# Qualification of labeled Endothelial Progenitor cells for tracking in the context of tissue engineering

Noelie Thébaud, Audrey Aussel, Robin Siadous, Jérôme Toutain, Reine Bareille, Alexandra Montembault, Laurent David, Laurence Bordenave

► To cite this version:

Noelie Thébaud, Audrey Aussel, Robin Siadous, Jérôme Toutain, Reine Bareille, et al. Qualification of labeled Endothelial Progenitor cells for tracking in the context of tissue engineering. Journées RITS 2015, Mar 2015, Dourdan, France. pp 108-109. inserm-01154946

HAL Id: inserm-01154946

https://inserm.hal.science/inserm-01154946

Submitted on 25 May 2015

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Qualification of labeled Endothelial Progenitor cells for tracking in the context of tissue engineering

NB. Thébaud<fnref n="1" />,<fnref n="2" />,<fnref n="3" />*, A. Aussel<fnref n="1" />,<fnref n="2" />, R. Siadous<fnref n="1" />,<fnref n="2" />, J Toutain<fnref n="3" />, R. Bareille<fnref n="1" />,<fnref n="2" />, A Montembault<fnref n="4" />, L David<fnref n="4" />, L. Bordenave<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="5" />

## Abstract

In order to track location and distribution of endothelial cells (ECs) within scaffolds, we chose lentiPGK-TdTomato transduction of human Endothelial Progenitor Cells (EPCs). Because transduction could have a functional impact on cell behaviour, we checked different parameters for qualification of labeled-EPCs. After isolation and expansion, EPCs were transduced with the lentiviral vector containing the TdTomato protein gene. Conventional karyotyping, differentiation capacity, viability, proliferation assays and functional assays were performed with labeled and unlabeled EPCs. Results show that cell labeling did not affect cell adhesion nor induce cell death. Cell labeling did not induce more chromosomal aberrations. Phenotypical characterization was not affected. In the context of tissue engineering applications, labeled EPCs maintained their ability to line scaffolds, withstand physiological arterial shear stress and form tubular networks in co-cultures with human osteoblast progenitor cells. So it is possible to label human EPCs with TdTomato without affecting their behaviour by the transduction procedure. This creates an important tool for vascular and bone tissue engineering.

key-words: Biomaterials, Molecular Imaging, Optical Imaging.

## I. INTRODUCTION

Vascularization is the key challenge in tissue engineering [1]. For that purpose, stem cells and progenitor cells may hold the key to repair and could be used for tissue engineering and regenerative medicine. Endothelial progenitor cells can be easily isolated from cord blood and differentiated in Endothelial Progenitor Cells (EPCs) [2]. However, the location of endothelial cells in a 3D matrix raises question of traceability. To fulfill these requirement, cell fluorescent labeling by lentiviral transduction is a method used to track cell location and distribution within a matrix. We chose lentiPGK-TdTomato transduction for cell labeling. However, transduction could influence the viability, proliferation,

differentiation capacity and chromosomal stability of EPCs, which would limit its use. Therefore, the aim of this study was to *in vitro* evaluate the influence of lentiPGK-TdTomato transduction in view to qualify human labeled-EPCs for cell tracking experiments in the context of tissue engineering.

## II. MATERIALS AND METHODS

### II.1 Isolation and expansion of human cells derived from mononuclear cell (MNC) cultures

EPCs were isolated from cord blood and cultured as previously described by [3].

### II.2 EPCs lentiviral transduction (EPCs labeling)

The lentiviral vector contained the TdTomato protein gene under the control of the Phosphoglycerate kinase (PGK) promoter. For viral transduction, 2.10⁵ freshly trypsinized EPCs in suspension were mixed with 6.10⁶ viral particles (MOI: 30). Expression of TdTomato was observed under fluorescent microscope (excitation 554 nm and emission maxima 581 nm).

### II.3 Cytogenetic analysis

labeled and unlabeled EPCs were cultured to evaluate chromosomal abnormalities at early and late passages using conventional karyotyping.

### II.4 Assessment of endothelial phenotype in EPCs

The following criteria were used for characterization and to control the stability of the endothelial phenotype during the expansion of labeled or not EPCs: i) cellular uptake of UEA-1 lectin, ii) immunofluorescent stainings and flow cytometric analysis for CD31 and vWF according to [2]. For analysis of capillary tube formation, labeled or not EPCs were seeded onto Matrigel® and observed after 48 h in culture.

### II.5 Viability assay

At days 1, 2, 3, 6 and 9 neutral red and MTT assays were performed with cells from 3 different donors at the same passages labeled or not.

