Neural differentiation from pluripotent cells

Abstract

The present invention relates to the field of cellular biology and more particularly that of cell culture. The invention thus relates to a method for differentiating pluripotent cells into neural cells that is suitable and optimized for three-dimensional cell culture. The neural cells thus obtained can in particular be used in cell therapy for treating a neurodegenerative disease, for example Parkinson's disease.

Description

Neural differentiation from pluripotent cells Technical field

[0001] The invention relates to the field of cell biology and more particularly to that of cell culture. The invention thus relates to a method for differentiating pluripotent cells into neural cells, adapted and optimized for three-dimensional cell culture. The plurality of neural cells obtained, in particular in the form of microtissue, can in particular be used in the context of cell therapy in order to treat a neurodegenerative disease, for example Parkinson's disease. State of the art

[0002] Pluripotent embryonic stem (ES) cells have proliferation and differentiation properties that make them a promising tool for cell therapy, but they raise many ethical questions. Indeed, these ES cells are mainly obtained from aborted fetal tissue. Their clinical use is therefore difficult to support, which is why many governments prohibit their use for clinical purposes.

[0003] The discovery in 2006 of induced pluripotent stem cells (iPS or iPSCs) by Professor Yamanaka gave new impetus to the field, overcoming most of the ethical problems associated with the use of these ES cells. Clinical trials were quickly launched, with the aim of treating diseases that are incurable by traditional medicine, such as neurodegenerative diseases. The transplantation of neural cells, preferably in the form of micro-tissue, obtained from iPSCs makes it possible to envisage the development of new curative treatments.

[0004] For example, according to the World Health Organization (WHO), more than 8.5 million people worldwide have Parkinson's disease, and cases are increasing faster than any other neurological disorder. However, due to the aging of the global population, the number of people with Parkinson's disease is expected to double between 2005 and 2030, and consequently, the societal cost, in the absence of a cure, will continue to grow worldwide.

[0005] Indeed, to date, there is no curative treatment to treat these diseases, only treatments which at best slow down its progression with very nuanced results, have been developed. For Parkinson's disease, L-Dopa and dopamine agonists can be mentioned.

[0006] The new discoveries and technologies deployed in recent years in the field of cell culture seem to be the most promising solution to date for developing a curative treatment, unlike traditional drug treatments, which also induce strong undesirable side effects.

[0007] In this context, the advances made in the field of cell culture of pluripotent cells allowing tissue regeneration, bring real hope for repairing damaged neuronal tissues and thus maintaining or even restoring neuronal activity. Methods for differentiating pluripotent cells into neural cells, in two dimensions or in three dimensions, are already known. However, these have the following disadvantages, namely very high cell mortality, or low differentiation efficiency. As an example, we can cite the differentiation method described in application US 2017 / 0130199.

[0008] Although these methods are adaptable to three-dimensional cell culture, they need to be perfectly optimized in order to overcome the aforementioned drawbacks, namely to reduce cell mortality and improve the yield and differentiation rate of neural cells, and also to improve the efficiency of the graft. Indeed, it is well known from the prior art the fragility of neural cells, in particular mature cells, for example dopaminergic neurons, requiring significant precautions in a clinical context. In addition, the simple transposition of a two-dimensional differentiation method into a three-dimensional system certainly makes it possible to overcome the problem linked to high cell mortality, nevertheless, at the expense of differentiation yield. Conversely, the known two-dimensional methods make it possible to obtain a good differentiation yield, at the expense of cell mortality.

[0009] Therefore, there is no known differentiation method specific to three-dimensional cell culture that overcomes all of these drawbacks, namely reducing cell mortality, minimizing as much as possible the risk of mortality during transplantation, and offering better differentiation yields than those currently known in two-dimensional cell culture.

[0010] There is therefore a need for a new method of differentiating pluripotent cells into neural cells that is particularly suitable and optimized for culture. three-dimensional cell, making it possible to overcome the drawbacks of two-dimensional (2D) differentiation methods known from the prior art, namely low production yield, high cell mortality, and a low differentiation rate. Summary of the invention

[0011] Also, to meet this need, the invention proposes a new method for differentiating pluripotent cells into neural cells, implemented by means of at least one three-dimensional cellular microcompartment, in which a step of exposing said pluripotent cells to a mixture of several factors, known to be involved in the differentiation of pluripotent cells into neural cells, namely at least one SMAD inhibitor, at least one SHH activator, one FGF activator, and one Wnt activator is implemented.

[0012] Although known taken individually or in combination in differentiation methods of the prior art, the present invention, through the kinetics of exposure of cells to said factors in a three-dimensional cellular microcompartment, allows to resolve the aforementioned drawbacks.

[0013] Indeed, the inventors have observed that the specific exposure of the pluripotent cells associated with a constant increase in SMAD-type factors for a determined period, prior to the addition and therefore the exposure of the cells with the other differentiation factors, makes it possible to improve the yield, the differentiation rate, in particular the proportion of dopaminergic neurons in the mixture obtained comprising a plurality of neural cells, reduce cell mortality, and improve the success of the grafting of this plurality of neural cells obtained by the method according to the invention.

[0014] Contrary to the teaching of the prior art which aims to expose the pluripotent cells with all the factors of interest, in particular the SMAD factors, at a given concentration and at a given time, the inventors have discovered that increasing the concentration in the culture medium of at least one SMAD inhibitor continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells with said SMAD inhibitor, makes it possible to induce a reduction in the quantity of neural stem cells in the mixture of neural cells obtained by the method according to the invention (reduction of the PAX6+ and / or SOX1+ marker), and an increase in the quantity of cells engaged in differentiation, such as dopaminergic progenitors. (FOXA2 and / or OTX2) in said mixture obtained, thus overcoming the aforementioned drawbacks.

[0015] Thus, the invention relates to an in vitro method for differentiating pluripotent cells into neural cells, implemented by means of at least one three-dimensional cellular microcompartment in a suitable culture medium, said microcompartment comprising said pluripotent cells, said method comprising at least one step of exposing said pluripotent cells to: at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator, in which the concentration in the medium of at least one SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells to said SMAD inhibitor, by at least 20%, preferably at least 30%, more preferably at least 40% relative to the initial concentration of said SMAD inhibitor, to obtain a plurality of neural cells, at least 30%,preferably at least 40% of the cells expressing at least the FOXA2 and / or OTX2 markers. The mixture obtained, consisting of a plurality of neural cells, at the end of the method thus comprises a greater quantity of cells positive for FOXA2 markers than with the 3D culture methods of the prior art, and a lower quantity of cells positive for PAX6 and / or SOX1 markers than with the 3D culture methods of the prior art.,

[0016] According to a preferred object of the invention, the increase of at least one SMAD inhibitor continuously or discontinuously is an exponential increase. Also, the concentration of at least one SMAD inhibitor is preferentially increased exponentially.

[0017] Advantageously, the method according to the invention aims to expose the pluripotent cells to at least two SMAD inhibitors, in which the concentration of the two SMAD inhibitors is increased, exponentially or logarithmically, compared to the initial exposure of the pluripotent cells to said SMAD inhibitors.

[0018] According to another preferred object, the concentration of at least one SMAD inhibitor is increased for at least 3 days and for at most 4 days, preferably at most 5 days, after the initial exposure of the pluripotent cells with said SMAD inhibitor, more preferably two SMAD inhibitors.

[0019] According to another particularly advantageous object, the method according to the invention exposes the pluripotent cells with two SMAD inhibitors, each SMAD inhibitor being capable of acting respectively on distinct cell signaling pathways. Thus, the first SMAD inhibitor (i) is capable of acting on the BMP-2,4,7 pathway. In particular, it prevents the activation of transcription by the SMAD-1,5,8 / SMAD-4 complex, in particular by preventing the interaction of SMAD-1,5,8 with the cofactor SMAD4, in particular by preventing the phosphorylation of SMAD-1,5,8. The second SMAD inhibitor (ii) is capable of acting on the TGFbeta, Activin, Nodal pathway. In particular, it prevents the activation of transcription by the SMAD-2,3 / SMAD-4 complex, in particular by preventing the interaction of SMAD-2,3 with the cofactor SMAD4, in particular by preventing the phosphorylation of SMAD-2,3.

[0020] Advantageously, the concentration of the first SMAD inhibitor (i) is increased by at least 20% and the concentration of the second SMAD inhibitor (ii) is increased by at least 450%, 1 day after the initial exposure of the pluripotent cells to said SMAD inhibitors, more preferably the concentration of the first SMAD inhibitor (i) is increased by at least 40% and the concentration of the second SMAD inhibitor (ii) is increased by at least 900%.

[0021] According to another preferred object of the invention, the concentration of the first SMAD inhibitor (i) is increased by at least 33% and the concentration of the second SMAD inhibitor (ii) is increased by at least 4950%, 2 days after the initial exposure of the pluripotent cells to said SMAD inhibitors, more preferably the concentration of the first SMAD inhibitor (i) is increased by at least 67% and the concentration of the second SMAD inhibitor (ii) is increased by at least 9900%.

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

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

[0024] According to another preferred object of the invention, the FGF activator, in particular FGF-8b, is also added at least 3 days and at most 5 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, advantageously at the same time as the SHH activator, i.e. during the initial exposure with at least one SHH activator.

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

[0026] Thus, the method according to the invention advantageously comprises the exposure of said pluripotent cells, in which: - the at least one SHH activator is added at least 3 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and / or - the FGF activator, in particular FGF-8b, is added at least 3 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and / or - at least one Wnt activator is added at least 6 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells.

[0027] When at least one SMAD inhibitor is added in order to expose the pluripotent cells with said SMAD inhibitor, these are preferentially in isolated form or in the form of aggregates. Furthermore, the cell culture is a three-dimensional cell culture and the cells are encapsulated in three-dimensional cellular microcompartments. Said microcompartments are well known and in particular described in patent application WO 2018 / 096277.

