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a Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan;
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: c7 x3 @7 ?! M# \# {b R&D Laboratories,Asahi Kasei Medical Co., Ltd., Tokyo, Japan# S. z* j9 M5 J- Q9 e5 C$ s( a4 Q
9 r! e+ z* y$ j/ }$ pKey Words. Angiogenesis ? Cord blood ? Endothelium ? Progenitor cell; F a; m# \) _9 B
" F8 f* F9 R: _' W0 @4 ?Correspondence: Toyoaki Murohara, M.D., Ph.D., Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. Telephone: 81-52-744-2149; Fax: 81-52-744-2157; e-mail: muro-hara@med.nagoya-u.ac.jp1 M0 ?" q( z: V7 B/ v) @# ?- X% i
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ABSTRACT
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% Z! \7 h, J. \. ]1 {9 y. a2 rPostnatal neovascularization has been considered to result from proliferation, migration, and remodeling of pre-existing mature endothelial cells, a process referred to as angiogenesis . On the other hand, at the early embryonic stage, neovascularization results from vasculogenesis, the de novo formation of blood vessels from endothelial progenitor cells (EPCs) or angioblasts . EPCs and hematopoietic stem cells (HSCs) are believed to derive from common precursor cells (i.e., hemangioblasts), because EPCs and HSCs share cell-surface antigens, including KDR, Tie-2, and CD34 . We and other investigators recently discovered that peripheral blood of adult species contains EPCs derived from CD34 mononuclear cells . More recently, EPCs have been isolated from adult bone marrow and human cord (placental) blood .
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6 \ i: z' B& R4 H A" b- S" uTransplantation of EPCs or bone marrow mononuclear cells (MNCs) containing EPCs into severely ischemic tissues has been shown to induce angiogenesis and to increase functional blood supply . This strategy is termed therapeutic angiogenesis by cell transplantation. However, the method of the isolation of EPCs is still complicated. In particular, isolation of MNCs, the initial step of cell isolation, requires several centrifuge processes of blood samples through a Ficoll density gradient . These methods are often time-consuming and are accompanied by a higher chance of contamination. To overcome these issues, we recently developed a novel MNC isolation device for the processing of human cord blood (StemQuickTME, Asahi Kasei Medical, Tokyo, Japan) . This filter system was proven to be easy in handling, and the blood processing was performed within a completely closed system inside an ordinary clean bench. However, there has been no report demonstrating that any cell filter–processed human MNC could give rise to EPCs. Accordingly, we examined whether functional angiogenic EPCs would differentiate from culture of MNCs obtained by our new cell-filtration device, StemQuickTME.$ T q0 `4 z; B
( C5 k( e3 b8 W' o# F7 t" L# FMATERIALS AND METHODS- O- Z7 F8 B; d
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Efficacy of MNC Isolation0 d6 ]* c. I* `' X% ?8 D
+ u+ v% n2 l& x& fWe first calculated the recovery rates of total WBCs and MNCs and compared those between the Ficoll centrifuge method and the StemQuickTME filtration method. The recovery rates of WBCs and MNCs were significantly greater in samples isolated by the StemQuickTME filtration method than in those isolated by the Ficoll centrifuge method (p 5 n' B# @2 I" W0 W8 b4 c5 P
i" C S1 ]; }Figure 2. (A): The recovery rates of WBCs and MNCs were significantly greater in MNC samples isolated by the StemQuick TME filtration method than in those isolated by the Ficoll centrifuge method. (B): The percent reduction rates of RBCs, platelets, and granulocytes were significantly lower in MNC samples isolated by the StemQuickTME filtration method than in those isolated by the Ficoll centrifuge method. (C): The recovery rates of CD34 , CD133 , and CD34 CD133 cells were significantly greater in MNC samples isolated by the StemQuick TME filtration method than in those isolated by the Ficoll centrifuge method (p
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- [3 W [; D5 E7 hEfficacy of Progenitor Cell Recovery
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Circulating EPCs have been reported to be derived from CD45lowCD34 CD133 MNCs in human blood. We thus used flow cytometry to analyze the number of CD34 , CD133 , and CD34 CD133 cells in cord blood MNCs isolated by the StemQuickTME filtration method and the Ficoll centrifuge method and compared those between the two isolation methods. The recovery rates of CD34 , CD133 , and CD34 CD133 cells with CD45low were significantly greater in the StemQuickTME filtration method than in the Ficoll centrifuge method (p
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# V, E- n" [6 L& e0 eDifferentiation of Spindle-Shaped Attaching EPC-Like Cells
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+ J- Q8 H: O) P4 KWhen cord blood MNCs isolated by the StemQuickTME filter were cultured on gelatin-coated plastic plates, several cell clusters appeared within 48 hours, and numerous spindle-shaped attaching cells differentiated and sprouted (Fig. 3A). The spindle-shaped attaching cells were observed as isolated cells, cell clusters, or linear cord-like structures. Such morphological appearance resembled that of endothelial progenitor cells developed from the culture of adult human peripheral blood, as reported previously .
