Exploiting induced pluripotent stem cell-derived retinal pigment epithelium to unravel host-pathogen interaction in ocular tuberculosis: a reverse translational in vitro model

Abstract

Mycobacterium tuberculosis (Mtb) can infect the retinal pigment epithelium (RPE) cells. Current in vitro research models for ocular tuberculosis (OTB) only rely on RPE cell culture approaches. Until now it remains unclear why only a minority of patients with active systemic tuberculosis (TB) develops concurrent OTB. There is significant variation in the clinical manifestations of OTB, which is potentially influenced by ethnic differences and diversity in mycobacterial strains. To better understand the immunopathobiology of OTB, particularly an individual's susceptibility to Mtb-infection and the specific host response, cell culture systems utilizing induced pluripotent stem cells (iPSC)-derived RPE cells offer a promising in vitro model to better mimic the disease. With this technology, RPE cells can be generated from specific patients of interest, enabling to test hypotheses in a bench to bedside or reverse manner. In this current study, we explore the utility of iPSC-derived RPE cells as an in vitro model for OTB. Such an approach would overcome drawbacks associated with the currently commonly used "general" RPE cell lines as disease model. The application of iPSC-derived RPE cells offers promising options for the identification of novel biomarkers and to study individualized drug screening methods for host-directed therapy of OTB, in order to restore and maintain vision in OTB patients with sight-threatening disease.

Keywords: induced pluripotent stem cells; personalized medicine; retinal pigment epithelium; tuberculosis; uveitis.

Figure 1

Generation of iPSC-derived RPE and its utilization as in vitro OTB model. (A) Schematic of iPSC-derived RPE generation using chetomin (CTM) and nicotinamide (NIC) treatment during the differentiation protocol, (B) Bright field microscopy images (10x magnification) of OZR1, ARPE-19, and iPSC-derived RPE cells at different time points, (C) Flow-cytometric analysis of the expression of RPE markers (RPE-65 and PMEL17) in iPSC-derived RPE compared to OZR1 cells, ARPE-19 cells, and undifferentiated iPSCs. iPSC-derived RPE cells (passage 2), ARPE-19 cells, and OZR1 cells were cultured for 21 days. Fraction (%) of viable cells within the sample for each marker were presented. Thresholds were determined from unstained controls. Data were generated from a single experiment, (D) fluorescence microscope images with similar 40x magnification in all slides of iPSC-derived RPE (passage 2), ARPE-19, and OZR1 cells immunostained for ZO-1 (t = 21 days), (E) Microscopy image of iPSC-derived RPE and OZR1 cells at 48 hours after infection with live H37Rv Mtb (AFB Kinyoun staining: 20x magnification (left) and 63x magnification (right)), (F) Bar graphs represent the level of proteins measured in the culture supernatants of Mtb-infected (strain H37Rv, red bars) versus non-infected controls (white bars) iPSC-derived RPE and OZR1 cells (t = 48 hours). Data were generated from two independent experiments.

Figure 2

Potential application of iPSC-derived cells (e.g., RPE) as a reverse translational in vitro model in OTB. Testable hypotheses arising from questions in clinical care can be explored using this in vitro concept. iPSC-derived cells can be sourced from patients/individuals with diverse characteristics, such as different clinical phenotypes, ethnic backgrounds, or disease manifestations (e.g., active pulmonary TB with and without OTB). These cells can then be expanded to create a “village on a dish”. High-throughput analysis can be employed to characterize the disease, identify novel biomarkers, or discover therapeutic targets. This technology can be utilized to perform “clinical trials in a dish.” Clinical-grade of patient-derived cells (autologous or allogeneic) also hold promise as candidates for cellular therapy in restoring damaged ocular structure due to OTB. (Figure created with Biorender.com).