[0028] In the context of the invention, said three-dimensional microcompartments are suitable for the culture of pluripotent cells, comprising in particular said cells, a layer of extracellular matrix or extracellular matrix substitute. For example, said layer may be of the Matrigel® type or of the fibrin type and the microcompartment comprises an external layer of hydrogel, for example alginate.

[0029] Since the exposure of the pluripotent cells with at least one SMAD inhibitor can be carried out simultaneously, or after the encapsulation of said pluripotent cells, said pluripotent cells can thus be in the form of isolated cells, in the form of aggregates, or a mixture comprising isolated forms and aggregates.

[0030] According to another object of the present invention, the pluripotent cells can be of any type and capable of being differentiated into cells of interest, in particular neural cells, very preferably said pluripotent cells are induced pluripotent stem cells (iPSCs). It is particularly well documented in the prior art, the different cell culture techniques making it possible to obtain induced pluripotent stem cells (iPSCs), in particular in the articles 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 particular object of the invention, the method according to the invention may comprise an additional step of exposure with at least the factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination, to continue the maturation of the neural cells and obtain neural cells expressing at least FOXA2 and / or OTX2, allowing in particular the appearance of dopaminergic neurons expressing in particular the FOXA2 and TH+ markers.

[0032] Advantageously, the method according to the invention allows an increase in cells expressing FOXA2 and TH+ and a decrease in cells expressing PAX6 / SOX1, demonstrating respectively an increase in the number of mature neural cells, namely at least dopaminergic cells (neurons and dopaminergic progenitors), and a decrease in neural stem cells, which results in a better commitment of pluripotent cells in differentiation into neuronal cells. Finally, the absence of pluripotency markers, in particular TRA-1-60+ / OCT+, confirms the absence of pluripotent stem cells within the final product obtained at the end of the method, i.e. the plurality of neural cells in the form of micro-tissue. Such an absence reduces the risks of toxicities or uncontrolled differentiation.

[0033] Preferably, said factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination, are added for at least 5 days, more preferably, for at least 13 days.

[0034] According to another preferred subject, said factors selected from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination, are added at least 12 days after the initial exposure of the pluripotent cells with the SMAD factors.

[0035] According to another object of the invention, said method for differentiating pluripotent cells into neural cells comprises an intermediate step for at least 1 day during which the medium does not comprise inhibitors of the TGF-Beta pathway, when the microcompartment comprises at least 50% of neural cells expressing at least the FOXA2+ / OTX2+ factors, to eliminate the differentiation factors from the medium.

[0036] Finally, according to another aspect, the invention also relates to the plurality of neural cells obtained by the method according to any of the preceding embodiments. Preferably, the final product obtained by the method is a mixture of neural cells, including in particular dopaminergic neurons, dopaminergic progenitors, glial cells. Thus, the invention also relates to a heterogeneous population of neural cells, particularly of interest for the transplantation of neural cells. This heterogeneous population composed of a plurality of types of neural cells, preferably a striatal and / or cortical population, is very preferably in the form of an aggregate, or micro-tissue.

[0037] According to another aspect, the invention also relates to the plurality of neural cells, preferably in the form of a micro-tissue, for its use as a medicament, more preferably for its use in the prevention and / or treatment of Parkinson's disease.

[0038] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow. Brief description of the Figures

[0039] [Fig. 1] represents the steps of the differentiation method according to a particular embodiment of the invention.

[0040] [Fig. 2] represents the kinetics of appearance of the markers during the neurodifferentiation process according to the invention in a bioreactor, measured by flow cytometry. The selected differentiation days correspond to the day of encapsulation of the iPSCs (D0) and to the end of the phases of neural induction (D5), ventralization (post-rinsing) (D12), maturation (D17) and post-maturation (D24). The data are presented in histogram form and compile data from 16 different and independent bioreactors (n = 16).

[0041] [Fig. 3] represents the transcriptomic expression of the tyrosine hydroxylase (TH) gene relative to the expression of the housekeeping gene ACT-B (left panel), and the transcriptomic expression of the Engrailed (EN-1) gene relative to the expression of the housekeeping gene ACT-B (right panel), by RT-qPCR at different differentiation timings, namely at the end of the neural induction phase (D5), at the end of the ventralization (post-rinsing) phase (D12), at the end of the maturation phase (D17) and at the end of the post-maturation phase (D24) of the differentiation method according to the invention and of a method outside the invention not comprising the step of adding at least one SMAD inhibitor increased continuously or discontinuously for at least 3 days.

[0042] [Fig. 4] is a comparative analysis by flow cytometry (FACS) of the mixture of neural cells obtained at the end of a differentiation method outside the invention and by the differentiation method according to the invention (neuronal micro-tissue at D24). The results are presented in the form of a histogram. The increase in cells positive for the FOXA2 marker indicates an increase in the number of dopaminergic cells (neurons and dopaminergic progenitors), conversely the decrease in PAX6+ / SOX1+ positive cells indicates a decrease in the number of neural stem cells within the mixture of cells obtained. At D24, the neuronal micro-tissue obtained according to the method of the invention comprises less than 40%, more preferably less than 30% of cells expressing FOXA2.

[0043] [Fig. 5] represents the mRNA expression levels of the cells obtained by the method according to the invention composing the final product at D24, by RT-qPCR. The values ​​are expressed in proportion to those of the iPSCs (D0) by comparing the average of the Ct values ​​with that of 5 different housekeeping genes (ACTB, PSMB4, NONO, Clorf43 and YWHAZ). The experiments were carried out in duplicate and are represented as Tuckey Boxplot (n=17).

[0044] [Fig. 6] describes the ability of the plurality of neural cells, in the form of micro-tissue (lightless variant), obtained by the differentiation method according to the invention to restore motor symmetry in a hemi-Parkinsonian rat model, using the amphetamine-induced rotation test (rotometer). The graft transplantation is carried out unilaterally in the rat striatum, after lesion. The lesion was previously carried out by injection of a neurotoxin (6-OHDA) into the medial forebrain bundle (MFB). This lesion reproduces severe destruction of dopaminergic neurons and induces a unilateral motor deficit, similar to the motor symptoms of Parkinson's disease.

[0045] [Fig. 7] is an image of a section of a post-mortem graft, 20 weeks after transplantation of neural cells obtained according to the method according to the invention. A staining for TH, a marker of dopaminergic neurons (B), a staining for the human marker Steml21 (C), a section of the graft including both markers. The rectangle indicates the magnification area shown in the right panel (E). Scale bars, 2.5 mm (B, C and D) and 50 µm (E). Panel D represents reinnervation from the graft.

[0046] [Fig. 8] is a representation of a variant of a neural tissue unit obtained with the method according to the invention, with light, whose cells are stained for the markers FOXA2 and OTX2, as well as Ki67

[0047] [Fig. 9] is a representation of a variant of a neural tissue unit obtained with the method according to the invention, the cells of which are stained for the markers SOX2 and NESTIN

[0048] [Fig. 10] A and B) Hematoxylin-eosin-safranin (HES) staining of microtissues according to the invention at D24. A) Microtissue with lumen obtained by implementing the method according to the invention, showing a dense zone of nuclei, the Cl layer (dotted lines) organized radially around a lumen (“L”). B) Microtissue without lumen, obtained by implementing the method according to the invention. Scale bars: 50 pm.

[0049] [

[0050] [Fig. 11] Image of a solid neural microtissue variant without lumen in a microcompartment.

[0051] [Fig. 12] Immunofluorescence images of a microtissue variant according to the invention with lumen at D24. Neural microtissues were immunolabeled for the dopaminergic neuron marker TH (A), the dopaminergic progenitor marker FOXA2 (B) and counterstained with the nuclear dye DAPI (C). Scale bar: 50 pm. * indicates the presence of the lumen. Cells organized radially around the lumen are predominantly FOXA2 positive, and correspond to the C1 layer, while TH expression is localized to the periphery of the cells surrounding the lumen, corresponding to the C2 layer.

[0052] [Fig. 13] Immunofluorescence images of a microtissue variant obtained with the method according to the invention with light at D24. Neural microtissues were immunolabeled for the proliferative cell marker Ki67 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 pm. * indicates the presence of the lumen. Cells organized radially around the lumen are KI67 positive (approximately less than 50% of the cells).

[0053] [Fig. 14] Immunofluorescence images of microtissues obtained with the method according to the invention at D24. Neural microtissues were immunolabeled for the dopaminergic progenitor marker OTX2 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 pm. Asterisks (*) indicate the presence of the lumen. Cells organized radially around the lumen are predominantly OTX2 positive, corresponding to the Cl layer.

[0054] [Fig. 15], Size distribution of microtissues (light variant) obtained with the method according to the invention at D24. The microtissues were imaged using a wide-field microscope and their size was measured using image analysis software. The average diameter size of the microtissues is 176.8 ± 60.48 pm.

[0055] [Fig. 16], Post-mortem histological analysis of rats transplanted with microtissues according to the invention with light (Lot 1) and without light (Lot 2). A and B) represent brain sections of animals 20 weeks after transplantation with lot 1 (A) and lot 2 (B), immunostained for TH and the human marker Steml21. Scale bars: 2.5 mm. C, D) Quantification of the number of cells expressing TH per volume of injected microtissue (C) and the number of cells expressing TH per number of injected microtissues (D). The non-parametric Mann-Whitney test was performed. ns= p>0.05; * p<0.05. Detailed description of the invention

[0056] Definition

[0057] For the purposes of the invention, the term "microcompartment" or "capsule" means a partially or totally closed hollow three-dimensional structure containing one or more cells. The structure consists of an external hydrogel layer, preferably stiffened, and a hollow internal part comprising at least one cell and / or at least one aggregate of cells and / or cellular microtissue (or tissue unit), and optionally an extracellular matrix and / or extracellular matrix substitute suitable for cell culture and growth of said cells.

[0058] For the purposes of the invention, “human cells” means human cells or immunologically humanized non-human mammalian cells. Even where not otherwise specified, cells, pluripotent cells, progenitor cells and differentiated cells, including neural cells, are obtained or derived from human cells or immunologically humanized non-human mammalian cells.

[0059] By "pluripotent" cells within the meaning of the invention is meant cells which have the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such, given that they have already undergone a first step of differentiation, they can only generate the cells of the embryonic, endodermal, mesodermal, and ectodermal layers but can no longer generate the cells of the trophectoderm. Human pluripotent cells can be called hPSC or ES in the context of the present invention. They can in particular be induced pluripotent stem cells (iPSC or hiPSC for human induced pluripotent stem cells). The pluripotency of these cells can be evaluated by the presence of markers such as the 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, the pluripotent cells obtained from the embryonic stem cells are obtained without destruction of the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, said human embryonic stem cells may be excluded.

[0060] For the purposes of the invention, the term "induced pluripotent stem cell" or "iPSC" or "hiPSC" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles 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).

[0061] For the purposes of the invention, “neural” cells mean all cells of the nervous system. These may be mature cells, for example dopaminergic or GABAergic neurons, etc., or support cells known as glial cells (astrocytes, oligodendrocytes, etc.); or progenitor cells (dopaminergic progenitors, GABAergic etc.); or stem cells that give rise to neural tissue (neural stem cells). It can also be a mixture of neural-type cells, in particular a heterogeneous population of neural cells comprising several cell types, for example neurons, dopaminergic neurons, progenitors, neural stem cells, possibly glial cells.

[0062] For the purposes of the invention, the term "progenitor" cells means cells that are in the process of differentiation, i.e. already engaged in a differentiation pathway, but not yet differentiated. In the context of the invention, progenitor cells are, for example, radial glial cells, or radial glial progenitor cells (RGPCs), but also dopaminergic progenitors expressing at least the factor FOXA2.

[0063] For the purposes of the invention, "differentiated" cells are understood to mean cells that exhibit a particular phenotype, as opposed to pluripotent cells that are not differentiated or progenitor cells that are in the process of differentiating. In this context, differentiated cells are mature cells, for example neuronal cells, i.e. neurons, for example dopaminergic neurons, expressing at least the factors FOXA2 and TH.

[0064] For the purposes of the invention, the term "cell layer" or "cell layer" means several cells forming a layer or layer structured around a lumen; for example, it may be a set of cells cooperating with each other and grouped in three dimensions. The thickness of the cell layer or layer may be variable. This layer or layer is organized in three dimensions in the neural tissue unit.

[0065] By "micro-tissue", or "microtissue" or "neural tissue unit" or "tissue unit" within the meaning of the invention is meant at least one neural tissue unit comprising a plurality of neural cells obtained by the differentiation method according to the invention, the plurality of neural cells comprising for example dopaminergic neurons, progenitors, in particular dopaminergic progenitors, glial cells, said cells are organized in a three-dimensional network possibly in an extracellular matrix. Said micro-tissue can be encapsulated in a three-dimensional cellular microcompartment or decapsulated and capable of being implanted in the nervous system of a mammal, preferably a human.

[0066] For the purposes of the invention, "exponential increase" or "exponentially increased" means the exponential growth of a quantity, namely in the context of the invention, of a concentration of a differentiation factor, for example a SMAD inhibitor. This means the mathematical definition known to those skilled in the art characterized in that the growth follows an exponential law over time.

[0067] For the purposes of the invention, the term "logarithmic increase" or "logarithmically increased" means the logarithmic growth of a quantity, namely in the context of the invention, of a concentration of a differentiation factor, for example a SMAD inhibitor. This means the mathematical definition known to those skilled in the art, characterized in that the growth follows a logarithmic law over time.

[0068] For the purposes of the invention, the term "SMAD inhibitor" means a family of molecules involved in the signal transduction of transforming growth factor-beta (TGF-beta) and its analogues. There are at least 8 types of SMAD proteins in mammals, numbered from SMAD 1 to SMAD 8. When TGF-beta binds to its cell surface receptor, the latter phosphorylates SMAD 2 and SMAD 3, which can form a complex with SMAD 4 and then migrate into the nucleus. The SMAD complex thus formed binds to the promoter sequence of target genes to activate their transcription and thus mediate the biological effect of TGF-beta. Conversely, SMAD 6 and SMAD 7 proteins inhibit TGF-beta signal transduction. When BMP-2 binds to its cell surface receptor, it phosphorylates SMAD 1, SMAD 5, and SMAD 8, which can form a complex with SMAD 4 and then migrate into the nucleus.The SMAD complex thus formed binds to the promoter sequence of target genes to activate their transcription and thus mediate the biological effect of BMP-2. Conversely, SMAD 6 and SMAD 7 proteins inhibit BMP-2 signal transduction. For example, the SMAD inhibitor may be selected from Noggin, LDN193189, Dorsomorphin, DMH1, and A83-1, SB431542, SB505124, LY2157299, LY550410, and their combination.

[0069] For the purposes of the invention, the term "SHH activator" means a molecule capable of activating the Sonic Hedgehog signaling pathway. The Sonic hedgehog protein is, in mammals, one of the three proteins involved in the signaling pathway called Hedgehog; the SHH protein is the ligand of the Hedgehog signaling pathway which plays a key role in the regulation of vertebrate organogenesis, such as the growth of digits on limbs and the organization of the brain. The Sonic Hedgehog (SHH) signaling pathway has long been known to play a major role during embryonic development in vertebrates. Preferably, the SHH activator is chosen from SHH, SHH C25II, SAG (Smoothened Agonist), and Purmorphamine.

[0070] For the purposes of the invention, the term "FGF activator" means a molecule capable of activating the signal of the FGF receptor. The FGF receptor belongs to the family of transmembrane receptors whose intracellular part has tyrosine protein kinase activity. When the tyrosine kinase receptor binds its extracellular ligand, it becomes capable of phosphorylating intracellular proteins or other transmembrane receptors on certain tyrosine amino acids, thus allowing the transduction of the signal from the outside to the inside of the cell. The main tyrosine kinase receptors are the receptors for polypeptide growth factors (EGF, FGF, PDGF, VEGF, etc.). Preferably, the FGF activator is rhFGF-8b.

[0071] For the purposes of the invention, the term "Wnt activator" or "activator of the Wnt signaling pathway" means a molecule capable of activating the Wnt signaling pathway, which plays various roles in animal development and the maintenance of stem cells. Preferably, the Wnt activator is chosen from CHIR 99021, XAV939 and BIO.

[0072] By "prevention" within the meaning of the invention is meant the reduction to a lesser degree of the risk or probability of occurrence of a given phenomenon, for example, in the context of the present invention, Parkinson's disease.

[0073] For the purposes of the invention, the term "treatment" means a reduction in the progression of the disease, a stabilization, a reversal or regression, or even an interruption or inhibition of the progression of a disease, for example Parkinson's disease.

[0074] For the purposes of the invention, the term “Feret diameter” means the distance, in particular “d” or “D”, between two tangents, these two tangents being parallel, such that the entire projection is between these two parallel tangents.

[0075] For the purposes of the invention, "the largest dimension" of X means the value of the largest Feret diameter of X.

[0076] For the purposes of the invention, the term "light" or "lumen" means a substantially acellular volume, containing an aqueous solution, topologically surrounded by cells, in particular by at least one layer of cells forming a barrier to the circulation of fluids, characterized by the presence of tight junctions positive for the marker ZONULA OCCLUDENS 1 or ZO-1 bordering said light.

[0077] Process of differentiation of pluripotent cells into neural cells

[0078] The present invention therefore relates to an in vitro method for differentiating pluripotent cells into neural cells, implemented by means of at least one three-dimensional cellular microcompartment in a suitable culture medium, said microcompartment comprising said pluripotent cells, said method comprising at least one step of exposing said pluripotent cells to: - at least two SMAD inhibitors, - at least one SHH activator, - at least one FGF activator, and - at least one Wnt activator, wherein the concentration in the culture medium of at least one SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells to said SMAD inhibitor, by at least 20%, preferably at least 30%, more preferably at least 40%, relative to the initial concentration of said SMAD inhibitor, to obtain a plurality of neural cells, at least 30%, preferably at least 40% of the neural cells expressing at least the following factors FOXA2 / OTX2.

[0079] In the context of the invention, the pluripotent cells are encapsulated in three-dimensional microcompartments or capsules allowing three-dimensional cell culture, making it possible to approach as closely as possible the physiological conditions in vivo. The advantages of three-dimensional cell culture are now well described and well known to those skilled in the art.

[0080] The method may comprise a prior step of cell culture of the pluripotent cells, followed by the encapsulation of said pluripotent cells in three-dimensional cellular microcompartments. The three-dimensional cellular microcompartments are notably described in patent application WO 2018 / 096277. Briefly, the encapsulation may advantageously comprise the following steps: a. encapsulating the mixture comprising the pluripotent cells, a culture medium and an extracellular matrix or extracellular matrix substitute, in an external hydrogel layer, the encapsulation comprising the following sub-steps: i. bringing said mixture into contact with a hydrogel solution intended to form said external layer to form at least one drop, and ii. collecting the drop obtained in a calcium bath capable of stiffening said hydrogel solution to form the outer layer of each microcompartment. b. culturing the capsules obtained in the previous step in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days, and c. recovering the cellular microcompartments obtained, comprising the pluripotent cells of interest.

[0081] Preferably, the encapsulation step is carried out by simultaneous co-injection of the hydrogel solution intended to form the outer layer, of the mixture comprising in particular the pluripotent cells and possibly the extracellular matrix or extracellular matrix substitute, and optionally of an intermediate solution. Said co-injection is carried out concentrically via a microfluidic or millifluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking up into drops. The drops are then recovered in the calcium bath, by gravity, making it possible to stiffen the hydrogel solution to form the outer layer and, consequently, the cellular microcompartment comprising the pluripotent cells of interest. Said pluripotent cells may be in isolated form or in the form of cell aggregates or clusters.Also, the encapsulated pluripotent cells are suspended, in the capsule, in the form of single or isolated cells and / or clusters or aggregates of cells. Preferably, the isolated cells represent less than 50% in number of the total encapsulated cells, more preferably the isolated cells are iPSCs.

