Volume 18, Issue 12, Pages 2164-2172 (December 2010) Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain  Lone Fjord-Larsen, Philip Kusk, Jens Tornøe, Bengt Juliusson, Malene Torp, Carsten R Bjarkam, Mette S Nielsen, Aase Handberg, Jens Christian H Sørensen, Lars U Wahlberg  Molecular Therapy  Volume 18, Issue 12, Pages 2164-2172 (December 2010) DOI: 10.1038/mt.2010.154 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 NGC-0295 produced NGF. (a) NGF western blotting of conditioned medium from NGC-0295 cells, with purified recombinant NGF and proNGF as references. The mature monomer is indicated by an arrow. A band, corresponding to the dimer and the double band, likely to represent intermediate partially processed forms (pp NGF) are also shown by arrows. (b) Survival and (c) neurite outgrowth in PC12 induced by NGC-0295 produced NGF. Data shown are the means ± SEM (n = 3) from representative experiments and *indicates a significant difference (P < 0.05) from the respective control with no addition. (d) Quantification of neurite outgrowth induced by NGC-0295 produced NGF in porcine DRG cultures. Data shown are the means ± SEM (n = 4) from a representative experiment, and *indicates a significant difference (P < 0.05) from the control wells. Example of porcine DRG neurons (arrows) cultured in the presence of devices with (e) parental RPE cells or (f) NGC-0295 cells. Bar = 150 µm. DRG, dorsal root ganglion; NGF, nerve growth factor; pp NGF, partially processed NGF. Molecular Therapy 2010 18, 2164-2172DOI: (10.1038/mt.2010.154) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 NsG0202 devices. (a) The clinical NsG0202 configuration with a tether part for retrieval linked to the PES fiber containing the NGF-secreting NGC-0295 cells growing on PVA matrix. (b) NGF release (mean ± SEM) measured in medium incubated with devices before implantation (for all devices, n = 36) and after explantation, either after 7 weeks (n = 4), 6 months (n = 19, as one device was damaged after explantation) or 12 months (n = 12) in vivo. (c) Von Kossa staining of device after 12 months in vivo. Examples of areas with darkly stained calcium deposits, associated with the PVA matrix (white arrows) or in direct association with cells (black arrows) are indicated. Surrounding cells are healthy and viable. (d) NGF release (mean ± SEM) measured from devices (n = 10) kept in vitro. (e) Von Kossa staining of device with healthy and viable cells and no calcium deposits after 12 months in vitro. Bar = 300 µm. NGF, nerve growth factor; PES, polyethersulfone; PVA, polyvinylalcohol. Molecular Therapy 2010 18, 2164-2172DOI: (10.1038/mt.2010.154) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Histopathology. (a) Tissue reaction scores in slabs with NsG0202 (n = 20), empty devices (n = 6), and sham surgery (n = 6), taken after 6 months (6 m) and NsG0202 (n = 12) after 12 months (12 m). Data shown are the mean score ± SEM and *indicates a significant difference (P < 0.05) between the gliosis responses in NsG0202 groups after 6 and 12 months. Photomicrographs of (b) capillary hyperplasia (white arrow) and pigment deposits (black arrows), i, implant site (c) lymphocytic infiltration (white arrows) and swollen axons indicated by black arrows (d) tissue with spongiosis (black dotted arrow) and gliosis (dark gray arrows). Bar = 75 µm. Molecular Therapy 2010 18, 2164-2172DOI: (10.1038/mt.2010.154) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Cholinergic cells (examples of p75NGFR-immunoreactive cells pointed out by arrows) surrounding the implantation sites. Most cells are in front of the rear implant site. Areas, where punches were taken (in other slabs) for NGF ELISA analyses, are indicated in white. The same areas were used for image analyses. Bar = 2,500 µm. ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor. Molecular Therapy 2010 18, 2164-2172DOI: (10.1038/mt.2010.154) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions