Search

JP-7856562-B2 - Methods for the production of Müller cells and cell products

JP7856562B2JP 7856562 B2JP7856562 B2JP 7856562B2JP-7856562-B2

Inventors

  • リム，グロリア アストリッド
  • カウ，ペン ティー
  • イーストレイク，カレン
  • マーレイ‐ダニング，セリア
  • ピント デ カルヴァーリョ，カーラ パトリシア

Assignees

  • ユーシーエル ビジネス リミテッド

Dates

Publication Date
20260511
Application Date
20201030
Priority Date
20191031

Claims (9)

  1. Müller cells derived from RC-9 human embryonic stem cells, (a) expressing detectable levels of CD29, vimentin, CD44, and nestin, and not expressing detectable levels of Tra-1-60, (b) Müller cells capable of secreting neurotrophins BDNF and PEDF.
  2. A purified, substantially homogeneous population of two or more Müller cells as described in claim 1.
  3. A method for producing therapeutic-grade human Müller cells, a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Adding synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culturing the cells for at least three days. d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 200 to 300 days until the retinal organoids are visible. e) A method comprising the step of dissociating the retinal organoids and isolating Müller cells.
  4. A pharmaceutical composition comprising Müller cells as described in claim 1, or a population of Müller cells as described in claim 2, or Müller cells that can be derived from claim 3, and a pharmaceutically acceptable carrier.
  5. (a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least three days. (d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2 to 300 days until the retinal organoids are visible. (e) A pharmaceutical composition comprising a population of Müller cells obtained from a method comprising the step of dissociating the retinal organoid and isolating the Müller cells, and a pharmaceutically acceptable carrier.
  6. A pharmaceutical composition according to claim 4 for treating a disease or condition of the retina, or a composition comprising Müller cells according to claim 1, a population of Müller cells according to claim 2, or Müller cells that can be derived from claim 3 for treating a disease or condition of the retina.
  7. A pharmaceutical composition for treating a disease or condition of the retina, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of Müller cells, the Müller cells being: (a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least three days. (d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2 to 300 days until the retinal organoids are visible. (e) A pharmaceutical composition obtained by a method comprising the step of dissociating the retinal organoid and isolating Müller cells.
  8. The pharmaceutical composition or composition according to claim 6, wherein the disease or condition of the retina is loss of vision, blindness, glaucoma, optic nerve injury, optic nerve degeneration, a disease causing damage or degeneration of the optic nerve, dominant ocular atrophy, Leber hereditary optic neuropathy, congenital amaurosis, optic neuritis, mitochondrial disorder causing optic nerve injury, ganglion cell disease, optic nerve cell disease, or ischemic optic neuropathy.
  9. The aforementioned glaucoma: (a) Primary glaucoma, including primary open-angle glaucoma, acute primary closed-angle glaucoma, chronic primary angle glaucoma, normal-tension glaucoma, childhood glaucoma, and juvenile glaucoma; or (b) Developmental glaucoma, such as Axenfeld anomaly, Rieger anomaly, Reiger syndrome, aniridia, and Peters anomaly, traumatic glaucoma, steroid-induced glaucoma, pseudo-epidermal exfoliative glaucoma, pigmentary glaucoma, uveitis glaucoma, neovascular glaucoma, mixed-mechanism glaucoma, iris-corneal endothelial syndrome, diseases causing optic nerve damage, Posner-Schlossman syndrome, juvenile chronic rheumatoid arthritis, and ankylosing spondylitis with secondary uveitis .

Description

This invention relates to a novel process for producing therapeutic GMP-grade Müller cells derived from stem cells using a product free of animal-derived components, and to Müller cells obtained therefrom. The Müller cells are suitable for the treatment of eye diseases, including glaucoma. Cell culture media are also provided. Glaucoma is the most frequent cause of irreversible blindness worldwide, and is estimated to affect approximately 111,000,000 people by 2040. Retinal ganglion cell (RGC) loss is a characteristic feature of ophthalmic neuropathy, including glaucoma, where damage to RGC axons occurs at the level of the optic nerve head (ONH). Under normal conditions, RGCs receive visual signals from photoreceptors through two anterior layers of neuronal cells (bipolar and non-axonal cells) and transmit this information to the brain through axons exiting the eyeball via the ONH and optic nerve. Damage to RGCs results from physical and molecular mechanisms, including mechanical compression, reduced paracrine neurotrophic factor support, glial activation, oxidative stress/reduction in the antioxidant defense system, immune system dysregulation, and mitochondrial dysfunction/metabolic deficiencies. These established mechanisms of RGC dysfunction, along with the evidence from the literature, indicate underlying multifactorial, metabolic defects that lead to the loss of RGC function and subsequent RGC degeneration and death in glaucoma. While currently approved treatments for glaucoma aim to slow disease progression, ultimately these treatments do not halt ongoing damage to the RGC, and therefore the disease continues to progress, with many patients losing sight in one or both eyes. Several studies highlight the importance and crucial role of Müller cells in providing functional and metabolic support to the retinal glycemic system (RGC). Under homeostatic conditions, Müller cells offer numerous beneficial functions, including: supplying nutrients and protection against the neurotoxic glutamate; ion and water homeostasis; buffering mechanical stimuli; structural stabilization of the retina; modulation of immune and inflammatory responses; antioxidant production; glucose metabolism; exhibiting considerable metabolic activity/support due to the presence of a significant number and large mitochondria; and promoting significant neuroprotective levels of adenosine triphosphate (ATP), all of which are disrupted in glaucoma. Based on the known role of Müller cells, Müller cell therapy is expected to promote the repair, survival, and function of RGCs, and therefore improve visual function for patients suffering from optic nerve damage. To evaluate this hypothesis, several studies were conducted in relevant glaucoma-like animal models using Müller cells generated from different sources, including human adult cadaveric donor retina (Singhal et al, Stem Cells Translational Medicine, 2012); cat donor sources (Becker et al, Stem Cells Translational Medicine, 2016); and human iPSC lineage (Eastlake et al, Stem Cells Translational Medicine, 2019). Each of these pharmacological studies demonstrated that single intravitreal administration of Müller cells could significantly improve RGC function. The generation of embryoid bodies and the formation of retinal organoids were initially based on Nakano's protocol. Nakano et al. (Cell Stem Cell 10, 771-785, June 14, 2012) found that fetal bovine serum (FBS) is an effective enhancer for retinal differentiation of stem cells; however, fetal bovine serum and other animal-based products are unacceptable in cell-based therapeutics administered to humans. Therefore, there is a global challenge to develop methods for culturing and differentiating Müller cells in accordance with Good Manufacturing Practice (GMP) standards for clinical application without negatively impacting cell scalability, yield, morphology, or biological properties. This invention provides an improved method for culturing GMP-compliant stem cell-derived Müller cells suitable for cell therapy, and Müller cells derived therefrom. Müller cells. A schematic diagram showing Müller cells with other retinal cell types. GCL = Ganglion cell layer. INL = Inner granular layer. ONL = Outer granular layer.Stage 2 of the cell culture process – neuroretinal differentiation. A schematic diagram illustrating the initial process for stem cell differentiation to generate mature retinal organoids. The diagram shows embryoid body formation, neural differentiation, and induction of maturation from stem cells.Stage 3 of the cell culture process – Dissociation and proliferation of Müller cells from retinal organoids. Schematic diagram illustrating the cell dissociation and proliferation process. Müller cells are isolated from retinal organoids. A single-cell suspension can be prepared using dissociation with a weak cell dissociation reagent (GCDR), followed by centrifugation and plate culture on a fibronectin-coated flask. Next, proliferation