[0082] Once the cells of interest are encapsulated, advantageously in isolated and / or aggregate form, said cells will be 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. Said differentiation agents are added directly into the culture medium comprising said microcompartments, preferably in a bioreactor. Said differentiation agents diffuse through the outer layer of the cellular microcompartments, the latter being permissive to their diffusion. Also, said differentiation agents diffuse inside each cellular microcompartment, within the culture medium present in said cellular microcompartment.

[0083] In this context, the inventors have developed a process to improve the yield in three-dimensional cell culture, to improve the differentiation rate, thus improving the success of the grafting of the mixture of neural cells obtained by said method according to the invention, and to reduce cell mortality compared to the teaching of the prior art.

[0084] To achieve this, the inventors observed that, exposing the encapsulated pluripotent cells with at least one SMAD inhibitor added gradually, i.e. its concentration is increased gradually before the addition of the other differentiation factors, namely the SHH activators, the FGF activator and the Wnt activator, which overcomes the drawbacks of the prior art.

[0085] Thus, the invention relates to the addition of at least one SMAD inhibitor, preferably two SMAD inhibitors, progressively before the start of neural differentiation, initiated by the addition of the other factors, namely the SHH activators, the FGF activator and the Wnt activator.

[0086] Conversely, the prior art teaches us that after the step of culturing the pluripotent cells with the objective of their amplification, said pluripotent cells obtained are exposed to all the differentiation factors at high concentration, in particular the combination of the following factors: SMAD inhibitors, SHH activators and FGF activator. The exposure of the pluripotent cells certainly allows differentiation into neural cells but at the cost of high cell mortality. Indeed, sudden exposure of cells not yet expressing the receptors of the pathway induced by the differentiation factor, to a high concentration, induces significant cellular stress which induces a high rate of cell mortality. In addition, the production yield is not satisfactory with a low rate of differentiation.Conversely, a progressive increase in these factors promotes the compliance and resilience of cells to the differentiation process, i.e. the capacity of cells to respond to a signal, thus reducing cell mortality.

[0087] Thus, the present invention aims to expose said pluripotent cells, expressing in particular OCT / NANOG, SSEA5 / SSEA4, TRA-1-60 / OCT, encapsulated, with at least one SMAD inhibitor at T0. Said SMAD inhibitor is added at a lower concentration than the concentration known in the prior art, in order not to cause cellular stress and significant cellular mortality. Advantageously, the concentration of said SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the exposure initial concentration of pluripotent cells to said SMAD inhibitor, more preferably at least 40% relative to the initial concentration of said SMAD inhibitor.

[0088] By "continuous increase" within the meaning of the invention, we mean an increase in the concentration of the differentiating factor of interest in a linear manner over time, preferably in a given period, for example over 3 days.

[0089] Conversely, by "discontinuous increase" within the meaning of the invention, we mean an increase in the concentration of the differentiating factor of interest in stages until the final concentration of interest is obtained, over a given period, for example 3 days.

[0090] The increase in the concentration of the SMAD inhibitor can take several forms. Thus, the increase can follow an exponential law or a logarithmic scale. Very preferably, the increase in the concentration of at least one SMAD inhibitor is exponential. Also, the concentration of at least one SMAD inhibitor is, preferably, increased exponentially, making it possible to reduce the cell mortality of the encapsulated cells of interest and to improve the differentiation rate.

[0091] Preferably, the concentration of at least one SMAD inhibitor is increased for at most 4 days, more preferably at most 5 days after the initial exposure of the pluripotent cells to said SMAD inhibitor.

[0092] According to another object of the invention, the pluripotent cells are exposed with at least two SMAD inhibitors, at least a first SMAD inhibitor (i) is increased continuously or discontinuously, preferably exponentially, and the pluripotent cells are exposed with a second SMAD inhibitor (ii), the concentration of said SMAD inhibitor (ii) may be constant over time or increased continuously or discontinuously. Thus, the concentration of at least two SMAD inhibitors is preferentially increased, compared to the initial exposure concentration of the pluripotent cells to said SMAD inhibitors.

[0093] When the concentration of the second SMAD inhibitor (ii) is increased continuously or discontinuously, the increase in concentration may be exponential or logarithmic. Preferably, the increase in the concentration of the second SMAD inhibitor (ii) is logarithmic, this has the effect of maintaining the pluripotent cells and preventing the pluripotent cells from engaging in an undesired pathway, for example the pathway for engaging the pluripotent cells towards the mesendoderm. According to a particularly preferred subject, 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.

[0094] Preferably, the concentration of the two SMAD inhibitors is increased for at most 4 days, more preferably at most 5 days after the initial exposure of the pluripotent cells to said SMAD inhibitor.

[0095] Also, the present invention relates to a differentiation method comprising a step of exposing the encapsulated pluripotent cells with at least one, preferably at least two SMAD inhibitors, in which the concentration of each SMAD inhibitor is increased for a given period, advantageously between 3 and 5 days. During this period, the concentration of a first SMAD inhibitor (i) is advantageously increased exponentially, while the concentration of a second SMAD inhibitor (ii) can be stable or increased, preferably increased according to a logarithmic scale.

[0096] According to another object of particular interest, 1 day after the initial exposure of the pluripotent cells to said SMAD inhibitors, the concentration of a first SMAD inhibitor (i) is increased by at least 40% and the concentration of the second SMAD inhibitor (ii) is increased by at least 900%, compared to the initial concentration of said SMAD inhibitors, i.e. at 0 day, i.e. the initial exposure of the SMAD inhibitors with the encapsulated pluripotent cells of interest.

[0097] Preferably, 2 days after the initial exposure of the pluripotent cells to said SMAD inhibitors the concentration of the first SMAD inhibitor (i) is increased by at least 67% and the concentration of the second SMAD inhibitor (ii) is increased by at least 9,900%, compared to the initial concentration.

[0098] According to a preferred object of the invention, the SMAD inhibitor may be chosen from a SMAD inhibitor (i) capable of acting on the BMP-2,4,7 pathway and a SMAD inhibitor (ii) capable of acting on the TGFbeta, Activin, Nodal pathway.

[0099] The SMAD inhibitor (i) capable of acting on the BMP-2,4,7 pathway advantageously makes it possible to prevent the activation of transcription by the SMAD-1,5,8 / SMAD-4 complex, in particular by preventing the interaction of SMAD-1,5,8 with the cofactor SMAD4, in particular by preventing the phosphorylation of SMAD-1,5,8.

[0100] The SMAD inhibitor (ii) capable of acting on the TGFbeta, Activin, Nodal pathway advantageously prevents the activation of transcription by the SMAD- complex 2,3 / SMAD-4, in particular by preventing the interaction of SMAD-2,3 with the cofactor SMAD4, in particular by preventing the phosphorylation of SMAD-2,3.

[0101] Preferably, the SMAD inhibitor may be chosen from Noggin, LDN193189, Dorsomorphin, DMH1, and A83-1, SB431542, SB505124, LY2157299, LY550410, and their combination.

[0102] Particularly preferably, the first SMAD inhibitor (i) is selected from the factors Noggin, LDN193189, Dorsomorphin, DMH1, A83-1, and their combination. Particularly preferably, the second SMAD inhibitor (ii) is selected from the factors SB431542, SB505124, LY2157299, LY550410, and their combination.

[0103] By "Noggin" as used herein is meant a secreted homodimeric glycoprotein that binds to and inactivates members of the transforming growth factor beta (TGF-) signaling protein superfamily, such as bone morphogenetic protein 4 (BMP4). Noggin protein 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) Nat Methods 2, 185-190; Wang, et al. (2005) Biochem Biophys Res Commun 330, 934-942).

[0104] For the purposes of the invention, “LDN193189” or “LDN-193189” means a chemical analog of the Noggin protein, an inhibitor of the bone morphogenetic pathway (BMP), which inhibits ALK1, ALK2, ALK3 and ALK6. It is a derivative of dorsomorphin which is generally used at concentrations approximately 100 times lower (Sanvitale et al.; Vogt et al.). This compound notably promotes the differentiation of neural progenitor cells from human pluripotent stem cells (Chambers et al.; Kriks et al.), promotes the differentiation of neural crest cells from human pluripotent stem cells (Kreitzer et al.), promotes the differentiation of foregut endoderm from definitive endoderm derived from human and mouse pluripotent stem cells (Kearns et al.), promotes the differentiation of inner ear sensory epithelial cells from mouse embryonic stem cells (Koehler et al.). The compound is notably accessible via its CAS number 1062368-24-4.

[0105] For the purposes of the invention, "Dorsomorphin" means an AMPK inhibitor, of formula C24H25N5O, under the name 6-[4-[2-(l-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[l,5-a]pyrimidine dihydrochloride. Dorsomorphin inhibits the BMP pathway by targeting type I receptors ALK2, ALK3 and ALK6. It is a compound analogous to the Noggin protein and compound LDN193189. It is accessible via CAS number 866405-64-3.

[0106] For the purposes of the invention, “DMH1” means a small molecule inhibitor of the highly selective BMP pathway promoting neurogenesis in hiPSCs, accessible via CAS number 1206711-16-1.

[0107] For the purposes of the invention, "A83-1" means a selective inhibitor of the TGF-β type I ALK receptor, of the formula 3-(6-Methyl-2-pyridinyl)- / V-phenyl-4-(4-quinolinyl)-l / 7-pyrazole-l-carbothioamide. In particular, it inhibits TGF-β-induced epithelial-mesenchymal transition (EMT) via inhibition of Smad2 phosphorylation. Previous studies have shown that A83-01 significantly promotes somatic cell reprogramming.

[0108] For the purposes of the invention, “SB431542” or “SB-431542” means an inhibitor of activin receptor-like kinases, ALK5, ALK4 and ALK7. It is used in particular in combination with LDN193189, CHIR99021 and DAPT to transform astrocytes into neurons. It is accessible via CAS number 301836-41-9.

