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Adv Biochem Engin/Biotechnol (2010) 123: 201–217

DOI: 10.1007/10_2009_65

Springer-Verlag Berlin Heidelberg 2010

Published online: 18 February 2010

Outgrowth Endothelial Cells: Sources,

Characteristics and Potential Applications in

Tissue Engineering and Regenerative Medicine

Sabine Fuchs, Eva Dohle, Marlen Kolbe, and Charles James Kirkpatrick

Abstract Endothelial progenitor cells from peripheral blood or cord blood are

attracting increasing interest as a potential cell source for cellular therapies aiming

to enhance the neovascularization of tissue engineered constructs or ischemic

tissues. The present review focus on a speciﬁc population contained in endot helial

progenitor cell cultures designated as outgrowth endothelial cells (OEC) or endo-

thelial colony forming cells from peripheral blood or cord blood. Special attention

will be paid to what is currently known in terms of the origin and the cell biological

or functional characteristics of OEC. Furthermore, we will discuss current concepts,

how OEC might be integrated in complex tissue engineered constructs based on

biomaterial or co-cultures, with special emp hasis on their potential application in

bone tissue engineering and related vascularization strategies.

Keywords Bone tissue engineering, Co-culture models, Endothelial progenitor

cells, Vascularization

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

2 Endothelial Progenitor Cells in the Neovascularization Process .............................202

3 Diversity of Endothelial Progenitor Cell Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

4 Origin of OEC and Enrichment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

5 In Vivo Evaluations of Angiogenic Potential of OEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

6 OEC for Vascularization of Complex Tissue Engineered Constructs . . . . . . . . . . . . . . . . . . . . . 209

7 Self-Endothelialization Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

S. Fuchs (*), E. Dohle, M. Kolbe, and C.J. Kirkpatrick

Institute of Pathology, Universita

tsmedizin der Johannes Gutenberg-Universita

t, Langenbeck-

strasse 1, Mainz, Germany

e-mail: fuchss@uni-mainz.de

1 Introduction

The creation of adequate tissue equivalents and other therapeutical products in tissue

engineering and regenerative medicine is, for several reasons, a highly challenging

task. First of all, tissues reveal a highly complex structure, usually consisting of more

than a single cell type and a cellular organization in an individual, tissue speciﬁc

manner. In addition, tissue function is regulated by a close interaction of the individ-

ual cell types controlled via matrix components and cell to cell communication

mechanisms also underlying the control through physiological conditions such as

oxygen tension, state of inﬂammation, or mechanical stimulation. Last but not least,

tissues are integrated into the body and linked to central body functions, such as

vascularization, ensuring the supply with oxygen and nutrients, as well as the removal

of waste products. Furthermore, adequate vascularization is an essential prerequisite

allowing stem cells to approach the sites of tissue repair [1–4]. The development of

new therapies leading to a fast and successful vascularization is therefore one of the

most central and highly discussed subjects in tissue engineering and regenerative

medicine. In this context, the use of endothelial progenitor cells for proangiogenic

cell therapies has been proposed as a potential means to overcome the current

problems in the neovascularization of bioengineered or harmed tissues [4, 5]. Despite

high expectations, the use of endothelial progenitor cells is currently not yet feasible

for broad clinical applications, due to a series of open questions regarding the

deﬁnition of the relevant cell types and the transfer into practicable approaches,

which meet the clinical requirements.

2 Endothelial Progenitor Cells in the Neovascularization Process

For many years angiogenesis, the generat ion of blood vessels from the existing

vasculature through activation of proliferation and sprouting mechanisms in adult

endothelial cells, was considered as the exclusive pathway for blood vessel forma-

tion in an adult organism.

The understanding of blood vessel formation has changed in recent years,

mainly due to the discovery of so-called endothelial progenitor cells by Asahara

et al. [6]. They postulated that in the adult organism endothelial progenitor cells

contribute to de novo formation of blood vessels in a process described as vascu-

logenesis. Since then, vasculogenesis, the contribution of stem cells or endothelial

progenitor cells to de novo vascularization, has been discussed as a collateral

mechanism in neovascularization. Although t he deﬁnition clearly differenti ates

the two processes in the academic sense, both mechanisms seem to work hand

in hand in blood vessel formation and remodeling. Both pathway s are coupled

through a series of mostly unknown signaling mechanisms guiding endothe-

lial progenitor cells to the sites of repair and inducing their differentiation and

functional integration into the vasculature [7].

