Neural differentiation of pluripotent cells

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of cell biology, and more particularly to the field of cell culture. Therefore, this invention relates to a method for differentiating pluripotent cells into neural cells, a method suitable for and optimized for three-dimensional cell culture. The resulting variety of neural cells, particularly neural cells in microtissue form, can be used specifically in cell therapy for treating neurodegenerative diseases such as Parkinson's disease. Background Technology

[0002] Pluripotent embryonic stem cells (ES cells) possess proliferative and differentiation properties, making them a promising tool in cell therapy, but this raises numerous ethical concerns. In reality, these ES cells are primarily obtained from tissues of aborted fetuses. Therefore, their clinical application is difficult to accept, which is why many countries prohibit their use for clinical purposes.

[0003] Professor Shinya Yamanaka's discovery of induced pluripotent stem cells (iPSCs) in 2006 injected new momentum into the field, resolving most of the ethical issues associated with the use of ES cells. Clinical trials were quickly initiated, aiming to treat diseases incurable by conventional medicine, such as neurodegenerative diseases. Transplantation of nerve cells obtained from iPSCs (preferably in the form of micro-tissues) has offered promise for developing new curative treatments.

[0004] For example, according to the World Health Organization (WHO), more than 8.5 million people worldwide suffer from Parkinson's disease, and the number of cases is growing faster than any other neurological disorder. However, with the world's population aging, the number of people with Parkinson's disease is projected to triple between 2005 and 2030, and therefore, in the absence of a cure, the societal costs will continue to rise globally.

[0005] In fact, to date, no curative treatment has been developed for these diseases; only treatments that at best slow disease progression and have inconsistent efficacy have been developed. For Parkinson's disease, levodopa (L-Dopa) and dopaminergic agonists can be mentioned.

[0006] Recent discoveries and technologies in the field of cell culture appear to be the most promising solutions for developing curative treatments, in contrast to conventional drugs which not only fail to cure the disease but also cause strong and undesirable side effects.

[0007] In this context, advances in the field of pluripotent cell culture for tissue regeneration offer genuine hope for repairing damaged neuronal tissue and thus maintaining or even restoring neuronal activity. Methods for differentiating pluripotent cells into two-dimensional or three-dimensional neural cells are known. However, these methods each suffer from drawbacks, namely, extremely high cell death rates or extremely low differentiation yields. The differentiation method disclosed in application US 2017 / 0130199 can be cited as an example.

[0008] While these methods are applicable to three-dimensional cell culture, they require perfect optimization to overcome the aforementioned drawbacks: reducing cell death rate and increasing neuronal yield and differentiation rate, as well as improving transplantation efficiency. Indeed, it is well-known in the prior art that the fragility of neuronal cells (especially mature cells such as dopaminergic neurons) requires extreme caution in clinical settings. Furthermore, simply transplanting two-dimensional differentiation methods into three-dimensional systems, while overcoming the high cell death rate, sacrifices differentiation yield. On the other hand, known two-dimensional methods offer good differentiation yields, but at the cost of cell death.

[0009] Therefore, there is currently no known differentiation method specifically for three-dimensional cell culture that can overcome all these drawbacks, namely, reducing cell death rate, minimizing the risk of graft death, and providing better differentiation yield than currently known two-dimensional cell culture.

[0010] Therefore, there is a need for a new method for differentiating pluripotent cells into neural cells, particularly a method suitable for and optimized for three-dimensional cell culture, which should avoid the disadvantages of known two-dimensional (2D) differentiation methods in the background art, namely, low yield, high cell death rate and low differentiation rate. Summary of the Invention

[0011] Furthermore, to meet this need, the present invention proposes a novel method for differentiating pluripotent cells into neural cells through at least one three-dimensional cellular microcompartment, wherein the step of exposing the pluripotent cells to a mixture of several factors known to participate in the differentiation of pluripotent cells into neural cells is carried out, namely, at least one SMAD inhibitor, at least one SHH activator, FGF activator, and Wnt activator.

[0012] Although the use of these factors alone or in combination is known in the differentiation methods of the prior art, the present invention overcomes the above-mentioned disadvantages by regulating the kinetics of cell exposure to said factors in three-dimensional cellular microcompartments.

[0013] In fact, the inventors have observed that, prior to adding other differentiation factors to cells and thus exposing them to other differentiation factors, specifically exposing pluripotent cells to a continuously increasing SMAD-type factor for a defined period of time can improve yield, differentiation rate (particularly the proportion of dopaminergic neurons in the resulting mixture containing multiple nerve cells), reduce cell death rate, and improve the transplantation success rate of multiple nerve cells obtained by the method according to the invention.

[0014] In contrast to the teachings of the prior art which aim to expose pluripotent cells to all factors of interest (especially SMAD factors) at a given concentration and for a given time, the inventors have discovered that increasing the concentration of at least one SMAD inhibitor in the culture medium in a continuous or discontinuous manner for at least 3 days from the initial exposure of pluripotent cells to the SMAD inhibitor can induce a reduction in the amount of neural stem cells (reduction of PAX6+ and / or SOX1+ markers) in the neural cell mixture obtained by the method according to the invention, and an increase in the amount of cells involved in differentiation (such as dopaminergic progenitor cells (FOXA2 and / or OTX2) in the obtained mixture), thereby overcoming the aforementioned disadvantages.

[0015] Therefore, the present invention relates to an in vitro method for differentiating pluripotent cells into neural cells by means of at least one three-dimensional cellular microcompartment in a suitable culture medium, the microcompartment containing the pluripotent cells, the method comprising at least one step of exposing the pluripotent cells to a substance: -At least two SMAD inhibitors, -At least one SHH activator, - at least one FGF activator, and -At least one Wnt activator, The method involves increasing the concentration of at least one SMAD inhibitor in the culture medium, either continuously or discontinuously, for at least 3 days from the initial exposure of pluripotent cells to the SMAD inhibitor, by at least 20%, preferably at least 30%, more preferably at least 40%, relative to the initial concentration of the SMAD inhibitor, to obtain a variety of neural cells, wherein at least 30%, preferably at least 40%, of the cells express at least FOXA2 and / or OTX2 markers. Therefore, at the end of the method, the resulting mixture of various neural cells contains a higher amount of FOXA2 marker-positive cells and a lower amount of PAX6 and / or SOX1 marker-positive cells than the prior art 3D culture method.

[0016] According to a preferred embodiment of the invention, the at least one SMAD inhibitor is increased exponentially, either continuously or discontinuously. Furthermore, the concentration of the at least one SMAD inhibitor preferably increases exponentially.

[0017] Advantageously, the method according to the invention aims to expose pluripotent cells to at least two SMAD inhibitors, wherein the concentrations of the two SMAD inhibitors increase exponentially or logarithmically relative to the initial exposure of the pluripotent cells to the SMAD inhibitors.

[0018] According to another preferred purpose, after pluripotent cells are initially exposed to the SMAD inhibitor, more preferably two SMAD inhibitors, the concentration of the at least one SMAD inhibitor increases for at least 1 day, at least 3 days, and at most 4 days, preferably at most 5 days.

[0019] According to another particularly advantageous purpose, the method according to the invention exposes pluripotent cells to two SMAD inhibitors, each of which acts on a different cell signaling pathway. Thus, the first SMAD inhibitor (i) acts on the BMP-2,4,7 pathway. Specifically, it inhibits the activation of transcription by the SMAD-1,5,8 / SMAD-4 complex, particularly by inhibiting the interaction of SMAD-1,5,8 with the SMAD4 cofactor, and especially by inhibiting the phosphorylation of SMAD-1,5,8. The second SMAD inhibitor (ii) acts on the TGFβ / activin / Nodal pathway. Specifically, it inhibits the activation of transcription by the SMAD-2,3 / SMAD-4 complex, particularly by inhibiting the interaction of SMAD-2,3 with the SMAD4 cofactor, and especially by inhibiting the phosphorylation of SMAD-2,3.

[0020] Advantageously, one day after the initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 20% and the concentration of the second SMAD inhibitor (ii) increases by at least 450%, more preferably, the concentration of the first SMAD inhibitor (i) increases by at least 40% and the concentration of the second SMAD inhibitor (ii) increases by at least 900%.

[0021] According to another preferred objective of the invention, two days after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 33% and the concentration of the second SMAD inhibitor (ii) increases by at least 4950%, more preferably, 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 9900%.

[0022] More preferably, the method according to the invention includes exposing pluripotent cells to two SMAD inhibitors, wherein the first SMAD inhibitor (i) is selected from Noggin factor, LDN193189, Dorsomorphin, DMH1, A83-1 and combinations thereof; and the second SMAD inhibitor (ii) is selected from SB431542, SB505124, LY2157299, LY550410 and combinations thereof.

[0023] According to another objective, the method according to the invention also exposes pluripotent cells to at least one SHH factor. Preferably, at least one SHH activator is added at least 3 days and at most 5 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor. Furthermore, contrary to methods known in the prior art, cell exposure to at least one SHH activator is not simultaneous with the initial exposure of pluripotent cells to the SMAD inhibitor. Very preferably, at least two SHH activators are added at least 3 days and at most 5 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor.

[0024] According to another preferred objective of the invention, an FGF activator, particularly FGF-8b, is added at least 3 days and at most 5 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor, advantageously added simultaneously with an SHH activator, i.e., during the initial exposure to at least one SHH activator.

[0025] According to another preferred objective of the invention, at least one Wnt activator is added at least 6 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor, more preferably at least 3 days after the initial exposure of cells to SHH and / or FGF activators.

[0026] Therefore, the method according to the invention advantageously includes exposing the pluripotent cells, wherein: - At least 3 days after initial exposure of pluripotent cells to at least one SMAD inhibitor, add at least one SHH activator, and / or - Add an FGF activator, particularly FGF-8b, at least 3 days after initial exposure of pluripotent cells to at least one SMAD inhibitor, and / or - At least 6 days after initial exposure of pluripotent cells to at least one SMAD inhibitor, add at least one Wnt activator.

[0027] When at least one SMAD inhibitor is added to expose pluripotent cells to the SMAD inhibitor, these pluripotent cells are preferably in isolated or aggregated form. Furthermore, the cell culture is a three-dimensional cell culture, and the cells are encapsulated in three-dimensional cell microcompartments. These microcompartments are well known and specifically described in patent application WO 2018 / 096277.

[0028] In the context of this invention, the three-dimensional microcompartments are suitable for culturing pluripotent cells, particularly comprising the cells, an extracellular matrix layer, or an extracellular matrix substitute layer. By way of example, the layer may be Matrigel. ® Or it may be a fibrous type, and the microcompartments contain an outer layer of hydrogel (e.g., alginate).

[0029] Since the exposure of pluripotent cells to at least one SMAD inhibitor can be performed simultaneously with or after the encapsulation of the pluripotent cells, the pluripotent cells can therefore be in the form of isolated cells, aggregates, or a mixture of isolated and aggregate forms.

[0030] According to another object of the invention, the pluripotent cells can be of any type and are capable of differentiating into cells of interest, particularly neural cells, and very preferably, the pluripotent cells are induced pluripotent stem cells (iPSCs). Various cell culture techniques for obtaining induced pluripotent stem cells (iPSCs) are particularly well described in the background art, especially in the articles of Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 207, 131(5): 861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0031] According to another specific object of the invention, the method according to the invention may include an additional step of exposure to at least one of the following factors: rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof, to promote further maturation of nerve cells and obtain nerve cells expressing at least FOXA2 and / or OTX2, particularly dopaminergic neurons that are specifically expressing FOXA2 and TH+ markers.