[0109] As used herein, "SB505124" or "SB-505124" also means a selective inhibitor of transforming growth factor beta type I receptors ALK4, ALK5 and ALK7. It selectively and concentration-dependently inhibits ALK4-, ALK5- and ALK7-dependent activation of downstream cytoplasmic signal transducers Smad2 and Smad3 and components of the TGF-beta-activated protein kinase pathway, but does not impair ALK1-, ALK2-, ALK3- or ALK6-mediated Smad signaling. SB-505124 is three to five times more potent than the previously described related ALK5 inhibitor, SB-431542.

[0110] For the purposes of the invention, "LY2157299" or "galunisertib" means a small molecule inhibitor of the TGF-beta signaling pathway, in particular of the TGF-β receptor I, which specifically regulates the phosphorylation of SMAD2, thereby abrogating the activation of the canonical pathway.

[0111] For the purposes of the invention, the term “LY550410” is also understood to mean a small molecule inhibitor of the TGF-beta signaling pathway, in particular of the TGF-β receptor I.

[0112] Thus, very preferentially, the concentration of the first SMAD inhibitor (i) chosen from the factors Noggin, LDN193189, Dorsomorphin, DMH1, A83-1, and their combination, is increased exponentially for a period of between 3 and 5 days after the initial exposure of said SMAD inhibitor with the encapsulated pluripotent cells, i.e. at T0.

[0113] According to another highly preferred subject, the concentration of the second SMAD inhibitor (ii) selected from factors SB431542, SB505124, LY2157299, LY550410, and their combination, is increased logarithmically for a period of between 3 and 5 days after the initial exposure of said SMAD inhibitor with the encapsulated pluripotent cells.

[0114] According to another preferred subject of the invention, the first SMAD inhibitor (i) is the Noggin factor, the concentration of which during the initial exposure with the encapsulated pluripotent cells is between 50 and 70 ng / mL. The concentration of Noggin is then advantageously increased exponentially for at least 3 days after the initial exposure with the encapsulated pluripotent cells to a concentration of between 50 and 70 ng / mL. Preferably, the maximum added concentration of the Noggin factor is 100 ng / mL.

[0115] According to another preferred object of the invention, the second SMAD inhibitor (ii) is the factor SB431542, the concentration of which during the initial exposure with the encapsulated pluripotent cells is between 0.1 and 0.3 pM. The concentration of SB431542 is then advantageously increased exponentially or logarithmically for at least 3 days after the initial exposure with the encapsulated pluripotent cells to a concentration of between 50 and 70 ng / mL until a maximum added concentration of the factor SB431542 of 20 pM is reached.

[0116] Thus, unlike known 2D or 3D methods, the exposure of encapsulated pluripotent cells with at least one SMAD inhibitor in 3D culture, preferably two inhibitors, and the gradual increase in the concentration of said inhibitors prior to the addition of the other differentiation factors, in particular the SHH activator and the FGF activator, makes it possible to overcome the drawbacks of the prior art, namely reducing cell mortality and concomitantly improving the differentiation yield. By "prior to the addition of the other differentiation factors" within the meaning of the invention, is meant the exposure of at least one SMAD inhibitor at least 3 days before the addition of the other differentiation factors, in particular the SHH activator and the FGF activator.

[0117] This delayed exposure over time of the different differentiation factors with the pluripotent cells, combined with an increase for at least 3 days and at most 5 days, of the concentration of at least one SMAD inhibitor, preferably at least two SMAD inhibitors, allows to improve the yield, obtained both by the reduction of cell mortality as well as a better rate of differentiation of the cells of interest, facilitating in addition, the synchronization of the different cell populations obtained in the capsule. Indeed, pluripotent cells are constantly cycling. However, the phase of the cycle impacts the cellular response to a given signal. Therefore, the exposure of an increasing concentration over a characteristic cell cycle time (24h) optimizes the probability that the cells receive an optimized signal at the time of the peak of their competence.

[0118] At least 3 days and at most 5 days after the initial exposure of the pluripotent cells with at least one SMAD inhibitor, the differentiation method comprises a step of exposing the pluripotent cells engaged in the differentiation process, with at least one SHH activator, and at least one FGF activator. Preferably, the method comprises exposure to at least two SHH activators. Finally, the exposure of the pluripotent cells with the SMAD inhibitors is maintained throughout the exposure period with at least one SHH activator and at least one FGF activator.

[0119] Thus, at least 3 days and at most 5 days after the initial exposure of the pluripotent cells with at least two SMAD inhibitors, the pluripotent cells are exposed with the SMAD inhibitors at the maximum concentration to which the cells at D3-D5 are subjected.

[0120] When the SMAD inhibitors are Noggin and SB431542 respectively, the concentration to which the cells are exposed after a period of between 3 and 5 days of differentiation is respectively 100ng / mL and 20 pM for a period of between 9 and 12 days.

[0121] The SHH activator is particularly useful for activating the Sonic Hedgehog signaling pathway. The canonical Shh protein signal is mediated by a multicomponent receptor that includes Patched (PTCHI, PTCH2) and Smoothened (SMO). Binding of Shh protein to PTCH inactivates the basal repression of SMO by PTCH. Shh protein, SHH-C25II, SAG (Smoothened Agonist), and Purmorphamine can be used to activate this pathway. This pathway is involved in the patterning of the developing central nervous system. SHH protein regulates the fate of neural stem cells through tissue morphogenesis.

[0122] FGF-8b is a member of the fibroblast growth factor family. FGF-8b is widely expressed during embryogenesis and regulates epithelial-mesenchymal transitions. FGF-8b plays a role in organizing and triggering the gastrulation, and is also involved in the structuring of the midbrain / hindbrain.

[0123] Unlike SMAD inhibitors, the concentration of said SHH activator and FGF activator is stable over time. Preferably, the duration of exposure of said SHH activator and said FGF activator is thus between 9 and 12 days, from the exposure of the SHH activator and the FGF activator with the cells already engaged in differentiation expressing in particular a lower quantity of the OCT / NANOG and TRA-1-60 / OCT markers than the pluripotent cells.

[0124] Thus, at least one SHH activator is advantageously added at least 3 days and at most 5 days after the initial exposure of at least one SMAD inhibitor and encapsulated pluripotent cells. Preferably, at least two SHH activators are added.

[0125] According to an object of the invention, at least one FGF activator is advantageously added between 3 and 5 days after the initial exposure of at least one SMAD inhibitor and encapsulated pluripotent cells, very preferably concomitantly or simultaneously with the exposure of the cells with at least one SHH activator, more preferably two SHH activators. Thus, the FGF activator is preferentially added between 3 and 5 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells.

[0126] According to a particularly preferred subject of the invention, at least one SHH activator is the SHH factor C25II, at a set concentration of 100 ng / mL.

[0127] According to another particularly preferred subject of the invention, at least one SHH activator is the Purmorphamine factor, at a set concentration of 2 pM.

[0128] According to a preferred subject of the invention, the FGF activator is the rhFGF-8b factor, at a concentration of 100 ng / mL.

[0129] The method according to the invention also comprises a step of exposing the cells undergoing differentiation with a Wnt activator. This is preferably added in order to expose the cells undergoing differentiation, namely the cells expressing at least the PAX6 / SOX1 factors, i.e. radial glial cells, or radial glial progenitor cells (RGPCs), which are bipolar progenitor cells responsible for the production of all the neurons of the cerebral cortex and also produce certain glial cell lines, in particular astrocytes and oligodendrocytes.

[0130] Preferably, the Wnt activator is the factor CHIR 99021, at a set concentration of 3 pM.

[0131] According to another object, the concentration of at least one of the factors chosen from the SHH activator, the FGF activator, and the Wnt activator, is between 30 and 300% of the set concentration, preferably between 50 and 200% of the set concentration, more preferably between 90 and 110% of the set concentration.

[0132] According to a preferred subject of the invention, the Wnt activator is added at least 6 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells. According to a variant, the Wnt activator is added at least 3 days after exposure with at least one SHH activator and one FGF activator and the pluripotent cells engaged in differentiation expressing at least the PAX6 / SOX1 factors. According to another variant, at least one Wnt activator is added when the cells are radial glial progenitor cells.

[0133] After a period of between 12 and 17 days of exposure of the encapsulated pluripotent cells with at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator, a heterogeneous population of neural cells is obtained. This population comprises a plurality of neural-type cells, of which at least 30%, preferably 40%, express the FOXA2 and / or OTX2 marker.

[0134] Preferably, the method may include an intermediate step to eliminate the differentiating factors from the mixture. Advantageously, the intermediate step lasts between 12 and 48 hours, very advantageously 24 hours.

[0135] According to another object of the invention, the differentiation method may comprise an additional maturation step, during which the cells obtained are exposed with at least the differentiation factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination.

[0136] The present invention therefore also has as another subject, an in vitro method for differentiating pluripotent cells further comprising the exposure of the neural cells obtained after a period of between 12 and 17 days of exposure of the encapsulated pluripotent cells with at least two SMAD inhibitors, with at least the factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination.

[0137] Also, after a period of between 12 and 17 days of exposure of the encapsulated pluripotent cells with at least one SMAD inhibitor, the obtained neural cells are exposed with the differentiation factors (called maturation differentiation factors or maturation factors) chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination. Said maturation factors are added for at least 5 days at a concentration stable over the at least 5 days of exposure.

[0138] According to another object, said maturation factors are added for at most 30 days, preferably at most 11 days, even more preferably at most 5 days, at a stable concentration.

[0139] Advantageously, the rhBDNF factor is added for at least 12 days at a concentration. According to a particularly preferred subject, rhBDNF is added at a set concentration of 10 ng / mL

[0140] According to a particularly preferred object, L-Ascorbic acid is added at a set concentration of 200 pM.

[0141] According to a particularly preferred subject matter, rhGDNF is added at a set concentration of 10 ng / mL

[0142] According to a particularly preferred subject matter, rhTGF-Beta3 is added at a set concentration of 1 ng / mL.

[0143] According to a particularly preferred subject matter, rhFGF-20 is added at a set concentration of 5 ng / mL

[0144] According to a particularly preferred object, dbcAMP is added at a set concentration of 0.5 mM

[0145] According to a particularly preferred object, DAPT is added at a set concentration of 10 pM

[0146] According to a particularly preferred object, Compound E is added at a set concentration of 1 pM.

