Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

The authors acknowledge the support of the Barts and the London Charity, the Multiple Sclerosis Society of Great Britain and Northern Ireland, the National Multiple Sclerosis Society, USA, notably the National Centre for the Replacement, Refinement & Reduction of Animals in Research, and the Wellcome Trust (grant no. 092539 to ZA). The siRNA was provided by Quark Pharmaceuticals. The funders and Quark Pharmaceuticals had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Systematic Evaluation of Integration Free Reprogramming Methods for Deriving Clinically Relevant Patient Specific Induced Pluripotent Stem (iPS) Cells

<div><p>A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells. </p> </div

Pluripotency of established iPS lines and detecting the genomic integration of episomal plasmids.

<p>A) Representative images of embryoid bodies (EBs) generated from BJ-pla-iPS (left) and BJ-mRNA-iPS (right). B) Repsentative image of karyotype 46, XY. BJ-pla-iPS cells at passage 15 is shown. C) H&E stained slides of teratomas formed from injections of iPS cells into the testis capsule of NOD-SCID mice. Tissue derivatives indicative of the three germ lineages were observed. D) Human pluripotent stem cell scorecard assay results comparing BJ-pla-iPS and BJ-mRNA-iPS lines.</p

Characterisation of established iPS lines derived from BJ fibroblasts using retrovirus, plasmids and mRNA.

<p>A) Representative phase images of established iPS and H1 cell lines. Immunostaining with pluripotency markers (green) and counter staining with DAPI (blue). Scale bar is 200µm. B) Alkaline phosphatase staining of whole plates/wells at days 30 (retrovirus and plasmid) and day 20 (mRNA). C) Flow cytometry analysis with pluripotency markers SSEA-4 and TRA-1-81. SSEA-1 is a negative marker of human pluripotent stem cells. Green line denotes H1, red line denotes BJ-RV-iPS, green line denotes BJ-Pla-iPS and black line denotes BJ-mRNA-iPS. D) RT-PCR analysis for expression of key pluripotency genes.</p

Comparison of different culture conditions during plasmid based reprogramming.

<p>A) Genomic integration of episomal plasmids. EBNA-1 and OriP sequences could not be detected in established iPS lines by passage 10. B) Morphology of RDP2-iPS cell lines derived in E8 medium, using different extracellular matrices. Scale bar is 400µm. C) Flow cytometric analysis of SSEA-4, TRA-1-81 and TRA-1-60 expression in RDP2-iPS cell lines derived on different matrices. Grey shaded areas denote the secondary antibody alone control. D) Whole well alkaline phosphatase staining of BJ, RDP2, PD1 and RP2 fibroblasts at day 30 of reprogramming using E8 medium and vitronectin.</p

OCT imaging during ON in MOG^TCRxThy1CFP mice

<p>(A) Histological changes in the thickness of RGC layer in control mice (n=5) and MOG^TCRxThy1CFP mice (n=5) at day 21. Results represent the mean ± SEM of RGC layer thickness. **P<0.01 and ***P<0.001 between control mice and MOG-specific TCR mice at the same distance from optic nerve head. (B) Multiline OCT, a Spectralis® HRA + OCT modified by Heidelberg Engineering Inc to allow imaging of CFP-expressing RGC and adapted to comply with mouse optics with a +25D lens and fitted animal mount. Mouse is ABH strain. (C) OCT images of retina were acquired using a circular OCT scan surrounding the optic nerve head on (D) day 0 and (E) day 21 of disease induction and RNFL thickness was calculated. (F) Decrease in RNFL thickness after immunisation of MOG^TCRxThy1CFP mice to develop ON (n=10). (G) Correlation between RGC density (measured using retinal flatmounts) and RNFL thickness (measured using OCT). Results represent the mean ± SEM of RNFL thickness. * P<0.05, **P<0.01, ***P<0.001 compared to control.</p

Neuroprotection with oxcarbazepine and caspase-2 siRNA in MOG^TCRxThy1CFP mice.

<p>(A) MOG^TCRxThy1CFP transgenic mice received the following: optic neuritis control (n=7), 100µl daily dose of vehicle (1:1 PBS and DMSO) (n=7) or 100μl 10mg/kg OXC (dissolved in 1:1 PBS and DMSO) (n=7). Mice were sacrificed on day 21. Eyes were flatmounted and RGC were counted using stereology software. Results represent mean ± SEM of RGC density. (B) Cross sections of the retina stained with total caspase-2 and (C) active caspase-2 and both were secondary labelled with Alex Fluor 568 (Red). RGC are shown in green (arrow), but degenerating RGC with lower CFP expression could be detected (dashed arrow). (D) RNFL thickness after ON induction and treatment with either nonsense or caspase-2 siRNA. (E) Quantification of RGC density after ON induction and treatment with either a nonsense (n=7) or caspase-2 siRNA (n=8). Plots show mean ± SEM of RGC density and RNFL thickness.</p