[0032] Advantageously, the method according to the invention increases the number of cells expressing FOXA2 and TH+ and decreases the number of cells expressing PAX6 / SOX1, respectively demonstrating an increase in the number of mature neural cells (i.e., at least dopaminergic cells (neurons and dopaminergic progenitor cells)) and a decrease in neural stem cells, which makes pluripotent cells more prone to differentiating into neural cells. Finally, the absence of pluripotency markers (especially TRA-1-60+ / OCT+) confirms the absence of pluripotent stem cells in the final product obtained at the end of the process (i.e., various neural cells in microtissue form). This absence reduces the risk of toxicity or uncontrolled differentiation.

[0033] Preferably, the factor selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trogostatin A, and combinations thereof is added for at least 5 days, more preferably for at least 13 days.

[0034] According to another preferred purpose, at least 12 days after initial exposure of pluripotent cells to SMAD factors, the factors selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof are added.

[0035] According to another object of the invention, the method for differentiating pluripotent cells into neural cells includes an intermediate step lasting at least one day, during which the culture medium is free of TGF-β pathway inhibitors in order to remove differentiation factors from the culture medium when the microcompartment contains at least 50% neural cells expressing at least FOXA2+ / OTX2+ factors.

[0036] Finally, according to another aspect, the invention also relates to a variety of nerve cells obtained by a method according to any of the foregoing embodiments. Preferably, the final product obtained by this method is a mixture of nerve cells, particularly including dopaminergic neurons, dopaminergic progenitor cells, and glial cells. Therefore, the invention also relates to a heterogeneous population of nerve cells, particularly a heterogeneous population of nerve cells for nerve cell transplantation. This heterogeneous population composed of multiple nerve cell types, preferably striatal and / or cortical populations, is very preferably in aggregate or microtissue form.

[0037] According to another aspect, the present invention also relates to a variety of nerve cells preferably in the form of micro-tissues, which are used as drugs, more preferably for the prevention and / or treatment of Parkinson's disease.

[0038] Other features and advantages will become apparent from the specific embodiments, examples and the following figures of the invention. Attached Figure Description

[0039] [ Figure 1 The diagram illustrates the steps of a differentiation method according to a specific embodiment of the present invention.

[0040] [ Figure 2The diagram illustrates the kinetics of biomarker expression, measured by flow cytometry, during the neural differentiation method according to the invention in a bioreactor. Selected differentiation days correspond to the day of iPSC encapsulation (day 0), and the end of the neural induction phase (day 5), the end of the ventralization phase (after flushing) (day 12), the end of the maturation phase (day 17), and the end of the post-maturation phase (day 24). Data are presented as histograms and pooled from 16 different and independent bioreactors (n=16).

[0041] [ Figure 3 The transcriptomic expression of tyrosine hydroxylase (TH) relative to ACT-B housekeeping gene expression, as detected by RT-qPCR at different differentiation times, is shown in the left subplot, and the transcriptomic expression of Engrailed gene (EN-1) relative to ACT-B housekeeping gene expression is shown in the right subplot. These different differentiation times are the end of the neural induction phase (day 5), the end of the ventralization phase (after rinsing) (day 12), the end of the maturation phase (day 17), and the end of the post-maturation phase (day 24) of the differentiation method according to the present invention and methods outside the scope of the present invention (excluding the step of adding at least one SMAD inhibitor continuously or discontinuously for at least 3 days).

[0042] [ Figure 4 This is a comparative flow cytometry (FACS) analysis of neural cell mixtures obtained at the end of a differentiation method outside the scope of this invention and neural cell mixtures obtained according to the differentiation method of this invention (neuronal microtissue at day 24). The results are presented in the form of histograms. An increase in FOXA2-positive cells indicates an increase in the number of dopaminergic cells (neurons and dopaminergic progenitor cells), while a decrease in PAX6+ / SOX1+-positive cells indicates a decrease in the number of neural stem cells in the obtained cell mixture. At day 24, the neuronal microtissue obtained according to the method of this invention contained less than 40%, more preferably less than 30%, of cells expressing FOXA2.

[0043] [ Figure 5 The figures show the mRNA expression levels of cells obtained according to the method of the present invention, which constituted the final product on day 24, as detected by RT-qPCR. The values ​​are expressed as a proportion of the Ct values ​​relative to the Ct values ​​of iPSCs (day 0) by comparing the mean Ct value with the Ct values ​​of five different housekeeping genes (ACTB, PSMB4, NONO, C1orf43, and YWHAZ). The experiments were performed in duplicate, and the results are shown in Tukey box plots (n=17).

[0044] [ Figure 6This paper describes the ability of various nerve cells in microtissue forms (lumenless variants) obtained through the differentiation method according to the invention to restore motor symmetry in a rat model of hemilateral Parkinson's disease when evaluated using an amphetamine-induced rotation test (rotameter). The graft was unilaterally transplanted into the striatum of the affected rats. Prior to the experiment, a lesion model was established by injecting a neurotoxin (6-OHDA) into the medial forebrain tract (MFB). This lesion mimics severe damage to dopaminergic neurons and induces unilateral motor deficits, producing motor symptoms similar to those of Parkinson's disease.

[0045] [ Figure 7 [Image 1] is a cross-sectional image of a post-graft containing nerve cells 20 weeks after transplantation, obtained according to the method of the present invention. Staining results for the dopaminergic neuron marker TH (B), staining results for the human marker Stem121 (C), and sections of the graft containing both markers. The area marked by the rectangle is a magnified field of view shown in the right-hand subplot (E). Scale bars: 2.5 mm (B, C, and D) and 50 μm (E). Subplot D shows the reinnervation phenomenon induced by the graft.

[0046] [ Figure 8 The illustration shows a variant of a luminous neural tissue unit obtained by the method according to the invention, with cells stained against markers FOXA2 and OTX2 and Ki67.

[0047] [ Figure 9 The image shows a variant of a neural tissue unit obtained using the method according to the invention, with cells stained against the markers SOX2 and NESTIN.

[0048] [ Figure 10 Microtissues according to the invention stained with hematoxylin-eosin-safranin (HES) on day 24. A) Luminous microtissues obtained by implementing the method according to the invention, exhibiting densely packed nucleated regions, i.e., layer C1 (dashed lines), which are radially distributed around the lumens (“L”). B) Luminous microtissues obtained by implementing the method according to the invention. Scale bar: 50 μm.

[0049] [ Figure 11 Images of solid neural microtissue variants without lumen in microcompartments.

[0050] [ Figure 12 Immunofluorescence image of a tubular microtissue variant according to the invention on day 24. Neural microtissue was immunostained against dopaminergic neuronal marker TH (A) and dopaminergic progenitor cell marker FOXA2 (B), and counterstained with the nuclear dye DAPI (C). Scale bar: 50 μm. This indicates the presence of a lumen. The cells radially distributed around the lumen are mainly FOXA2 positive and correspond to the C1 layer, while TH expression is located on the periphery of the cells surrounding the lumen and corresponds to the C2 layer.

[0051] [ Figure 13 Immunofluorescence images of luminous microtissue variants obtained by the method according to the invention on day 24. Neural microtissue was immunostained against the proliferating cell marker Ki67 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 μm. This indicates the presence of a lumen. Cells radially distributed around the lumen are positive for KI67 (approximately less than 50% of the cells).

[0052] [ Figure 14 Immunofluorescence images of microtissue obtained according to the method of the present invention on day 24. Neural microtissue was immunostained against the dopaminergic progenitor cell marker OTX2 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 μm. Asterisks ( The presence of a lumen is indicated by the number 0. The cells radially distributed around the lumen are predominantly OTX2-positive, corresponding to layer C1.

[0053] [ Figure 15 Size distribution of microtissues (a luminal variant) obtained using the method according to the invention on day 24. The microtissues were imaged using a wide-field microscope, and their dimensions were measured using image analysis software. The average diameter of the microtissues was 176.8 ± 60.48 μm.

[0054] [ Figure 16 Postmortem histological analysis of rats transplanted with microtissues according to the invention (with lumen (batch 1) and without lumen (batch 2)). A) and B) show brain sections from animals 20 weeks after transplantation with batch 1 (A) and batch 2 (B), immunostained for TH and the human marker Stem121. Scale bar: 2.5 mm. C, D) Quantitative analysis of the number of TH-expressing cells per unit volume of injected microtissue (C) and the number of TH-expressing cells per unit number of injected microtissues (D). Nonparametric Mann-Whitney test was performed. ns = p > 0.05; p<0.05. Detailed Implementation

[0055] definition For the purposes of this invention, "microcompartment" or "capsule" refers to a partially or completely enclosed hollow three-dimensional structure that contains one or more cells. The structure comprises a preferably hardened hydrogel outer layer and a hollow inner portion containing at least one cell and / or at least one cell aggregate and / or cellular micro-organism (or tissue unit), and optionally an extracellular matrix and / or extracellular matrix substitute suitable for cell culture and said cell growth.

[0056] For the purposes of this invention, "human cell" means human cell or immune-humanized non-human mammalian cell. Even if not explicitly stated otherwise, these cells, pluripotent cells, progenitor cells, and differentiated cells (including nerve cells) are obtained or derived from human cells or immune-humanized non-human mammalian cells.

[0057] For the purposes of this invention, "pluripotent" cells refer to cells that have the ability to form all tissues present in a complete primitive organism but cannot form a complete organism on its own because they have undergone the initial differentiation steps and can only produce cells of the germ layer, endoderm, mesoderm, and ectoderm, but cannot produce cells that trophoblast the ectoderm. In the context of this invention, human pluripotent cells may be referred to as hPSC cells or ES cells. Specifically, these cells may be induced pluripotent stem cells (iPSC or hiPSC refers to human induced pluripotent stem cells). The pluripotency of these cells can be evaluated by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA4 / 5, Tra-1-60, and Tra-1-81. Optionally, according to a very specific embodiment, pluripotent cells obtained from embryonic stem cells are obtained without destroying the embryo from which they are derived, for example using the techniques described by Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, said embryonic stem cells from humans may be excluded.

[0058] For the purposes of this invention, “induced pluripotent stem cells” or “iPSC” or “hiPSC” means pluripotent stem cells that have been induced to become pluripotent through genetic reprogramming of differentiated somatic cells. These cells are particularly positive for pluripotency markers, such as staining with alkaline phosphatase and expression of proteins NANOG, SOX2, OCT4, and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 207, 131(5): 861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0059] For the purposes of this invention, "nerve" cells refer to all cells of the nervous system. These cells can be mature cells, such as dopaminergic or GABAergic neurons, so-called glial supporting cells (astrocytes, oligodendrocytes, etc.); or progenitor cells (dopaminergic or GABAergic progenitor cells, etc.); or neural tissue stem cells (neural stem cells). They can also be mixtures of neural cells, particularly heterogeneous populations of neural cells containing several cell types, such as neurons, dopaminergic neurons, progenitor cells, neural stem cells, and optionally glial cells.

[0060] For the purposes of this invention, "progenitor cell" refers to a cell in the process of differentiation, which has participated in the differentiation pathway but has not yet differentiated. In the context of this invention, progenitor cells are, for example, radial glial cells or radial glial progenitor cells (RGPCs), and dopaminergic progenitor cells expressing at least the FOXA2 factor.

