Cell Biology International 31 (2007) 950e957www.elsevier.com/locate/cellbi

Antigen expression of cord blood derived stem cells underosteogenic stimulation in vitro

Marcus Jager*, Rudiger Krauspe

Research Lab for Regenerative Medicine and Biomaterials, Department of Orthopaedics, Heinrich-Heine University Medical School,Moorenstr. 5, D-40225 Duesseldorf, Germany

Received 7 December 2006; revised 17 January 2007; accepted 12 March 2007

Abstract

Tissue engineering strategies have become a promising option for treating musculoskeletal defects in future. Cord blood includes mesen-chymal stem cells which are able to differentiate into several cell lines under lineage specific stimulation including osteoblasts, chondroblastsand adipoblasts. In this study the antigen pattern of cord blood stem cells cultivated onto a porous porcine collagen I/III scaffold is investigated.The cultures were stimulated with osteogenic mixture (dexamethasone, ascorbic acid, glycerolphosphatate [DAG]) over 21 days in vitro. Thefollowing antigens and markers served for immuncytochemical evaluation: bone sialoprotein, osteocalcin, osteonectin, osteopontin, cartilageproteoglycan, chondrogenic oligomeric matrix protein, collagen I/II/III/X, CD13, CD 31, CD34, CD44, CD45, CD105, fibroblast growth factorreceptor 2, vascular endothelial growth factor, vimentin and von-Kossa and HE stainings. We showed that a collagen I/III scaffold is an appro-priate cellular carrier for cord blood progenitor cells and allows for an osteoblastic differentiation. Moreover there were differences in antigenpattern, dependent on the location of the adherent cells. CD105 and VEGF were only expressed at the bottom of the biomaterial. Futureinvestigations should show the role of cytomechanical forces in the differentiation of cord blood derived progenitor cells and also if a cell-loaded collagen I/III scaffold is appropriate to stimulate bone regeneration in vivo.� 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Osteoblast; Cord blood; Biomaterial; Collagen

1. Introduction

In the past, several authors have shown that mononuclearcells isolated from human cord blood exhibit a mesenchymalin vitro multipotency. It was demonstrated that cord blood pro-genitor cells are capable of differentiating into the osteogenic,chondrogenic, myogenic and adipogenic lines under lineagespecific stimulation (Bradstock et al., 2006; Gang et al.,2004; Jager et al., 2006a; Jager et al., 2003; Kakinumaet al., 2003; Kim et al., 2004; Kogler et al., 2004, 2006; Leeet al., 2004; Mareschi et al., 2001; Wagner et al., 2005). In ad-dition some authors report a lower immunogenic reaction in

* Corresponding author. Tel: þ49 (0)211 81 16036; fax: þ49 (0)211 81

16693.

E-mail address: jaeger@med.uni-duesseldorf.de (M. Jager).

1065-6995/$ - see front matter � 2007 International Federation for Cell Biology

doi:10.1016/j.cellbi.2007.03.004

patients after unrelated or non-HLA-matched allogenic cordblood transplantation compared to bone marrow derived cells(Bradstock et al., 2006; Kleen et al., 2005). Owing to thesetwo biological aspects, cord blood may be an attractive sourcefor stem cell-based therapy of musculoskeletal defects and tis-sue engineering in the future (Ghen et al., 2006). For cellbased and tissue engineering strategies the use of appropriatescaffolds that allow for cellular adherence and also for line-age specific differentiation is prerequisite for success. In thepast collagen and ceramic biomaterials such as tricalcium-phosphatate (TCP) and hydroxyapatite (HA) have provedexcellent promoters of osteoblast differentiation and biominer-alization of human bone marrow cells in vitro (Jager et al.,2005, 2006b; Jager and Wilke, 2003; Wang et al., 2003).However, there is a lack of data in the literature relating tothe antigen expression in cord blood derived cells culturedonto collagen scaffolds under osteogenic stimulation in vitro.

. Published by Elsevier Ltd. All rights reserved.

951M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

Because the osteogenic potency of mesenchymal progenitorcells cultivated onto bioresorbable scaffolds is of major inter-est for future clinical applications to treat bone defects, thegrowth and differentiation of cord blood progenitor cellsseeded onto a porous collagen sponge was investigated inthis study.

2. Materials and methods

2.1. Cell culture

After isolation of cord blood derived mononuclear cells from one healthy

newborn by density gradient centrifugation, the cells were cultivated and ex-

panded in DMEM-low glucose media (1 g/L), supplemented with 30% FCS

and 100 nM dexamethasone in 5% CO2 at 37 �C. After a confluent monolayer

formed, cells were passaged, supported by 0.05% trypsin/0.02% EDTA as

described previously (Jager et al., 2005). According to the Declaration of Hel-

sinki in its present form, the parents of the donor provided written informed

consent before delivery of the offspring.

2.2. Scaffold and osteogenic stimulation

Human cord blood derived progenitor cells were cultivated onto a porous,

porcine-derived collagen I/III scaffold (ACI-Maix�, Fa. Matricel, GmbH,

Germany). According to the manufacturer’s information, ACI-Maix� consists

mainly of type I collagen, contains low amounts of other natural fiber-forming

proteins like elastin and collagen type III and only traces of non-collagenous

proteins (cysteine <0.5%, tryptophane <0.1%, hexosamines <0.5%). ACI-

Maix� is sterile (gamma irradiation), endotoxin-free and has a CE-mark for

use as a covering membrane in autologous chondrocyte implantation tech-

niques. Based on an analysis of microscopic sections of the collagen scaffold

the maximal distance between the single fibers is about 50 mm and the esti-

mated average of porosity is about 20 mm.

Then, 106 cord blood cells were pipetted onto the central surface of

a 5.0 � 5.0 mm collagen sponge and cultivated under osteogenic stimulation

(10�7 M dexamethasone, 5 � 10�6 M ascorbic acid, 0.01 M b-glycerolphos-

phate) in DMEM-low glucose, 30% FCS at 5 vol% CO2 and 37 �C over

21 days. To inhibit cell adherence onto culture polystyrene flasks we used culture

dishes which do not allow for cellular adherence according to the manufacturer’s

information.

2.3. Morphology and immunocytochemistry

To evaluate the expression of different antigens we performed immuno-

cytochemical stainings against the following different markers using 10 mm

frozen sections (Kyostat, Zeiss, Germany), which were cut transversally and

fixed onto slides:

e bone sialoprotein (BSP) (anti-rabbit/1:250 in PBS-TA, Chemicon Int.,

Hampshire, UK)

e osteocalcin (OC) (ani-mouse IgG3/1:50 in PBS-TA, Zymed, Munchen,

Germany)

e osteonectin (ON), (anti-rabbit/1:50 in PBS-TA, Chemicon Int.)

e osteopontin (OP), (rabbit anti-human OP polyclonal antibody, AB1870,

Chemicon Int./1:50 in PBS-TA)

e cartilage proteoglycan (CP) (anti-mouse IgG1/1:250 in PBS-TA, Chemi-

con Int.)

e chondrogenic oligomeric matrix protein (COMP) (anti-rat IgG1, Serotec

GmbH, Dusseldorf, Germany)

e collagen I (anti-collagen I mouse IgG1, 1:100 in PBS-TA, Dako-Cytoma-

tion, Hamburg, Germany)

e collagen II (anti-collagen II rabbit, 1:20 in PBS-TA, Chemicon Int.)

e collagen III (anti-collagen III rabbit, 1:40 in PBS-TA, Chemicon Int.)

e collagen X (anti-collagen X rabbit, 1:40 in PBS-TA, Calbiochem/Merck

KGaG, Germany)

e CD13 (aminopeptidase N) (anti-mouse IgG1, 1:100 in PBS-TA, Dako-

Cytomation)

e CD105 (endoglin) (anti-mouse IgG1, 1:20 in PBS-TA, Dako-Cytomation)

e fibroblast growth factor receptor 2 (FGF-R2), (anti-mouse IgG1, 1:500 in

PBS-TA, Oncogene)

e CD31 (PECAM-I, GP-IIa) (anti-mouse IgG1, 1:50 in PBS-TA, Dako-

Cytomation)

e CD34 (anti-mouse IgG3/1:20 in PBS-TA, Dako-Cytomation)

e CD44 (H-CAM, Pgp-1) (anti-mouse IgG1, 1:20 in PBS-TA, Dako-

Cytomation)

e CD45 (leukocyte common antigen) (anti-mouse IgG1, 1:100 in PBS-TA,

Dako-Cytomation)

e vascular endothelial growth factor (VEGF) (anti-rabbit, 1: 50 in PBS-TA,

Zymed)

e vimentin (anti-mouse IgG2a/1:200 in PBS-TA, Dako-Cytomation)

In addition we performed conventional HE stainings. Deposition of calcium

phosphates as a sign of biomineralization was assessed by von-Kossa staining

and alkaline phosphatase (ALP) activity was measured after direct substrate

incubation (SK-5.200, Vector, Burlingame CA, USA) for 30 min at RT.

After an incubation period of 21 days the collagen samples were fixed in

5% paraformaldehyde at 4 �C for 30 min, rinsed in phosphate-buffered saline

(PBS) and dehydrated in graded alcohols. Endogenous peroxidases of the

specimen were blocked by 3% perhydrol-isopropanol solution.

After rinsing in Tris-buffer, the cell culture dishes were incubated with

primary antibodies against different antigens mentioned above with further

incubation at 4 �C for 12 h. A second antibody system (avidin-biotin-com-

plex and 3,3-diaminobenzidine) was used for optical visualization. The cell

cultures were analyzed blinded by an independent observer using episcopic

light microscopy (Axiovert 200, Zeiss, Germany) in combination with a com-

puter-supported imaging picture analysis system (Axiovision, Zeiss).

Based on light microscopy we used the following scoring system for semi-

quantitative evaluation of antigen expression after 3 weeks of osteogenic

Fig. 1. (a) Shrinking and elevation of the scaffold’s margins after an in vitro incubation period with human cord blood derived cells of 21 days. (b) The negative

control without cells showed no significant changes in morphology.

952 M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

Table 1

Semiquantitative antigen expression in dependency of the localization of the cord blood derived cells after 21 days in vitro

Antigen/marker Detection/amount Antigen/marker Detection/amount Antigen/marker Detection/amount

ALP þþþ (margin/bottom),

þ (center/top)

CD44 þþþ (margin/bottom),

þþþ (center/top)

Cartilage proteoglycan (CP) þ (margin/bottom),

þþþ (center/top)

v. Kossa þþþ (margin/bottom),

þþþ (center/top)

CD105 þþ (margin/bottom),

� (center/top)

cartilage oligomeric

protein (COMP)

þþþ (margin/bottom),

þþþ (center/top)

Bone sialoprotein

(BSP)

þþþ (margin/bottom),

þþþ (center/top)

Collagen I þþ (margin/bottom),

� (center/top)

FGF-R2 � (margin/bottom),

� (center/top)

CD13 þþþ (margin/bottom),

þþþ (center/top)

Collagen II � (margin/bottom),

� (center/top)

osteocalcin (OC) � (margin/bottom),

� (center/top)

CD31 � (margin/bottom),

� (center/top)

Collagen III þþþ (margin/bottom),

þþþ (center/top)

osteonectin (ON) þþ (margin/bottom),

þþþ (center/top)

CD34 � (margin/bottom),

� (center/top)

Collagen X þþþ (margin/bottom),

þþþ (center/top)

osteopontin (OP) þþ (margin/bottom),

þþþ (center/top)

VEGF þþ (margin/bottom),

� (center/top)

Vimentin þþþ (margin/bottom),

þþ (center/top)

Fig. 2. Immuncytochemical stainings of different antigens of human cord blood derived stem cells cultured onto a collagen I/III membrane and stimulated over 21

with osteogenic mixture (DAG).

953M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

Fig. 2 (continued).

stimulation: no antigen expression (�), detection of single positive cells (þ),

detection of numerous positive cells which showed a direct contact to each

other (þþ), dense layer of antigen positive cells (þþþ). All experiments

were performed in triplicate.

3. Results

Starting at 1 week after cultivation we observed a shrinkingof the collagen mesh but also a lifting of its margins with

further cultivation (Fig. 1). In contrast, negative controls donot show these morphological changes of the scaffold. Mostcells were located in the center and on the top of the collagenscaffold, where cord blood cells migrated into deeper layers ofthe three-dimensional fibrous collagen network. In contrast, themargins of the scaffold showed significant less number of cells.

Corresponding to an osteoblastic differentiation all osteo-blastic antigens excepted OC were expressed by the cordblood cells after 21 days in vitro (OP, ON, BSP, collagen I).

954 M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

Fig. 2 (continued).

Furthermore, we found significant amounts of ALP and alsosigns of a beginning biomineralization as shown by von-Kossastaining. In contrast to these findings both hematopoietic anti-gens CD34 and CD45 were negative in all cultures. We foundno expression of CD31 and FGF-R2, but vimentin, VEGF,CD13, CD44 and CD105. Although collagen II was not ex-pressed we also found an expression of chondroblast-typicalmarkers such as COMP and CP.

For some antigens the expression by cord blood derivedcells was dependent on the localization of the cells on the scaf-fold (bottom/margin vs. top/center of the scaffold, Table 1,Fig. 2). CD105 was only expressed by a single monolayer ofcells located on the bottom and at the margins of the matrix,whereas biomineralization only occurred in the central area.In the latter region BSP was also expressed in higher amountscompared to the bottom side and at the biomaterial’s margins.

ALP showed a reciprocal type of expression: a weak ALPactivity in the center of the collagen membrane and a highactivity level at the bottom side of the scaffold. VEGF wasexpressed only at the bottom side of the biomaterial.