<footnote n="1">¹INSERM, U1026, F-33000 Bordeaux, France</footnote>

<footnote n="2">²Univ. Bordeaux, Bioingénierie tissulaire, U1026, F-33000 Bordeaux, France</footnote>

<footnote n="3">³CHU Bordeaux, F-33000 Bordeaux, France</footnote>

<footnote n="4">⁴IMP UMR CNRS 5223, 15 boulevard Latarjet, 69000 Villeurbanne Cedex</footnote>

<footnote n="5">⁵CHU Bordeaux, CIC-IT Biomaterials, F-33000 Bordeaux, France</footnote>

<footnote>*noelie.thebaud@inserm.fr</footnote>
## II.6 Applications of labeled EPCs for tissue engineering

Several features were checked relating potential applications of EPCs in tissue engineering. To determine whether labeled EPCs maintained their ability to line prosthesis, we used the synthetic vascular graft INTERGARD™ or biological Chitosan-based hydrogels [4]. These prostheses were filled with the labeled or not EPCs. Prosthesis samples endothelialized with unlabeled EPCs were observed in epifluorescence (Live/Dead). To determine the effects of laminar shear stress (1.2 Pa) on labeled EPCs, we used a parallel plate culture flow chamber seeded with the labeled or not EPCs. The angle deviation of cells was quantified. Then, cells were co-cultured with Human Osteoprogenitors (HOPs). Intracellular alkaline phosphatase (ALP) and immunofluorescent stainings for vWF were checked respectively in HOPs and EPCs.

## II.7 Statistical analysis

Comparisons between unlabeled and labeled cells were performed by GraphPad Prism ®5: two way ANOVA and Bonferroni post test for proliferation assay and angle deviation, Wilcoxon test for cytometry analysis and Mann-Whitney test for cytogenetic analysis. P values less than 0.05 were considered significant.

# III. RESULTS

## III.1 Assessment of endothelial phenotype in labeled or not EPCs

According to immunofluorescent stainings and cytometry, there was no difference between labeled and unlabeled EPCs (UEA-1+, vWF+, CD31+). Labeled and control cells formed tube-like structures on Matrigel®.

## III.2 Cytogenetic analysis

Chromosomal abnormalities were noticed for TdTomo cells but labeling did not induce more chromosomal aberrations.

## III.3 Viability assay

Proliferative capacity is not significantly affected by TdTomo labelling.

## III.4 Applications of labeled EPCs for tissue engineering

*Ability to line prosthesis*

Labeling did not alter the propensity of TdTomo EPCs to cover the luminal side of Chitosan-based hydrogels and INTERGARD™ prosthesis (Figure 1).

<figure><img src="image_2.png" /><figcaption>Figure 1: Confocal microscopy of vascular prosthesis INTERGARD™ seeded with labeled EPCs and unlabeled cells revealed by Live/Dead test.</figcaption></figure>

*Effects of shear stress*

After 24 hours under shear stress conditions, cells elongated and became orientated compared with static conditions(a), confirmed with angle deviation(b).

<figure><img src="image_3.png" /><figcaption>Figure 2: microscopic appearance (A to H) of the TdTomo-labeled EPCs (A, C, E, G) and unlabeled EPCs (B, D, F, H) under static (A, B, E, F) of shear stress conditions (C, D, G, H) after 6 hours (A, B, C, D) or 24 hours (E, F, G, H). Angle deviation analysis in each condition (I). **: p< 0.01.</figcaption></figure>

*Functionality*

In direct co-culture conditions between labeled or unlabeled EPCs and HOPs, a tubular-like network occurred.

# IV. DISCUSSION – CONCLUSION

From the present study, it is possible to label human EPCs with TdTomo without affecting the viability and capacities of these cells, suggesting no gross modification of the cell-specific properties by the transduction procedure. This creates an important tool for numerous applications. Our results provide a qualification of labeled EPCs for vascular and bone tissue engineering.

# ACKNOWLEDGMENTS

Agence Nationale pour la Recherche (ANR) for grant TECSAN Collaborative Research Program ChitoArt (ANR2010-TECS017).

# REFERENCES

[1] Novosel, E.C., C. Kleinhans, and P.J. Kluger, Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011. **63**(4-5): p. 300-11.

[2] Thebaud, N.B., et al., Human progenitor-derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study. J Tissue Eng Regen Med, 2010. **4**(6): p. 473-84.

[3] Thebaud, N.B., et al., Whatever their differentiation status, human progenitor derived - or mature - endothelial cells induce osteoblastic differentiation of bone marrow stromal cells. J Tissue Eng Regen Med, 2012. **6**(10): p. e51-60.

[4] Rami L, Malaise S, Delmond S, et al. Physicochemical modulation of chitosan-based hydrogels induces different biological responses: Interest for tissue engineering. J Biomed Mater Res A. 2013;102(10):3666-3676.