[0061] For the purposes of this invention, "differentiated" cells refer to cells with a specific phenotype, as opposed to undifferentiated pluripotent stem cells or progenitor cells undergoing differentiation. In this context, differentiated cells are mature cells that express at least FOXA2 and TH factors, such as neuronal cells, i.e., neurons, such as dopaminergic neurons.

[0062] For the purposes of this invention, "cell layer" or "cell bed" refers to a layered or bed-like structure formed by multiple cells surrounding a lumen; for example, it can be a group of cells working synergistically and aggregated in a three-dimensional manner. The thickness of the cell layer or cell bed can be variable. The cell layer or cell bed is arranged in an orderly three-dimensional manner within a neural tissue unit.

[0063] For the purposes of this invention, "microtissue" or "neural tissue unit" or "tissue unit" means at least one neural tissue unit comprising a variety of neural cells obtained by the differentiation method according to the invention, including, for example, dopaminergic neurons, progenitor cells (especially dopaminergic progenitor cells), and glial cells, said cells optionally arranged in a three-dimensionally ordered manner in the extracellular matrix. The microtissue may be encapsulated in three-dimensional cellular microcompartments or decapsulated, and is suitable for implantation into the nervous system of mammals (preferably humans).

[0064] For the purposes of this invention, "exponential increase" or "exponentially increasing" means that a certain quantity grows exponentially; that is, in the context of this invention, it refers to the exponential increase in the concentration of a differentiation factor (e.g., a SMAD inhibitor). Therefore, a characteristic of the mathematical definition known to those skilled in the art is that this growth follows an exponential law over time.

[0065] For the purposes of this invention, "logarithmic increase" or "logarithmically increasing" means that a quantity increases logarithmically; that is, in the context of this invention, it refers to the logarithmic increase in the concentration of a differentiation factor (e.g., a SMAD inhibitor). Therefore, a characteristic of the mathematical definition known to those skilled in the art is that this increase follows the logarithmic law over time.

[0066] For the purposes of this invention, "SMAD inhibitor" refers to a factor involved in transforming growth factor. -β ( TGF-β The SMAD family of molecules that signal transduction, including SMAD proteins and their analogues, exists in mammals. At least eight SMAD proteins are identified, numbered SMAD 1 through SMAD 8. When TGF-β binds to its cell surface receptor, the receptor phosphorylates SMAD 2 and SMAD 3, which then form a complex with SMAD 4 and migrate to the nucleus. This SMAD complex binds to the promoter sequences of target genes to activate their transcription, thereby mediating the biological activity of TGF-β. Conversely, the proteins SMAD 6 and SMAD 7 inhibit TGF-β signal transduction. When BMP-2 binds to its cell surface receptor, the receptor phosphorylates SMAD 1, SMAD 5, and SMAD 8, which then form a complex with SMAD 4 and migrate to the nucleus. This SMAD complex binds to the promoter sequences of target genes to activate their transcription, thereby mediating the biological activity of BMP-2. Conversely, proteins SMAD 6 and SMAD 7 inhibit BMP-2 signaling. SMAD inhibitors can be selected, by way of example, from Noggin, LDN193189, Dorsomorphin, DMH1 and A83-1, SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

[0067] For the purposes of this invention, "SHH activator" refers to a molecule capable of activating the Sonic Hedgehog signaling pathway. The Sonic Hedgehog protein is one of three mammalian proteins involved in the Hedgehog signaling pathway; the SHH protein is a ligand for the Hedgehog signaling pathway and plays a crucial role in regulating vertebrate organogenesis, such as the growth of limbs and toes and the formation of brain tissue. The Sonic Hedgehog (SHH) signaling pathway has long been known to play a major role during vertebrate embryonic development. Preferably, the SHH activator is selected from SHH, SHH C25II, SAG (Smoothened agonist), and Purmorphamine.

[0068] For the purposes of this invention, "FGF activator" refers to a molecule capable of activating FGF receptor signaling. FGF receptors belong to a family of transmembrane receptors with intracellular protein tyrosine kinase activity. When a tyrosine kinase receptor binds to its extracellular ligand, it becomes capable of phosphorylating other transmembrane receptors on intracellular proteins or certain tyrosine amino acids, thereby enabling signal transduction from the extracellular to the intracellular space. The main tyrosine kinase receptors are receptors for polypeptide growth factors (EGF, FGF, PDGF, VEGF, etc.). Preferably, the FGF activator is rhFGF-8b.

[0069] For the purposes of this invention, "Wnt activator" or "Wnt signaling pathway activator" refers to a molecule capable of activating the Wnt signaling pathway, which plays various roles in animal development and stem cell maintenance. Preferably, the Wnt activator is selected from CHIR 99021, XAV939, and BIO.

[0070] For the purposes of this invention, "prevention" means reducing the risk or probability of a particular phenomenon (e.g., Parkinson's disease in the context of this invention) to a low level.

[0071] For the purposes of this invention, "treatment" means slowing the progression of a disease, stabilizing, reversing or eliminating the condition, or even interrupting or inhibiting the progression of a disease (such as Parkinson's disease).

[0072] For the purposes of this invention, "Feret diameter" refers to the distance between two tangents, specifically "d" or "D", which are parallel such that the entire projection is contained between these two parallel tangents.

[0073] For the purposes of this invention, the “maximum size” of X means the value of the maximum Feret diameter of X.

[0074] For the purposes of this invention, "lumen" means a substantially cell-free volume containing an aqueous solution, topologically surrounded by cells (particularly at least one layer of cells forming a fluid circulation barrier), characterized by the presence of a positive tight junction of a ZONULA OCCLUDENS 1 or ZO-1 marker adjacent to the lumen.

[0075] Methods for differentiating pluripotent cells into nerve cells Therefore, the object of the present invention is an in vitro method for differentiating pluripotent cells into neural cells by means of at least one three-dimensional cellular microcompartment in a suitable culture medium, the microcompartment containing the pluripotent cells, the method comprising at least one step of exposing the pluripotent cells to a substance: -At least two SMAD inhibitors, -At least one SHH activator, - at least one FGF activator, and -At least one Wnt activator, The concentration of at least one SMAD inhibitor in the culture medium is increased continuously or discontinuously for at least 3 days from the initial exposure of pluripotent cells to the SMAD inhibitor, relative to the initial concentration of the SMAD inhibitor by at least 20%, preferably at least 30%, more preferably at least 40%, to obtain a variety of nerve cells, wherein at least 30%, preferably at least 40%, of the nerve cells express at least the following factor FOXA2 / OTX2.

[0076] In the context of this invention, pluripotent cells are encapsulated in three-dimensional microcompartments or capsules, so that three-dimensional cell culture closely approximates in vivo physiological conditions. The advantages of three-dimensional cell culture have now been fully described and are well known to those skilled in the art.

[0077] This method may include a preliminary step of culturing pluripotent cells, followed by encapsulating the pluripotent cells in three-dimensional cell microcompartments. Three-dimensional cell microcompartments are described in detail in patent application WO 2018 / 096277. In short, encapsulation may advantageously include the following steps: a. Encapsulating a mixture comprising pluripotent cells, culture medium, and extracellular matrix or an extracellular matrix substitute in the outer layer of a hydrogel, the encapsulation comprising the following sub-steps: i. Contact the mixture with a hydrogel solution intended to form the outer layer to form at least one droplet, and ii. The obtained droplets are collected in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microchamber.

[0078] b. The capsules obtained in the preceding steps are cultured in a culture medium, preferably in a bioreactor, preferably for at least 1 day, more preferably for 3 to 50 days, and c. Recover the obtained cell microcompartments containing pluripotent cells of interest.

[0079] Preferably, the encapsulation step is performed by simultaneously co-injecting the following substances: a hydrogel solution intended to form the outer layer, a mixture containing, in particular, pluripotent cells and optionally an extracellular matrix or extracellular matrix substitute, and optionally an intermediate solution. The co-injection is performed concentrically via a microfluidic or microfluidic injector, forming a jet of the mixture of solutions at the injector outlet, which breaks into droplets. The droplets are then collected in a calcium bath, where gravity hardens the hydrogel solution to form the outer layer, and thus creates cellular microcompartments containing the pluripotent cells of interest. The pluripotent cells may be in isolated form or in the form of cell aggregates or clusters. The encapsulated pluripotent cells are also suspended in the capsule as single or isolated cells and / or cell clusters or aggregates. Preferably, isolated cells comprise less than 50% of the total number of encapsulated cells; more preferably, the isolated cells are iPSC cells.

[0080] Once the cells of interest are encapsulated, they are advantageously presented in isolated and / or aggregated form, and the cells are exposed to several differentiation agents, such as at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator. The differentiation agents are added directly to the culture medium containing the microcompartments, preferably in a bioreactor. The differentiation agents diffuse through the outer layer of the cell microcompartments, which allows them to diffuse further. Furthermore, the differentiation agents diffuse within each cell microcompartment and within the culture medium present in the cell microcompartments.

[0081] In this context, the inventors have developed a method for improving the yield of three-dimensional cell culture, which enhances the differentiation rate, thereby increasing the transplantation success rate of neural cell mixtures obtained by the method according to the invention, and reducing cell death rate relative to the teachings of the prior art.

[0082] To achieve this, the inventors have observed that exposing encapsulated pluripotent cells to gradually added concentrations of at least one SMAD inhibitor, i.e., gradually increasing the concentrations before adding other differentiation factors (i.e., SHH activators, FGF activators, and Wnt activators), overcomes the disadvantages of the prior art.

[0083] Therefore, the present invention relates to the gradual addition of at least one SMAD inhibitor, preferably two SMAD inhibitors, prior to the initiation of neural differentiation, which is initiated by the addition of other factors (i.e., SHH activator, FGF activator and Wnt activator).

[0084] Conversely, background techniques teach that after the expansion culture of pluripotent cells, the resulting pluripotent cells are exposed to high concentrations of differentiation factors, particularly a combination of SMAD inhibitors, SHH activators, and FGF activators. While this exposure does enable pluripotent cells to differentiate into neural cells, it comes at the cost of high cell death. In fact, sudden exposure to high concentrations of differentiation factors in cells that do not yet express receptors associated with the differentiation factor-induced pathway induces significant cellular stress, leading to high cell death. Furthermore, yields are suboptimal, and differentiation rates are low. Conversely, gradual increases in these factors promote cellular compliance and tolerance to the differentiation process, i.e., the cells' responsiveness to signals, thereby reducing cell death.

[0085] Therefore, the present invention aims to expose the encapsulated pluripotent cells (particularly expressing OCT / NANOG, SSEA5 / SSEA4, TRA-1-60 / OCT) to at least one SMAD inhibitor at T0. The SMAD inhibitor is added at a lower concentration than known in the background art to avoid cellular stress and significant cell death. Advantageously, the concentration of the SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells, more preferably by at least 40% relative to the initial concentration of the SMAD inhibitor.

[0086] For the purposes of this invention, "continuous increase" means that, preferably within a given time period, such as 3 days, the concentration of the differentiation factor of interest increases linearly with time.

[0087] Conversely, for the purposes of this invention, "discontinuous increase" means that within a given time period, such as 3 days, the concentration of the differentiation factor of interest is gradually increased until the final concentration of interest is reached.

[0088] The increase in SMAD inhibitor concentration can take several forms. Therefore, the increase can follow an exponential or logarithmic proportion. Most preferably, the increase in the concentration of at least one SMAD inhibitor is exponential. Furthermore, the concentration of at least one SMAD inhibitor preferably increases exponentially to reduce cell death rate in the encapsulated cells of interest and increase differentiation rate.

