Derivation of Endothelial Cells from CD34– Umbilical Cord Blood - 《stemcells》2004-2005文献干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 24 下一页

返回列表

查看: 307300\|回复: 236	go Derivation of Endothelial Cells from CD34– Umbilical Cord Blood [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA6 u* V/ I5 e. K5 u

Key Words. Endothelial progenitor cells ? Vasculogenesis ? Cord blood ? CD343 Z0 T# S' b3 [, p0 d

Matilde Murga, Ph.D., Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 12C207, MSC 1907, 10 Center Drive, Bethesda, Maryland 20892, USA. Telephone: 301-594-9599; Fax: 301-594-9585; e-mail: costama@mail.nih.gov

ABSTRACT; ^8 W5 b7 y, s" A& _' u& T' [

Isolation of endothelial cell precursors and expansion of mature endothelial cells in vitro are limiting steps for their use as biomaterial to produce vascular grafts and to mediate therapeutic neovascularization . Once inoculated in vivo, purified endothelial cells can home to sites of ischemia and contribute to angiogenesis and vasculogenesis . While there is evidence that this process is beneficial in the context of ischemic vascular disease , endothelial cell precursors may participate in tumor angiogenesis and favor tumor growth . Thus, the potential contribution of endothelial cell precursors to tumor growth may limit the therapeutic value of stem cell transplantation. Key to isolation of endothelial cell precursors for therapeutic use or their removal from grafts is their characterization and identification of strategies for their removal or expansion and differentiation into functional endothelial cells.0 u/ R3 |. T4 c' C5 U; U8 ^
3 D; Y4 @5 ^2 T8 [$ V' q
Previously, mature endothelial cells and endothelial cell precursors have been reported in adult peripheral blood, bone marrow, and umbilical cord blood . During development, hematopoietic and endothelial cells develop in close proximity to each other in the blood islands within the yolk sac and in a region of the embryonic dorsal aorta . This suggests that both cell lineages derive from a common precursor, named the hemangioblast . Phenotypically, common progenitors for hematopoietic stem cells and endothelial cells are thought to express CD34, KDR/vascular endothelial growth factor receptor-2 (VEGFR-2), and Tie-2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains, also named TEK) . Whereas most endothelial cells maintain CD34 expression , hematopoietic stem cells lose CD34 expression once differentiated into mature blood cells . Thus, the CD34 antigen is broadly accepted as an appropriate marker for isolation of endothelial cells and their precursors, and selection of cells that bear the CD34 marker has been used successfully to isolate endothelial cell precursors from different sources . However, recent studies suggest the existence of a CD34–, CD14  cell population with angioblast-like properties in the peripheral blood .

Here, we demonstrate the existence of CD34– endothelial cell precursors in umbilical cord blood, which can differentiate into endothelial cells with long-term proliferative capacity, and propose a new model for endothelial cell ontogeny after birth.5 P( [, U* y% k3 r

MATERIALS AND METHODS; r# R- h7 |1 m5 z

Isolation and Culture of CD34– Cord Blood-Derived Mononuclear Cells/ p; g1 |& v4 m  u0 I/ J# O
, u" @0 O$ X; K- e
Cord blood-derived mononuclear cells were depleted of CD34  cells by negative selection over immunomagnetic beads or FACS cell sorting. Flow cytometry analysis before and after purification confirmed that the negative selection step performed by either method had removed the CD34  cells present in the cord blood mononuclear cell pool (see results below from immunomagnetic beads separation, Fig. 1B). The purified CD34– cells were subsequently incubated (2 x 106 cells/ml) on fibronectin-coated culture dishes in culture medium (Iscove’s modified) supplemented with heat-inactivated sera (10% fetal bovine and 10% horse), VEGF-A (20 ng/ml), endothelial cell growth supplement (ECGF, a crude extract of bovine neural tissue containing basic and acidic FGF; 15 μg/ml), and hydrocortisone (1 μM). To eliminate mature endothelial cells that might be present in the cord blood and would be expected to attach rapidly to the plate , adherent cells were discarded after 4 days of initial incubation. Nonadherent cells were saved and replated onto new fibronectin-coated dishes in the same culture medium. Fibronectin-adherent cells were kept in culture changing the medium every 4–5 days. Daily observation revealed little change in cell morphology and density during the initial 3 weeks of culture (Fig. 2A). Change of culture medium to "HUVEC medium" produced no apparent growth over a 9-day period of observation (not shown). By contrast, when the culture medium was changed to a "differentiation medium" consisting of M199 culture medium supplemented with FBS (10%), VEGF-A (10 ng/ml), EGF (10 ng/ml), IGF-1 (20 ng/ml), bFGF (4 ng/ml), ascorbate (50 ng/ml), and porcine heparin (25 ng/ml), small clusters of adherent, flat, and elongated cells were noticeable throughout the plate within three days (Fig. 2B). Over the following 5–8 days, cells with this elongated morphology grew to form a monolayer that filled the plate (Fig. 2C and 2D). After reaching confluence, cells were trypsinized and re-plated under the same culture conditions. These cells were maintained in continuous culture with periodic splitting for over 80 days from the time of cell separation (i.e., over 60 days after differentiation).0 c' E+ s6 i- S' p1 @" m% v

