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US-12618045-B2 - Automated method for preparing retinal pigment epithelium cells

US12618045B2US 12618045 B2US12618045 B2US 12618045B2US-12618045-B2

Abstract

Disclosed are methods for preparing retinal pigment epithelium (RPE) cells from pluripotent stem cells (PSCs). More particularly, it represents an automated method that combines in a sequential manner three differentiating agents to direct the differentiation of human PSCs into RPE cells.

Inventors

  • Christelle Monville
  • Florian REGENT
  • Lise MORIZUR
  • Karim Ben M′Barek

Assignees

  • CENTRE D'ETUDE DES CELLULES SOUCHES (CECS)

Dates

Publication Date
20260505
Application Date
20200619
Priority Date
20190621

Claims (14)

  1. 1 . An automated method for large-scale production of retinal pigment epithelium (RPE) cells and for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells, wherein the method comprises the use of only three differentiating agents to direct the differentiation of human pluripotent stem cells into RPE cells in the sequential steps of: (a) culturing human pluripotent stem cells in a medium supplemented with a first differentiating agent consisting of one Nicotinamide (NA) mimetic compound to generate differentiating cells, for at least 3 days; (b) culturing said differentiating cells obtained in step a) in a medium supplemented with a second differentiating agent, consisting of one member of transforming growth factor β (TGF β) superfamily to further differentiating said differentiating cells, for at least 3 days; (c) culturing said further differentiating cells obtained in step b) in a medium supplemented with a third differentiating agent, consisting of one activator of the Wnt canonical pathway to induce said further differentiating cells to differentiate into RPE cells, for 20 to 50 days, wherein the Nicotinamide (NA) mimetic compound is Nicotimamide, wherein the one member of transforming growth factor β (TGF β) superfamily is selected from the group consisting of the TGFβ subfamily, Activin, Nodal, growth and differentiation factors (GDF), bone morphogenetic protein (BMP), and antiMullerian hormone (AMH), wherein the one activator of the Wnt canonical pathway is a GSK-3 inhibitor selected from the group consisting of 3F8, 1-Azakenpaullone, 10Z-Humenialdisine, Alsterpaullone, A-1070722, AR-A014418, AZD1080, AZD2858, Bikinin, BIO, Cazpaullone, CT98014, CT98023, CT99021 (Chir99021), Chir98014, Dibromocantharelline, GSKJ2, HMK-32, Hymenialdesine, Indirubin, Indirubin-3′-oxime, IM-12, Kenpaullone, L803, L803-mts, Lithium carbonate, LY2090314, Manzamine A, Meridianin, NSC693868, NP031115, Palinurine, SB216763, SB415286, TCS21311, TC-G-24, TCS2002, TDZD-8, Tideglusib, Tricantine and TWS119, and wherein the automated method uses an apparatus for large-scale production of cells comprising: a) robotic means for handling culture vessels; b) means for inoculating cells into a culture; c) means for changing or adding medium to a culture; and d) programmable control means.
  2. 2 . The automated method of claim 1 , wherein the medium in step (b) is free of the Nicotinamide (NA) used in step a).
  3. 3 . The automated method of claim 1 , wherein the medium in step (c) is free of the Nicotinamide (NA) and the one member of transforming growth factor β (TGF β) superfamily respectively used in steps a) and b).
  4. 4 . The automated method of claim 1 , wherein the method comprises the sequential steps of: (a) culturing human pluripotent stem cells in a medium supplemented with Nicotinamide to generate differentiating cells; (b) culturing said differentiating cells obtained in step a) in a medium supplemented with Activin A to further differentiating said differentiating cells; (c) culturing said further differentiating cells obtained in step b) in a medium supplemented with CHIR99021 to induce said further differentiating cells to differentiate into RPE cells.
  5. 5 . The automated method of claim 1 , wherein the method further comprises the step of: (d) treating the population of cells obtained in step c) to remove the non-adherent cells.
  6. 6 . The automated method of claim 5 , wherein the step d) is a two-step dissociation procedure comprising or consisting of washing and treating the cells enzymatically.
  7. 7 . The automated method of claim 1 , wherein the method further comprises the step of (e) expanding the cells obtained in step d) over at least two passages.
  8. 8 . The automated method of claim 7 , wherein the passages comprises (i) dissociating the RPE cells and/or the differentiating cells in a first vessel to form a suspension; (ii) transferring the RPE cells and/or the differentiating cells to at least two further culture vessels; and (iii) culturing the RPE cells and/or the differentiating cells until the RPE cells and/or the differentiating cells are 50 to 100% confluent, wherein the passages does not comprise a centrifugation step.
  9. 9 . The automated method of claim 1 , wherein the method uses a) robotic means for handling culture vessels; b) means for inoculating cells into a culture; c) means for changing or adding medium to a culture; and d) programmable control means; wherein the apparatus is adapted to the phase of directed differentiation of hPSCs toward RPE cells and the phase of passage the cells when they reach a predetermined percentage confluence.
  10. 10 . The automated method of claim 1 , wherein culturing said human pluripotent stem cells in a medium supplemented with Nicotinamide (NA) is carried out for 7 days.
  11. 11 . The automated method of claim 1 , wherein culturing said differentiating cells obtained in step a) in a medium supplemented with the one member of transforming growth factor β (TGF β) superfamily is carried out for 3 to 10 days.
  12. 12 . The automated method of claim 1 , wherein culturing said differentiating cells obtained in step a) in a medium supplemented with the one member of transforming growth factor β (TGF β) superfamily is carried out for 7 days.
  13. 13 . The automated method of claim 1 , wherein culturing said human pluripotent stem cells in a medium supplemented with Nicotinamide (NA) is carried out for 7 days, wherein culturing said differentiating cells obtained in step a) in a medium supplemented with Activin A is carried out for 7 days, wherein culturing said differentiating cells obtained in step b) in a medium supplemented with Chir99021 is carried out for 21-28 days.
  14. 14 . The automated method of claim 1 , wherein the method comprises the sequential steps of: (a) culturing human pluripotent stem cells in a medium supplemented with Nicotinamide is carried out for 7 days, wherein the amount of Nicotinamide is 10 mM, (b) culturing said differentiating cells obtained in step a) in a medium supplemented with Activin A is carried out for 7 days, wherein the amount of Activin A is 10 ng/ml, (c) culturing said further differentiating cells obtained in step b) in a medium supplemented with CHIR99021 is carried out for 21-28 days, wherein the amount of Chir99021 is 10 ng/ml.

Description

This application is the U.S. national phase of International Application No. PCT/EP2020/067177 filed 19 Jun. 2020, which designated the U.S. and claims priority to EP patent application Ser. No. 19/305,807.0 filed 21 Jun. 2019, the entire contents of each of which are hereby incorporated by reference. FIELD OF THE INVENTION The present invention relates to methods for preparing retinal pigment epithelium (RPE) cells from pluripotent stem cells (PSCs). More particularly, the present invention relates to an automated method that combines in a sequential manner three differentiating agents to direct the differentiation of human PSCs into RPE cells. BACKGROUND OF THE INVENTION The retinal pigment epithelium (RPE) is a monolayer of pigmented cells localized between the neuroretina and the choroids. The RPE cells play roles in the maintenance and function of the retina and its photoreceptors. These include the formation of the blood-retinal barrier, absorption of light and protection against photooxidation, transport of nutrients to the neural retina, regeneration of visual pigment, and phagocytosis of shed photoreceptor membranes. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and their ability to differentiate into any cell type. Due to these properties, extensive efforts have been done to use them as a source material for cell therapy to repair damaged tissues. At the forefront of cell therapy, the replacement of the retinal pigment epithelium, acts as a proof of concept. RPE cells play crucial roles in sight and their dysfunction or their loss may engender the secondary loss of photoreceptors. RPE cells are altered in 5-6% of Retinitis Pigmentosa cases and in Age-related Macular Degeneration (AMD). AMD is the leading cause of blindness in developed countries with more than 150 million people affected worldwide, a figure that will increase in the coming years. It can be classified into two groups, dry (atrophic) or wet (exudative), which is based on the presence of a choroidal neovascularization. There is still no treatment for dry AMD and for most of RPs. As such, the transplantation of RPE cells derived from human pluripotent stem cells (hPSC-RPE) represents an attractive strategy for treating retinal degenerative diseases. hPSCs spontaneously differentiate into RPE cells after removal of basic fibroblast growth factor (bFGF), used to maintain the pluripotency state, from the culture medium. The distinctive cobblestone morphology of RPE cells as well as their pigmentation allow to manually collect pigmented areas that appear upon differentiation of hPSCs to obtain a pure population of hPSC-RPE cells. Such approach of RPE cell production is used as cell replacement material in on going and planned clinical trials. However, this spontaneous method remains fastidious, inefficient and time consuming (8 to 12 weeks of hPSCs differentiation) making it incompatible with the industrial large-scale production which is required to treat the potential millions of patients. During the last ten years, several teams have developed improved differentiation protocols by combining the use of an increasing number of cytokines and small molecules selected on the basis of results obtained from developmental studies. One of the quickest and most efficient protocol was published in the publication “Canonical/β-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 56, 1002-1013 (2015). Following data demonstrating that RPE and neural retina progenitors (NRPs) have the same embryonic origin, they combined a protocol allowing the efficient differentiation of NRPs with previously described RPE inducing factors such as Nicotinamide, Activin A, in addition to many others including bFGF, Noggin, DKK1 (Dickkopf WNT signaling pathway inhibitor 1), Insulin Growth Factor (IGF)-1. The previous protocol was modified to include Chir99021 and SU5402 from day 8 to 14. Using this method, they obtained cells expressing the pigmentation marker PMEL17 after 14 days of differentiation allowing bypassing manual enrichment of pigmented cells. Background art also includes WO2017021973 and WO2008129554 which disclose two-step methods of generating retinal pigment epithelial (RPE) cells comprising culturing a population of human pluripotent stem cells in the presence of Nicotinamide; and further subjecting the cells to another stage of differentiation in the presence of activin A, with or without Nicotinamide. Although the differentiation of hPSCs into RPE cells became more efficient during the last years, it still remains a long and laborious process requiring meticulous manipulations from hPSCs thawing to hPSCRPE cell banking. Many cell culture parameters, such as seeding homogeneity, the time spent by the cells out of the incubator or th