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Figure 3. (A): Cord blood mononuclear cells isolated by StemQuickTME filter were cultured on gelatin-coated plates. Attaching cells formed clusters or linear cord-like structures. (B):At day 7 of culture, differentiated endothelial progenitor cell–like attaching cells were positively stained with Ulex europaeus agglutinin-1 lectin and took up DiI-acLDL. Three photographs of (B) are in the same microscopic field. Abbreviation: DiI-acLDL, 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine–labeled acetylated low-density lipoprotein.- L; {* U s' a! ?
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Fluorescence Detection of EPCs
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( R+ a: Z( ~, P5 L3 CTo further examine whether spindle-shaped attaching cells have endothelial characteristics, these cells were subjected to fluorescence chemical detection for EPCs. At day 7 of culture, more than 90% of the attaching cells were positively stained with UEA-1 lectin, and more than 95% of attaching cells took up DiI-acLDL (Fig. 3B), two characteristic features of cells in endothelial lineage.9 ~# d5 G* K% E- j4 F V" t
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RT-PCR Analysis of Endothelial Lineage-Related Genes
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We performed RT-PCR to analyze the expression of endothelial lineage-related mRNAs in MNCs and attaching cells during the culture of MNCs differentiating into EPC-like attaching cells (Fig. 4). RT-PCR analysis showed that CD31, Lox-1, and eNOS were persistently expressed on these cells during culture for up to 21 days. The expression of CD31 and Lox-1 mRNA had a tendency to decrease during culture. In contrast, another stem cell marker, CD133 mRNA, was expressed early after starting cell culture (~day 7), but its expression was markedly suppressed thereafter. In contrast, KDR mRNA was little expressed initially and gradually appeared thereafter. Because attaching cells at days 7–14 of MNC culture had multiple endothelium-related markers and functions, we defined such spindle-shaped attaching cells as EPC fraction in the present study.* R' [( Q; O0 m! m' V
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Figure 4. CD31, Lox-1, and eNOS were persistently expressed on attaching cells during culture for up to 21 days. CD133 mRNA was expressed early after starting cell culture (~ day 7), but its expression was markedly suppressed thereafter. KDR mRNA was little expressed initially and gradually appeared thereafter. Abbreviation: eNOS, endothelial nitric oxide synthase.% V4 y5 k: m3 [2 ]
; M' T6 ?; H* {8 kCord Blood–Derived EPCs Participated in Angiogenesis-Like Endothelial Networks In Vitro( ]: R0 Q5 P2 U% ]
4 G O. {; ~+ JWe next examined whether EPCs obtained with MNC culture isolated by the StemQuickTME filter would have angiogenic function. To examine whether EPCs participated in endothelial network formation in vitro, EPCs were collected at day 7 of culture, green fluorescence labeled, and cocultured with red fluorescence–labeled HUVECs on basement membrane matrix gel. At 6 hours of coculture, EPCs started to contact HUVECs (Fig. 5A). Within 24 hours, incorporation of labeled EPCs was identified within the angiogenesis-like HUVEC networks (Fig. 5B).1 o8 J& T t, e' P# g8 {1 C* w9 |$ ^$ ]
9 `1 F/ }+ B% N8 o* ]8 p' @Figure 5. Participation of cord blood–derived endothelial progenitor cell–like attaching cells in neovascularization in vitro. (A): The phase-contrast and fluorescent photomicrographs at the same microscopic field are shown. Green fluorescence–labeled attaching cells contacted red fluorescence–labeled HUVECs on matrix gel at 6 hours of coculture. (B): Similarly, labeled attaching cells were incorporated into the nonlabeled HUVEC network on basement matrix gel at 24 hours of coculture. Abbreviation: HUVEC, human umbilical vein endothelial cell.0 q$ A) U% x( `. G8 ~0 P2 K z
