitro. It should be noted that non-VEGF-induced cells also showed low mRNA expression levels of endothelial cell markers by RT-PCR after 14 days of culture. This suggests that prolonged culture itself may induce a modest level of differentiation that was not present in these cells prior to prolonged culture. Finally, the possibility exists that endothelial differentiation in our purified Sca-1 /CD31¨C cell suspension resulted from differentiation of a small subpopulation of contaminating Sca-1 /CD31  cells. Evidence against this experimental artifact is the finding that a purified suspension of Sca-1¨C/CD31¨C cells (which would be as likely to suffer contamination by other subpopulations of cells) did not differentiate into endothelial-like cells (data not shown).
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To induce cardiomyogenic differentiation, Sca-1 /CD31¨C cells were plated at 1¨C2 x 103 cells per cm2 on fibronectin-coated surfaces. We tested several permutations of differentiation-inducing cytokines and drugs. The most effective was the presence of DKK-1, DMSO, BMP2, FGF4, FGF8, and 5-azacytidine in the medium during a 14-day culture period. This induction protocol induced expression of cardiac-specific genes GATA-4 and NKx2.5 (Fig. 2A, 2B). However, spontaneous contractions were not seen in these partially differentiated cells. LacZ-labeled cardiac Sca-1 /CD31¨C cells cocultured with neonatal rat cardiomyocytes showed further differentiation, as evidenced by positive staining for troponin T and phospholamban (Fig. 2C), suggesting that coculture induces more complete Sca-1 /CD31¨C cell differentiation. These data indicate (not surprisingly) that the stem cell microenvironment plays an important role in determining cardiomyocyte differentiation.

Figure 2. Sca-1 /CD31 cells were induced to differentiate into cardiomyocyte-like cells. Sca-1 /CD31¨C cells were cultured in the basal medium containing 10 ng/ml Dickkoff-1, 0.75% dimethyl sulfoxide, 10 ng/ml BMP2, 100 ng/ml fibroblast growth factor 4 (FGF4), and 10 ng/ml FGF8 in combination (with the addition of 10 µM 5'-azacytizine for the initial 3 days); total time in culture was 14 days. (A): Immunofluorescence staining of induced Sca-1 /CD31¨C cells showed nuclear expression of NKx2.5-stained (left, red) and DAPI-stained (middle) nuclei. Right, merged view of NKx2.5-stained and DAPI-stained nuclei. (B): GATA-4-stained (left, red) and DAPI-stained (middle) nuclei. Right, merged view of GATA-4-stained and DAPI-stained nuclei. (C): Sca-1 /CD31¨C cells cocultured with neonatal rat cardiomyocytes were induced to express cardiac specific markers. Top row of photomicrographs shows troponin T (red), DAPI (light blue), X-gal (dark blue), and a merged view of the preceding photomicrographs. Arrows point to triple-positive cells. The bottom photomicrograph shows an X-gal-positive cell (blue) costained for phospholamban (brown). Abbreviations: BMP, bone morphogenetic protein; DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; DKK1, Wnt antagonist, Dickkoff-1; NKx2.5, Homeobox protein NKx2.5; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.' c& j! [( R6 s8 v

In Vivo Studies

Sca-1  /CD31¨C Cells Increase Significantly in Myocardium After Infarction. Initial experiments were designed to compare endogenous Sca-1 /CD31¨C cell fractions in populations of myocyte-free muscle cell populations of mouse heart and skeletal muscle. FACS analyses showed that normal hearts have threefold more Sca-1 /CD31¨C cells than does skeletal muscle (Fig. 3A).* v( `5 ^% V& m$ M% L9 U1 c
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Figure 3. A significant increase of Sca-1 /CD31¨C cell population after myocardial infarction. (A): Representative fluorescence-activated cell sorting analyses of cardiomyocyte-depleted cell suspensions obtained from a normal heart (left), a normal skeletal muscle (middle), and a heart that was infarcted 7 days prior to cell harvest (right). Significantly more endogenous Sca-1 /CD31¨C cells (right bottom quadrant) are present in normal myocardium than in skeletal muscle. The number of Sca-1 /CD31¨C cells in myocardium increased after myocardial infarction. (B): The time course of the increase of the myocardial Sca-1 /CD31¨C cell population following infarction. (C): Numerous endogenous Sca-1  cells distributed in the peri-infarct region 7 days after MI. Arrows point to Sca-1 (red) positive cells in upper photomicrograph and double-positive cells in merged picture of the same section stained with DAPI (blue). (D): Representative Western blot demonstrating enhanced Sca-1 protein expression in hearts 7 days after MI (top); same blot reprobed with GAPDH antibody as controls for equal loading (bottom). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction.
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FACS analysis was also used to determine whether the Sca-1 /CD31¨C cell population increased in hearts following infarction; this would suggest a role for Sca-1 /CD31¨C cells in normal cardiac repair and remodeling processes. Seven days after myocardial infarction, the Sca-1 /CD31¨C cell fraction of the cardiomyocyte-free cardiac cell population increased from 10.8% ¡À 2.40% in normal hearts to 21.8% ¡À 1.6% in the myocardial infarction group (n = 6, p , K/ E& D* ], E  {3 F