The elevated edges of the scaffold showed low cell numbersthat had a similar antigen profile corresponding to the cellsgrown in the center area of the biomatrix.

4. Discussion

In this study we showed that human cord blood derivedmononuclear cells cultured onto a porous collagen I/III mem-brane can differentiate into osteogenic cell lines under lineagespecific stimulation.

The properties of a collagen I/III sponge to allow for anosteoblastic differentiation corresponds to other investigators

955M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

Fig. 2 (continued).

(George et al., 2006) and also to our own previously publisheddata (Jager et al., 2005) for bone marrow derived mesenchymalstem cells. All osteoblastic markers except OC were expressedby cord blood derived cells. Because OC is expressed inadvanced stages of an osteoblastic differentiation and matureosteoblasts, the detection of the beginning of biomineralizationin the von-Kossa staining is a contradiction to the absence ofOC expression. The expression of osteoblastic proteins bycord blood derived mesenchymal cells after DAG stimulation

agrees with data from other investigators (Hutson et al.,2005; Kang et al., 2006; Kogler et al., 2004; Lee et al., 2004;Wild et al., 2004).

One possible explanation for the elevation of the scaffold’smargins and also for the differences in antigen pattern of thecells, depending on the location, are cytomechanical forces.We hypothesize that the initial high number of cells in the cen-tre and on the top of the scaffold promotes intercellular adher-ence and communication with further cultivation. This may

956 M. Jager, R. Krauspe / Cell Biology International 31 (2007) 950e957

F2

F1

day 0 day 21

Fig. 3. Theory of cytomechanical forces leading to a deformation of the collagen membrane with distension on the bottom side and compression on the top side

(F1 > F2, F, force).

lead to an elevation of the biomaterial’s margins where thenumber of cells is lower. The initial application of the cellsin the center area of the scaffold by pipetting also supportsthis hypothesis. As a consequence of the geometric changesof the collagen membrane those cells adhered onto the bottomof the scaffold may be elongated and stretched by distensionforces (Fig. 3). It would appear that cytomechanical forcesand cell deformations significantly influence cellular prolifer-ation and differentiation (Stephanou and Tracqui, 2002).

We found that VEGF, a typical angiogenic marker which isexpressed by sheer stressed endothelial cells in vivo (Witmeret al., 2002), was only expressed at the bottom of the mem-brane. In addition it was shown that VEGF is also expressedin other cells in organs elongated to yield stress, including en-dometrium (Goteri et al., 2004), bladder and gastrointestinalorgans. Hou et al. (2006) demonstrated using a rat modelthat mesenchymal stem cells from bone marrow express VEGFafter injection into infarcted myocardium. Also CD105 (endo-glin), a typical marker for mesenchymal stem cells (Dominiciet al., 2006), was only expressed at the bottom of the collagenmembrane. Besides the theory of cytomechanical forces it ispossible that the cord blood cells upon the collagen carrierunderwent a further osteoblastic differentiation whereas atthe bottom side of the membrane cells persist to expressCD105. The expression of vimentin in cord blood derived cellsalso agrees with results by Kogler et al. (2004) and Rosadaet al. (2003).

Although the cell cultures were stimulated osteogenically,we found CP and COMP as typical markers for a chondrogenicdifferentiation. In addition CD44 with its hyaluronan bindingfunction is also a marker of mesenchymal cord blood cellswhich is expressed in vitro as shown by other investigators(Lee et al., 2004; Lu et al., 2005). The absence of CD31,CD34 and CD45 expression by mesenchymal cord blood cellsafter osteogenic stimulation agrees with Lee et al. (2004).

However, although the high porosity of the collagen carriermay allow for migration and spreading of the cells, it is un-clear if the low cell number at the edges and at the bottomof the scaffold is based on a lack of migration potency com-bined with an initial high cell number in the center of the bio-membrane or if migration effects are responsible for local celldistribution onto the biocarrier’s surface at the end of study.

Further studies should investigate the role of cytomechani-cal forces in cord blood derived mesenchymal progenitor cells,

as well as elucidate if a collagen I/III carrier is a valuable toolfor the in vivo application of these cells.

5. Conclusions

The current study showed that mononuclear cells derivedfrom cord blood are able to adhere, proliferate and differenti-ate into osteoblasts onto a collagen I/III scaffold under osteo-genic stimulation. In addition, the expression of antigens wasdependent on the localization of the cells within the collagencarrier.

Acknowledgements

The authors thank G. Kogler, Institute for Cell Therapeuticsand Transplantation Diagnostics, UKD for technical support.

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