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a Cardiothoracic Surgery, Falk Research Center, and9 }7 P% j- ^- w2 e
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b Department of Pathology, Stanford University Medical School, Stanford, California, USA# f. M. Z2 X! h& k
5 H& P( N& E5 _) @. M2 NKey Words. Growth substances ? Myocardial infarction ? Cell transfer ? Embryonic stem cells% T! {# p: N3 w) `) D
7 T1 n& z& F0 m) R. k* I3 WCorrespondence: Theo Kofidis, M.D., Cardiothoracic Surgery, Falk Research Center, 2nd Floor, Stanford University Medical School, 300 Pasteur Dr., Stanford, CA 94305. Telephone: 650-723-5408; Fax: 650-725-846; e-mail: tkofidis@stanford.edu* v$ {: m1 f' t% N
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ABSTRACT
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8 {; K7 J6 J# G9 P/ O. [Severe acute myocardial ischemia may result in irreparable loss of cardiomyocytes and heart function . Current therapeutic strategies focus on revascularization or recanalization procedures to prevent further tissue loss. With the advent of cell transfer for myocardial restoration, a number of different cell types and administration routes have been proposed . Most of the reported approaches involve autologous bone marrow stem cells or myoblasts, but these cells have limited plasticity and impaired viability of the transferred cells in the area of ischemic lesion . Moreover, the complex cell by microenvironment interactions, which are critical for cellular commitment and function, need to be further studied.
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6 R h$ e; _8 ?: A! K I, TA major determinant of engraftment and host organ–specific differentiation of donor primordial cells is the array of growth factors that are known to promote cardiogenesis and proliferation and to prevent apoptosis and cell death . A distinct role is displayed by insulin-like growth factor-1 (IGF-1); it promotes cell mitogenesis and proliferation in early developmental stages and causes eccentric myocardial hypertrophy in the adult. After binding to its receptor on embryonic stem cells (ESCs), IGF-1 induces expression of a number of cardiac-specific transcription factors such as the zinc finger GATA proteins and Nkx-2.5, a coactivator of GATA-4. GATA-4 and Nkx-2.5 are essential for heart development . In parallel to adult cardiomyocytes, ESC-derived cardiomyocytes developmentally express cardiac-specific proteins and ion channels. ESC-derived cardiomyocytes have been successfully purified up to 70% from native ESC populations through multiple duplication steps . The stimulation of asymmetric progeny generation toward the cardiac phenotype has been demonstrated recently .
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0 ]8 @2 W- f8 I( G9 h3 D5 jExperience with ESC injection into myocardium is very limited, and past efforts have faced the issue of tumorigenicity of these cells in vivo. Their survival and differentiation potential to cardiac-specific cells after transfer in an area of infarction is largely unknown. In the study reported here, we hypothesized that injection of undifferentiated ESCs along with IGF-1 will enhance their potential to harness microenvironmental input in order to survive, differentiate, and improve function of the ischemic heart.$ f2 q Y7 X. P+ `' `1 ^
+ T/ ?% j) W! B( G: G7 bMATERIALS AND METHODS+ R$ O- V- |9 H9 m6 F- V
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Extension of GFP Graft and Scale of Myocardial Restoration" W; {$ E6 K, o- `: V
9 n. \- q' s- U! | z0 oThe engrafted cells formed an easily distinguishable GFP voluminous graft in all animals (Fig. 1A). The ratio of restored area to infarcted area ranged between 27% and 68%, with an average of 50% ± 18.10% versus 39.3% ± 7.2% in the ESC-only group (Fig. 1A). The cells formed dense conglomerates, which in many cases did not allow for distinguishing individual cells under the GFP filter. This was often possible via comparison to the corresponding H&E, trichrome, or otherwise stained section or by colocalization studies using GFP and the Texas red signal of the individual cell marker such as connexin-43.
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Figure 1. (A): The injected cells form extensive conglomerates within the scar tissue (x 50, arrows). (B): Conn-43 is being expressed abundantly between donor and recipient cells, as depicted by arrows (confocal laser scan x 630). (C): -Sarc actin (p = .015) and MHC I expression (p = .01) are significantly higher in the IGF-1–treated group. (D): CD3 cell populations were similar in both treated groups. Also for CD11, scar formation, cellular atypia, and nuclear polymorphism findings were similar for the two groups. Vascularity was significantly higher in the ESC-only group (p = .028). Abbreviations: Conn-43, connexin-43; ESC, embryonic stem cell; IGF-1, insulin-like growth factor-1; MHC, major histocompatibility complex; -sarc, -sarcomeric.
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Engraftment and Differentiation- d% L5 D- N% @1 _* u* i: t
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This study was not designed to provide proof of cell fusion between donor and recipient cells. Even though cell-by-cell count was frequently not possible in the dense conglomerate, the degree of connexin-43 expression on GFP cells reached up to 25% of the field-in-focus, without specific topographic preference〞that is, in the core or in the periphery and border zones of the graft. Connexin-43 was expressed at various sites of individual cells or at intercellular junction points (Fig. 1B). The area of connexin-43 expression–contained cells aligned in multiple directions, which in only a few cases displayed the typical sarcolemma and myotubular orientation that are morphologically attributable to cardiac cells. Fewer than 10% of the connexin-43 or -sarcomeric actin cells or cell islets in the cardiomyocyte morphology displayed a myotubular form. There was a trend toward stronger expression of connexin-43 (area ratio) in the IGF-1 group without statistical significance. The -sarcomeric actin pattern displayed striations on GFP elongated cells (Fig. 1C). The overall area of expression of -sarcomeric actin ranged from 20%–40% of the GFP graft area (mean, 31.6% ± 10.4%) versus 4%–8% (mean, 6.5% ± 2.2%) in the ESC-only group. In most, though not in all, cases, connexin-43 and -actinin–positive cells (signals) colocalized on sequential sections. The -sarcomeric actin expression was more pronounced in the IGF-1 group (p = .015) (Fig. 2A). SMA was expressed in the GFP cells that participated in vessel formation. The overall expression of SMA in the GFP graft was only very minor and mostly present in the border zones of the GFP cellular conglomerate. d# j& u5 g; Y; r# q2 q1 R
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Figure 2. Assessment of cardiac diameters, wall thickness, and function. (A): Fractional shortening was highest in the IGF-1–treated group, compared with untreated controls (p = .015). All groups treated with cells displayed better cardiac function than the animals that had undergone left anterior descending artery ligation only. (B): EDD diameter was significantly increased in the untreated group (controls, p = .038). The left PWT was found to be thicker in the ESCs plus IGF (ESC IGF)–treated group (p = .032). The SWT was similar in all three groups. Abbreviations: EDD, end diastolic; ESC, embryonic stem cell; IGF-1, insulin-like growth factor-1; PWT, ventricular posterior wall; SWT, septal wall thickness.! m5 m" X4 e% q
, _$ W4 i& r8 l fImmune Response and Tumorigenicity$ p' H/ J( ]. X
+ y- Z; ~* L; wVascularity was higher in the IGF-1–treated group (p = .028). MHC I was expressed more extensively in the IGF-1 group (p = .01). CD3 cells accumulated along the borders of the graft, forming conglomerates. Their presence within the graft was rare, and their population was similar in the groups treated with IGF ESC and ESC only (Fig. 1D). CD11 cells were detected primarily within the GFP grafts. Their distribution did not follow a specific pattern. None of the inflammatory cells stained for GFP, indicating they were of host origin. In many occasions, the inflammatory cells have infiltrated and surrouded the donor cells. Their number was similar in the two treated groups. Cellular atypia, nuclear polymorphism, and a shift of the ratio of cytoplasmic to nuclear cells was evident in all animals 2 weeks after cell injection. The animals treated with IGF-1 did not show more cellular atypia than those treated with ESC only. Nuclear polymorphism was similar in both treated groups (Fig. 1D).
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9 P# |, a. A1 t- R1 F7 O- rFunctional Effects of ESC and IGF-1 Treatment in Postischemic Hearts
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Echocardiography revealed significantly larger EDD diameters in the control group than in the group treated with ESC only (p = .038) and in the IGF ESC–treated group. PWT was higher in the IFG-1–treated group (p = .032) (Fig. 2B). FS was higher in the IGF-1 and ESC-only groups than in the controls. There was no significant difference between IGF-1–treated animals and ESC controls, but a clear trend for IGF ESC–treated mice to display better FS was noted (Fig. 2A).
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We thank Eliana Martinez, M.D., for the thorough review of the manuscript, insightful comments, and adjustments for language and style. Theo Kofidis and Jorg L. de Bruin contributed equally to this work.
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REFERENCES
0 U& E5 P6 V3 T
" _7 s7 |+ D& y: P1 v7 V8 O4 hAnversa P, Leri A, Kajstura J et al. Myocyte growth and cardiac repair. J Mol Cell Cardiol 2002;34:91–105.
; ?1 B. |6 q& g% L% F
0 Z* p, T6 ^& u3 f; e( a% j1 KEl Oakley RM, Ooi OC, Bongso A et al. Myocyte transplantation for myocardial repair: a few good cells can mend a broken heart. Ann Thorac Surg 2001;71:1724–1733.7 c: R- J |1 \6 u) ~2 \* f/ b
; l: I$ Z) r2 }8 j8 A' a) t9 JHassink RJ, Brutel de la Riviere A, Mummery CL et al. Transplantation of cells for cardiac repair. J Am Coll Cardiol 2003;41:711–717.
% Q6 X/ q& C4 o5 {4 a( l- Y9 x D8 G: W/ T8 [$ z! y
Shake JG, Gruber PJ, Baumgartner WA et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 2002;73:1919–1925.
/ n; y' `$ ~9 o4 O1 O+ F9 e/ d- V; H
Sakai T, Li RK, Weisel RD et al. Autologous heart cell transplantation improves cardiac function after myocardial injury.Ann Thorac Surg 1999;68:2074–2080.
( e3 {( z% d9 l' v! y- G( Q+ _% u4 f
Orlic D, Kajstura J, Cimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
9 [ ^+ C! G; S1 e% i0 C6 N Y# d8 t# w9 r2 R; Q
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597–600.
0 V4 y6 ?4 ^; z/ E
& S) |/ d: g! g7 a7 L7 H* ?Yang Y, Min JY, Rana JS et al. VEGF enhances functional improvement of postinfarcted hearts by transplantation of ESC-differentiated cells. J Appl Physiol 2002;93:1140–1151.
- z9 E- [+ W) s, k8 E$ r3 @6 T# X2 h2 Z
Engelmann GL, Boehm KD, Birchenall-Roberts MC et al. Transforming growth factor-beta 1 in heart development. Mech Dev 1992;38:85–97.% S( B/ o# Z! j. z8 i
! E2 u: {' X4 b1 U7 [$ B) TWang L, Mang W, Markowitch R et al. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res 1998;83:516–522.
; D5 D0 P3 T: O6 \4 Y4 ^& g- r& Y& G6 B/ v2 {
Pampusch MS, Kamanga-Sollo E, White ME et al. Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I. J Endocrinol 2003;176:227–235.' q" O% N. T, i6 I0 q
4 P6 F+ g; ^" A+ O
Ren J. Short-term administration of insulin-like growth factor I (IGF-1) does not induce myocardial IGF-1 resistance. Growth Horm IGF Res 2002;12:162–168.# e9 t% W9 I( Y/ S1 H
' Q# r0 S6 Z$ f6 m
Sachinidis A, Kolossov E, Fleischmann BK et al. Generation of cardiomyocytes from embryonic stem cells. Herz 2002;27:589–597. `& J) S$ p6 F: d% f& S
4 V1 h: S9 T W3 ^) }! D, \* r9 b# V
Behfar A, Zingman LV, Hodgson DM et al. Stem cell differentiation requires a paracrine pathway in the heart. FASEB J 2002;16:1558–1566.; V& _1 U' Y5 W6 G5 {! `( n
+ q: u2 A5 b* b! n9 s6 ~% b$ NChao W, Matsui T, Novikov MS et al. Strategic advantages of insulin-like growth factor-I expression for cardioprotection. J Gene Med 2003;5:277–286.
9 _& K% n# G& B9 m( k: \ a; Z' M( C6 w. ^* I
Sundgren NC, Giraud GD, Schultz JM et al. Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes. Am J Physiol Regul Integr Comp Physiol 2003;Aug 28 .
0 X* V7 @, Z7 B
7 ?9 v2 Y" V9 u% |Welch S, Plank D, Witt S et al. Cardiac-specific IGF-1 expression attenuates dilated cardiomyopathy in tropomodulin-overexpressing transgenic mice. Circ Res 2002;90:641–648.
0 t) R% t: H( c$ ~2 S) s* G
# X* K; k9 Q9 _Strom TB, Field LJ, Ruediger M. Allogeneic stem cells, clinical transplantation and the origins of regenerative medicine. Curr Opin Immunol 2002;14:601–605.& }% I0 O) B# e+ m8 o/ w' k+ c
2 p3 H3 j3 O. G0 }5 r
Vogel G. Embryonic stem cells: stem cells not so stealthy after all. Science 2002;297:175–177.
6 O' W: t) [. U' m3 A3 C; d; ^5 @- X" {
Kaufman DS, Thomson JA. Human ES cells: haematopoiesis and transplantation strategies. J Anat 2002;200(Pt 3):243–248.
1 e7 R' V. D" d' k2 Y) M: Q
' t7 Z0 H. Q) D! x, IWagers AJ, Christensen JL, Weissman IL. Cell fate determination from stem cells. Gene Ther 2002;9:606–612.* g1 e/ |# q+ H, K/ J3 K4 N
* L. i* L: @0 ^' V9 TWagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
+ e ]& [7 x4 [+ ^# U9 }7 o4 e4 |4 V& K$ J+ Y6 K
Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.
! j+ ], M9 D" B9 m2 T8 c/ w: x% m0 D- L4 Y: X* ^
Rubin H. The disparity between human cell senescence in vitro and lifelong replication in vivo. Nat Biotechnol 2002;20:675–681.+ v' _; t& ^1 `) w+ e* ^
. r& @- l, u/ N+ S( _- Y1 o
Sachs T. Collective specification of cellular development. Bioessays 2003;25:897–903.(Theo Kofidisa, Jorg L. de) |
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发表于 2009-3-5 10:35
发表于 2015-6-3 16:43