[0089] Preferably, after the pluripotent cells are initially exposed to the SMAD inhibitor, the concentration of at least one SMAD inhibitor increases for up to 4 days, more preferably up to 5 days.

[0090] According to another object of the invention, pluripotent cells are exposed to at least two SMAD inhibitors, wherein at least a first SMAD inhibitor (i) is increased in a continuous or discontinuous manner, preferably exponentially, and the pluripotent cells are exposed to a second SMAD inhibitor (ii), the concentration of which may remain constant over time or increase in a continuous or discontinuous manner. Therefore, it is preferable to increase the concentration of at least two SMAD inhibitors relative to the initial exposure concentration at which the pluripotent cells are initially exposed to the SMAD inhibitors.

[0091] When the concentration of the second SMAD inhibitor (ii) is increased in a continuous or discontinuous manner, the increase can be exponential or logarithmic. Preferably, the increase in the concentration of the second SMAD inhibitor (ii) is logarithmic, which has the effect of maintaining the undifferentiated state of pluripotent cells and preventing pluripotent cells from differentiating into undesirable pathways (e.g., pathways that predispose pluripotent cells to differentiate into mesoderm). According to a particularly preferred purpose, the increase in the concentration of the first SMAD inhibitor (i) is exponential, and the increase in the concentration of the second SMAD inhibitor (ii) is logarithmic.

[0092] Preferably, after initial exposure of pluripotent cells to the SMAD inhibitor, the concentrations of the two SMAD inhibitors increase for up to 4 days, more preferably up to 5 days.

[0093] Furthermore, the present invention aims to provide a differentiation method comprising the step of exposing encapsulated pluripotent cells to at least one, preferably at least two, SMAD inhibitors, wherein the concentration of each SMAD inhibitor is increased over a given time period (advantageously 3 to 5 days). During this period, the concentration of the first SMAD inhibitor (i) advantageously increases exponentially, while the concentration of the second SMAD inhibitor (ii) may be stable or increasing, preferably increasing in a logarithmic proportion.

[0094] According to another objective of particular interest, one day after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 40% and the concentration of the second SMAD inhibitor (ii) increases by at least 900% relative to the initial concentration of the SMAD inhibitor (i.e., on day 0, i.e., when the SMAD inhibitor was initially exposed to the encapsulated pluripotent cells of interest).

[0095] Preferably, two days after the initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 67% relative to the initial concentration, and the concentration of the second SMAD inhibitor (ii) increases by at least 9900%.

[0096] According to a preferred purpose of the present invention, the SMAD inhibitor may be selected from SMAD inhibitors (i) capable of acting on the BMP-2,4,7 pathway and SMAD inhibitors (ii) capable of acting on the TGFβ / activin / Nodal pathway.

[0097] SMAD inhibitors (i) that can act on the BMP-2,4,7 pathway advantageously enable the inhibition of transcriptional activation through the SMAD-1,5,8 / SMAD-4 complex, particularly by preventing the interaction of SMAD-1,5,8 with the SMAD4 cofactor, and especially by preventing the phosphorylation of SMAD-1,5,8.

[0098] SMAD inhibitors (ii) that can act on the TGFβ / activin / Nodal pathway advantageously enable the inhibition of transcriptional activation through the SMAD-2,3 / SMAD-4 complex, particularly by preventing the interaction of SMAD-2,3 with the SMAD4 cofactor, and especially by preventing the phosphorylation of SMAD-2,3.

[0099] Preferably, the SMAD inhibitor can be selected from Noggin, LDN193189, Dorsomorphin, DMH1 and A83-1, SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

[0100] In a particularly preferred embodiment, the first SMAD inhibitor (i) is selected from Noggin, LDN193189, Dorsomorphin, DMH1, A83-1 factor, and combinations thereof. In a particularly preferred embodiment, the second SMAD inhibitor (ii) is selected from SB431542, SB505124, LY2157299, LY550410 factor, and combinations thereof.

[0101] For the purposes of this invention, "Noggin" refers to a secreted homodimeric glycoprotein that binds to and inactivates members of the transforming growth factor β (TGF-β) signaling protein superfamily, such as bone morphogenetic protein 4 (BMP4). Noggin is typically a 65 kDa protein expressed in human cells as a glycosylated, disulfide-linked dimer. (Groppe et al., (2002). Nature 420, 636-642; Xu et al. (2005) NatMethods 2, 185-190; Wang et al. (2005) Biochem Biophys Res Commun 330, 934-942).

[0102] For the purposes of this invention, "LDN193189" or "LDN-193189" refers to a compound similar to Noggin protein (a bone morphogenetic pathway (BMP) inhibitor) that inhibits ALK1, ALK2, ALK3, and ALK6. It is a derivative of Dorsomorphin, which is typically used at concentrations approximately 1 / 100th that of Dorsomorphin (Sanvitale et al.; Vogt et al.). This compound significantly promotes the differentiation of human pluripotent stem cells into neural progenitor cells (Chambers et al.; Kriks et al.), promotes the differentiation of human pluripotent stem cells into neural crest cells (Kreitzer et al.), promotes the differentiation of the final endoderm from human and mouse pluripotent stem cells into foregut endoderm (Kearns et al.), and promotes the differentiation of mouse embryonic stem cells into inner ear sensory epithelial cells (Koehler et al.). This compound is specifically obtained through its CAS number 1062368-24-4.

[0103] For the purposes of this invention, "Dorsomorphin" refers to an AMPK inhibitor of formula C24H25N5O, named 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride. Dorsomorphin inhibits the BMP pathway by targeting type I receptors such as ALK2, ALK3, and ALK6. It is a compound similar to Noggin protein and compound LDN193189. It is available through CAS number 866405-64-3.

[0104] For the purposes of this invention, "DMH1" refers to a small molecule inhibitor of the highly selective BMP pathway that promotes hiPSC neurogenesis and can be obtained through CAS number 1206711-16-1.

[0105] For the purposes of this invention, "A83-1" refers to formula 3-(6-methyl-2-pyridyl)- N -Phenyl-4-(4-quinolinyl)-1 H 1-Pyrazole-1-thiocarboxamide is a selective inhibitor of the TGF-β type I ALK receptor. Specifically, it inhibits TGF-β-induced epithelial-mesenchymal transition (EMT) by inhibiting SMAD2 phosphorylation. Previous studies have shown that A83-01 significantly promotes somatic cell reprogramming.

[0106] For the purposes of this invention, "SB431542" or "SB-431542" refers to a receptor kinase inhibitor of activin receptor types ALK5, ALK4, and ALK7. It is specifically used in combination with LDN193189, CHIR99021, and DAPT to convert astrocytes into neurons. It is available through CAS number 301836-41-9.

[0107] For the purposes of this invention, "SB505124" or "SB-505124" also refers to a selective inhibitor of the ALK4, ALK5, and ALK7 receptors of transforming growth factor-β type I. It selectively and in a concentration-dependent manner inhibits the activation of ALK4, ALK5, and ALK7-dependent downstream cytoplasmic signaling molecules Smad2 and Smad3, as well as components of the TGF-β-activated protein kinase pathway, without altering Smad signaling induced by ALK1, ALK2, ALK3, or ALK6. SB-505124 is three to five times more potent than the previously described related ALK5 inhibitor SB-431542.

[0108] For the purposes of this invention, "LY2157299" or "galuniseti" refers to a small molecule inhibitor of the TGF-β signaling pathway, particularly TGF-β receptor I, which specifically regulates SMAD2 phosphorylation, thereby eliminating activation of the classical pathway.

[0109] For the purposes of this invention, “LY550410” is also used herein to refer to a small molecule inhibitor of the TGF-β signaling pathway, particularly TGF-β receptor I.

[0110] Therefore, very preferably, the concentration of the first SMAD inhibitor (i) selected from Noggin, LDN193189, Dorsomorphin, DMH1, A83-1 factor and combinations thereof increases exponentially during a period of 3 to 5 days after initial exposure of the SMAD inhibitor to encapsulated pluripotent cells (i.e., at T0).

[0111] According to another highly preferred purpose, after initial exposure of the SMAD inhibitor to encapsulated pluripotent cells, the concentration of a second SMAD inhibitor (ii) selected from factors SB431542, SB505124, LY2157299, LY550410 and combinations thereof increases logarithmically for a period of 3 to 5 days.

[0112] According to another preferred objective of the invention, the first SMAD inhibitor (i) is a noggin factor, which is present at a concentration of 50 ng / mL to 70 ng / mL upon initial exposure to encapsulated pluripotent cells. Subsequently, following initial exposure to encapsulated pluripotent cells at a concentration of 50 ng / mL to 70 ng / mL, the concentration of noggin advantageously increases exponentially for at least 3 days. Preferably, the maximum supplemental concentration of the noggin factor is 100 ng / mL.

[0113] According to another preferred objective of the invention, the second SMAD inhibitor (ii) is factor SB431542, which is initially exposed to encapsulated pluripotent cells at a concentration of 0.1 μM to 0.3 μM. Then, following initial exposure to encapsulated pluripotent cells at a concentration of 50 ng / mL to 70 ng / mL, the concentration of SB431542 advantageously increases exponentially or logarithmically for at least 3 days until the maximum supplemental concentration of factor SB431542, 20 μM, is reached.

[0114] Therefore, unlike known 2D or 3D methods, exposing encapsulated pluripotent cells to at least one SMAD inhibitor, preferably two inhibitors, in a 3D culture, and gradually increasing the concentration of said inhibitors before adding other differentiation factors (especially SHH activators and FGF activators), overcomes the shortcomings of the prior art, namely, improving differentiation yield while reducing cell death. For the purposes of this invention, "before adding other differentiation factors" means exposing the cells to at least one SMAD inhibitor for at least 3 days before adding other differentiation factors (especially SHH activators and FGF activators).

[0115] This method of exposing pluripotent cells to various differentiation factors in a time-delayed manner, combined with increasing the concentration of at least one SMAD inhibitor (preferably at least two SMAD inhibitors) for at least 3 days and at most 5 days, enables improved yield by reducing cell death and increasing the differentiation rate of cells of interest, and further promotes the synchronization of various cell populations obtained in the capsule. In fact, pluripotent cells are constantly in the cell cycle. However, the phase of the cycle affects the cell's response to a given signal. Therefore, exposure to increasing concentrations within the characteristic cell cycle time (24 hours) increases the probability that the cells will receive optimized signals at their peak capacity.

[0116] The differentiation method comprises exposing pluripotent cells to at least one SMAD inhibitor for at least 3 days and at most 5 days after initial exposure to at least one SMAD inhibitor. Preferably, the method includes exposure to at least two SHH activators. Finally, the pluripotent cells are continuously exposed to the SMAD inhibitor for the duration of exposure to at least one SHH activator and at least one FGF activator.

[0117] Therefore, after initial exposure of pluripotent cells to at least two SMAD inhibitors for at least 3 days and at most 5 days, the pluripotent cells were exposed to the maximum concentration of SMAD inhibitors experienced by the cells from day 3 to day 5.

[0118] When these SMAD inhibitors were Noggin and SB431542, their exposure concentrations were 100 ng / mL and 20 μM, respectively, 3 to 5 days after cell differentiation, for a period of 9 to 12 days.

