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EP-4735585-A1 - ENHANCING NEURONAL DIFFERENTIATION OF NEURAL PROGENITOR CELLS

EP4735585A1EP 4735585 A1EP4735585 A1EP 4735585A1EP-4735585-A1

Abstract

The present invention relates to a method for directing differentiation of neural cells into neurons by obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014.

Inventors

  • NICLIS, Jonathan
  • DJORDJEVIC, Djordje
  • CASTIGLIONI, Veronica

Assignees

  • Novo Nordisk A/S

Dates

Publication Date
20260506
Application Date
20240628

Claims (15)

  1. 1. A method for directing differentiation of neural cells into neurons comprising obtaining a cell population comprising neural cells and contacting said cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF- 03084014.
  2. 2. The method according to the preceding claim, wherein the inhibitor of NOTCH signaling is LY411575.
  3. 3. The method according to any one of the preceding claims, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells.
  4. 4. The method according to any one of the preceding claims, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 35 days.
  5. 5. The method according to any one of the preceding claims, wherein the neural cells are specific to a region selected from forebrain, midbrain, and hindbrain/spinal cord.
  6. 6. The method according to the preceding claim, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the midbrain region, preferably ventral midbrain region, and wherein the cell population is not contacted with the inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells.
  7. 7. The method according to the preceding claim, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 18 to 35 days, and wherein the neural cells are differentiated into neural cells specific to the forebrain region, preferably dorsal forebrain region, and wherein the cell population is not contacted with the inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells.
  8. 8. The method according to any one of the preceding claims, wherein at least 70% of the cell population express SOX2 and wherein 5-35% of the cell population are INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling.
  9. 9. The method according to any one of the preceding claims, wherein the neural cells are specific to the ventral midbrain region, and wherein 20-75% of the cell population are ASCL1+ at the time of contacting the cell population with the inhibitor of NOTCH signaling.
  10. 10. The method according to any one of claims 1 to 7, wherein the neural cells are specific to the forebrain region, and wherein 5-40% of the cell population are TBR2+ at the time of contacting the cell population with the inhibitor of NOTCH signaling.
  11. 11. The method according to any one of the preceding claims, wherein the cell population is contacted with the inhibitor of NOTCH signaling from 12 hours to 2 days.
  12. 12. The method according to any one of the preceding claims, wherein the concentration of the inhibitor of NOTCH signaling is from 0.2 pM to 1000 pM.
  13. 13. The method according to any one of the preceding claims, wherein the cell population is contacted with the NOTCH inhibitor 48h prior to cryopreservation.
  14. 14. The method according to any of the preceding embodiments, wherein the cell population has a density of 0.5 x 10 6 cells/cm 2 - 2.0 x 10 6 cells/cm 2 at the time of contacting the cell population with the inhibitor of NOTCH signaling.
  15. 15. An in vitro cell population comprising neural cells obtained by the method according to any of the preceding claims or a composition comprising said in vitro cell population comprising neural cells, wherein said neural cells are specific to a region selected from forebrain, midbrain and hindbrain/spinal cord.

Description

ENHANCING NEURONAL DIFFERENTIATION OF NEURAL PROGENITOR CELLS TECHNICAL FIELD The present invention relates generally to the field of stem cells, such as human embryonic stem cells. Methods are provided for obtaining stem cell-derived neural cells. Specifically, methods are provided for directing differentiation of stem cell-derived neural cells into neurons of the forebrain, midbrain and hindbrain/spinal cord region. BACKGROUND The prospect of using human pluripotent stem cells (hPSCs) in the treatment of various conditions seems very promising. Treatments include cell-replacement therapy of neurological conditions such as Parkinson’s disease, stroke, paralysis, epilepsy and other such disorders. For such treatments to become viable, however, it requires the development of in vitro methods to artificially produce the stem cell-derived products for their delivery to the central nervous system (CNS). The differentiation of hPSCs into defined cell types is a difficult process to control; in many cases the progeny generated from in vitro protocols are heterogenous. Typically, when differentiating into neural cells, the mixture of cell types produced includes neurons (and within this a variety of neuronal subtypes such as glutamatergic and dopaminergic neurons), glia, neural stem cells (NSCs) as well as other non-neuronal cells (e.g., meningeal stromal cells). Such heterogenous cultures are suboptimal for disease modelling studies or many cell replacement therapies. Cellular replacement therapy for Parkinson’s disease is a prime example. In Parkinson’s disease, the A9 ventral midbrain dopaminergic neurons (vmDAs) are lost and arguably the sole cell type that must be transplanted to restore lost function. Presently, however, all academic scientific publications and all human clinical trials underway are transplanting a multipotent progenitor population that results in mixed populations of cells in vivo, in addition to vmDAs. This mixed population includes cell types such as proliferative NSCs, stromal cells, such as vascular leptomeningeal cells (VLMCs), and glial cells, such as astrocytes. Non-dopaminergic neurons do not restore function in Parkinson’s disease and some (i.e., serotonergic neurons) produce negative gain of function behaviors in patients as seen in clinical trials transplanting human fetal cells, and more generally these non-vmDAs carry unknown safety and efficacy risks. Consequently, there is a need to provide methods which ensure that patients are treated with ventral midbrain neurons or progenitors thereof. In the case of stroke or paralysis due to damage of the spinal cord, it is likely that only specific neuronal regional identity cell types and specific subtypes of neurons are required to restore lost function in both cases, while glial cells, stromal cells or other types of neurons are not necessary. As such for these indications it is also valuable to have maximal control over a differentiation process to have a refined cell product for maximal efficacy and safety. It is therefore an object of the present invention to overcome the aforementioned challenges, in particular to provide methods which can direct the differentiation of neural cells into neurons. SUMMARY The object as outlined above is achieved by the aspects of the present invention. In addition, the present invention may also solve further problems, which will be apparent from the disclosure of the exemplary embodiments. In a first aspect the present invention has provided a method comprising obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014, preferably LY411575. Specifically, the method is for directing differentiation of neural cells into neurons, meaning that the developmental fate of the neural cells is affected towards a certain outcome, i.e., neurons. However, according to the method the differentiation into neurons is not necessarily completed and the cells may not necessarily develop into the final fate during the method as disclosed herein. Preferably, the cells are directed towards differentiation towards neurons in vitro, only to be suitable for later administration into a patient wherein the further development in vivo results in a neuronal fate. The present inventors have found that the outcome of protocol for differentiating a cell population, such as human PSCs, towards or into neurons can be improved with the inhibition of the NOTCH signaling pathway using alternative compounds than usually used in the field of neural differentiation, namely the small molecule DAPT. The present invention has surprisingly found that the compound LY411575 demonstrates improved upregulation of neuronal genes and downregulation of NPC genes during the differentiation compared to DAPT. The present inventors have further identified the alternative c