& G D; C% H9 r- s5 d2 q+ t) i# F+ qTransplanted Cord Blood–Derived EPCs Participated in Neovascularization in the Ischemic Hindlimb of Immunodeficient Nude Rats In Vivo& S" n. o7 _! S! I
- d7 Z* ]1 c9 h6 }5 \We examined whether transplanted cord blood–derived EPCs participated in postnatal neovascularization in immunodeficient animals in vivo. EPCs were isolated at day 3 of culture and were green fluorescence labeled. Unilateral hindlimb ischemia was surgically induced in the nude rats. On the day of surgery, rats (n = 3) received injection of green fluorescence–labeled cord blood–derived EPCs (5 x105 cells/animal) in the ischemic thigh skeletal muscles. At day 14 after induction of limb ischemia and cell implantation, frozen tissue sections were prepared from the ischemic tissues. Fluorescence microscopic examination revealed that labeled EPCs were distributed among the preserved skeletal myocytes in the ischemic hindlimbs. Adjacent section was stained with PE-conjugated anti-CD31 mAb to detect capillary endothelial cells. Side-by-side analysis of the fluorescence microscopic view revealed that some of the transplanted EPCs were costained with anti-CD31 mAb, indicating that implanted EPCs were arranged into endothelial capillaries among the skeletal myocytes in the ischemic hindlimbs (Fig. 6A).
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& C4 J2 U, T- K1 ?- \Figure 6. (A): Tissue-transplanted EPC-like attaching cells were incorporated into the microvasculature in the ischemic hindlimb tissues. Some green fluorescence–positive transplanted attaching cells were costained with the endothelial marker CD31. (B): Augmented neovascularization by local transplantation of EPC-like attaching cells in the ischemic hindlimb of immunodeficient nude rats. LDBF analysis at day 14 showed increased blood flow in the ischemic hindlimb in EPC-transplanted animal. (C): LDBF analyses revealed significantly augmented ratios of the ischemic/normal hindlimb blood flow in the EPC-transplanted group compared with saline-treated control group. ***p
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Transplantation of EPCs Quantitatively Augmented Ischemia-Induced Neovascularization in Nude Rats
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Finally, we examined whether in vivo transplantation of EPCs might quantitatively augment postnatal neovascularization and blood flow in the ischemic hindlimb in immunodeficient nude rats. Unilateral (left) hindlimb ischemia was created in seven additional animals, and EPCs isolated at day 3 of culture were locally transplanted into the ischemic thigh muscle area (5 x 105 EPCs/rat) at four different injection points. LDBF analyses revealed significantly augmented ratios of the ischemic to normal hindlimb blood flow in the EPC-transplanted group compared with saline-treated control group (Figs. 6B, 6C). Typical LDBF images obtained at day 14 of hindlimb ischemia are shown in Figure 6B. Therefore, the transplantation of EPCs (3 x 105 cells/animal) augmented ischemia-induced neovascularization and blood flow in vivo./ d2 t5 }4 }* v+ i8 z3 H1 V
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DISCUSSION
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We thank Dr. Kazuo Matsui for preparing cord blood samples. This research was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Japan Heart Foundation, and the Terumo Research Foundation (to T.M.).
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REFERENCES
9 e5 t( X+ S+ x; i6 C
6 w" a" C+ ` W$ I: U6 R) iFolkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31.
6 U+ A7 {9 s6 b! H/ Y: H& t2 G4 h# w
# d. v W: r+ }2 R$ G+ dRisau W, Sariola H, Zerwes HG et al. Vasculogenesis and angiogenesis in embryonic stem cell-derived embryoid bodies. Development 1988;102:471–478., n' G- w9 `1 T0 ^* l
& q( R0 ?4 n. z( m
Risau W. Differentiation of endothelium. FASEB J 1995; 9:926–933.
. }0 y$ c+ J; E+ {1 T- @
, ?; p7 g: V% sFlamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro. Development 1992; 116:435–439.
: d2 h- K) E' i5 b
: v% m- D% t# j* W6 m" }. |Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275:965–967.
1 _3 S. r+ f# }. U0 ?, E, E& b6 a9 O$ W: R% ?4 O
Shi Q, Rafii S, Wu MH et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362–367.: S- U( \% @, @2 d& ~% y/ Z0 H+ o
% C9 I6 [0 h; ]9 g! m H2 YMurohara, Ikeda H, Duan J et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000;105:1527–1536.
1 t4 n1 i( o& N( I6 E. [) M2 E( G. ]
7 M$ ~7 p, ?9 X" DGunsilius E, Duba HC, Petzer AL et al. Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells. Lancet 2000; 355:1688–1691.; A4 ?% w0 `9 v! B
3 H7 X# b+ N& v
Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 1999;103:1231–1236.
8 ]; ]7 [8 F( o$ D9 l6 p# R+ C% ~2 {. f5 u
Yasutake M, Sumita M, Terashima S et al. Stem cell collection filter system for human placental/umbilical cord blood processing. Vox Sang 2001;80:101–105." {% p5 l) d' C; H4 |, S7 `1 i
8 {/ I4 I( u0 i. `Eichler H, Kern S, Beck C et al. Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration. STEM CELLS 2003;21:208–216.
9 s# X6 f. z, N9 K( d3 S2 _+ M* b# H; l ~5 C/ I' v& U
Salven P, Mustjoki S,Alitalo R et al. VEGFR-3 and CD133 identify a population of CD34 lymphatic/vascular endothelial precursor cells. Blood 2003;101:168–172.
% _- u5 } `+ q5 I9 q% A' a
0 Y3 r; f/ F3 T8 o: L( {8 _5 N2 bKondo T, Hayashi M, Takeshita K, Numaguchi Y, Kobayashi K, Iino S, Inden Y, Murohara T. Smoking cessation rapidly increases circulating progenitor cells in periperal blood in chronic smokers. Arterioscler Thromb Vasc Biol 2004; 24:1442–1447.
- _* `' b% K. ?. O* j- S& ?
" o: T7 l( C* e8 i% k8 wTakahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–438., F+ @, ?% n. @
2 P8 H6 @0 ~, @1 N RBaumgartner I, Pieczek A, Manor O et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114–1123.
; |6 `, \; r) e: F4 c) F2 B9 |% L
Yanagisawa-Miwa A, Uchida Y, Nakamura F et al. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 1992;257:1401–1403.
; r( n1 x2 p! @+ X" c- j# C
, j2 S _6 ~! s2 J0 BSawamura T, Kume N, Aoyama T et al. An endothelial receptor for oxidized low-density lipoprotein. Nature 1997; 386:73–77./ N& m! f$ V. x- c
$ H/ l7 K) }- L4 R, _' iChen CH, Cartwright J Jr, Li Z et al. Inhibitory effects of hypercholesterolemia and ox-LDL on angiogenesis-like endothelial growth in rabbit aortic explants: essential role of basic fibroblast growth factor. Arterioscler Thromb Vasc Biol 1997;17:1303–1312.. v1 U Q$ c! F
- u/ h6 _. j; u. m) c
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;125:1747–1757.(Mika Aokia, Mikitomo Yasu) |
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发表于 2009-3-5 10:36
发表于 2015-5-30 13:59
