Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells
Inventors
Bharti, Kapil • Chase, Lucas • Xuezhu, Feng • Jha, Balendu Shekhar
Assignees
Fujifilm Cellular Dynamics Inc • US Department of Health and Human Services
Publication Number
US-12065671-B2
Publication Date
2024-08-20
Expiration Date
2036-09-07
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Abstract
Provided herein are methods of producing an RPE cell population from a starting cell suspension, such as a single cell suspension, of pluripotent stem cells (PSCs). Such a method may comprise culturing the starting single cell suspension of PSCs in differentiation media to produce human RPE cells.
Core Innovation
The invention provides methods for producing retinal pigment epithelial (RPE) cell populations from a starting cell suspension of pluripotent stem cells (PSCs), specifically utilizing essentially single cell suspensions rather than embryoid bodies. These methods involve culturing dissociated human induced pluripotent stem cells (iPSCs) in defined differentiation media to induce stepwise differentiation into mature, functional RPE cells. The process includes culturing in retinal induction medium to initiate retinal lineage differentiation, further culturing in retinal differentiation medium, followed by retinal medium to form differentiating RPE cells, and finally culturing in RPE maturation medium to produce mature RPE cells.
The problem addressed by the invention stems from limitations of existing methods that rely on embryoid bodies for producing RPE cells from pluripotent stem cells. Such methods suffer from a lack of reproducibility, variability in efficiency, and poor scalability necessary for commercial scale production. The formation of embryoid bodies is itself not reproducible and inefficient, creating a bottleneck for the large-scale generation of clinical-grade RPE cells needed for therapeutic applications, screening assays, and retinal disease modeling.
The innovation therefore lies in bypassing the embryoid body formation step by starting with a single cell suspension of PSCs, providing a more reproducible, efficient, and scalable differentiation process. The methods utilize defined culture media compositions comprising specific pathway inhibitors and growth factors that facilitate retinal lineage commitment and RPE maturation. Additionally, methods include culturing on defined extracellular matrix components like recombinant human laminin, vitronectin, or fibronectin, culturing in feeder-free and xeno-free systems, and optionally cryopreserving the resultant RPE cells. Optional depletion of non-RPE contaminating cells using cell surface markers enhances purity and maturity of the produced RPE population.
Claims Coverage
The patent comprises 20 independent claims detailing methods of producing human retinal pigment epithelial (RPE) cells from dissociated human induced pluripotent stem cells (iPSCs). Key inventive features cover the starting cell preparation, defined culture conditions, media compositions, differentiation steps, and maturation protocols avoiding embryoid body formation.
Method of producing RPE cells from singly dissociated iPSCs without embryoid bodies
A method comprising obtaining a starting population of iPSCs dissociated to essentially single cells; culturing the iPSCs at an initial specified cell density in retinal induction medium to initiate differentiation; further culturing in retinal differentiation medium; culturing in retinal medium to form differentiating RPE cells; and culturing in RPE maturation medium, thereby producing RPE cells, explicitly excluding embryoid body formation.
Use of defined matrix substrates during iPSC culture
Culturing iPSCs on a matrix comprising at least one recombinant cellular adhesion protein such as laminin, vitronectin, or fibronectin, preferably human-derived, in retinal induction medium.
Defined media compositions with pathway inhibitors and growth factors
Retinal induction medium comprising WNT pathway inhibitors, BMP pathway inhibitors, TGFβ pathway inhibitors, and insulin growth factor 1 (IGF1); retinal differentiation medium comprising WNT pathway inhibitors, BMP pathway inhibitors, TGFβ pathway inhibitors, MEK inhibitors, and IGF1; and RPE maturation medium optionally containing primary cilium inducers such as prostaglandin E2 (PGE2) or aphidicolin.
Post-maturation dissociation and reseeding steps
Following differentiation and maturation, dissociating the RPE cells, reseeding the dissociated cells, and culturing further in RPE maturation medium to enhance maturity and functionality.
Culture conditions including feeder-free, fully-defined, and xeno-free media
Culturing iPSCs at initial cell densities between 1,000 to 40,000 cells/cm2; without feeder layers; in fully-defined and xeno-free culture media to enhance reproducibility and clinical compliance.
Cryopreservation of RPE cells
Optionally cryopreserving the differentiated RPE cells after maturation for storage and later use.
Selection of starting iPSC populations
Starting with pre-confluent iPSCs dissociated into single cells, with preferred seeding densities stated to optimize differentiation efficiency.
The claims collectively disclose a defined, reproducible method for producing clinical-grade mature RPE cells from single cell suspensions of human iPSCs using specific media formulations, culture conditions, and differentiation steps, all while avoiding embryoid body formation, thereby overcoming prior limitations related to reproducibility and scalability.
Stated Advantages
Provides a highly efficient and reproducible method for differentiating PSCs into mature retinal pigment epithelial cells without using embryoid bodies.
Enables scalable production suitable for commercial and clinical applications due to improved reproducibility and efficiency.
The use of defined, feeder-free, and xeno-free media and matrices enhances safety and consistency for clinical-grade cell therapy.
Incorporation of primary cilium inducers such as PGE2 improves RPE cell maturity, functionality, and purity.
MACS depletion of contaminating non-RPE cells improves purity of the final RPE cell population to near homogeneity.
Suitable for cryopreservation, facilitating storage and distribution for therapeutic use.
Applicable across different iPSC lines and operators with high reproducibility and consistency.
Documented Applications
Production of clinical-grade RPE cells for cell therapy to treat retinal degenerative diseases such as age-related macular degeneration, Best disease, Stargardt's macular dystrophy, retinitis pigmentosa, and other retinal disorders.
Screening for pharmaceutical compounds affecting RPE cell function, including toxicity and pharmacological testing.
Research applications including disease modeling and studying retinal pigment epithelial biology.
Transplantation therapy including autologous and MHC-matched allogeneic RPE grafts to enhance ocular tissue maintenance and repair.
Formulation of pharmaceutical compositions comprising purified RPE cells with scaffolds or matrices for transplantation.
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