Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format which enhanced generation of retinal organoids with retinal pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. © AlphaMed Press 2018

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine

<div><p>Introduction</p><p>The use of patients’ own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs.</p><p>Methods</p><p>MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists.</p><p>Results</p><p>We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs.</p><p>Conclusions</p><p>We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.</p></div

Treatment of lung cancer MEs with the TKI erlotinib based on their genomic profile.

<p>Only the effusion with the activating <i>EGFR</i> gene mutation (A) responded to treatment with the tyrosine kinase inhibitor erlotinib, but not the effusion with wild-type <i>EGFR</i> (B).</p

Determining tumor cell content in cultured malignant effusions.

<p>A-C. Second passage (P2) of a malignant effusion of a metastatic ovarian carcinoma. BerEP4 (B) and Calretinin (C) were used as epithelial and mesothelial markers, respectively. In this case, the number of mesothelial cells in passage 2 was very low (1–10%) (This case corresponds to the light blue line in the histogram of 2G, Ovarian Ca.). D-E. First passage (P1) of a malignant pleural effusion of an adenocarcinoma of the lung. TTF1 (nuclear) was used as specific marker for epithelial cells. F. In most of the effusions, relative tumor cell content decreased during the first passages in culture. Y-axis denotes relative tumor content as determined by the pathologist. G. Example of the tumor cell decrease in a malignant effusion from a patient with lung adenocarcinoma. In P0 and P1 most of the cells were from a pulmonary adenocarcinoma, as shown by TTF1 nuclear positivity (small box). In contrast, cells in P2 were mostly of non-epithelial origin (no specific nuclear TTF1 staining). Of note, mesothelial cells can change their morphology in culture and show pronounced atypia, and may be mistaken for tumor cells (green arrowhead). H. Substantial increase in tumor cells (EpCAM positive) and decrease in mesothelial cells (Calretinin positive) after enrichment with EpCAM antibody coated magnetic beads (MACS). Red arrowheads point towards epithelial tumor cells, green arrowheads towards mesothelial (non-neoplastic) effusion cells.</p

Treatment of effusions with cytotoxic agents.

<p>Treatment of a malignant pleural effusion from pulmonary adenocarcinoma with different concentrations of cisplatin (A), pemetrexed (B) and a combination thereof (C).</p

Measurement of the growth inhibitory effect of the TKI crizotinib in real-time.

<p>A. H522 cells, which are wild-type for ALK do not respond to crizotinib treatment. B. The ALK rearranged H3122 lung cancer cells show high sensitivity for even low doses of crizotinib. Arrow head points towards time point used for normalization.</p

Discovery of Risdiplam, a Selective Survival of Motor Neuron‑2 (<i>SMN2</i>) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA)

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound <b>2</b> was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of <b>1</b> (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA

Discovery of Risdiplam, a Selective Survival of Motor Neuron‑2 (<i>SMN2</i>) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA)

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound <b>2</b> was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of <b>1</b> (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA