Mesenchymal Cells Metalloproteinases And Adhesion On Microcarriers

C. Mosbeux1, A.R. dos Santos Pedregal1, D. Ribeiro de Sousa1 , Vincianne Hendrick1, N. Joseph, M. Bensellam1, D. Blankaert1, T. Marique1, C. Alloin1, D. Parent2, C. Liesnard2, J.P. Van Vooren2, S. Lowagie1 and J. Werenne1

1Laboratory of Animal Cell Biotechnology. Dept. of Bioengineering,

Faculty of Sciences, Université Libre de Bruxelles, Belgium and 2Erasme Hospital,, Brussels, Belgium.

Abstract: The purpose of this work was to approach the development of a safe and validable process to grow the amounts of adult Mesenchymal Stem Cells (MSC) necessary for future biomedical applications. Using a single use Vue Life bag system integrated in a simple agitations bioreactor we constructed, we have studied the adhesion, migration and growth properties of MSC on different carriers (Cytodex and Bionoc) in comparison to other cells (endothelial/Kaposi sarcoma cells, CHO and HeLa) in relation to the expression of endogenous Metalloproteinases.

Keywords: Mesenchymal Stem Cells; Kaposi sarcoma; endothelial cells; CHO; HeLa; Adhesion; Migration; Mobility; Metalloproteinases; Single use bioreactor.

1. INTRODUCTION

Despite a number of controversial issues including ethical ones, stem cells represent a very important challenge for future therapeutic approaches. Especially adult Mesenchymal Stem Cells (MSC) derived from mesoderm, since it has been documented that they could lead to differentiation of tissues of the other germ layers (-ecto and -endoderm), showing previously unexpected "plasticity", offer important promises.

As MSC proliferate extensively in vitro without obvious senescence or loss of differentiation potential, they may be an ideal source for therapy of inherited, and also for degenerative diseases.

In parallel to the necessary development of conditions securing appropriate differentiation of MSC, one of the numerous challenges in the field of cell based therapy and tissue engineering strategies, is to obtain enough progenitor cells (e.g. 500 millions cells) and to deliver them efficiently to the repair or regeneration site in the body.

This will need the development of culture conditions and devices that

could increase the ability of MSC to proliferate in vitro and would permit their migration towards the appropriate location for therapeutic tissue transplantation.

First steps have been taken in this direction using adult rat mesenchymal cells (MSC) as previously characterised and briefly by us (Bensellam et al. 2003).

We compared here the adhesion and aggregation properties of Endothelia, Kaposi Sarcoma and Mesenchymal Stem Cells on different solid surfaces.

Their mobility were evaluated in relation to the expression of different metalloproteinases (MMPases after treatment with different factors (PMA,TNF and other Cytokines).

2. MATERIAL AND METHODS

2.1 Cells and culture system

The control of the expansion of Mesenchymal Stem cells in order they can maintain their pluripotentiality and differentiation without the risk of tumoral like invasivity being not a straightforward problem, we started to approach it, by evaluating the interest of using either Cytodex 3 or Bionoc II carriers, and using VueLife bags, given the interest of developing for the future a process that could be easily validated for medical applications.

We first compared in a parallel investigation rat MSC cultivated as adapted (Bensellam et al. 2003) from a paper of Wislet-Gendebien et al. (2003), to endothelial cells of different origins, including Human Umbilical Endothelial cells (HUVEC) and Human Kaposi Sarcoma cells (Blankaert et al. 1998), and to HeLa as well as CHO cells (Hendrick et al. 2001).

2.2 Metalloproteinases Zymography

Studying cell adhesion and migration properties in relation to the expression of active MMPases required zymographic analysis as previously described by our group (Blankaert et al. 1998).

As MMPases expression was shown to be strongly regulated by MPA under API control in conditions related to properties related to metastasis disseminations (Marique and Werenne, 2001), we have studied the effect of this tumor promotor as well as the effect of TNF and "conditioned" medium (medium in which cells have been cultivated) or "activated conditioned" medium (same but treated at pH 2 with 10 M HCl and then quickly neutralized with 10 M NaOH) on expression of these enzymes by the different cells, using zymography.

3. RESULTS

3.1. MMPases expression

The following Zymograms (Fig. 1) showed that in HUVEC, after 48h treatment with PMA , the 92 kD MMPases is maximally produced, while in absence of the factor it was nearly undetectable. In Kaposi Sarcoma cells which are, as we have established, of endothelial origin, the 92 kD MMPases is expressed without stimulation, PMA 1nM just increasing it only marginally (Fig. 2). TNF acts synergistically strongly with HUVEC, and much less on Kaposi Sarcoma cells (data not shown), in relation to the basal invasivity properties of this AIDS related tumor.