[0119] SHH activators significantly activate the Sonic Hedgehog signaling pathway. Typical SHH protein signaling occurs via a multi-component receptor containing patched (PTCH1, PTCH2) and smoothed (SMO). SHH protein binding to PTCH inactivates the basal inhibitory effect of PTCH on SMO. SHH protein, SHH-C25II, SAG (smoothed agonist), and purmorphamine can be used to activate this pathway. This pathway is involved in the development of the central nervous system. SHH proteins regulate the fate of neural stem cells through tissue morphogenesis.

[0120] FGF-8b is a member of the fibroblast growth factor family. FGF-8b is widely expressed during embryogenesis and regulates the epithelial-mesenchymal transition. FGF-8b plays a role in ordered alignment and triggering gastrulation, and is also involved in the construction of the midbrain / hindbrain.

[0121] Unlike SMAD inhibitors, the concentrations of the SHH activator and FGF activator become stable over time. Preferably, the exposure time of the SHH activator and FGF activator, calculated from the start of exposure to cells already involved in the differentiation process, is therefore 9 to 12 days, during which the levels of the markers OCT / NANOG and TRA-1-60 / OCT expressed by these cells are significantly lower than those in pluripotent cells.

[0122] Therefore, at least one SHH activator is advantageously added at least 3 days and at most 5 days after the encapsulated pluripotent cells are initially exposed to at least one SMAD inhibitor. Preferably, at least two SHH activators are added.

[0123] According to one object of the invention, it is advantageous to add at least one FGF activator 3 to 5 days after initial exposure of encapsulated pluripotent cells to at least one SMAD inhibitor, and very preferably, to add it simultaneously or concurrently with cell exposure to at least one SHH activator (more preferably two SHH activators). Therefore, it is preferable to add the FGF activator 3 to 5 days after initial exposure of pluripotent cells to at least one SMAD inhibitor.

[0124] According to a particularly preferred purpose of the present invention, at least one SHH activator is SHH factor C25II, with a target concentration of 100 ng / mL.

[0125] According to another particularly preferred purpose of the invention, at least one SHH activator is a Purmorphamine factor with a target concentration of 2 μM.

[0126] According to a preferred embodiment of the present invention, the FGF activator is rhFGF-8b with a concentration of 100 ng / mL.

[0127] The method according to the invention further includes the step of exposing differentiating cells to a Wnt activator. Preferably, the activator is added to expose differentiating cells to the activator, namely cells expressing at least the factor PAX6 / SOX1, namely radial glial cells or radial glial progenitor cells (RGPCs), which are bipolar progenitor cells responsible for generating all neurons in the cerebral cortex and also generating certain glial cell lineages (particularly astrocytes and oligodendrocytes).

[0128] Preferably, the Wnt activator is factor CHIR 99021, with a target concentration of 3 μM.

[0129] According to another purpose, the concentration of at least one factor selected from SHH activator, FGF activator and Wnt activator is 30% to 300% of the target concentration, preferably 50% to 200% of the target concentration, and more preferably 90% to 110% of the target concentration.

[0130] According to a preferred embodiment of the invention, a Wnt activator is added at least 6 days after initial exposure of pluripotent cells to at least one SMAD inhibitor. According to one variant, a Wnt activator is added at least 3 days after exposure of pluripotent cells (which tend to differentiate into cells expressing at least PAX6 / SOX1) to at least one SHH activator and one FGF activator. According to another variant, at least one Wnt activator is added when the cells are radial glial progenitor cells.

[0131] Heterogeneous neural cell populations were obtained by exposing encapsulated pluripotent cells to at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator for a period of 12 to 17 days. This population contained multiple neural cell types, with at least 30%, preferably 40%, of the cells expressing the FOXA2 and / or OTX2 markers.

[0132] Preferably, the method may include an intermediate step of removing differentiation factors from the mixture. Advantageously, this intermediate step lasts from 12 hours to 48 hours, and very advantageously 24 hours.

[0133] According to another objective of the invention, the differentiation method may include an additional maturation step, wherein the obtained cells are exposed to at least one of the following differentiation factors: rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof.

[0134] Therefore, another object of the present invention is an in vitro method for differentiating pluripotent cells, the method further comprising exposing nerve cells obtained after encapsulated pluripotent cells to at least two SMAD inhibitors for a period of 12 to 17 days to at least a factor selected from the following: rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof.

[0135] Furthermore, after exposing encapsulated pluripotent cells to at least one SMAD inhibitor for a period of 12 to 17 days, the resulting neural cells were exposed to differentiation factors (so-called maturation differentiation factors or maturation factors) selected from the following: rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof. The maturation factors were added at a stable concentration for at least 5 days during the exposure period.

[0136] For another purpose, the maturation factor is added at a stable concentration for up to 30 days, preferably up to 11 days, or even more preferably up to 5 days.

[0137] Advantageously, rhBDNF is added at a concentration for at least 12 days. According to a particularly preferred embodiment, rhBDNF is added at a target concentration of 10 ng / mL.

[0138] L-ascorbic acid was added at a target concentration of 200 μM for a particularly preferred purpose.

[0139] According to a particularly preferred embodiment, rhGDNF is added at a target concentration of 10 ng / mL.

[0140] For a particularly preferred purpose, rhTGF-β3 was added at a target concentration of 1 ng / mL.

[0141] According to a particularly preferred embodiment, rhFGF-20 is added at a target concentration of 5 ng / mL.

[0142] According to a particularly preferred embodiment, dbcAMP is added at a target concentration of 0.5 mM.

[0143] According to a particularly preferred embodiment, DAPT is added at a target concentration of 10 μM.

[0144] Compound E is added at a target concentration of 1 μM for a particularly preferred purpose.

[0145] According to a particularly preferred purpose, trogostatin A is added at a target concentration of 10 nM.

[0146] According to another purpose, the concentration of at least one factor selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E and trichostatin A is 30% to 300% of the target concentration, preferably 50% to 200% of the target concentration, and more preferably 90% to 110% of the target concentration.

[0147] Preferably, at the end of nerve cell exposure to a maturation factor selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof, the method includes an additional flushing step. This step is designed to avoid simultaneous activation and inhibition of the TGF-β pathway.

[0148] In fact, for another purpose, when the microcompartment contains at least 50% of nerve cells expressing at least FOXA2 and / or OTX2 markers, the method preferably includes an additional flushing step lasting at least 1 day.

[0149] Micro-tissues obtained by the method according to the invention At the conclusion of the differentiation method according to any one of the foregoing embodiments, a plurality of nerve cells are obtained, which are arranged in a three-dimensionally ordered manner and thus form micro-tissues or neural tissue units. Therefore, according to another aspect, the invention also relates to micro-tissues composed of a plurality of nerve cells arranged in a three-dimensionally ordered manner and extracellular matrix, said nerve cells being obtainable by the differentiation method of the invention. These plurality of nerve cells advantageously include at least dopaminergic neurons, progenitor cells (particularly dopaminergic progenitor cells), and glial cells.

[0150] The various neural cells expressed several markers specific to the cell types contained within the diverse neural cells obtained through differentiation. Preferably, the diverse neural cells constituting the microtissue exhibited a larger number of cells positive for the FOXA2 marker and a smaller number of cells positive for the PAX6 / SOX1 marker, indicating an increased number of dopaminergic cells and a decreased number of neural stem cells within the microtissue. This reflects a greater tendency for cells to differentiate into dopaminergic pathways, resulting in a higher differentiation rate. Furthermore, higher expression levels of the EN1 marker (a population proven to play a key role in transplantation efficiency (Kirkeby et al., 2017)) correlated with better functionality of the obtained microtissue. Additionally, higher expression levels of the TH marker (a marker of dopaminergic neurons) reflected a greater tendency for cells to differentiate into dopaminergic pathways, resulting in a higher differentiation rate.

[0151] According to one embodiment, the neural tissue unit can be solid and without lumens. This variant is preferably obtained when each capsule encapsulates more than 20 cells before differentiation.

[0152] According to another variant, the neural tissue unit may include at least one lumen. This variant is preferably obtained when each capsule encapsulates no more than 20 cells before differentiation.

[0153] According to a particular variant, the object of the present invention is a neural tissue unit arranged in a three-dimensional order, comprising at least dopaminergic neurons and neural progenitor cells, and optionally neural stem cells, said unit having a maximum size of less than 600 μm and comprising at least one lumen defined by a C1 cell layer, said cell layer primarily comprising neural progenitor cells expressing at least SOX2. Preferably, the C1 layer comprises neural progenitor cells expressing OTX2. Preferably, the neural progenitor cells in the C1 layer express at least SOX2 and OTX2.

[0154] Therefore, the neural tissue unit comprises at least one lumen defined by a C1 cell layer mainly containing neural progenitor cells expressing at least SOX2. In other words, the neural tissue unit comprises a C1 cell layer forming a boundary adjacent to the lumen. The C1 cell layer is a concentric layer surrounding the lumen.

[0155] The neural tissue unit contains lumens, which significantly improves cell survival and preserves the specific physiological cellular structure of this tissue. During transplantation, this micro-tissue helps to enhance the differentiation potential and / or survival rate of TH+ cells (i.e., dopaminergic neurons), thereby improving the survival and transplantation rate of the neural tissue unit according to the invention.

[0156] According to a preferred purpose, the C1 layer located at the edge of the lumen is itself surrounded by a C2 cell layer or cell bed containing dopaminergic neurons. The C2 cell layer or cell bed advantageously contains at least 3%, at least 5%, at least 10%, or at least 15% of dopaminergic neurons. Thus, the C2 cell layer forms a second concentric layer surrounding the C1 layer. Very advantageously, the C2 cell layer contains 3% to 50%, or even more advantageously 15% to 50%, of dopaminergic neurons expressing at least the tyrosine hydroxylase (TH) marker.

[0157] Furthermore, preferably, several concentric layers are thus formed around the lumen, namely: - Peripheral cell layer (C1 cell layer), and - There is also at least one concentric layer (C2 layer) containing dopaminergic neurons around the edge, which is different from the C1 layer.

[0158] According to one variant, the microtissue according to the invention may comprise a plurality of lumens, wherein at least one lumen is surrounded by a marginal cell layer (C1 cell layer), and at least one concentric layer (C2 layer) containing dopaminergic neurons is also present around the periphery, the concentric layer being different from the C1 layer. Preferably, if the microtissue comprises a plurality of lumens, each lumen is surrounded by a marginal cell layer (C1 cell layer), and at least one concentric layer (C2 layer) containing dopaminergic neurons is also present around the periphery, the concentric layer being different from the C1 layer.

[0159] Very preferably, dopaminergic neurons are largely excluded from the pericaval region (i.e., the region containing the lumen and layer C1). In fact, dopaminergic neurons are located in the second layer, a cell bed that is topologically more outer, which can be described as the cortical region of the second cell bed.

[0160] For the purposes of this invention, "substantially exclude" means that the pericavitary region may contain some dopaminergic neurons. In other words, we cannot rule out the presence of at least one dopaminergic neuron in the pericavitary region, particularly the presence of 1 to 50 dopaminergic neurons.

[0161] For the purposes of this invention, "cortical region of the second cell bed" specifically refers to the C2 cell layer, excluding any region directly adjacent to the lumen.

[0162] Preferably, layer C2 therefore contains at least 15% tyrosine hydroxylase (TH) positive cells. The TH gene is a marker of dopaminergic neurons. Therefore, layer C2 contains at least 15% cells expressing this marker, and thus at least 15% dopaminergic neurons are present in layer C2.

