Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells

Abstract

Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.

Keywords: cell therapy; doxycycline; gene therapy; glial cell line-derived neurotrophic factor; induced pluripotent stem cell; inducible reversible transgene; luciferase; non-invasive imaging; piggyBac; tetracycline.

Graphical abstract

Figure 1

Description of pB-RTP-Tet-GDNF/memClover-FLuc Vector (A) pB-RTP-Tet-GDNF/memClover-FLuc plasmid that is designed to stably integrate into genome when transfected in combination with pBase plasmid. (B) pBase plasmid. (C) Transgenes constitutively expressed or expressed only in the presence of doxycycline. (D) Live unstained fluorescent imaging of human iNPCs nucleofected with pB-RTP-Tet-GDNF/memClover-FLuc and grown as neurospheres. (E) Firefly luciferase activity normalized to renilla luciferase in iNPCs. N = 4 independent experiments. (F) Real-time firefly luciferase activity in live culture of iNPCs. N = 3 independent experiments. Error bars represent ± SEM. ^∗∗∗p < 0.001.

Figure 2

Doxycycline Regulates GDNF Expression from pB-RTP-Tet-GDNF/memClover-FLuc Nucleofected iPSC-Derived NPCs (A and B) Nucleofected iNPCs grown in the (A) absence or (B) presence of dox. (C) Nucleofected iNPCs grown in the absence of dox after dox was added and removed. (D) ELISA of 24 hr incubated medium from cells in (A–C), as well as in a culture with GDNF protein accumulation for 7 days. N = 3 independent experiments. Error bars represent ±SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

Reporter Transgene Expression Is Inducible and Reversible in iPSC-Derived NPC Transplants (A) Experimental design for nucleofection, expansion, FACS, transplant, treatment, and post-mortem analysis. (B) Weekly bioluminescence imaging of one animal from each group (animal no. 9 for group A and animal no. 3 for group B) over a 7-week in vivo experimental period. ON/OFF buttons indicate if animal received dox during the week prior to imaging. (C) Summary of weekly bioluminescence activity for all animals (N = 14). One animal (no. 13) was excluded from this analysis due an abnormally high bioluminescence signal observed and no inducible transgene signal observed by post-mortem immunohistochemistry. Error bars represent ± SEM.

Figure 4

Doxycycline Mediates GDNF Expression in Transplanted iPSC-Derived NPCs (A and B) Striatal region of (A) animal no. 1 at experimental endpoint [Started OFF→ON→OFF→Ended ON] and (B) Animal no. 7 at experimental endpoint [Started ON→OFF→ON→Ended OFF]. (C) High magnification image of squared region in (A). Pink arrows indicate GDNF expression in areas distant from TagBFP⁺ cells, suggesting that host cells uptake secreted GDNF. (D) High magnification image of squared region in (B). Note: Clover signal and GDNF signal are likely background given the overlap (B2/B3 and D2/D3). See also Figure S2.