Figure 1. Characterization of cord blood-derived mononuclear cells depleted of CD34  cells. A) RT-PCR analysis of CD34 and CD31 mRNA expression. RNA was isolated from cord blood CD34– MNCs prior to culture (0) and after 9-day culture in isolation medium (9). RNA from HUVECs and from murine embryonal fibroblasts (MEF) were used as controls. NT: no RNA template. B) Flow cytometric analysis of surface antigen expression in cord blood MNCs before (total cord MNC) and after CD34  cell depletion (CD34– cord MNC). C) Comparative flow cytometric analysis of surface antigen expression on CD34– cord blood MNCs prior to culture; fibronectin-adherent cord blood CD34– MNCs after 12 days incubation; and cord blood-derived endothelial cells (ECs) obtained 12 days after addition of differentiation medium.

Figure 2. Microscopic morphology of cord blood-derived CD34– adherent mononuclear cells cultured in vitro. Phase contrast images of cell cultures. A) Adherent CD34– mononuclear cells plated over fibronectin-coated dishes in isolation medium; day 6 of culture. B) Cluster formation after 3 days in differentiation medium; day 24 of culture. C) Cell cluster sprouting; day 27 of culture. D) Confluent endothelial-like monolayer; day 30 of culture.

Characterization of CD34– Cord Blood-Derived Cell Populations

The cord blood-derived mononuclear cell population negatively selected for CD34  cells did not express CD34 mRNA as detected by RT-PCR, but expressed CD31 (Fig. 1A). CD31 identifies platelet endothelial cell adhesion molecule (PECAM) present on endothelial cells and in non-mesoderm-derived cells . Expression of CD34 continued to be absent from adherent cells after culture for 9 days in Iscove’s medium (Fig. 1A), at which time the cells appeared not to be proliferating and had the morphology of the cells depicted in Figure 2A. FACS analysis of the CD34– cell population prior to culture confirmed that the negative selection step had removed the CD34  cells present in the cord blood mononuclear cell population, and showed expression of CD31 on most CD34– cells (Fig. 1B and 1C). Additionally, this CD34– cell population showed expression of the monocyte/macrophage markers CD14 and the common leukocyte antigen CD45 (Fig. 1B). The expression of CD14 on a proportion of CD34– cells isolated from cord blood prompted us to examine the importance of cells bearing the CD14 marker to the generation of endothelial cell precursors. CD14–CD34– cells isolated from the cord blood did not give rise to colonies of endothelial cell precursors under the culture conditions described here. In addition, the percentages of CD34–CD14  cells derived from CD34– cord blood mononuclear cells were 29.2% ± 8.8% on day 0 and 97% ± 1.4% on day 9 of culture in isolation medium. These results suggest that endothelial cell precursors derive from CD34–CD14  cells. However, we cannot formally exclude the possibility of a derivation from CD34–CD14– cell precursors that require CD34–CD14  cells to expand and mature.
- P' |% `, e) a
Patterns of Endothelial Cell Gene Expression in a CD34– Adherent Cell Population Derived from Cord Blood, y- b/ i! x. y0 _: H  ?( g% @3 a5 I
9 h  Z9 ]: `9 J5 N8 s, Z% X/ |
We analyzed the expression of a wide panel of vascular markers in the endothelial cell-like population (Fig. 2D) derived from cord blood and compared these with the CD34– cord blood mononuclear cells prior to culture, the adherent cell population after a 12-day incubation on fibronectin-coated plates (prior to addition of differentiation medium), and the HUVECs (Fig. 1C and Fig. 3). The endothelial-cell-specific marker VE-cadherin ; the endothelial cell receptors Tie-1 , TEK/Tie-2 , VEGFR-1/Flt-1 , and VEGFR-2/KDR ; ec-NOS ; vWF stored and released by endothelial cells ; the endothelial-specific Notch ligand Delta4 ; and FGFR-1  were expressed in these cells. None of these RNAs could be amplified from the CD34– cord blood mononuclear cells, whereas all were expressed by HUVECs (Fig. 3A). These results strongly suggest that the CD34– cells isolated from cord blood had undergone a complete endothelial cell differentiation process.6 f" e4 D- i9 b% X1 _  y