[0163] According to another objective of the invention, up to 50% of the cells in the neural tissue unit express the Ki-67 marker, preferably 5% to 50% of said cells express the Ki-67 marker. Advantageously, layer C1 has a higher percentage of cells expressing Ki67 than layer C2.

[0164] According to another particularly preferred purpose, the neural tissue unit arranged in a three-dimensional order comprises at least dopaminergic neurons and neural progenitor cells, the neural tissue unit having a maximum size of less than 600 μm, and comprising at least one lumen defined by a C1 cell layer, the cell layer being substantially composed of neural progenitor cells expressing at least SOX2.

[0165] Preferably, the microtissue obtained according to the present invention has a substantially spherical shape.

[0166] Alternatively, the neural tissue units obtained according to the present invention have an oval, spherical, spherical, or teardrop shape, or a substantially oval, substantially spherical, or substantially teardrop shape.

[0167] Preferably, the neural tissue units obtained according to the present invention have a larger size of 10 μm to 600 μm ± 10%, more preferably 150 μm to 400 μm ± 10%, more preferably 100 μm to 300 μm ± 10%, and even more preferably 200 μm ± 10%. These sizes are particularly beneficial to the survival of neurons within the neural tissue units and optimize post-implantation remodeling and angiogenesis.

[0168] According to another preferred objective of the invention, the neural tissue unit obtained according to the invention comprises neural progenitor cells, particularly dopaminergic progenitor cells, which express FOXA2 and / or OTX2 and / or LMX1A in addition to expressing the SOX2 marker. Very preferably, the C1 cell layer comprises neural progenitor cells that express FOXA2 and / or OTX2 and / or LMX1A in addition to expressing the SOX2 marker. Even more preferably, in the case where the tissue unit comprises a lumen and C1 and C2 layers, the C1 cell layer comprises neural progenitor cells that express FOXA2 and / or OTX2 and / or LMX1A and SOX2.

[0169] According to one aspect of the invention, in the case where the tissue unit comprises a lumen and layers C1 and C2, the C1 cell layer comprises neural progenitor cells expressing SOX2 and FOXA2, or SOX2 and OTX2, or SOX2 and LMX1A. According to one variant, the C1 cell layer comprises neural progenitor cells expressing SOX2 and FOXA2 and OTX2, or SOX2 and FOXA2 and LMX1A, or SOX2 and OTX2 and LMX1A. According to another variant, the C1 cell layer comprises neural progenitor cells expressing both SOX2 and FOXA2 and OTX2 and LMX1A.

[0170] According to another preferred objective of the invention, the neural tissue unit obtained according to the invention comprises nerve cells, particularly dopaminergic neurons expressing EN1 and / or GIRK2 or PAX6. Optionally, the nerve cells may also express SOX1.

[0171] According to one variant, the neural tissue unit according to the invention comprises neurons expressing EN1 and GIRK2. In practice, dopaminergic neurons significantly express TH and / or GIRK2, while dopaminergic progenitor cells significantly express LMX1A and / or OTX2 and / or FOXA2 and / or EN1 and / or CORIN.

[0172] In fact, the higher the expression level of the EN1 marker (a population that has been shown to play a key role in transplantation efficiency (Kirkeby et al., 2017)), the better the functionality of the obtained neural tissue units.

[0173] According to another purpose, the neural tissue unit according to the invention may comprise glial cells.

[0174] In the context of this invention, neural tissue units are obtained in three-dimensional cellular microcompartments, particularly cellular microcompartments.

[0175] Therefore, the neural tissue unit according to the invention is preferably obtained in a hollow hydrogel cell microcompartment (or capsule). Such cell microcompartments may contain one or more neural tissue units according to the invention. Preferably, the cell microcompartment contains a single neural tissue unit according to the invention.

[0176] According to a preferred embodiment of the invention, the neural tissue unit is obtained by neural differentiation of cells capable of differentiating into neural cells within hollow hydrogel cell microcompartments. The stem cells are preferably pluripotent stem cells. This can be any neural differentiation method known to those skilled in the art, or one of the methods described herein. Cells capable of differentiating into neural cells include stem cells and / or neural progenitor cells. Stem cells may preferably be pluripotent stem cells and / or neural stem cells. Preferably, these cells are human cells. According to a preferred embodiment, the cells capable of differentiating into neural cells are immunocompatible with the human body intended to receive the neural tissue unit; According to a preferred embodiment, the stem cells are pluripotent stem cells, more preferably induced pluripotent stem cells, and even more preferably induced human pluripotent stem cells. According to another embodiment, the pluripotent cells are pluripotent stem cells that do not include human or animal embryonic stem cells. Regardless of whether embryonic stem cells are used, the embryo is not damaged.

[0177] According to one variant, the neural tissue unit comprises a lumen and is obtained in a microcompartment from up to 20 cells capable of differentiating into neural cells (i.e., stem cells and / or progenitor cells), more preferably from up to 10 differentiateable cells, and even more preferably from up to 5 differentiateable cells. As mentioned above, this number of differentiateable cells encapsulated in the cellular microcompartment is particularly advantageous for reducing the internal pressure within the microcompartment, which facilitates the formation of the neural tissue unit according to the invention, i.e., a unit comprising a lumen and at least one cell layer adjacent to the lumen. Conversely, excessive internal pressure results in a neural tissue unit lacking a lumen and a biomimetic cellular structure, namely, the C1 cell layer advantageously adjacent to the lumen and the C2 cell layer surrounding the C1 cell layer.

[0178] The present invention also relates to a variant of a method for preparing and obtaining a neural tissue unit comprising a lumen, the neural tissue unit being intended for implantation into the nervous system of a human mammal, the method comprising the following steps: a. Encapsulating up to 20, preferably up to 10, and more preferably up to 5 cells capable of differentiating into nerve cells within hollow hydrogel microcompartments. b. Induce cell differentiation into cells capable of differentiating into nerve cells existing in cellular microcompartments to obtain at least one neural tissue unit with at least one lumen. c. Optionally, at least partially remove the outer layer of the hydrogel from the microcompartment to recover the neural tissue unit according to the invention.

[0179] Micro-tissue as a drug According to the last aspect, the present invention relates to the use of micro-tissue or neural tissue units obtained by implementing the method according to the invention as medicines, preferably for the prevention and / or treatment of neurodegenerative diseases, and even more preferably for the prevention and / or treatment of Parkinson's disease.

[0180] In fact, the inventors have demonstrated that neural tissue units according to the invention can be injected and thus transplanted, particularly those described in [the original text]. Figure 7 (Micro-tissues without lumen) and Figure 16 (In the micro-tissue with lumens).

[0181] According to one variant, the invention also relates to the use of cellular microcompartments comprising at least one microorganism according to the invention as a drug.

[0182] The neural tissue unit according to the invention can then be implanted into the nervous system of a subject suffering from a neurodegenerative disease (particularly Parkinson's disease) to at least partially replace the subject's defective neurons.

[0183] The invention will now be illustrated by non-limiting embodiments of the method according to the invention, the obtained microstructures, and the corresponding results.

[0184] Example Example 1 — Differentiation scheme according to the present invention The generation of neural microtissues 2D culture of hiPSCs All hiPSC lines were maintained on fibronectin and cultured in mTeSR1 medium. Cultures were fed daily and passaged every 3 to 4 days at 37°C for 6 minutes using the enzyme-free ReLeSR reagent (approximately 80% confluence). Cells were then reseeded in small clusters (100 μm to 200 μm) at a density of approximately 20,000 to 40,000 cells / cm². Cells were then incubated at 37°C in a humidified environment containing 5% CO₂.

[0185] hiPSC's 3D encapsulation Prior to encapsulation, 2D stem cell colonies were isolated. HiPSCs were resuspended in mTeSR1 medium supplemented with 10 μM Y-27632. Cells were then mixed with human fibrinogen and Y-27632 at a 1:1 volume ratio to achieve a final fibrinogen concentration of 14 mg / mL and a Y-27632 concentration of 10 μM. Therefore, the final cell concentration in the cell / matrix solution was 7.1 × 10^6 viable cells / mL to 7.4 × 10^6 viable cells / mL, referred to as the encapsulation density. Tubes were connected to the three inlets of a 3D glass-printed microfluidic colayer flow apparatus. 3D glass-printed microcapillary tips were attached to the nozzle outlet for better flow control. The cell / matrix suspension was loaded into the internal channels of the three-way apparatus. Sodium alginate SDS solution was injected into the external channels. To prevent alginate gelation within the microfluidic device due to calcium release from suspended cells, a calcium-free solution (sorbitol) is used in the central channel of the co-extruded chip, acting as a barrier against calcium diffusion. This solution is also supplemented with thrombin to a final concentration of 0.02 U / mL to allow fibrin to cross-link within the capsule. The following three solutions are typically flowed at 80 mL / h in the three channels: alginate solution, sorbitol solution, and cell + matrix suspension. At these rates, the composite solution forms a liquid jet, which then breaks down into droplets. The outer layer of alginate readily gels when the droplets come into contact with a 100 mM calcium bath. Therefore, the inner cell / matrix solution remains trapped within closed, spherical, and permeable microcompartments. Each capsule encapsulates more than 20 cells. Within 5 minutes of encapsulation, the capsules are flushed with DMEM / F-12 supplemented with 2.9 mM CaCl2 and containing 15 mM HEPES to reduce the basal calcium concentration. Finally, they were transferred to mTeSR1 medium supplemented with 10 μM Y-27632, which was used as the initial suspension medium for neural differentiation.

[0186] 3D Neural Differentiation of hiPSCs in Static T-Flavor Flasks or Bioreactors Further neural differentiation can be achieved in several culture systems, including static suspension culture using T-flasks or well plates and stirred suspension culture using 30 mL or 500 mL bioreactors.

[0187] Under static conditions, encapsulated hiPSCs were cultured in suspension using T-flasks (5 mL to 30 mL) in a cell culture incubator maintained at 37°C and 5% CO2, with the culture medium changed daily.

[0188] Stirred suspension cultures were conducted in various bioreactors under stirred conditions. Benchtop rBSTs, including 30 mL and 500 mL bioreactors, were used. In the 500 mL and 30 mL rBSTs, the stirring speeds were set as follows: 150 rpm for days 0-7, 200 rpm for days 7-18, and 250 rpm for days 18-24, or 55 rpm for days 0-24. In both cases, the bioreactors were inoculated with capsules at a volume-based concentration of 25% (v / v). In the 500 mL bioreactor, the culture volume was maintained at 300 mL during the culture period. On day 1, the medium was completely replaced with fresh medium supplemented with ROCK inhibitor. On day 2, the medium was not replaced. On day 3, the medium was completely replaced with fresh medium without ROCK inhibitor. After that, the medium was replaced by infusion. On days 12, 13, and 18, the medium was completely replaced with fresh medium. The capsules were 22% to 25% of the final volume relative to the culture medium, and the pH was maintained at 7.2 ± 0.2. In an empty bioreactor, dissolved oxygen levels (day 0) were calibrated to 100% before and after autoclaving; and recalibrated under initial conditions (with culture medium added). Oxygen levels were monitored and controlled during the test. Oxygen was controlled to 50% and adjusted accordingly.

