Research article

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

Mark W Ronsyn^1,2 , Jasmijn Daans² , Gie Spaepen³ , Shyama Chatterjee⁴ , Katrien Vermeulen² , Patrick D'Haese³ , Viggo FI Van Tendeloo^2,6 , Eric Van Marck⁴ , Dirk Ysebaert^5,6 , Zwi N Berneman^2,6 , Philippe G Jorens^1,6 and Peter Ponsaerts^2,6

¹Division of Clinical Pharmacology, University of Antwerp, Antwerp, Belgium

²Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (VIDI), University of Antwerp, Antwerp, Belgium

³Laboratory of Physiopathology, University of Antwerp, Antwerp, Belgium

⁴Laboratory of Pathology, University of Antwerp, Antwerp, Belgium

⁵Laboratory of Experimental Surgery, University of Antwerp, Antwerp, Belgium

⁶Centre for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium

author email corresponding author email

BMC Biotechnology 2007, 7:90doi:10.1186/1472-6750-7-90

Published:	14 December 2007

Abstract

Background

Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord.

Results

First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants.

Conclusion

In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo.