Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells.

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.

Basic fibroblast growth factor support of human embryonic stem cell self-renewal.

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.