Abstract With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of individual patients with defined factors, have unlimited potential in cell therapy and in modeling complex human diseases. Significant progress has been achieved to improve the safety of iPSCs and the reprogramming efficiency. To avoid the cancer risk and spontaneous reactivation of the reprogramming factors associated with the random integration of viral vectors into the genome, several approaches have been established to deliver the reprogramming factors into the somatic cells without inducing genetic modification. In addition, a panel of small molecule compounds, many of which targeting the epigenetic machinery, have been identified to increase the reprogramming efficiency. Despite these progresses, recent studies have identified genetic and epigenetic abnormalities of iPSCs as well as the immunogenicity of some cells derived from iPSCs. In addition, due to the oncogenic potential of the reprogramming factors and the reprogramming-induced DNA damage, the critical tumor suppressor pathways such as p53 and ARF are activated to act as the checkpoints that suppress induced pluripotency. The inactivation of these tumor suppression pathways even transiently during reprogramming processes could have significant adverse impact on the genome integrity. These safety concerns must be resolved to improve the feasibility of the clinic development of iPSCs into human cell therapy.

Purpose

Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).

Methods

We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of α(v)β₅ integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.

Results

Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.

Conclusions

Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.

The breakthrough of induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific iPSCs may become a renewable source of autologous cells for cell therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood. Using a humanized mouse model (denoted Hu-mice) reconstituted with a functional human immune system, we demonstrate that most teratomas formed by autologous integration-free hiPSCs exhibit local infiltration of antigen-specific T cells and associated tissue necrosis, indicating immune rejection of certain hiPSC-derived cells. In this context, autologous hiPSC-derived smooth muscle cells (SMCs) appear to be highly immunogenic, while autologous hiPSC-derived retinal pigment epithelial (RPE) cells are immune tolerated even in non-ocular locations. This differential immunogenicity is due in part to abnormal expression of immunogenic antigens in hiPSC-derived SMCs, but not in hiPSC-derived RPEs. These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.