Figure 3. Endothelial cell phenotype of cord blood-derived adherent cells. A) RNA, isolated from cord blood CD34– MNCs prior to culture (cord MNC) and after 10-day culture in "differentiation" medium (cord EC), was subjected to RT-PCR analysis. RNA from HUVECs and from murine embryonal fibroblasts (MEF) were used as controls. NT: no RNA template. B) Flow cytometric analysis of surface antigen expression on CD34-depleted cord blood MNCs after 9-day culture in "differentiation" medium. C) Ace-LDL uptake by cord blood-derived adherent cells obtained after 80 days (line bar = 100 μm).

By FACS analysis, most endothelial-like cells obtained after 9-day culture in differentiation medium (31 days total culture time, depicted in Fig. 2D) expressed CD31, VEGFR-1, VEGFR-2, VE-cadherin, and Tie-2, but did not express CD45 (Fig. 3B), CD90 (Thy-1), CD117 (c-Kit), or AC133 (Fig. 1C). In addition, a small proportion (5%) of cells expressed the monocyte/macrophage or dendritic cell markers CD14, CD1a, CD83, CD86, and CD68 and the CD34 marker (Fig. 1C and Fig. 3B). Noteworthy, the CD34– cell population prior to culture and the adherent cell population (prior to differentiation) expressed high levels of CD45, HLA-DR, CD14, and CD86, which were not detected or were detected at very low levels in the endothelial-cell like population (Fig. 1 and Fig. 3). In addition, the endothelial cell-like cells incorporated acetylated-LDL, a characteristic of endothelial cells (Fig. 3C).

Proliferative Capacity of Cord Blood-Derived Endothelial Cells/ {% T' Z  W  X# T
9 }/ J6 F8 p6 }  X2 I
The growth curve of CD34– cord blood-derived endothelial cells incubated in differentiation medium is depicted in Figure 4A. Over a period of 60 days, during which the cultures were maintained in differentiation medium with periodic splitting, no detectable change in cell morphology, expression of cell surface markers, or gene expression was detected (not shown).! `9 z: k  f2 c& ]- V

Figure 4. Proliferative capacity of cord blood-derived CD34– mononuclear cells and endothelial cells. A) Cell expansion of CD34– mononuclear cells during culture in vitro. The increase in cell population doubling level (PDL) was calculated according to the formula PDL =log (nf/n0)/log2, where n0 is the initial cell number of viable cells and nf is the final number of cells. B) Cord blood-derived endothelial cells and HUVECs (2,000 cells/well) were cultured for 48 hours in culture medium supplemented with 10% heat-inactivated FBS, 10 ng/ml VEGF, 10 ng/ml EGF, and 4 ng/ml FGF, individually or in combination. HUVEC growth medium is described in Materials and Methods. Results represent the mean (±SD) of three experiments, each performed in triplicate.' H8 c/ w- L; [+ ?, S1 c2 o. z3 i5 N

We compared proliferative responses of cord blood-derived endothelial cells with those of HUVECs (Fig. 4B). Importantly, in the presence of 10% serum alone, cord-derived endothelial cells failed to proliferate and died, indicating their absolute dependency on exogenous growth factors and primary nature. Both primary endothelial cells (cord-derived and HUVECs) displayed similar patterns of response to the endothelial growth factors VEGF-A, bFGF, and EGF. However, HUVECs proliferated significantly better (p

