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CN-121986159-A - Neural differentiation of pluripotent cells

CN121986159ACN 121986159 ACN121986159 ACN 121986159ACN-121986159-A

Abstract

The present invention relates to the field of cell biology, and more particularly to the field of cell culture. The present invention thus relates to a method for differentiating pluripotent cells into neural cells, which is suitable for and optimized for three-dimensional cell culture. The nerve cells thus obtained can be used in cell therapies for the treatment of neurodegenerative diseases such as parkinson's disease.

Inventors

  • Maxim Feyo
  • Nicholas Prud'hon
  • Lucia Cordero Espinosa

Assignees

  • 树蛙疗法公司

Dates

Publication Date
20260505
Application Date
20240721
Priority Date
20230721

Claims (14)

  1. 1. An in vitro method for differentiating pluripotent cells into neural cells carried out in a suitable medium by at least one three-dimensional cellular micro-compartment comprising said pluripotent cells, comprising at least one step of exposing said pluripotent cells to: at least two SMAD inhibitors, which are, At least one SHH activator and at least one of the at least one SHH activators, -At least one FGF activator, and -At least one Wnt activator, and-at least one Wnt activator, Wherein the concentration of at least one SMAD inhibitor in the culture medium is increased in a continuous or discontinuous manner by at least 30% relative to the initial concentration of the SMAD inhibitor for at least 3 days from initial exposure of the pluripotent cells to the SMAD inhibitor to obtain neural cells expressing at least FOXA 2.
  2. 2. The method of the preceding claim, wherein the concentration of the at least one SMAD inhibitor increases exponentially, preferably the concentration of at least two SMAD inhibitors increases exponentially.
  3. 3. The method of one of the preceding claims, wherein the concentration of the at least one SMAD inhibitor increases up to 4 days after initial exposure of the pluripotent cells to the SMAD inhibitor.
  4. 4. The method of the preceding claim, wherein the concentration of first SMAD inhibitor (i) increases by at least 40% and the concentration of second SMAD inhibitor (ii) increases by at least 900% 1 day after initial exposure of the pluripotent cells to the SMAD inhibitor.
  5. 5. The method of the preceding claim, wherein the concentration of the first SMAD inhibitor (i) increases by at least 67% and the concentration of the second SMAD inhibitor (ii) increases by at least 9,900% 2 days after the initial exposure of the pluripotent cells to the SMAD inhibitor.
  6. 6. The method of one of the preceding claims, wherein the first SMAD inhibitor (i) is capable of acting on BMP-2,4,7 pathway and the second SMAD inhibitor (ii) is capable of acting on tgfβ/activin/Nodal pathway.
  7. 7. The method of the preceding claim, wherein the first SMAD inhibitor (i) is selected from Noggin factor, LDN193189, dorsomorphin, DMH1, a-83-01, and combinations thereof, and the second SMAD inhibitor (ii) is selected from SB431542, SB505124, LY2157299, LY550410, and combinations thereof.
  8. 8. The method of the preceding claim, wherein the Noggin factor is at a concentration of 50ng/mL to 70ng/mL upon initial exposure.
  9. 9. A method according to one of claims 7 or 8, wherein the factor SB431542 is at a concentration of 0.1 μm to 0.3 μm at initial exposure.
  10. 10. The method according to one of the preceding claims, wherein A. At least 3 days after initial exposure of the pluripotent cells to the at least one SMAD inhibitor, adding the at least one SHH activator, and/or B. At least 3 days after initial exposure of the pluripotent cells to the at least one SMAD inhibitor, adding the at least one FGF activator, and/or C. The at least one Wnt activator is added at least 6 days after the pluripotent cells are initially exposed to the at least one SMAD inhibitor.
  11. 11. The method according to one of the preceding claims, wherein the pluripotent cells are in isolated form or in aggregate form.
  12. 12. The method according to one of the preceding claims, wherein the pluripotent cells are induced pluripotent stem cells (ipscs).
  13. 13. The method of one of the preceding claims, further comprising exposing the obtained neural cells to at least one factor selected from the group consisting of rhBDNF, L-ascorbic acid, rhGDNF, rhTGF- β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin a, and combinations thereof, to obtain neural cells expressing at least the FOXA2/OTX2 marker.
  14. 14. The method of the preceding claim, wherein the method comprises an additional intermediate step for at least 1 day during which the medium is free of TGF- β pathway inhibitors when the micro-compartments comprise at least 50% of nerve cells expressing at least the FOXA2/OTX2 marker.