[0189] Under static and agitated conditions, the molecules were added as follows. Starting from day 0, mTeSR1 medium was supplemented with 10 μM Y-27632 during the first 72 hours of culture to achieve ROCK inhibition. mTeSR1 medium was used until day 3. GMP recombinant human Noggin protein was initially added at a concentration of 60 ng / mL on day 0 (encapsulation day), and gradually increased to 84 ng / mL and 100 ng / mL on days 1 and 2, respectively. Similarly, SB431542 GMP was initially added at a concentration of 0.2 μM on day 0, and gradually increased to 2 μM and 20 μM on days 1 and 2, respectively. On day 3, the medium was replaced with Neurobasal medium. ™ CTS ™ GlutaMAX was supplemented with DMEM / F-12 at a 1:1 ratio. ™ And supplemented with N-2 CTS ™ and B-27 ™ GMP supplements. From day 3 to day 11, add 100 ng / mL GMP human Noggin recombinant protein, 20 μM SB431542 GMP, 100 ng / mL SonicHedgehog / Shh (C24II) N-Term GMP human recombinant protein, and 100 ng / mL HumanKine. ®FGF-8b recombinant human protein and 2 μM StemMACSPurmorphamine were added. CHIR99021 GMP was added from day 6 to day 12. Subsequently, from day 13 to day 17, 200 μM ascorbic acid, 10 ng / mL GMP recombinant human GDNF protein, and 1 ng / mL recombinant human TGF-β3 protein (HumanKine) were added. ® The regimen consisted of 5 ng / mL recombinant human FGF-20 protein, 0.5 mM bibutyryl cAMP, 10 μM DAPT RMU, 1 μM compound E, and 10 nM trichostatin A. Finally, 20 ng / mL HumanKine recombinant BDNF protein was added from day 13 to day 24. This differentiation protocol is described in [reference needed]. Figure 1 middle.

[0190] Images of neural microtissue obtained on day 24 are shown in Figure 11 and Figure 10 B in.

[0191] Example 2 — Observational dynamics of different cell types during differentiation according to the present invention .

[0192] plan The method for preparing the micro-tissues is the same as that in Example 1.

[0193] Flow cytometry analysis Neural microtissue was harvested at different stages of neural differentiation. Samples with a final concentration of 20% capsules (V / V) in RelesR were incubated at room temperature for 5 minutes to remove alginate capsules. After washing with RelesR, the neural microtissue was dissociated using a neurosphere dissociation kit at 37°C with stirring (150 rpm) for 40 minutes, and manually resuspended every 10 minutes using a micropipette. Cells were then fixed and permeabilized using a 1:4 ratio of fixation / permeabilization concentrate and fixation / permeabilization diluent to a maximum cell density of 5.10^6 cells / mL. Cells were then resuspended in flow cytometry staining buffer at a cell density of 833,333 cells / mL. Cells were centrifuged at 500g for 5 minutes at room temperature, and the supernatant was removed. The samples were then incubated at room temperature in the dark for 30 minutes with a specific antibody diluted 1:50 in 1×permeabilization buffer (Invitrogen). Finally, the sample was washed twice, centrifuged at 500g for 5 minutes at room temperature, and the cells were resuspended in staining buffer. (Using MACSQuant) ®Samples were analyzed using Analyzer 10 (Miltenyi Biotec). Allotype control experiments were performed to determine the positive limit; samples exceeding this limit were considered positive, and antibody specificity was verified. Compensation controls were set up for each fluorescent dye to eliminate interference from spectral overlap. Data post-processing was performed using FlowJo analysis software. Unstained cells were used as negative controls to distinguish between stem cell-specific or neuron-specific antibody staining and non-specific background signals. Therefore, significant signals from the NANOG+ / OCT4+, SSEA5+ / SSEA4+, and TRA-1-60+ / OCT+ populations were used to identify and calculate the percentage of pluripotent cells. Signals from the PAX6+ / SOX1+ or FOXA2+ / OTX2+ populations were used to identify and calculate the percentage of neural stem cells and dopaminergic progenitor cells in the samples.

[0194] RNA extraction and RT-PCR analysis Samples were homogenized in Tri reagent (Euromedex), and RNA was isolated using the standard chloroform / isopropanol protocol. RNA processing and analysis were performed according to a modified version of a published method. cDNA was synthesized from 2 μg of total RNA using Maxima reverse transcriptase (FisherScientific). LightCycler was used. ® qPCR was performed using a 480 real-time PCR system (Roche). Transcription-specific primers were used to generate cDNA (4 ng) and qPCR reactions were performed in duplicate for each sample in a final volume of 10 μL using a LightCycler 480 SYBR Green I Master (Roche). Reference genes were identified using the RefFinder method. If no specific reference gene was mentioned, relative expression analysis was normalized to the following five reference genes: proteasome subunit β4 (Psmb4), octamer-binding protein containing a non-POU domain (Nono), chromosome 1 open reading frame 43 (C1orf43), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activator protein ζ (Ywhaz), and actin β (Actb). PCR data were exported and analyzed using software tools developed by NeuroCentre Magendie. Relative expression levels were assessed by calculating 2 - ΔCt = 2 - (Ct(gene of interest) - Ct(mean of the five reference genes)).

[0195] Statistical analysis All statistical analyses were performed using GraphPad Prism 8. For behavioral analyses (in vivo), two-way ANOVA and Tukey's multiple comparison test were used to compare with the solvent group for up to 20 weeks. p<0.0001). From week 24 to week 32, two-way ANOVA and Sidak multiple comparison tests were used (…). p<0.05).

[0196] result The results are shown in Figure 2 In this embodiment, the selected differentiation days correspond to the day of iPSC encapsulation (day 0), the end of the neural induction phase (day 5), the end of the ventralization phase (after washing) (day 12), the end of the maturation phase (day 17), and the end of the post-maturation phase (day 24). On day 0, almost all encapsulated cells expressed the characteristic markers of pluripotent iPSC cells: OCT / NANOG, SSEA5 / SSEA4, and TRA-1-60 / OCT. On day 5, the significant decrease in OCT / NANOG and TRA-1-60 / OCT markers and the increase in FOXA2 / OTX2 markers indicated the beginning of cell differentiation. On day 12, almost all cells expressed FOXA2 / OTX2, a characteristic marker of dopaminergic progenitor cells.

[0197] Example 3 — Comparison of the method according to the invention and methods outside the scope of the invention .

[0198] plan The research plan is the same as that disclosed in Examples 1 and 2.

[0199] result Figure 3 The results presented show the transcriptomic expression of the tyrosine hydroxylase (TH) gene as detected by RT-qPCR at different differentiation times. The results correspond to the relative expression levels of the TH gene relative to the ACT-B housekeeping gene. The TH gene is a marker of dopaminergic neurons, thus indicating that the greater the number of cells expressing this marker in the microtissues obtained according to the method of the present invention, the higher the proportion of dopaminergic neurons.

[0200] Figure 3The results presented show the transcriptomic expression of the Engrailed (EN-1) gene as detected by RT-qPCR at different differentiation times. The results correspond to the relative expression levels of the EN-1 gene relative to the ACT-B housekeeping gene. The EN-1 gene is a marker of dopaminergic progenitor cells; therefore, this indicates that higher expression levels of this marker in microtissues obtained according to the method of the present invention correlate with a higher proportion of dopaminergic progenitor cells. Furthermore, this cell type is of particular interest because previous studies have shown that EN-1 gene expression can serve as a predictor of transplant success when using dopaminergic progenitor cells (Kirkeby et al., 2017). The method according to the present invention increases EN-1 gene expression within microtissues compared to microtissues obtained using methods outside the scope of this invention.

[0201] Figure 4 The results shown describe the results of flow cytometry analysis (FACS) of dissociated microtissue obtained using the method according to the invention, compared to microtissue obtained using standard methods outside the scope of the invention.

[0202] An increase in the number of dopaminergic cells (FOXA2+) was associated with a decrease in the number of neural stem cells (PAX6+ / SOX1+), reflecting a greater tendency for cells to differentiate into dopaminergic pathways. The absence of the pluripotency marker (TRA-1-60+ / OCT+) confirmed the absence of pluripotent stem cells in the microtissues obtained using the method according to the invention. These results indicate that cells exhibit a better differentiation tendency and thus a higher differentiation yield when using the method according to the invention.

[0203] Example 4 — Characterizing microtissues composed of various nerve cells according to the present invention .

[0204] plan The research plan is the same as that disclosed in Examples 1 and 2.

[0205] result Characterization of multiple cells constituting the microtissue by qPCR revealed the presence of numerous mature or differentiated cells. Results are shown in... Figure 5 In particular, the presence of the following cells can be observed: such as neurons (MAP2, TUBB3), dopaminergic neurons (TH, GIRK2), dopaminergic progenitor cells (LMX1A, OTX2, FOXA2, EN1, CORIN), astrocytes (GFAP), GABAergic neurons (GAD1), and oligodendrocytes (OLIG2).

[0206] Therefore, the microtissue contains a variety of neural cell types, including a greater number of mature cells or cells at the end of differentiation, which confirms that the neural cells obtained by the method according to the invention have a better differentiation tendency.

[0207] Example 5 — Injection of micro-tissue according to the invention for the treatment of Parkinson's disease .

[0208] plan Induction of a rat model of hemilateral Parkinson's disease Rowett nude rats (Rnu+) were first anesthetized in an induction chamber containing 4% isoflurane (induction chamber parameters: i4 Oxy1Air1). The rats were then fitted with an anesthesia mask and given approximately 2.5% isoflurane (induction chamber parameters: i2.5 Oxy0.4Air0.4). The concentration could be slightly adjusted by periodically checking paw and body temperature manually. Analgesia was achieved by subcutaneous injection of buprenorphine and lidocaine at concentrations of 0.05 mg / kg and 5 mg / kg, respectively. At least 20 minutes prior to injection of 6-hydroxydopamine hydrobromide (6-OHDA / HBr), the rats received an additional intraperitoneal injection of desipramine hydrochloride at a concentration of 25 mg / kg. Using a stereotactic frame with the following coordinates (anterior fontanelle as reference), 2.5 μl of freshly prepared 0.5% (w / v) 6-OHDA / HBr was perfused into the medial forebrain tract (MFB): AP: -3.8; ML (right): 1.6; DV: -8 to -7. Rat behavior was assessed using an amphetamine-induced rotation test 3 weeks after lesion stabilization.

[0209] Amphetamine-induced rotational test (rotameter) Amphetamine-induced rotational behavior was assessed before transplantation (3 weeks after 6-OHDA injection) and every 4 weeks post-transplantation. Rotational behavior was recorded starting 10 minutes after intraperitoneal injection of amphetamine (2.5 mg / kg) and continued for 40 minutes. Results are expressed as rotations per minute. Behavioral analysis was performed only on rats that exhibited more than 3 rotations per minute after lesion stabilization.

[0210] Injecting neuronal microtissue into a rat model of hemilateral Parkinson's disease Rowett nude rats (Rnu+) were first anesthetized in an induction chamber containing 4% isoflurane (induction chamber parameters: i4 Oxy1Air1). The rats were then fitted with an anesthesia mask and given approximately 2.5% isoflurane (induction chamber parameters: i2.5 Oxy0.4Air0.4). The concentration was slightly adjusted by periodic manual checks of paw and body temperature. Analgesia was achieved by subcutaneous injection of buprenorphine and lidocaine at concentrations of 0.05 mg / kg and 5 mg / kg, respectively. Neuronal microtissue was then injected into the right striatum of the rats using a 25 μL Hamilton syringe (Hamilton, ref. 1702 CX SYR) with a custom-made needle (glass cannula). The injection medium consisted of a custom-made administration medium (CMC-DA70) composed of carboxymethyl cellulose and 70 kDa dextran. Injections were performed along two tracks using a stereotactic frame. At the end of the procedure, meloxicam (Metacam) was administered subcutaneously at a concentration of 1 mg / kg for postoperative analgesia management.