Cord Blood-Derived Endothelial Cells Form Extracellular Matrix-Dependent Networks In Vitro
5 {( s; d$ Q1 t' p- L5 I/ \0 j, N6 Y
Endothelial cells plated on extracellular matrix preparations, such as Matrigel, can assemble into a capillary-like network . We examined the capacity of cord blood-derived endothelial cells to form these networks in vitro. Within 24 hours of incubation on a Matrigel-coated surface, cord blood-derived endothelial cells formed characteristic tube-like structures assembled in a branching reticular network (Fig. 5A), indistinguishable from those generated by HUVECs under the same conditions (not shown) .5 c- `5 a# _7 b' @- j6 p! W

Figure 5. Participation of cord blood-derived endothelial cells in extracellular matrix-dependent morphogenic processes. A) Cord blood-derived endothelial cells were plated over Matrigel-coated wells and incubated in vitro for 24 hours. The representative image reflects tube formation detected by phase contrast microscopy (original magnification 5x). B) Consecutive sections from Matrigel plugs removed from SCID mice 8 days after inoculation with bFGF were immunostained for mouse and human CD31. C) SCID mice were injected subcutaneously with cord blood-derived endothelial cells (after 70-day culture) mixed with Matrigel and bFGF. Plugs were removed after 8 days. Images reflect immunohistochemical staining using mouse anti-human CD31 primary antibody or control IgG followed by a biotinylated horse anti-mouse secondary antibody. Human CD31 expression is marked by immunoperoxidase (brown) staining; counterstain is hematoxylin. Arrow points to vessel lined with CD31  endothelial cells containing red blood cells (original magnifications 20x and 63x). D) Human CD31 immunostaining of Matrigel plugs containing HUVECs or cord blood-derived endothelial cells. Plugs were removed after 20 days. Capillary structures containing red blood cells are shown. E) Quantitative analysis of human CD31  cells in histological sections of Matrigel plugs containing HUVECs or cord blood-derived endothelial cells removed after 20 days. Results reflect the mean surface area (expressed in mm2) occupied by CD31  cells within a surface area of 106 μm2.
' e4 y. r6 v: V+ C6 m5 J; J' h& E# y
Participation of Cord Blood-Derived Endothelial Cells in Neovascularization In Vivo% u/ {3 f3 p4 \" ?/ J" B; N
7 o: s2 Z% f1 ]& T
To examine further whether cord blood-derived endothelial cells could assemble into vascular structures, we studied their behavior in two distinct in vivo angiogenesis models. We utilized endothelial cells generated from cord blood CD34– cells after 70–80 days in culture. In the Matrigel model, cells were mixed with Matrigel plus bFGF and injected subcutaneously into the midabdominal region of SCID mice, which are T-cell immunodeficient and are not expected to reject human cells. Matrigel alone, with or without bFGF, and HUVECs were used as controls. All plugs were removed 8 or 20 days after inoculation. The presence of human endothelial cells in the plugs was evaluated by immunohistochemical analysis using antibodies directed at human CD31. As shown in Fig. 5B, anti-human CD31 antibodies are human specific and do not recognize murine endothelial cells, which are immunostained by antibodies directed at murine CD31. Confirming our previous results , Matrigel plugs impregnated with bFGF contained large numbers of cells staining for murine CD31, but not for human CD31, indicative of their murine endothelial cell derivation (not shown). By contrast, minimal cell infiltration was present in Matrigel plugs without bFGF (not shown). As shown, 8-day (Fig 5B) and 20-day (Fig. 5C) plugs from animals injected with cord blood-derived endothelial cells or HUVECs revealed the presence of scattered single cells and clearly identifiable microvessels that stained for human CD31, but not control IgG1. Most capillaries and isolated cells did not stain for human CD31 within the plugs, indicating their host origin. The presence of human CD31  cells and the frequency of these cells were consistent within animals from each group. Importantly, a few of the vascular structures lined with human CD31  cells contained red blood cells in their lumen (Fig. 5B and 5C), suggesting that they had anastomosed with the murine vasculature and had become functional blood-carrying microvessels. A comparison between cord blood-derived endothelial cells and HUVECs revealed that, at the 20-day time point, plugs with HUVECs contained greater numbers of CD31  cells than plugs with cord blood-derived endothelial cells (Fig. 5E). This result is consistent with the in vitro proliferation assays showing a greater proliferative capacity of HUVECs compared with cord blood-derived endothelial cells. Unlike the endothelial cells generated by in vitro culture (Fig. 5), freshly separated CD34– cord blood mononuclear cells (1 x 106) failed to generate vascular structures that stained for human CD31 when injected into SCID mice mixed with Matrigel plus bFGF over a 14-day period (not shown).1 l; Z+ U! M8 d) N) G
6 G4 I* a* \2 T' c5 k5 D# B
To examine whether cord blood-derived CD34– endothelial cells could contribute to tumor angiogenesis, we tested their ability to incorporate into tumor vasculature. The human Wilms tumor cell line SK-NEP-1 was selected based on its high tumorigenicity in athymic mice and failure to express human CD31 as determined by FACS (not shown) and by immunohistochemistry of tumor tissue (Fig. 6A). SK-NEP-1 cells were inoculated (10 x 106 cells) subcutaneously into groups of 4 week-old BALB/c athymic mice either alone or in conjunction with 1.5 x 106 HUVECs or cord blood-derived endothelial cells, and tumors were removed from the animals after 20 days. By immunohistochemistry, tumors were derived from inoculation of tumor cells alone did not contain cells that stained for human CD31 (not shown). Instead, tumors derived from inoculation of tumor cells plus HUVECs (Fig. 6) or with cord blood-derived endothelial cells (Fig. 6) contained human CD31  cells lining vascular structures (Fig. 6). The percent of cell staining for human CD31 was

Figure 6. Incorporation of cord blood-derived endothelial cells into tumor vasculature. Immunohistochemical analysis of human CD31 expression in tumors derived from inoculation of athymic mice with: SK-NEP-1 cells plus HUVECs, and SK-NEP-1 cells plus cord blood-derived endothelial cells. Original magnifications 20x and 63x.. y. i9 b8 i8 M" g( y5 D
+ x9 k; R0 W1 R( x
DISCUSSION4 M  v: F. r+ n1 v
6 m. }2 I( c: ?: [2 m
The authors thank Ms. L. Sierra, Dr. Y. Aoki, and Dr. O. Fernandez-Capetillo.: y/ N) s; g* e) t* b

REFERENCES

Scott SM, Barth MG, Gaddy LR et al. The role of circulating cells in the healing of vascular prostheses. J Vasc Surg 1994;19:585–593.# L: g% W: h# Q
/ [4 W1 }) m/ u/ d7 ?' x; S" r
Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.7 @1 h4 M4 r5 S, a
# k1 [& O1 e/ u( z0 o9 L
Folkman J, Watson K, Ingber D et al. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989;339:58–61.+ [/ `* k: Z8 |

Rafii S. Circulating endothelial precursors: mystery, reality, and promise. J Clin Invest 2000;105:17–19.- R" h7 o% S# v5 n; Q: y6 K
( t6 W) D/ ?  q, X9 H' v* _
Lyden D, Hattori K, Dias S et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194–1201.
0 S, H8 i+ Z- q7 h4 [9 I/ K- g
Stump MM, Jordan GL Jr., Debakey ME et al. Endothelium grown from circulating blood on isolated intravascular Dacron hub. Am J Pathol 1963;43:361–367.
4 G1 d. ?0 U2 ~
Nieda M, Nicol A, Denning-Kendall P et al. Endothelial cell precursors are normal components of human umbilical cord blood. Br J Haematol 1997;98:775–777.

Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228." t( d& F* Q6 K$ }3 g2 P' t' R$ z

Murohara T, Ikeda H, Duan J et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000;105:1527–1536.

Reyes M, Lund T, Lenvik T et al. CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625./ \( E7 A$ g1 |. \# s
  \+ x( @# _3 g$ I. }
Sabin F. Studies on the origin of blood vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Contrib Embryol Carnegie Inst of Washington 1920;9:214–262.- k7 N/ U* s& i4 S# P* O+ k, q

Murray PDF. The development in vitro of blood of the early chick embryo. Proc R Soc London 1932;111:497–521.
+ d; _) [  O  w  o% A* O; q
Hatzopoulos AK, Folkman J, Vasile E et al. Isolation and characterization of endothelial progenitor cells from mouse embryos. Development 1998;125:1457–1468.  B/ W& i% L4 p
5 v$ F6 T. f# V9 ~* \1 O
Nishikawa SI, Nishikawa S, Kawamoto H et al. In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos. Immunity 1998;8:761–769.& E5 s: C5 R: A5 \
+ F3 Y) U' p5 _* x+ ^; z  h
Fina L, Molgaard HV, Robertson D et al. Expression of the CD34 gene in vascular endothelial cells. Blood 1990;75:2417–2426.0 ?+ R/ z( t) r  O

Civin CI, Strauss LC, Brovall C et al. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1 cells. J Immunol 1984;133:157–165.

