Christopher Laver

Efficient Production of Photoreceptor Precursor Cells from Human Embryonic Stem Cells.

Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However, existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs, we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs, using size-controlled embryoid bodies (EBs), and addition of triiodothyronine (T3) and taurine to the differentiation medium, with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation, which will help understand retinal diseases and facilitate large-scale preclinical trials.

Structural divergence of essential triad ribbon synapse proteins among placental mammals – Implications for preclinical trials in photoreceptor transplantation therapy

Abstract As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false‐positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans‐synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans‐synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1‐to‐1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin‐G (LG)‐like domain and on its N‐terminal domain – a putative region of trans‐synaptic interaction. Localization of structural divergence to the N‐terminus of each putative post‐translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin – this would be consistent with a recent counter‐paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N‐terminal fragment can still mediate the trans‐synaptic bridge, while the C‐terminal fragment may act as a diffusible trophic or “homing” factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals – alongside known inter‐mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date – use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution. Graphical abstract Figure. No caption available. HighlightsProteins essential to photoreceptor synaptogenesis diverge among placental mammals.Key triad ribbon synapse protein, pikachurin, is predicted to diverge heavily.Pikachurin selection patterns are consistent with post‐translational cleavage.Divergence localized to the N‐terminus of cleavage products indicates dual roles.Testing of human photoreceptor grafts in distantly related mammals is cautioned.

Use of magnetism to enhance cell transplantation success in regenerative medicine

An increasingly important need in regenerative medicine is the efficient in vivo targeting and tracking of cells. Locating cells using magnetism and directing their passage along magnetic fields is evolving as a particularly useful noncontact approach in solving these problems. With this in mind, in recent years, a new class of molecules – magnetic nanoparticles (MNPs) – has been developed [1]. MNPs are particles with magnetic cores surrounded by various biological polymers that can be internalized by cells through endocytosis [1]. MNPs can be classified as superparamagnetic, paramagnetic, ferimagnetic or ferromagnetic depending on their magnetic cores, and contain manganese, gadolinium or, most commonly, iron oxide [2]. Significant advances have been made in targeting therapeutic drugs using these molecules and now MNPs are being used to target cells [1,3]. Magnetic fields and MNPs are being used in cell therapies in a number of ways. In most cases, solid magnets are localized to the disease site and then MNP-labeled cells are injected either into the vasculature or sometimes intrathecally to accumulate in the region of the solid magnet. In preclinical studies, magnetic targeting has been successful in directing cells to damaged tissues in many organs, such as the spinal cord [4], the muscle microvasculature of an ischemic limb [5], arteriosclerosis within larger blood vessels [6], the dystrophic retina [3] and directing natural killer cells to tumors [7]. MNPs have also been used to augment biomaterial scaffolds used in tissue regeneration [8]. In addition, magnetism has been used in tissue manufacture in preparation for in vivo work. For example, magnetism has been used to build sheets of mesenchymal stem cells [9]. Currently, however, the most successful role for MNPs in cell therapies has been their use in imaging and tracking cells. In vivo, MNPs

Influence of Iron Oxide Nanoparticles on Innate and Genetically Modified Secretion Profiles of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have well-established paracrine effects that are proving to be therapeutically useful. This potential is based on the ability of MSCs to secrete a range of neuroprotective and anti-inflammatory molecules. Previous work in our laboratory has demonstrated that intravenous injection of MSCs, treated with superparamagnetic iron oxide nanoparticle fluidMAG-D resulted in enhanced levels of glial-derived neurotrophic factor, ciliary neurotrophic factor, hepatocyte growth factor and interleukin-10 in the dystrophic rat retina. In this present study we investigated whether the concentration of fluidMAG-D in cell culture media affects the secretion of these four molecules in vitro. In addition, we assessed the effect of fluidMAG-D concentration on retinoschisin secretion from genetically modified MSCs. ELISA-assayed secretion of these molecules was measured using escalating concentrations of fluidMAG-D which resulted in MSC iron loads of 0, 7, 120, or 274 pg iron oxide per cell respectively. Our results demonstrated glial-derived neurotrophic factor and hepatocyte growth factor secretion was significantly decreased but only at the 96 hours time-point whereas no statistically significant effect was seen with ciliary neurotrophic factor secretion. Whereas no effect was observed on culture media concentrations of retinoschisin with increasing iron oxide load, a statistically significant increase in cell lysate retinoschisin concentration (p=0.01) was observed suggesting that increasing fluidMAG-D concentration did increase retinoschisin production but this did not lead to greater secretion. We hypothesize that higher concentrations of iron-oxide nanoparticle fluidMAG-D have an effect on the innate ability of MSCs to secrete therapeutically useful molecules and also on secretion from genetically modified cells. Further work is required to verify these in vitro finding using in vivo model systems.

Bimodal in vivo imaging provides early assessment of stem-cell-based photoreceptor engraftment.

PurposeSubretinal transplantation of stem-cell-derived photoreceptor precursor cells (PPCs) is a promising and innovative approach to treating a range of blinding diseases. However, common barriers to efficient preclinical transplantation comes in the form of suboptimal graft architecture, limited graft survival, and immune-rejection, each of which cannot be assessed using conventional in vivo imaging (ie, rodent ophthalmoscopy). With the majority of PPCs reported to die within the first few weeks after transplantation, understanding the mechanisms of graft failure, and ultimately devising preventative methods, currently relies on lengthy end point histology. To address these limitations, we hypothesized that combining two imaging modalities, optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (fcSLO), could provide a more rapid and comprehensive view of PPC engraftment.MethodsHuman ESC-derived PPCs were transplanted into 15 retinal dystrophic rats that underwent bimodal imaging at 0, 8, and 15 days posttransplant.ResultsBimodal imaging provided serial detection of graft: placement, architecture, and survival; each undetectable under ophthalmoscopy. Bimodal imaging determined graft placement to be either: subretinal (n=7), choroidal (n=4), or vitreal (n=4) indicating neural retinal perforation. Graft architecture was highly variable at the time of transplantation, with notable redistribution over time, while complete, or near complete, graft loss was observed in the majority of recipients after day 8. Of particular importance was detection of vitreal aggregates overlying the graft—possibly an indicator of host-site inflammation and rejection.ConclusionEarly real-time feedback of engraftment has the potential to greatly increase efficiency of preclinical trials in cell-based retinal therapeutics.