[0211] Post-mortem research Postmortem histological analysis Animals were first anesthetized by intraperitoneal injection of ketamine and toluidine. Then, intracardiac perfusion was performed: 150 mL of 0.9% NaCl was injected first, followed by 200 mL of 10% phosphate-buffered formalin. After extraction, the brain was post-fixed in 10% phosphate-buffered formalin at 4°C for 24 hours and finally stored in PBS at 4°C. Sections with a thickness of 40 μm were obtained using a vibratory microtome and stored in PBS containing 0.01% azide. The sections were washed three times with PBS. The sections were then incubated in PBS containing 0.1% Triton and 2% BSA at room temperature for 1 hour to permeabilize, and then washed three times with PBS. The sections were then stained overnight in PBS containing 0.05% Triton and 0.5% BSA with primary antibodies against tyrosine hydroxylase (TH; 1:1000) and human antigen (Stem121 1:1000) at 4°C. The sections were washed three times with PBS. The sections were then incubated with fluorescently conjugated secondary antibodies (Alexa 488, 568) (1:1000) in PBS Triton 0.05% BSA at room temperature for 2 hours. After rinsing three times with PBS, the sections were mounted using DAPI Fluoromount-G for counterstaining of cell nuclei. The slides were imaged using nanozoomers.

[0212] result The aim of this study was to verify the ability of neural microtissue according to the invention to restore motor asymmetry through a non-clinical efficacy study. Results are shown in… Figure 6 and Figure 7 middle.

[0213] The inventors observed the number of rotations induced by d-amphetamine before surgery and at 4, 8, 12, 16, and 20 weeks (n=43) and 24, 28, and 32 weeks (n=2) post-transplantation. The number of rotations was measured using a rotameter starting 10 minutes after injection of d-amphetamine (2.5 mg / kg ip) and continued for 40 minutes. For all Cryo 10K (p<0.0001), Cryo 21K (p<0.0001), Fresh 3K (p<0.01), and Fresh 10K (p<0.0001), neuronal microtissue transplantation achieved full functional recovery of motor deficits starting at 16 weeks post-surgery. A delay was observed in the Cryo 6K group, which achieved full functional recovery only at 20 weeks (p<0.0001). Long-term functional motor recovery was observed, with amphetamine-induced rotational counts stabilizing between 16 and 32 weeks (n=2). Cryo 6K, Cryo 10K, and Cryo 21K corresponded to cryopreserved products with an estimated total number of 6,000, 11,000, and 21,000 dopaminergic neurons (TH+ cells) initially transplanted, respectively. Fresh 6K and Fresh 10K corresponded to fresh products with an estimated total number of 3,000 and 10,000 dopaminergic neurons initially transplanted, respectively. Comparisons with the solvent group were performed using two-way ANOVA and Tukey's multiple comparison test, lasting up to 20 weeks ( (p < 0.0001). Two-way ANOVA and Sidak multiple comparison test were used from week 24 to week 32. Data are presented as mean ± SEM.

[0214] Figure 7 The results shown are fluorescence microscopic images of graft sections 20 weeks after transplantation of the microtissue according to the invention, immunostained against the TH marker (B) and the human marker Stem121 (C). The fusion of the two markers (D) is also shown, confirming the presence of dopaminergic neurons derived from the microtissue. The area marked by the rectangle is a magnified field of view shown in the right-hand subplot (E), indicating host tissue reinnervation caused by graft-derived axonal projections.

[0215] Example 6 — Preparation of microtissues containing lumens .

[0216] The scheme is the same as that described in Example 1, except that each capsule contains 2 to 20 HiPSC cells (preferably 5) to limit internal pressure and promote better cell organization during capsule differentiation. The final cell concentration in the cell / matrix solution is 2.2 × 10^6 live cells / mL to 3.7 × 10^6 live cells / mL, which is referred to as the encapsulation density.

[0217] Examples of the obtained neural tissue units are shown in Figure 10 In A.

[0218] Example 7 — Characterization of a microtissue (10A) comprising a lumen and composed of various nerve cells according to the invention and Units of various nerve cells according to the invention (10B) that do not contain lumen .

[0219] The purpose of this study is to characterize the microtissues of the present invention obtained after neural differentiation.

[0220] Hematoxylin-eosin-saffron (HES) staining and histological characteristic scoring analysis Microtissue samples were fixed with 4% paraformaldehyde at room temperature for 1 hour, followed by washing in phosphate buffer. The microtissues were centrifuged (10 min, 1200 rpm) and pre-coated with 3% agarose. After dehydration in ethanol, acetone, and xylene baths, the samples were embedded in paraffin. Sections were prepared to a thickness of 5 μm using a microtome and then adhered to pre-treated slides using an albumin-glycerol mixture. After dewaxing, the sections were immersed sequentially in Harris hematoxylin, eosin, and safranin solutions. Following dehydration, the sections were processed using Entellar. ® The slide is fixed between a glass slide and a coverslip. Hematoxylin-eosin-safranin staining can be used to observe the morphology and structure of the tissue. The cytoplasm appears pink, and the nucleus appears blue-purple. The extracellular matrix is ​​stained yellow to pink.

[0221] The stained microtissue was imaged using a slide scanner (NanoZoomer 2.0RS, Hamamatsu), and assessed by a neuropathologist for the presence of physiological features of the human embryonic brain: ventricles (referred to as lumen in this invention), ventricular regions (referred to as C1 region in this invention), intermediate regions, and mantle regions (referred to as C2 region in this invention) (Arenas et al., 2015, https: / / doi.org / 10.1242 / dev.097394). Abnormal histological features were also noted, including abnormal nuclear morphology and nuclear rupture.

[0222] These micro-organisms are shown Figure 10 A (with lumen) and Figure 10 In B (without lumen).

[0223] Immunofluorescence labeling, microscopy and image analysis At the end of the neural differentiation method (day 24), encapsulated 3D neuronal microtissues with lumen were harvested for confocal microscopy. Samples with a final concentration of 20% capsules (v / v) in RelesR were incubated at room temperature for 5 minutes to remove alginate capsules. The microtissues with lumen were fixed with 4% PFA in the dark at room temperature for 1 hour. After fixation, the samples were washed three times with PBS containing 0.1% Tween 20. The samples were permeabilized by stirring (170 rpm) for 30 minutes at room temperature in PBS containing 5% Triton X-100. The samples were washed three times with PBS containing 0.1% Tween 20. The samples were incubated with appropriate primary and secondary antibodies in PBS containing 0.1% Tween 20 at room temperature (170 rpm) for 72 hours. After each incubation, the samples were washed five times with PBS containing 0.1% Tween 20, with two washes performed by stirring at 170 rpm for 30 minutes. These micro-organisms are shown Figure 12 , Figure 13 and Figure 14 The images were then captured using a fluorescence microscope with deconvolution capabilities or a confocal microscope.

[0224] The results are shown in Figures 12 to 14 This study confirmed the presence of dopaminergic neurons, which were not radially distributed around the lumen. Instead, dopaminergic progenitor cells (progenitor cells) were radially distributed around the lumen. Figure 12 ].

[0225] Figure 13 The presence of KI67-labeled cells in the neural tissue unit was confirmed.

[0226] Figure 14 The presence of dopaminergic progenitor cells (progenitor cells) around the lumen was confirmed, and these dopaminergic progenitor cells were located in the C1 cell layer adjacent to the lumen.

[0227] Figure 15 The size distribution of microtissues according to the invention on day 24 is shown. The microtissues were imaged using a wide-field microscope, and their dimensions were measured using proprietary image analysis software. The average diameter of the microtissues was 176.8 ± 60.48 μm.

[0228] Example 8 — Injection of microtissue containing a lumen for the treatment of Parkinson's disease .

[0229] The scheme is the same as that in Example 5, except that the injected micro-tissue contains a lumen. .

[0230] result The results are shown in Figure 16The images show fluorescence microscopic images of graft sections 20 weeks after microtissue transplantation, immunostained for TH markers, the human marker Stem121, and the nuclear dye DAPI. In rats transplanted with batch 1 (lubricated microtissue according to the invention) compared to rats transplanted with batch 2 (lubricated microtissue according to the invention), microtissue-derived TH+ dopaminergic neurons were more numerous and more evenly distributed in the grafts.

[0231] Subplot C shows a stereoscopic quantitative analysis of the total number of TH-expressing cells in the graft at 20 weeks, with data normalized according to the volume of injected luminous or non-luminous microtissue.

[0232] Subplot D shows a stereoscopic quantitative analysis of the total number of TH-expressing cells at 20 weeks post-transplantation, with data normalized according to the number of injected luminous or non-luminous microtissues.

[0233] Each point corresponds to one rat. A nonparametric Mann-Whitney test was performed; ns = p > 0.05. p<0.05 (C, D).

Claims

1. An in vitro method for differentiating pluripotent cells into neural cells via at least one three-dimensional cellular microcompartment in a suitable culture medium, the microcompartment containing the pluripotent cells, the method comprising at least one step of exposing the pluripotent cells to a substance: -At least two SMAD inhibitors, -At least one SHH activator, - at least one FGF activator, and -At least one Wnt activator, Wherein, for at least 3 days from the initial exposure of the pluripotent cells to the SMAD inhibitor, 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, to obtain neural cells expressing at least FOXA2.

2. The method according to the preceding claims, wherein the concentration of the at least one SMAD inhibitor increases exponentially, preferably the concentrations of at least two SMAD inhibitors increase exponentially.

3. The method according to any one of the preceding claims, wherein the concentration of the at least one SMAD inhibitor increases at most 4 days after the pluripotent cells are initially exposed to the SMAD inhibitor.

4. The method according to the preceding claim, wherein one day after the pluripotent cells are initially exposed to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 40%, and the concentration of the second SMAD inhibitor (ii) increases by at least 900%.

5. The method according to the preceding claim, wherein two days after the pluripotent cells are initially exposed to the SMAD inhibitor, 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%.

6. The method according to any one of the preceding claims, wherein the first SMAD inhibitor (i) is capable of acting on the BMP-2,4,7 pathway, and the second SMAD inhibitor (ii) is capable of acting on the TGFβ / activin / Nodal pathway.

7. The method according to 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. The method according to the preceding claims, wherein the concentration of the Noggin factor at initial exposure is 50 ng / mL to 70 ng / mL.

9. The method according to any one of claims 7 or 8, wherein the concentration of factor SB431542 at initial exposure is from 0.1 μM to 0.3 μM.

10. The method according to any 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, add 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, add the at least one FGF activator, and / or c. At least 6 days after the pluripotent cells are initially exposed to the at least one SMAD inhibitor, the at least one Wnt activator is added.

11. The method according to any one of the preceding claims, wherein the pluripotent cells are in isolated or aggregated form.

12. The method according to any one of the preceding claims, wherein the pluripotent cell is an induced pluripotent stem cell (iPSC).

13. The method according to any one of the preceding claims, the method further comprising exposing the obtained nerve 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 nerve cells expressing at least the FOXA2 / OTX2 marker.

14. The method according to the preceding claim, wherein the method includes an additional intermediate step lasting at least one day, during which the culture medium is free of TGF-β pathway inhibitors when the microcompartment contains at least 50% of the neural cells expressing at least the FOXA2 / OTX2 marker.

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