Fernandez Pujol B, Lucibello FC, Zuzarte M et al. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol 2001;80:99–110.+ M5 o# |" n! T2 l9 T4 ^& [

Harraz M, Jiao C, Hanlon HD et al. CD34– blood-derived human endothelial cell progenitors. STEM CELLS 2001;19:304–312.4 A9 X0 ]- |$ l
* a. V# N6 W0 }  [2 q; N
Schmeisser A, Garlichs CD, Zhang H et al. Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovasc Res 2001;49:671–680.
& y# b1 j% {4 m- s+ @2 r8 E
Salvucci O, Yao L, Villalba S et al. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1. Blood 2002;99:2703–2711.
5 J# z: n# }" s' s, w& V7 Z" D6 n
Yao L, Sgadari C, Furuke K et al. Contribution of natural killer cells to inhibition of angiogenesis by interleukin-12. Blood 1999;93:1612–1621.5 `7 V1 c5 Z" @  n9 q

Yao L, Pike SE, Setsuda J et al. Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12. Blood 2000;96:1900–1905., O- P% i6 n* l7 ~3 \5 H$ V8 D3 W
# c, e! ~  L0 w! [$ {- `
Shi Q, Rafii S, Wu MH et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362–367.

Lin Y, Weisdorf DJ, Solovey A et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000;105:71–77.- J: D* q0 o; K3 u

Lampugnani MG, Corada M, Caveda L et al. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 1995;129:203–217.
7 g+ G# X' u$ S- w( g
Ayalon O, Sabanai H, Lampugnani MG et al. Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells. J Cell Biol 1994;126:247–258.

Partanen J, Armstrong E, Makela TP et al. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol 1992;12:1698–1707.) _5 S/ A3 m" `; q% \+ N
! a* D4 E0 Q! b! z( A
Dumont DJ, Yamaguchi TP, Conlon RA et al. tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene 1992;7:1471–1480.
2 O5 A$ E- r1 i( o' l
Matthews W, Jordan CT, Gavin M et al. A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proc Natl Acad Sci USA 1991;88:9026–9030.

Shibuya M, Yamaguchi S, Yamane A et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene 1990;5:519–524.7 L# N. X# a7 b- P7 S& B

Forstermann U, Pollock JS, Schmidt HH et al. Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991;88:1788–1792.

Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol 1982;95:355–360.
8 b) g, ~8 L! v( H
Shutter JR, Scully S, Fan W et al. Dll4, a novel Notch ligand expressed in arterial endothelium. Genes Dev 2000;14:1313–1318.1 ^4 [6 |& P6 f  _

Burger PE, Coetzee S, McKeehan WL et al. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells. Blood 2002;100:3527–3535.( E6 k; H' i2 t1 u" C
! V2 V7 H  X3 g- Y
Kubota Y, Kleinman HK, Martin GR et al. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 1988;107:1589–1598.( P4 P# ^. }) q8 l

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." w. s4 ^6 f" t2 C9 p3 _$ x; A
  G/ i3 P  ?& N+ W; B/ V8 ~2 \
Suzuki A, Andrew DP, Gonzalo JA et al. CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein. Blood 1996;87:3550–3562., X+ l+ ?& e$ H
: H2 E+ a- N) `, n6 j" G
Cheng J, Baumhueter S, Cacalano G et al. Hematopoietic defects in mice lacking the sialomucin CD34. Blood 1996;87:479–490.

Fackler MJ, Krause DS, Smith OM et al. Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells. Blood 1995;85:3040–3047.# Q/ k$ L; X9 z$ U

Baumheter S, Singer MS, Henzel W et al. Binding of L-selectin to the vascular sialomucin CD34. Science 1993;262:436–438.
, J* ?+ w1 U  J4 ], b' ?
Nakahata T, Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 1982;70:1324–1328.

Quirici N, Soligo D, Caneva L et al. Differentiation and expansion of endothelial cells from human bone marrow CD133( ) cells. Br J Haematol 2001;115:186–194.(Matilde Murga, Lei Yao, G)

收藏0 分享0 顶0 踩0