Description

Neural differentiation of pluripotent cells Technical Field The present invention relates to the field of cell biology, and more particularly to the field of cell culture. The present invention thus relates to a method for differentiating pluripotent cells into neural cells, which is suitable for and optimized for three-dimensional cell culture. The various neural cells obtained, in particular in the form of micro-tissues, can be used in particular in cell therapies for the treatment of neurodegenerative diseases (such as parkinson's disease). Background Pluripotent embryonic stem cells (ES cells) have proliferative and differentiation properties that make them a promising tool in cell therapies, but this involves many ethical issues. In fact, these ES cells are mainly obtained from the tissue of aborted fetuses. Therefore, their clinical use is not acceptable, which is why many countries prohibit their use for clinical purposes. Professor mountain extension (prof. Yamanaka) discovered in 2006 that induced pluripotent stem cells (iPS or iPSC) injected new power into this field, so that most of the ethical problems associated with the use of ES cells were solved. Clinical trials are rapidly initiated with the aim of treating diseases that are incurable by traditional medicine, such as neurodegenerative diseases. Transplanting neural cells (preferably in the form of micro-tissue) obtained from ipscs offers promise for developing new curative treatments. For example, according to World Health Organization (WHO) data, over 850 tens of thousands of people worldwide suffer from parkinson's disease and the rate of case growth is faster than any other neurological disorder. However, as the world population ages, the number of people suffering from parkinson's disease is expected to increase by a factor of two between 2005 and 2030, and therefore, the cost to society for this will continue to rise worldwide in the absence of curative treatment. In fact, to date, no curative treatment has been developed for treating these diseases, and only treatments which at best delay progression and have poor efficacy have been developed. For Parkinson's disease, mention may be made of L-Dopa (L-Dopa) and dopaminergic agonists. Recent findings and new technologies in the field of cell culture are now seen as the most promising solutions for developing curative treatments, in contrast to conventional drugs which are not only not radically curable but also cause strong and undesirable side effects. In this case, the progress made in the field of cell culture of pluripotent cells for tissue regeneration brings real hopes to repair damaged neuronal tissue and thus maintain or even restore neuronal activity. Methods for differentiating pluripotent cells into two-dimensional or three-dimensional neural cells are known. However, these methods have disadvantages in that cell death rate is extremely high or differentiation yield is extremely low, respectively. By way of example, mention may be made of the differentiation methods disclosed in application US 2017/013099. Although these methods are suitable for three-dimensional cell culture, they require perfect optimization to overcome the above-mentioned drawbacks, i.e., reduction of cell death rate and increase of yield and differentiation rate of neural cells, and increase of transplantation efficiency. Indeed, it is well known in the art that the vulnerability of nerve cells (particularly mature cells such as dopaminergic neurons) requires extreme caution in the clinical setting. In addition, the simple move of the two-dimensional differentiation method into the three-dimensional system overcomes the problem of high cell death rate, but sacrifices differentiation yield. On the other hand, the known two-dimensional methods provide good differentiation yields, but at the cost of cell death. Thus, there is no known differentiation method specific for three-dimensional cell culture that overcomes all of these drawbacks, i.e., reduces cell death rate, reduces the risk of graft death as much as possible, and provides better differentiation yields than currently known two-dimensional cell cultures. Thus, there is a need for a new method for differentiating pluripotent cells into neural cells, in particular a method suitable for three-dimensional cell culture and optimizing it, which avoids the drawbacks of the two-dimensional (2D) differentiation methods known in the background art, namely low yield, high cell death rate and low differentiation rate. Disclosure of Invention In addition, to meet this need, the present invention proposes a novel method of differentiating pluripotent cells into neural cells by at least one three-dimensional cellular compartment, wherein the step of exposing said pluripotent cells to a mixture of several factors known to be involved in differentiation of pluripotent cells into neural cells, i.e. at least one SMAD inhibitor, at least one SHH activator, FGF activat