202 S. Fuchs et al.

A signiﬁcant number of studies on endothelial progenitor cells have been

published over the last decade, deﬁning endothelial progenitor cells by surface

markerssuchasCD133andCD34[8–10], and a series of other characteristics

such as low density lipoprotein (LDL)-uptake [4] and binding of ulex europaeus

agglutinin (UEA) [6, 11]. The ability to form vascular structures in proangiogenic

matrices in vit ro, as well as the pot ential to contribute to t he v ascularization

in vivo have been deﬁned as functional key elements of endothelial progenitor

cells. Several sources, isola tion procedure s, and sugges ted marke r proﬁles for

endothelial progenitor cells have been de scribed, although there is still a lack of

consensus on which cell type would resemble the “true endothelial progenitor

cell” or might be preferred in t erms of a therapeutical application. From a

practical point of view, suchacellwouldhavetofulﬁllaseriesofprerequisites

including: (1) origin from an easy obtainablesourceinconnectionwithaminimal

invasive procedure and (2) a good expansion capability not interfering with the

therapeutical potential. Accordi ng to these requirements, endothelial progenitor

cells, in particular from peripheral blood or cord blood, have raised signiﬁcant

interest.

3 Diversity of Endo thelial Progenitor Cell Populations

Experimental evidence from studies by Lin [12], Gulati [13], Hur [14], and

Ingram [15] suggested that endothelial progenitor cells from the peripheral

blood are a heterogeneous population of cells. Endothelial progenitor cells

from peripheral blood were classiﬁed by several groups according to their

order of appearance and morphological characteristics in early or late endothe-

lial progenitor cells [ 14]. Other synonyms for late endothelial progenitor

cells include outgrowth endothelial cells (OEC) [12], late OEC, blood OEC,

and endothelial colony forming endothelial cells. Although the i solation proce-

dure or the nomination of these subsets differs slightly amongst the individual

studies, the phenotypic and functional key features described for those cells are

more or less identical and are summarized in Table 1. This rather simple classiﬁ-

cation was at least a basis for the deﬁnition of blood derived endothelial progenitor

cells for subsequent studies using these cells for applications in tissue engineering

and regenerative medicine as described in this review.

The isolation of mononuclear cells from the blood by ﬁcoll gradient centrifuga-

tion followed by culture in a commercial available cell culture medium resulted in

cellular colonies with cobble stone-like morphology showing remarkable simila-

rities to endothelial cells in terms of the marker proﬁle and functional key elements

(Table 1).

Those cells appear at relatively low frequency in the peripheral blood [15, 27,

28]. In contrast to these cells, so-called early endothelial progenitor cells make up

the majority of mononuclear cells derived from the peripheral blood and show a

more or less rounded to ﬁbroblast-like morphology. Although they express to some

Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications 203

Table 1 Overview of characteristics and functions

EPC OEC

Morphology Spindle shaped morphology [12, 16, 17]

Cobblestone-like morphology [13, 15, 18, 19]

Order of appearance After 7 days in culture [14, 16] After 2–3 weeks in culture [12–14]

Marker proﬁle CD31+ [6, 11, 17]

CD45+ [17, 20]

CD34+ [6, 11, 16]

CD14+ [13, 16, 17]

CD146 [21]

CD133+ [8, 9, 22]

Flt-1 [14]

eNos[14]

vWF [ 14, 19, 23]

VE-Cadherin [11, 14, 17]

KDR [11, 14, 23]

CD36+ [12]

Tie2+ [6, 13]

CD31+ [13, 19, 24]

CD45 [20]

CD34+ [8, 16, 20]

CD14 [13, 16]

CD146+ [21, 24]

CD133 [20]

Flt-1 [14]

eNos [13, 14]

vWF [ 14, 19, 24]

VE-Cadherin [12, 14, 24]

KDR [14, 16, 20]

CD36+ [12]

Tie-2+ [13]

Caveolin-1 [13, 24]

Characteristics

In vitro Low proliferative potential [14, 16, 17]

No tube formation on matrigel [16, 25]

High proliferative potential [14, 16]

Tube formation on matrigel [16, 25]

In vivo Vasculogenic potential through paracrine mechanisms [13]

Secretion of proangiogenic molecules [16]

High vasculogenic potential [13, 14, 26]

204 S. Fuchs et al.

extent relevant endothelial markers such as CD31 (compare Table 1), these cells do

not show the full marker proﬁle of endothelial markers or the functional features

typical for endothelial cells. Most of these cells still carry hematopoietic markers

such as CD45 (compare Table 1). Different studies have compared the early vs late

endothelial progenitor cells in terms of their characteristics. The isolation of OEC

has been reported from different sources such as human cord blood as well as adult

peripheral blood or from different species such as porcine [29], murine [30], and

canine [31].

Surprisingly, although only OEC showed the characteristics and angiogenic

potential of endothelial cells in vitro, both cell types contributed to the de novo

vascularization in vivo [14]. In a following study the same group provided addi-

tional data potentially explaining the discrepancies in the angiogenic potential of

early EPC in vitro and in vivo. In this study early EPC supported the angiogenic

activity of OEC in vitro [16] probably through paracri ne mechanisms based on the

production of IL-8 and VEGF by early EPC. This cytokine production improved the

angiogenic activity of OEC in vitro. In addition, the invasion of OEC into angio-

genic matrices in vitro was positively inﬂuenced when both cell type s were applied

mediated through activation of matrix metalloproteinases such as MMP-2 in OEC

and MMP-9 in early EPC. In the same study a synergistic effect was also docu-

mented for the neovascularization process in vivo by the application of both

populations in a hindlimb model. Subsequently, data from these studies are ques-

tioning the contribution of early endothelial progenitor cells to the neovasculariza-

tion process as a result of a true progenitor function in a strict sense including the

differentiation towards mature endothelial cells. It seems to be more adequate to

assume that only a small subset contained within this mixed population constitutes a

true endothelial stem progenitor cell with the ability to differentiate towards an

endothelial cell. On the other hand, the improvement of the vascularization pro-

cess by so-called early endothelial progenitor cells seems to be mainly the result of

other mechanism s such as the support of mature endothelial cells or true stem

cells by paracrine factors and other mechanisms which still need to be deﬁned

further.

4 Origin of OEC and Enrichment Strategies

Although it has been widely accepted that peripheral blood contains stem cells with

the potential to differentiate towards cells with endothelial phenotype there are still

a series of difﬁculties identifying their distinct origin. Several markers have been

suggested in attempts to deﬁne the origin of OEC or to isolate them in a more

speciﬁc manner from diverse mono nuclear cell fractions. Nevertheless, most of

these surface markers result only in an enrichm ent of cell populations containing

OEC. In addition, some of these applied surface markers are not suitable to

distinguish progenitor derived endothelial cells from mature endothelial cells

circulating in the blood stream, as discussed in the following section.

Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications 205

Gulati et al. [13] isolated and identiﬁed OEC from the CD 14 negative fraction of

mononuclear cells from the peripheral blood. Other approaches used CD146 to isolate

cells with the characteristics of OEC from the cord blood and adult peripheral blood

[12, 21]. The adhesion molecule CD146 is expressed in different types of endothelial

cells throughout the vascularization and is often used to identify circulating mature

endothelial cells (CEC) [32]. Delorme et al. used a combination of an adhesion step

in order to separate mature endothelial cells from the mononuclear fraction followed

by a magnetic separation step for CD146 positive cells. Using this approach they

identiﬁed two distinct populations in cord blood and in peripheral blood forming OEC

in culture CD146 positive EPC (CD146+, CD34+, CD45+, CD133+, or CD117+)

and CD146 CEC (CD146+, CD34+, CD45, CD133,orCD117).

The complexity of the problem of how to differentiate between endothelial cells

from the circulation and from cells which have been mobilized from the bone

marrow was highlighted by a study of Lin et al. [12]. This group investigated

OEC from recipients of gender-m ismatched bone marrow transplantations. By in

situ hybridization, Lin et al. were able to identify endothelial cells from the

recipient making up the major ity of endothelial cells in the early phases of the

culture. On the other hand, the donor derived endothelial cells constitute the major

population after 1 month of the cultures which was due to their high expansion

capacity (over 1,000-fold over 2 months). It was not possible to distinguish donor or

recipient derived OEC on the basis of a mark er proﬁle, whereas the capability of

expansion was one of the remar kable differences of the two endothelial cell

populations found in the peripheral blood. Both endothelial cell types were positive

for CD36, indicating their microvascular endothelial phenotype. This microvascu-

lar phenotype of OEC was also supported by ﬁndings from other groups. Microvas-

cular characteristics of OEC were recently also supported by microarray data,

indicating that OEC are different from macrovascular cells, share similarities

with microvascular cells or might be even considered as an individual subclass of

endothelial cells [33].

Although CD133 is one of the markers suggested for endothelial progenitor

groups, cell sorting experiments provided evidence that OEC are not derived from

CD133 or CD45 positive cells but seem to be enriched in the CD34 positive, CD45

negative fraction [20].