[0147] According to a particularly preferred object, Trichostatin A is added at a set concentration of 10 nM.

[0148] According to another object, the concentration of at least one of the factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E and Trichostatin A, is between 30 and 300% of the set concentration, preferably between 50 and 200% of the set concentration, more preferably between 90 and 110% of the set concentration.

[0149] Preferably, at the end of the exposure of the neural cells with the maturation factors chosen from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination, the method comprises a step additional rinsing. This step aims to avoid the activation and concomitant inhibition of the TGF-Beta pathway.

[0150] Indeed, according to another object, the method preferably comprises an additional rinsing step for at least 1 day when the microcompartment comprises at least 50% of neural cells expressing at least the FOXA2 and / or OTX2 markers.

[0151] Micro-fabric obtained by the process according to the invention

[0152] At the end of the differentiation method according to any of the embodiments previously described, a plurality of neural cells is obtained, this plurality of neural cells is organized in three dimensions and thus forms a micro-tissue or neural tissue unit. Thus, according to another aspect, the invention also relates to a micro-tissue consisting of a plurality of neural cells organized in three dimensions and the extracellular matrix, said neural cells are capable of being obtained by the differentiation method of the present invention. This plurality of neural cells advantageously comprises at least dopaminergic neurons, progenitor cells, in particular dopaminergic progenitors, and glial cells.

[0153] This plurality of neural cells expresses several markers, specific to the cell types included in the plurality of neural cells obtained by the differentiation process. Preferably, the plurality of neural cells thus constituting the microtissue presents a greater quantity of cells positive for the FOXA2 marker and a lower quantity of cells positive for the PAX6 / SOX1 markers, indicating an increase in the number of dopaminergic cells and a decrease in neural stem cells within the microtissue, which reflects a better commitment of differentiation towards the dopaminergic pathway and therefore a better differentiation rate. In addition, a greater expression of the EN1 marker, a population that has been demonstrated to play a key role in the efficacy of the graft (Kirkeby et al., 2017), reflects the obtaining of a microtissue with improved functionality.In addition, greater expression of the TH marker, a marker of dopaminergic neurons, reflects a better commitment of differentiation towards the dopaminergic pathway and therefore a better rate of differentiation.

[0154] Alternatively, the neural tissue unit may be solid, without lumen. This variant is preferentially obtained when, at the time of cell encapsulation before differentiation, more than 20 cells are encapsulated per capsule.

[0155] According to another variant, the neural tissue unit may comprise at least one light. This variant is preferentially obtained when at the time of encapsulation of the cells before differentiation, at most 20 cells are encapsulated per capsule.

[0156] According to a particular variant, the invention relates to a neural tissue unit organized in three dimensions, comprising at least dopaminergic neurons, and neural progenitor cells and possibly neural stem cells, said unit has a largest dimension less than 600 μm and comprises at least one lumen bordered by a layer C1 of cells comprising mainly neural progenitor cells expressing at least SOX2. Preferably, layer C1 comprises neural progenitor cells expressing OTX2. Preferably, the neural progenitor cells of layer C1 express at least SOX2 and OTX2.

[0157] Said neural tissue unit thus comprises at least one lumen which is bordered by the layer C1 of cells comprising predominantly neural progenitor cells expressing at least SOX2. In other words, the neural tissue unit comprises a layer C1 of cells forming a border juxtaposed to said lumen. Said layer C1 of cells being a concentric layer around said lumen.

[0158] The presence of light within said neural tissue unit allows in particular better cell survival and preserves the particular physiological cytoarchitecture of such tissue. During transplantation, such micro-tissue contributes to improving the differentiation potential and / or survival of TH+ cells, i.e. dopaminergic neurons, thus improving the survival rate and the grafting of the neural tissue unit according to the invention.

[0159] According to a preferred object, the C1 layer at the edge of the lumen is itself surrounded by a layer or layer of C2 cells which comprises dopaminergic neurons. The C2 layer or layer of cells advantageously comprises at least 3%, at least 5%, at least 10%, at least 15% of dopaminergic neurons. The C2 cell layer thus forms a second concentric layer around the C1 layer. Very advantageously, the C2 cell layer comprises between 3% and 50%, even more advantageously between 15% and 50% of dopaminergic neurons, these expressing at least the tyrosine hydroxylase (TH) marker.

[0160] Also, preferentially, several concentric layers are therefore formed around the light, namely: - a layer of border cells (the Cl cell layer), and - around said border still at least one concentric layer distinct from the layer Cl which includes dopaminergic neurons (layer C2).

[0161] According to a variant, the microtissue according to the invention may comprise several lumens, at least one lumen of which is surrounded by a layer of border cells (the cell layer C1), and around said border still at least one concentric layer distinct from the layer C1 which comprises dopaminergic neurons (the layer C2). Preferably, if it comprises several lumens, each lumen is surrounded by a layer of border cells (the cell layer C1), and around said border still at least one concentric layer distinct from the layer C1 which comprises dopaminergic neurons (the layer C2).

[0162] Very preferentially, dopaminergic neurons are substantially excluded from the periluminal zone, that is to say from the zone comprising the lumen and the Cl layer. Indeed, dopaminergic neurons are present within a second layer, topologically more external, which can be described as the second layer cortical zone.

[0163] By "substantially excluded", for the purposes of the invention, we mean that the periluminal area may contain a few dopaminergic neurons. In other words, we cannot exclude the presence of at least one dopaminergic neuron, in particular between 1 and 50 dopaminergic neurons in the periluminal area.

[0164] For the purposes of the invention, the term “second-layer cortical zone” means in particular the C2 cell layer, excluding any zone directly bordering the lumen.

[0165] Preferably, the C2 layer therefore comprises at least 15% of cells positive for tyrosine hydroxylase (TH). The TH gene is a marker for dopaminergic neurons. Thus, the C2 layer comprises at least 15% of cells expressing this marker and therefore at least 15% of dopaminergic neurons are present in the C2 layer.

[0166] According to another object of the present invention, at most 50% of the cells included in the neural tissue unit express the Ki-67 marker, preferably, between 5 and 50% of said cells express the Ki-67 marker. Advantageously, layer C1 has a ratio of cells expressing KI67 higher than layer C2.

[0167] According to another particularly preferred subject, the neural tissue unit organized in three dimensions, comprises at least dopaminergic neurons and neural progenitor cells, has a largest dimension less than 600 pm, and comprises at least one lumen bordered by a layer Cl of cells consisting essentially of neural progenitor cells expressing at least SOX2.

[0168] Preferably, the microtissue obtained according to the invention has a substantially spheroid shape.

[0169] Alternatively, the neural tissue unit obtained according to the invention has an ovoid, sphere, ball or teardrop shape or a substantially ovoid, substantially ball or substantially teardrop shape.

[0170] Preferably, the neural tissue unit obtained according to the invention has a larger dimension of between 10 pm and 600 pm plus or minus 10%, preferably between 150 pm and 400 pm plus or minus 10%, more preferably between 100 pm and 300 pm plus or minus 10%, even more preferably 200 pm plus or minus 10%. These dimensions are particularly favorable to the survival of neurons within the neural tissue unit and optimize the reorganization as well as the vascularization of the graft after implantation.

[0171] According to another preferred subject of the invention, the neural tissue unit obtained according to the invention comprises neural progenitor cells, in particular dopaminergic progenitors, which express FOXA2 and / or OTX2 and / or LMX1A, in addition to the marker SOX2. Very preferably, the cell layer C1 comprises neural progenitor cells which express FOXA2 and / or OTX2 and / or LMX1A, in addition to the marker SOX2, even more preferably, in the case of a tissue unit comprising a lumen and a layer C1 and C2, the cell layer C1 comprises neural progenitor cells which express FOXA2 and / or OTX2 and / or LMX1A and SOX2.

[0172] According to an object of the invention, in the case of a tissue unit comprising a lumen and a layer C1 and C2, the cell layer C1 comprises neural progenitor cells which express SOX2 and FOXA2, or SOX2 and OTX2, or SOX2 and LMX1A. According to a variant, the cell layer C1 comprises neural progenitor cells which express SOX2 and FOXA2 and OTX2, or SOX2 and FOXA2 and LMX1A, or SOX2 and OTX2 and LMX1A. According to a variant, the cell layer C1 comprises neural progenitor cells which express SOX2 and FOXA2 and OTX2 and LMX1A.

[0173] According to another preferred subject of the invention, the neural tissue unit obtained according to the invention comprises neural cells, in particular dopaminergic neurons, expressing EN1 and / or GIRK2 or PAX6. Optionally, the neural cells may also express SOX1.

[0174] According to a variant, the neural tissue unit according to the invention comprises neural cells expressing EN1 and GIRK2. Indeed, dopaminergic neurons express notably TH and / or GIRK2, dopaminergic progenitors express notably LMX1A and / or OTX2 and / or FOXA2 and / or EN1 and / or CORIN.

[0175] Indeed, greater expression of the EN1 marker, a population that has been shown to play a key role in graft efficacy (Kirkeby et al., 2017), reflects the achievement of a neural tissue unit with improved functionality.

[0176] According to another object, the neural tissue unit according to the invention may comprise glial cells.

[0177] In the context of the invention, the neural tissue unit is obtained in a three-dimensional cellular microcompartment, in particular a cellular microcompartment.

[0178] Thus, the neural tissue unit according to the invention is preferably obtained in a hollow cellular microcompartment (or capsule) made of hydrogel. Such a cellular microcompartment may comprise one or more neural tissue units according to the invention. Preferably, it comprises a single neural tissue unit according to the present invention.

