Mycoplasma Ocular Infection in Subretinal Graft Transplantation of iPS Cells-Derived Retinal Pigment Epithelial Cells

Abstract

PURPOSE. To report occurrence of acute severe inflammation after surgical implantation of mycoplasma-infected induced pluripotent stem cell-derived RPE (iPS-RPE) cells into the eyes of healthy primates, and determine the immunopathological mechanisms of the inflammation. METHODS.Ophthalmic allogeneic transplantation of iPS-RPE cells was performed in the subretina of major histocompatibility complex (MHC)-matched (two eyes) and MHCmismatched (one eye) healthy cynomolgus monkeys. The clinical course after transplantation was observed using color fundus photography, fluorescence angiography, and optical coherence tomography. After the animals were killed at 1 month after surgery, eyeballs were removed and pathologically examined. Microorganisms were analyzed by PCR methods and BLAST analysis using preserved graft iPS-RPE cells and the recipients' vitreous humor. Mixed lymphocyte-RPE assay was performed on the mycoplasma-infected and noninfected iPS-RPE cells in vitro. RESULTS.In tested eyes, abnormal findings were observed in the grafted retina 2 weeks after surgery. Here, we observed retinal vasculitis and hemorrhage, retinal detachment, and infiltration of inflammatory cells into the retina of the eyes. One month after surgery, animals were killed due to the severe immune responses observed. Using PCR methods, sequence analysis detected mycoplasma-DNA (Mycoplasma arginini species) in both the grafted RPE cells and the collected vitreous fluids of the monkeys. Mixed lymphocyte-RPE assay revealed that the infected iPS-RPE cells enhanced the proliferation of inflammatory cells in vitro. CONCLUSIONS.Transplantation of graft iPS-RPE cells contaminated with mycoplasma into the subretina caused severe ocular inflammation. Mycoplasma possesses the ability to cause immune responses in the host.

Distributed automated manufacturing of pluripotent stem cell products

“…3. This echoes recent observations made in the production of preclinical trials batches [28]. In order to mitigate this deviation, it is suggested that postprocess visual microscopic morphological comparison is performed, in this instance 'post-processing' refers to the completion of each process step, i.e.…”

A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models

“…First, we performed lymphocytes-grafts immune reaction (LGIR) tests [19][20][21][22], an in vitro system to predict immune rejection prior to transplantation by coculturing host immune cells and target explant cells. In the current study, for host immune cells, we used peripheral blood mononuclear cells (PBMC) from healthy monkeys (HM-1, -2, -3, and -4), and for target cells, we used human iPSC-RPE cells assuming iPSC-RPE transplantation.…”

“…In order to control immune attacks after RPE cell transplantation, we established an in vitro drug assay, termed "drug-lymphocytes-grafts immune reaction (Drug-LGIR)" tests in the current study. Previously, we established a similar test using peripheral blood lymphocytes of the recipient cocultured with iPSC-RPE cells of the explant to predict the immune rejections anticipated after RPE cell transplantation [19][20][21][22]. In the current study, we enhanced the test by applying commonly used medication for ocular inflammatory diseases in Japan.…”

“…The in vitro rejection assay LGIR tests were performed as previously described [19][20][21][22] using PBMC from healthy or transplanted monkeys as host immune cells (effector lymphocytes) and human iPSC-RPE cell lines as transplanted cells (target iPSC-RPE cells). They were cocultured for 5 days, and the responses of the effector lymphocytes against the target RPE cells in vitro was evaluated by Ki-67 FACS analysis.…”

“…They were cocultured for 5 days, and the responses of the effector lymphocytes against the target RPE cells in vitro was evaluated by Ki-67 FACS analysis. Procedure of Ki-67 staining and antibody information were described previously [19][20][21][22]. Briefly, PBMC cocultured with iPSC-RPE cells or allogenic B cells (positive control) were harvested, then fixed and permeabilized with 70% ethanol at −20 • C for 1 h, and then labeled with phycoerythrin (PE)-labeled anti-Ki-67 (BioLegend, CA, USA) together with either APC-labeled anti-CD4, APC-labeled anti-CD8a, APC-labeled anti-CD11b, FITC-labeled anti-CD20, or APC-labeled anti-NKG2A at room temperature for 40 min.…”