Finally, FACS analyses of bone marrow and circulatory blood cells did not show increased numbers of Sca-1 /CD31¨C cells at the early and late stages of myocardial infarction (data not shown). This suggests, but does not prove, that the increased numbers of Sca-1 /CD31¨C cells present in myocardium following infarction resulted from migration and proliferation of endogenous cardiac Sca-1 /CD31¨C cells rather than from increased homing of Sca-1 /CD31¨C cells derived from extracardiac tissues such as the bone marrow.

Sca-1 /CD31¨C Cells Transplanted into Myocardial Infarction Limit Post-MI LV Remodeling and Attenuate Contractile and Energetic Abnormalities. To further assess the role of Sca-1 /CD31¨C cells in cardiac repair/remodeling, saline-suspended, LacZ-labeled Sca-1 /CD31¨C cells were directly injected into five sites in the peri-infarct region immediately following coronary artery ligation (total number of cells injected = 1 x 106). Groups injected with the same number of LacZ-labeled Sca-1¨C/CD31¨C cells or the same volume of saline without suspended cells served as controls. Cell engraftment rates and echocardiograms were analyzed at 1, 2, and 3 weeks following transplantation.5 h4 ?8 T3 |2 E; g
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Significant rates of cell were found in the first 2 weeks following cell transplantation (1 week, Sca-1 /CD31¨C cells, 2.68% ¡À 0.08%; Sca-1¨C/CD31¨C cells, 2.92% ¡À 0.09%; 2 week, Sca-1 /CD31¨C cells, 1.04% ¡À 0.12%; Sca-1¨C/CD31¨C cells, 0.79% ¡À 0.06% ). However, after 3 weeks, the number of identifiable engrafted cells decreased (3 weeks, Sca-1 /CD31¨C cells, 0.42% ¡À 0.04%; Sca-1¨C/CD31¨C cells, 0.36% ¡À 0.06% (n = 5)). The current data do not permit a conclusion as to the reason for the disappearance of the labeled engrafted cells. They may have suffered apoptotic death, cleared the reporter gene viral vector, or proliferated sufficiently to dilute the viral vector content of the transfected cells and thereby limit the content of the reporter gene product.
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LVEDD, LVESD, and LVEF were measured by echocardiography. At 1 week following MI, no significant differences were seen in LVEDD in the infarct groups as compared with the noninfarcted group. In contrast, LVESD was significantly increased and LVEF was significantly decreased in all the infarct groups and the MI groups. However, at 2 and 3 weeks following myocardial infarction, hearts receiving Sca-1 /CD31¨C cells had significantly smaller LVEDD and LVESD (p " ]" R& C$ l5 d, ~6 Y4 t
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Figure 4. Sca-1 /CD31¨C cell transplantation into the peri-infarct region significantly attenuated the deterioration of cardiac function following myocardial infarction. (A): Left ventricular end-diastolic diameter was significantly smaller 2 and 3 weeks after Sca-1 /CD31¨C cell transplantation (n = 8, p ' g5 F5 H: e% [: l/ v

Our previous work using large animal models of heart disease demonstrated that myocardial energetic characteristics were abnormal in hearts with cardiac hypertrophy and that the severity of the myocardial energetic abnormality (as reflected by the ratio of phosphocreatine over ATP; PCr/ATP) was related to the severity of the hypertrophy and contractile dysfunction of remodeled post-MI and pressure overloaded hearts .

Figure 5. Myocardial bioenergetic characteristic deterioration in infarcted hearts was markedly attenuated after Sca-1 /CD31¨C cell transplantation. (A): Representative cardiac 31P nuclear magnetic resonance spectra in a control mouse and mice with MI or MI Sca-1 /CD31¨C cell transplantation or MI Sca-1¨C/CD31¨C cell transplantation. (B): PCr/ATP values in control, MI, MI Sca-1 /CD31¨C cell transplantation, and MI Sca-1¨C/CD31¨C cell transplantation (n = 9, p 6 Q! H3 ^; \% R5 t7 R4 u
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In agreement with the in vivo PCr/ATP data, the protein expression levels of the creatine kinase M isoform, the mitochondrial CK isoform, and the mitochondrial ATPase-ß subunit were all substantially preserved in MI hearts 3 weeks after Sca-1 /CD31¨C cell transplantation compared with MI hearts treated with saline (Fig. 5C). Taken together, these data indicate that partial preservation of LV structure and contractile function in hearts transplanted with Sca-1 /CD31¨C cells was accompanied by limitation of the development of myocardial energetic abnormalities.# g5 |6 l* I3 A

Transplanted Sca-1 /CD31¨C Cells Induce Myocardial Neovascularization and Differentiate into Cardiomyocytes and Endothelial Cells. To determine the mechanisms of the beneficial effects of Sca-1 /CD31¨C cell transplantation, we examined the effects of transplantation on neovascularization in post-MI hearts and also determined whether these cells (labeled with the adenovirus nuclear LacZ reporter gene) could undergo in vivo differentiation into endothelial cells and cardiomyocytes.

At 2 weeks after cell transplantation, immunofluorescence staining for vWF indicated significant angiogenesis in Sca-1 /CD31¨C cell treated hearts, with more vWF-expressing capillaries being present in peri-infarct regions of transplanted compared with saline-treated hearts (Fig. 6A). Quantitative evaluation of vWF-positive capillary numbers per high-power field (20x) indicated that capillarity was significantly greater in the Sca-1 /CD31¨C cell-treated group than the saline-treated group (MI saline, 189 ¡À 7; Sca-1 /CD31¨C cells, 253 ¡À 11; n = 5, p 9 B7 q. @: I" l/ h9 t
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Figure 6. Sca-1 /CD31¨C cell transplantation promoted angiogenesis following MI. (A): Photomicrographs of peri-infarction zone 14 days post-MI in untreated (top row) and Sca-1 /CD31¨C cell transplanted (bottom row) hearts. In each row, green immunofluorescence staining for vWF (left), blue DAPI staining of nuclei (middle), and a merged view of the preceding photomicrographs (right) are shown. (B): Mean number of vWF-stained capillaries in peri-infarct regions of untreated and Sca-1 /CD31¨C cell transplanted hearts 14 days post-MI. (C): Immunofluorescence staining showed infrequent transplanted Sca-1 /CD31¨C cells (with X-gal staining), which expressed vWF 14 days after transplantation. The photomicrograph on the left shows X-gal staining (dark blue), the next shows DAPI staining (light blue) of the same cell, the next shows vWF staining (red) of the same cell, and the photomicrograph on the right merges the preceding views. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; MI, myocardial infarction; vWF, von Willebrand factor; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.1 C& C8 y: p  z( X4 C* W6 M9 d6 d

To determine whether transplanted Sca-1 /CD31¨C cells underwent in vivo differentiation to cardiomyocytes, tissues sections were double-stained for ß-galactosidase and cardiac-specific markers, including troponin T, -sarcomeric actin, and N-cadherin (Fig. 7A, 7B). Among the cells that costained positive for ß-galactosidase and troponin T, we identified two distinct cell types residing in the myocardial peri-infarction zone. The first type was composed of small cells that contained these differentiation markers. The second group was composed of larger cells that stained for these differentiation markers and also had well-defined cross-striations characteristic of more mature cardiomyocytes (Fig. 7A). These observations suggest that transplanted Sca-1 /CD31¨C cell populations engraft successfully and can undergo in vivo transdifferentiation into endothelial cells (rarely) and cardiomyocyte-like cells (more frequently). However, the fraction of the engrafted cells that appeared to differentiate into cardiomyocytes was >20% of the total number detected; hence, the absolute number of newly differentiated cardiomyocytes was low and probably insufficient to make a substantial direct contribution to LV structure, function, or bioenergetic characteristics. It should be noted that the number of Sca-1 /CD31¨C cells that underwent differentiation to cardiomyocyte-like cells is overestimated. Some of the X-gal-stained nuclei detected may have been in small, undifferentiated cells that were positioned above or below neighboring cardiomyocytes rather than within these cardiomyocytes. If so, this would suggest that the cardiomyocytes that appeared to contain these nuclei were the product of Sca-1 /CD31¨C differentiation. The microscopic sections we examined were thin (8 µm), and an effort was made to determine that the stained nuclei were within cells with cardiac marker staining. However, in the absence of the study of a z-series of confocal images, we cannot be certain that some of these cells were misclassified as being newly differentiated. Importantly, even if this is the case, it does not affect the overall conclusion of this report that transdifferentaition of Sca-1 /CD31¨C most probably did not make a major contribution to the beneficial effects of cell transplantation (see Discussion).

Figure 7. Transplanted Sca-1 /CD31¨C cells differentiated into cardiomyocytes. (A): Immunofluorescence staining of engrafted Sca-1 /CD31¨C cells expressed cardiac-specific markers, including troponin T (green; left, top row) and -sarcomeric actin (green; left, bottom row), 14 days after transplantation. Subsequent photomicrographs in each row show DAPI nuclear staining (light blue), nuclear X-gal staining (dark blue), and merged views of the same cells. Arrows point to triple-positive cells, which also demonstrated striation. (B): Immunofluorescence staining shows transplanted Sca-1 /CD31¨C cells expressed the cardiomyocyte marker, N-cadherin. The photomicrograph on the left shows cells stained for nuclear X-gal (dark blue), the middle photomicrograph shows N-cadherin staining (orange), and the picture on the right merges the two preceding views. Arrows point to double-positive cells. Abbreviations: DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.9 H4 b5 L% s# F8 g2 ~
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DISCUSSION
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Several recent reports demonstrate the existence of endogenous cardiac stem cell-like populations that, when stimulated to migrate and proliferate, can contribute to an increase in LV mass after myocardial infarction in adult hearts . The present study presents evidence that 1) the number of Sca-1 /CD31¨C cells present in the LV increases following an MI; in consequence, they may participate in MI-induced repair/remodeling processes; and 2) transplantation of cardiac-derived Sca-1 /CD31¨C cells attenuates the development of adverse structural, functional, and energetic abnormalities associated with post-MI LV remodeling. These data raise the possibility that expansion and myocardial transplantation of autologous (cardiac biopsy-derived) Sca-1 /CD31¨C progenitor cells may be a useful therapeutic approach in post-MI patients.; X* V) P& t( \% G$ b* h: f

One of the key findings of this study is the early and significant increase of Sca-1 /CD31¨C cell numbers in the scar and periscar regions of infarcted hearts. The question of the origin of the increased number of Sca-1 /CD31¨C cells in the heart remains somewhat unsettled. There is evidence that cells residing in bone marrow, upon receiving stimulatory signals from injured myocardium, are released into the circulation for myocardial targeting , and as noted above, others have reported that local (i.e., intramyocardial) HGF and IGF stimulation can induce them to migrate to and proliferate in injured myocardial regions. Taken together, the current data further support the view that an endogenous Sca-1 /CD31¨C cell population exists in myocardium and responds to myocardial injury. A possible contribution of migration of this cell type from an extracardiac source is not absolutely excluded, although our data do not support this view.3 _: s" t& Z9 x& [) K

Nonetheless, in the absence of intramyocardial injection of growth factors, it remains to be determined precisely how endogenous cardiac Sca-1 /CD31¨C cells are recruited to the injured myocardial region and what signals regulate their proliferation following MI. Furthermore, although endogenous cardiac Sca-1 /CD31¨C cells proliferate in response to an MI (and have the capacity to differentiate to endothelial cells and cardiomyocytes), the magnitude of their participation in routine postinfarction remodeling/repair may be modest because their proliferation appears to be inadequate to prevent adverse LV remodeling. However, it is possible that without their participation, remodeling would be more severe. Only future studies with Sca-1 knockout mice (now under way) will be able to reveal the true contribution of these cells to normal remodeling processes.$ k; _) b3 h+ N* e  _, m% x" |$ W
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Importantly, the current findings clearly show that the substantial augmentation of intramyocardial Sca-1 /CD31¨C cells numbers by cell transplantation at the time of coronary ligation does attenuate structural and functional remodeling. The question that is unresolved is how this is accomplished. Several possible mechanisms come to mind. First, the transplanted cells may directly contribute to regeneration of cardiomyocytes and endothelial cells. The current findings indicate that the differentiation of Sca-1 /CD31¨C cells to these types of cells, although present, is modest. This suggests that in this cell transplant model, which does not have exogenous growth factor administration to increase progenitor cell survival, proliferation, and differentiation, the direct structural and functional contributions of the transplanted cells must be minimal.' n9 v; x1 r0 h6 V4 C2 F: s$ q

A second possibility is that inadequate angiogenesis in the viable mechanically overloaded peri-infarct myocardium limits the performance of this region, which stimulates global LV remodeling .
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Of interest, Thum et al. recently suggested that the local myocardial inflammatory response is in some way suppressed as a result of apoptosis of transplanted donor cells and that this suppression of inflammation may be beneficial to postinfarction cardiac function . We cannot exclude this possibility because a limited inflammatory response was observed following adenovirus protein-labeled cell transplantation and the marked progressive reduction of engrafted cells over time may have been due to apoptosis. However, our present work clearly shows that significant improvements of LV contractile function and myocardial energetics only occur in hearts transplanted with Sca-1 /CD31¨C cells. Therefore, these data clearly support the view that there are Sca-1 /CD31¨C cell-specific beneficial effects in infarcted hearts. However, it is possible that suppression of the inflammatory response induced by donor cell apoptosis may have facilitated the therapeutic responses resulting from Sca-1  cell transplantation.& u4 {+ B+ z; r& b
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Finally, a key observation of the present study was that the marked reductions of the protein expressions of key enzymes in energy production pathways in the untreated MI group were remarkably limited in the Sca-1 /CD31¨C cell-treated group and that this response was associated with marked attenuation of the severe reduction of PCr/ATP, also present in the untreated MI group. These data suggest that ATP synthetic capacity was better preserved in the treated group. However, whether the improved bioenergetic characteristics of the treated hearts caused limitation of structural and contractile abnormalities per se or whether preservation of energetic function occurred in parallel with the other beneficial effects of treatment remains to be determined.

The favorable bioenergetic responses to cell transplantation are notable because, in large animal models of postinfarction LV remodeling, we previously reported a significant reduction of myocardial PCr/ATP that was linearly correlated with the severity of LV hypertrophy and LV dysfunction and that these abnormalities were most severe in myocardium from animals with congestive heart failure .  X( S. [4 m( k  D/ d" a5 H

In conclusion, we have demonstrated that Sca-1 /CD31¨C cells possess cardiac stem cell characteristics. In response to myocardial infarction, the cardiac Sca-1 /CD31¨C cell population exhibited significant expansion, which likely resulted from proliferation of Sca-1 /CD31¨C cells endogenous to the heart. Myocardial transplantation of Sca-1 /CD31¨C cells (but not Sca-1¨C/CD31¨C cells) attenuated post-MI LV remodeling as manifested by 1) more favorable structural remodeling, 2) significant preservation of contractile performance, and 3) improved myocardial bioenergetic characteristics. Further investigations employing heart-derived Sca-1 /CD31¨C cells might lead to clinically useful therapies for myocardial infarction.
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DISCLOSURES4 C# r# s0 w6 K5 h( H* `) [# G' \
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS& [2 Z. F$ e! f7 m$ x

We thank John Fassett for providing us with rat neonatal cardiomyocytes and Atsushi Asakura for discussion on Sca-1 /CD31¨C cell isolation and endothelial cell differentiation. This work was supported by United States Public Health Service Grants HL50470, HL61353, HL67828, and HL71970 (to J.Z.). X.W. is supported by a Scientist Development Grant from American Heart Association (0435329Z). J.L. is supported by a Predoctoral Fellowship from the American Heart Association (0415468Z).5 R8 M; n6 k) q( k; G' p( D
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