[0179] According to a preferred subject of the invention, the neural tissue unit is obtained in a hollow cellular microcompartment made of hydrogel, by neural differentiation from cells capable of differentiating into neural cells. The stem cells are preferably pluripotent stem cells. This may be any neural differentiation or neurodifferentiation method known to those skilled in the art or one of the methods described in the present application. By cells capable of differentiating into neural cells is meant neural stem cells and / or progenitor cells. The stem cells may preferably be pluripotent stem cells and / or neural stem cells. Preferably the cells are human cells. According to a preferred embodiment, the cells capable of differentiating into neural cells are immunocompatible with the human being intended to receive the neural tissue unit;

[0180] According to a preferred embodiment, the stem cells are pluripotent stem cells, more preferably induced pluripotent stem cells, even more preferably induced human pluripotent stem cells. According to another embodiment, the pluripotent cells are pluripotent stem cells excluding human or animal embryonic stem cells. Whatever the embryonic stem cells, no destruction of the embryo is implemented.

[0181] Alternatively, the neural tissue unit comprises a lumen and was obtained in a microcompartment from at most 20 cells capable of differentiating into neural cells, i.e. stem cells and / or progenitors, more preferably from at most 10 cells capable of differentiating, even more preferably from at most 5 cells capable of differentiating. Such a quantity of cells capable of differentiating encapsulated in a cellular microcompartment as described above is particularly advantageous for reducing the internal pressure in said microcompartment which improves the formation of a neural tissue unit according to the present invention, namely a unit comprising a lumen and at least one cell layer bordering said lumen.Conversely, excessively high internal pressure results in the formation of a neural tissue unit devoid of lumen and devoid of biomimetic cellular organization, namely advantageously a layer of Cl cells bordering said lumen and a layer of C2 cells surrounding said layer of Cl cells.

[0182] The invention also relates to a method for preparing and obtaining a variant of a neural tissue unit comprising a lumen, intended to be implanted in the nervous system of a human mammal, said method comprising the following steps: a. encapsulation in a hollow hydrogel microcompartment of at most 20 cells capable of differentiating into neural cells, preferably at most 10, more preferably at most 5, b. induction of cell differentiation of the cells capable of differentiating into neural cells present in the cell microcompartment, so as to obtain at least one neural tissue unit with at least one lumen, c. optionally at least partially removing the external hydrogel layer from the microcompartment to recover the neural tissue unit according to the invention.

[0183] Micro-tissue as medicine

[0184] According to a final aspect, the invention relates to the micro-tissue or unit of neural tissue obtained by implementing a method according to the invention for its use as a medicament, preferably for the prevention and / or treatment of neurodegenerative disease, even more preferably for the prevention and / or treatment of Parkinson's disease.

[0185] Indeed, the inventors have demonstrated that it was possible to inject and therefore graft a unit of neural tissue according to the present invention, notably described in Figures 7 (with a microtissue without light) and in Figure 16. (with a microtissue with light)

[0186] According to a variant, the invention also relates to a cellular microcompartment comprising at least one micro-tissue according to the invention, for its use as a medicament.

[0187] The neural tissue unit according to the present invention can then be implanted into the nervous system of a subject suffering from a neurodegenerative disease, in particular Parkinson's disease, in order to at least partially replace the defective neurons of said subject.

[0188] The invention is now illustrated by non-limiting examples of the method according to the invention, of microtissues obtained and by results. Examples

[0189] Example 1 - Differentiation protocol according to the invention

[0190] Production of neural microtissues

[0191] 2D culture of hiPSCs

[0192] All hiPSC lines were maintained on Vitronectin and cultured in mTeSRl medium. Cultures were fed daily, passaged with enzyme-free reagent, ReLeSR for 6 min at 37 °C every 3-4 days (around 80% confluence), and replated as small clumps (between 100 and 200 µm) at a density of approximately 20,000-40,000 cells / cm 2 Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0193] 3D encapsulation of hiPSCs

[0194] Prior to encapsulation, 2D stem cell colonies were detached. HiPSCs were resuspended in mTeSRl medium supplemented with 10 μM Y-27632. The cells were then mixed in a 1:1 vol ratio with human fibrinogen and Y-27632 to achieve final fibrinogen and Y-27632 concentrations of 14 mg / mL and 10 μM, respectively. The final cell concentration in the cell / matrix solution was therefore between 7.1 x 10 A 6 and 7.4 x 10 A6 viable cells / mL, called encapsulation density. Tubes are connected to the three inlets of a 3D-printed glass co-laminar flow microfluidic device. A 3D-printed glass microcapillary tip is glued to the nozzle outlet for better flow control. The cell suspension is loaded into the inner channel of the three-way device. A sodium alginate SDS solution is injected into the outer channel. To prevent gelation of the alginate inside the microfluidic device due to calcium release from the suspended cells, a calcium-free solution (Sorbitol) is used in the channel. intermediate of the coextrusion chip and serves as a barrier against calcium diffusion. This solution is also supplemented with thrombin at a final concentration of 0.02 U / mL to allow crosslinking of the fibrin inside the capsules. Typical flow rates for the 3 solutions were around 80 mL / h for the three channels: the alginate solution, the sorbitol solution and the cell + matrix suspension. At these speeds, the composite solution forms a liquid jet that breaks up into droplets. When the droplets come into contact with the 100 mM calcium bath, the outer alginate layer gels easily. Therefore, the inner cell / matrix solution remains trapped in a closed, spherical and permeable microcompartment. More than 20 cells were encapsulated per capsule.Within 5 minutes of encapsulation, the capsules were rinsed with DMEM / F-12, 15mM HEPES supplemented with 2.9mM CaCl2, to reduce the basal calcium concentration. Finally, they were transferred to mTeSRl medium supplemented with 10pM Y-27632 as the initial suspension culture medium for neurodifferentiation.

[0195] 3D neurodifferentiation of hiPSCs in static T-flasks or bioreactors

[0196] Further neurodifferentiation can be achieved in several culture systems, including static suspension culture using T-flasks or well plates and stirred suspension cultures using 30 mL bioreactors or 500 mL bioreactors.

[0197] Under static conditions, suspension cultures of encapsulated hiPSCs were performed using T-flasks (5–30 mL) maintained in a cell culture incubator at 37 °C and 5% CO2, with daily medium changes.

[0198] Under shaking conditions, stirred suspension cultures were performed in different bioreactors. We used benchtop rBSTs, including 30 mL or 500 mL bioreactors. The shaking speed was set at 150 rpm from Day 0 to Day 7, 200 rpm from Day 7 to Day 18, and 250 rpm from Day 18 to Day 24 or 55 rpm from Day 0 to Day 24 in the 500 mL and 30 mL rBSTs, respectively. In both cases, the bioreactors were inoculated with 25% (V / V) capsules for a medium volume. In the 500 mL bioreactor, the culture volume was maintained at 300 mL throughout the culture period. On Day 1, the medium was completely renewed with fresh medium supplemented with ROCK inhibitor. On Day 2, no medium change was performed. On day 3, the medium was completely renewed with fresh medium without ROCK inhibitor. From this day on, the medium change was carried out by perfusion. On days 12, 13 and 18, the medium was completely renewed with。 fresh medium. The final capsule volume relative to the medium was between 22% and 25% and the pH was maintained at 7.2 ± 0.2. The dissolved oxygen (DO) level was calibrated to 100% before and after autoclaving in the empty bioreactor and at the starting conditions (filled with medium), respectively. During the test, the oxygen level was monitored and controlled. Oxygen was controlled at 50% and the oxygen level was regulated.

[0199] Under static and shaking conditions, the molecules were added as follows. Starting from day 0, the mTeSR1 medium was supplemented with 10 pM Y-27632 for ROCK inhibition during the first 72 hours of culture. The mTeSR1 medium was kept until day 3. The recombinant human protein Noggin GMP was initially added on day 0 (encapsulation day) at a concentration of 60 ng / mL and gradually increased on day 1 and day 2 to 84 ng / mL and 100 ng / mL respectively. Similarly, SB431542 GMP was initially added at a concentration of 0.2 pM on day 0, and was gradually increased to 2 pM and 20 pM on day 1 and day 2 respectively. On day 3, the medium was changed to Neurobasal™ CTS™ and DMEM / F-12, GlutaMAX™ supplement in a 1:1 ratio, supplemented with N-2 CTS™ supplement and B-27™ GMP supplement.From day 3 to day 11, 100 ng / mL of recombinant human Noggin GMP protein, 20 pM of SB431542 GMP, 100 ng / mL of recombinant human Sonic Hedgehog / Shh (C24II) N-Term GMP protein, 100 ng / mL of recombinant human FGF-8b HumanKine® protein and 2 pM StemMACS Purmorphamine were added. CHIR99021 GMP was added from day 6 to day 12. Then, 200 pM ascorbic acid, 10 ng / mL recombinant human GDNF GMP protein, 1 ng / mL recombinant human TGF-beta 3 protein HumanKine®, 5 ng / mL recombinant human FGF-20 protein, 0.5 mM Dibutyryl cAMP, 10 pM DAPT RMU, 1 pM Compound E and 10 nM Trichostatin A were added from day 13 to day 17. Finally, 20 ng / mL recombinant human BDNF protein HumanKine was added from day 13 to day 24. This differentiation protocol is described in Figure 1.

[0200] An image of neural microtissue obtained at D24 is shown in Figure 11 and Figure 10B.

[0201] Example 2 - Kinetics of appearance of different cell types during the differentiation process according to the invention.

[0202] Protocol

[0203] The microtissue preparation protocol is identical to that of example 1.