Besides these surface marker based isolation strategies, another approach to

enrich OEC from the mononuclear fraction has been described recently [28]. By

including a passaging step (Fig. 1a) in the early phase of the culture of mononuclear

cells, the formation of OEC colonies was signiﬁcantly improved in one group of

cultures classiﬁed as high colony forming cultures. This simple protocol modiﬁca-

tion resulted in a signiﬁcant enrichment of OEC (Fig. 1b) and in a higher number of

OEC gained per individual donor. Although the reason for this effect has to be

further deﬁned, the protocol modiﬁcation might exert a positive effect, speciﬁcally

on such OEC with a high clonogenic potential, which have been describ ed by

several groups as stated above.

Other groups modiﬁed the isolation protocols by using full blood preparations

omitting widely used centrifugation steps which resulted in an improved OEC

206 S. Fuchs et al.

colony formation. For the further expansion of these cells the same group estab-

lished culture conditions designed for a potential therapeutical use of OEC in

humans [34].

Isolation of OEC

blood

plasma

Centrifugation

Standard protocol

Ficoll

erythrocytes

Mononuclear cells

p1 p2… px

Appearance

of OEC Expansion

day 0

7 x28

Discard non-

adherent cells

Mononuclear cells

Modified protocol

Passaging step

Standard protocol Modified protocol

*of seeded MNC

generate OEC

colonies

0,0131 %*

Low colony-

forming culture

High colony-

forming culture

0,0010 %* 0,0014 %* 0,0024 %*

Low colony-

forming culture

High colony-

forming culture

Low colony-forming culture: < 0,004 %

of seeded MNC generate OEC colonies in

culture with passa

step (n = 23)

High colony-forming culture: > 0,004 %

of seeded MNC generate OEC colonies in

culture with passa

step (n = 23)

Fig. 1 (a) Schematic overview of the standard and the modiﬁed culture protocol according to

Kolbe et al. (b) Schematic overview on the effects of protocol modiﬁcation on the enrichment of

OEC from adult peripheral blood

Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications 207

Nevertheless, despite the advances in the understanding of endothelial progeni-

tor cell biology, the identiﬁcation and speciﬁc isolation of OEC from heterogeneous

peripheral blood cell populations remai ns also a technical problem due to their very

low frequencies in heterogeneous cell populations isolated from the peripheral

blood. Steurer et al. [35] recently compared real time PCR and ﬂow cytometry to

detect circulating endothelial cells and endothelial progenitor cells. Using deﬁned

numbers of circulating endothelial cells added to heterogeneous blood mononuclear

cells, detection limits were in the range of 0.001% for quantitative real time PCR or

0.01% for ﬂow cytometry. Despite the ten-times higher sensitivity of real time

PCR, the speciﬁcity of the real time PCR was lower due to the methodological

problems associated with this technology also due to the lack of markers exclu-

sively expressed on the relevant cell types.

5 In Vivo Evaluations of Angiogenic Potential of OEC

A series of studies investigated the in vivo potential of OEC for vascularization

strategies in tissue engineering and regenerative medicine. From these studies we

have learned that the successful formation of blood vessels depends strongly on the

experimental settings and the source of OEC as described in the following sectio n.

Melero-Martin et al. [36] co-implanted OEC from the cord blood or adult peripheral

blood together with smooth muscle cells in matrigel plugs. Only in co-implantion

approaches blood vessel formation by OEC was observed, reﬂecting an active role

of the smooth muscle cells in the blood vessel formation or stabilization. The

angiogenic activity of the blood derived cells reduced with increasing numbers of

passages that the cells underwent during their expansion in vitro. Consistent with

the ﬁnding that the therapeutical potential correlates with cellular aging, cord blood

derived endothelial cells had a higher angiogenic potential then those derived from

adult blood. This observation was also conﬁrmed by Au et al. [37] although this

group used different experimental settings and co-implanted the endothelial cells

with 10T1/2 mouse embryonic ﬁbroblasts as stabilizing perivascular-like cells in

collagen gels. In this study, co-implantation with other cell types such as 10T1/2 had

no effect on the blood vessel formation itself. Nevertheless, the stability of newly

formed blood vessels formed by adult peripher al blood derived cells was relatively

impaired, so that these vessels regressed within the 3 weeks. Compared to this cord

blood cell derived vessels revealed a stability lasting over months. In another study

by Au et al. the critical role of stabilizing cells for the in vivo outcome of

transplanted endothelial cells was proven in another experimental set up based on

HUVEC as endothelial cell source and on bone marrow mesenchymal stem cells

functioning as cells with perivascular potential. Using collagen gels based

approaches, the blood vessel formation without mesenchymal cells (MSCs) was

negligible whereas the addition of those or of 10T1/2 ﬁbroblasts leaded to blood

vessels with a long-term stability [38].

208 S. Fuchs et al.