Figure 1. MMPases expression in HUVEC FiSure MMPases expression in KAPOSI cells

In MSC conditioned medium, in presence of Foetal Calf Serum, a high molecular weight MMPases (97 kD) was detected , while in absence of Serum its molecular weight is reduced to 91 kD, indicating that a factor of the Serum modulated the enzyme properties (Fig. 3).

100 kD

50 kD

150 kD

75 kD

97 kD 91 kD

Figure 3. MMPases expression in MSC

97 kD 91 kD

Figure 3. MMPases expression in MSC

3.2. Adhesion and Growth of MSC

We have seeded in VueLife bags either Bionoc II carriers and Cytodex 3 microcarriers, and on both substratum, MSC attached and multiply.

In the Bionoc system (Fig. 4), it is not easy to observe the cells due to the lack of transparency of the material. Moreover, it is not easy to recover the cells by trypsinisation from these microcarriers.

On Cytodex 3, the behavior of MSC depended on the passage of the culture: Higher passage cells (over 45), we have observed that MSC are able to migrate from colonized to empty beads as observed also with HeLa cells. With MSC, cells forms bridges between beads (Fig. 5).

A difference is also observed between Higher and Lower passages MSC, as it was shown that while in both situations, cells migrated from Petri dish to microcarriers added to a monolayer, the Higher passage MSC (over 45) were loosely bound to microcarriers after trypsinisation while Lower passage cells (up to 33) spread happily after trypsinisation to Cytodex 3 (Fig. 6).

Moreover, Higher and Lower passage MSC, grown on Cytodex 3, are able to migrate to TC treated Petri dishes and colonize it, while only Higher passage MSC are able to do so on non TC treated Petri dishes, indicating that different interactions occurred between extracellular matrix and Cells of different passages (Fig. 7).

Figure 4. MSC on Bionoc II Figure 5. MSC on Cytodex 3

Figure 4. MSC on Bionoc II Figure 5. MSC on Cytodex 3

Cytodex Beads
Figure 6. Adhesion of Higher and Lower passage MSC on Cytodex after Trypsinisation
Microcarrier Cell Migration Assay

These properties altogether indicate that depending on their number of passage in culture, MSC undergo a striking evolution affecting their interaction with the extracellular matrix and that they are themselves participating in the production of material involved in their adhesion and mobility properties.

Finally (not shown), we observed that compared to a control culture, where migration from a confluent MSC on Petri dish to Cytodex 3 beads, which are trapped in the monolayer is quite limited, it is facilitated under PMA treatment and is inhibited by TGF beta.

4. CONCLUSIONS

It is known that in relation to the potential plasticity of adult MSC, medium composition and passage number, as well as a number of cytokines and related factors, affect their adhesion, aggregation, morphology and differentiation characteristics.

We have in the past shown that in Kaposi Sarcoma, mobility related to the invasivity of the tumor is correlated with a high 92 kD MMPase expression, which may be increased in normal endothelial cells by PMA and TNF. As it was the case in these endothelial cells we showed that these factors modulated the adhesion properties of MSC in a striking way.

Despite it appeared that the control of the expansion of Mesenchymal Stem Cells in order they can maintain their pluripotentiality and differentiation properties without the risk of tumoral like invasivity will not be a straightforward problem, our present results could usefully be taken into account for the development of a culture process in a simple, safe and easily validable bioreactor.

5. REFERENCES

Bensellam, M., Lowagie, S., Wislet-Gendebien, S., Rogister, B., and Werenne, J. (2003), unpublished, presented briefly at BELAC Meeting, Brussels, March 21.

Wislet-Gendebien, S. and Rogister, Bernard, J. Cell Science (2003).

Blankaert, D., Simonart, T., Van Vooren, J.P., Parent, D., Liesnard, C., Farber, C.M., Marique, T. and Werenne, J. Constitutive Release of Metalloproteinase-9 (92 kD Type IV Collagenase) by Kaposi's Sarcoma Cells (1998), J. Acquired Immune Deficiency Syndromes andRetrovirology, 18, 203-209.

Hendrick,V., Winnepenninckx, P., Abdelkafi, C., Vandeputte, O., Cherlet, M., Marique, T., Renemann, G., Loa, A., Kretzmer, G. and Werenne, J. (2001), Increased productivity of recombinant tissular plasminogen activator (tPA) by butyrate and shift of temperature: a cell cycle phase analysis, Cytotechnology, 36, 7183.

Marique, T. and Wérenne, J. (2001) Control of 92 kDa Collagenase secretion in mammalian cells by modulation of AP1 activity: an experimentally based theoretical study, J. Theor. Biol. 209, 3-8.

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