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


CONCLUSIONS
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.
Ronsyn
Mark W
MW
Division of Clinical Pharmacology, University of Antwerp, Antwerp, Belgium. mark.ronsyn@ua.ac.be
Daans
Jasmijn
J
Spaepen
Gie
G
Chatterjee
Shyama
S
Vermeulen
Katrien
K
D'Haese
Patrick
P
Van Tendeloo
Viggo Fi
VF
Van Marck
Eric
E
Ysebaert
Dirk
D
Berneman
Zwi N
ZN
Jorens
Philippe G
PG
Ponsaerts
Peter
P
D000818:Animals; D016026:Bone Marrow Transplantation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D015316:Genetic Therapy / Q000379:methods*; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D020933:Neurotrophin 3 / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics*; D051381:Rats; D013116:Spinal Cord / Q000378:metabolism* / Q000601:surgery*; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics
D020933:Neurotrophin 3