[0204] Flow cytometry analysis

[0205] Neural microtissues were harvested at several time points during neurodifferentiation. The alginate capsule was removed by incubating the sample for 5 min in RelesR at room temperature with a final concentration of 20% capsules (V / V). After washing with RelesR, neural microtissues were dissociated using a neurosphere dissociation kit for 40 min at 37 °C with shaking (150 rpm) and manual resuspension using a micropipette every 10 min. Then, cells were fixed and permeabilized using Fixation / Permeabilization Concentrate and Fixation / Permeabilization Diluent in a 1:4 ratio, at a maximum cell density of 5.10 A6 per mL. The cells were then resuspended in flow cytometry staining buffer at a cell density of 833,333 per mL. The cells were centrifuged at 500 g for 5 minutes at room temperature and the supernatant was removed. The samples were then incubated for 30 min in the dark at room temperature, with specific antibodies diluted 1:50 in 1X permeabilization buffer (Invitrogen). The samples were finally washed twice by performing centrifugations at 500 g for 5 minutes at room temperature and resuspending the cells with the staining buffer. The samples were analyzed using the MACSQUANT® Analyzer 10 analyzer (Miltenyi Biotec). Isotype controls were performed to determine the limit of positivity beyond which a sample will be considered positive and to validate the specificity of the antibodies.Compensation controls for each fluorochrome were used to eliminate spectral overlap. Data were post-processed with FlowJo analysis software. Unstained cells were used as a negative control to differentiate stem- or neuron-specific antibody staining from nonspecific background signal. Significant signal from the NANOG+ / OCT4+, SSEA5+ / SSEA4+, and TRA-1-60+ / OCT+ populations is therefore used to identify and calculate the percentage of pluripotent cells. Signal from the PAX6+ / SOX1+ or FOXA2+ / OTX2+ populations is used to identify and calculate the percentage of neural stem cells and dopaminergic progenitors in the sample.

[0206] RNA extraction and RT-PCR analyses

[0207] Samples were homogenized in Tri-reagent (Euromedex) and RNA was isolated using a standard chloroform / isopropanol protocol. RNA was processed and analyzed following an adaptation of published methods. cDNA was synthesized from 2 pg of total RNA using Maxima reverse transcriptase (Fisher Scientific). qPCR was performed using a LightCycler® 480 Real-Time PCR System (Roche). qPCR reactions were performed in duplicate for each sample, using transcript-specific primers, cDNA (4 ng), and LightCycler 480 SYBR Green I Master (Roche) in a final volume of 10 μl. For reference gene determination, the RefFinder method was used. If no specific reference gene was mentioned, relative expression analysis was normalized to five reference genes. The following genes were used: Proteasome subunit beta 4 (Psmb4), non-POU domain containing octamer binding (Nono), chromosome 1 open reading frame 43 (Clorf43), Tyrosine 3-Monooxygenase / Tryptophan 5-Monooxygenase Activation Protein Zeta (Ywhaz), and Actin beta (Actb). The PCR data were exported and analyzed in a computer tool developed at the NeuroCentre Magendie.The relative expression level was assessed by calculating 2-ACt = 2- (Ct(gene of interest) - Ct(average of the 5 reference genes)).

[0208] Statistical analysis

[0209] All statistical analyses were performed using GraphPad Prism 8. For behavioral analyses (in vivo), comparisons were made with the vehicle group using two-way ANOVA and Tuckey's multiple comparison test up to 20 weeks (**** p < 0.0001). From 24 to 32 weeks, two-way ANOVA and Sidak's multiple comparison test were used (* p < 0.05).

[0210] Results

[0211] The results are presented in Figure 2. In this example, the selected differentiation days correspond to the day of iPSC encapsulation (D0) and the end of the neural induction (D5), ventralization (post-rinsing) (D12), maturation (D17) and post-maturation (D24) phases. At D0, almost all encapsulated cells express the markers OCT / NANOG, SSEA5 / SSEA4, TRA-1-60 / OCT which are characteristic of iPSC-type pluripotent cells. At D5, we observe a strong decrease in OCT / NANOG and TRA-1-60 / OCT markers and an increase in FOXA2 / OTX2 markers, thus showing the beginning of cell differentiation. At D12, almost all cells express FOXA2 / OTX2, a characteristic marker of dopaminergic progenitors.

[0212] Example 3 - Comparison of the method according to the invention and a method outside the invention.

[0213] Protocol

[0214] The protocol is identical to that described in example 1 and 2.

[0215] Results

[0216] The results presented in Figure 3 show the transcriptomic expression of the Tyrosine Hydroxylase (TH) gene by RT-qPCR at different differentiation timings. The results correspond to the relative expression of the TH gene compared to the expression of the housekeeping gene ACT-B. The TH gene is a marker of dopaminergic neurons, thus demonstrating a greater quantity of cells expressing this marker and therefore a greater proportion of dopaminergic neurons in the microtissue, obtained with the method according to the invention.

[0217] The results presented in Figure 3, show the transcriptomic expression of the Engrailed (EN-1) gene by RT-qPCR at different differentiation timings. The results correspond to the relative expression of the EN-1 gene compared to the expression of the housekeeping gene ACT-B. The EN-1 gene is a marker of dopaminergic progenitors, thus demonstrating a better expression of this marker and therefore a greater proportion of dopaminergic progenitors in the micro-tissue, obtained with the method according to the invention. In addition, this cell type is particularly sought after and of interest because it has been shown that the expression of the EN-1 gene is a predictive marker of graft success in the context of the use of dopaminergic progenitors (Kirkeby et al., 2017). The use of the method according to the invention increases the expression of the EN-1 gene within the micro-tissue, compared to a micro-tissue obtained with a method outside the invention.

[0218] The results presented in Figure 4 describe the results of flow cytometry analysis (FACS) of the micro-tissues after dissociation obtained using the method according to the invention compared to the micro-tissues obtained with a standard method outside the invention.

[0219] The increase in the number of dopaminergic cells (FOXA2+), associated with the decrease in the number of neural stem cells (PAX6+ / SOX1+) reflects a better commitment of differentiation towards the dopaminergic pathway. The absence of pluripotency markers (TRA-1-60+ / OCT+) confirms the absence of pluripotent stem cells within the micro-tissue obtained with the method according to the invention. These results demonstrate a better commitment to differentiation and therefore an improvement in the differentiation yield using the method according to the invention.

[0220] Example 4 - Characterization of the micro-tissue consisting of a plurality of neural cells according to the invention.

[0221] Protocol

[0222] The protocol is identical to that described in example 1 and 2.

[0223] Results

[0224] The plurality of cells constituting the micro-tissue were characterized by qPCR, indicating the presence of numerous mature or differentiating cells. The results are presented in Figure 5. In particular, the presence of cell types, neurons (MAP2, TUBB3), dopaminergic neurons (TH, GIRK2), dopaminergic progenitors (LMX1A, OTX2, FOXA2, EN1, CORIN), astrocytes (GFAP), GABAergic neurons (GAD1) ​​and oligodendrocytes (OLIG2) can be observed.

[0225] Thus, the micro-tissue comprises a plurality of neural-type cells, comprising a greater number of mature cells or those at the end of differentiation, confirming a better commitment to the differentiation of the neural cells obtained with the method according to the invention.

[0226] Example 5 - Injection of micro-tissue according to the invention for the treatment of Parkinson's disease.

[0227] Protocol

[0228] Induction of the hemi-parkinsonian rat model

[0229] Rowett nude rats (Rnu+) were first anesthetized using a 4% isoflurane induction chamber (induction chamber settings: i4 Oxyl Airl). The rats were then placed on an anesthesia mask, using approximately 2.5% isoflurane (induction chamber settings: i2.5 OxyO.4 Air0.4). The concentration could be slightly adjusted by regularly 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. The rats received an additional intraperitoneal injection of Desipramine Hydrochloride at a concentration of 25 mg / kg at least 20 minutes before the injection of 6-hydroxydopamine hydrobromide (6-OHDA / HBr).2.5 μl of freshly prepared 6-OHDA / HBr at a concentration of 0.5% (w / v) was infused into the medial anterior brain bundle (MFB) using a stereotaxic frame with the coordinates (Bregma as reference): AP: -3.8; ML (right): 1.6; DV: from -8 to -7. After 3 weeks of lesion stabilization, behavior was assessed using the amphetamine-induced rotation test.

[0230] Amphetamine-induced rotation test (rotometer)

[0231] Amphetamine-induced turning behavior was assessed before (3 weeks after 6-OHDA injection) and every 4 weeks after transplantation. Turning was recorded 10 min after intraperitoneal injection of amphetamine (2.5 mg / kg mg kg-1) for 40 min. Results are presented in rotations / min. Only rats that showed more than 3 rotations per minute after lesion stabilization were included in the behavioral analysis.

[0232] Injection of neuronal microtissues into the hemi-Parkinsonian rat model

[0233] Rowett nude rats (Rnu+) were first anesthetized using a 4% isoflurane induction chamber (induction chamber settings: i4 Oxyl Airl). The rats were then placed on an anesthesia mask, using approximately 2.5% isoflurane (induction chamber settings: i2.5 OxyO.4 Air0.4). The concentration could be slightly adjusted by regularly 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. Rats were then injected with neural microtissues into the right striatum using a 25pL Hamilton (Hamilton, ref. 1702 CX SYR) with custom-made needles (glass cannula) and a custom-made delivery medium consisting of Carboxymethylcellulose and Dextran 70kDA (CMC-DA70). The injection was performed in two trajectories using a stereotaxic frame.At the end of surgery, Metacam was injected subcutaneously at a concentration of 1 mg / kg for postoperative pain management.

[0234] Post-mortem study

[0235] Post-mortem histological analysis

[0236] Animals were first anesthetized by intraperitoneal injection of ketamine and xylazine. Animals were then perfused intracardially with 150 ml of 0.9% NaCl, followed by 200 ml of 10% phosphate-buffered formalin. After extraction, brains were post-fixed in 10% phosphate-buffered formalin for 24 h at 4°C and finally stored in PBS at 4°C. 40 μm sections were collected using a vibratome and stored in PBS with 0.01% azide. Sections were washed 3 times with PBS. Sections were permeabilized by incubation for 1 h at room temperature in PBS with 0.1% Triton and 2% BSA, then washed 3 times with PBS. Sections were stained for tyrosine hydroxylase (TH; 1:1000) and human antigen (Stem121 1:1000) by incubating the primary antibodies in PBS 0.05% Triton 0.5% BSA overnight at 4 °C. Sections were washed 3 times with PBS.The sections were then incubated with fluorescent conjugated secondary antibodies (Alexa 488, 568) (1:1000) in PBS Triton 0.05% BSA for 2 hours at room temperature. After being rinsed 3 times with. PBS, sections were mounted on slides with DAPI Fluoromount-G, for counterstaining of nuclei. Slides were imaged using the nanozoomer.

[0237] Results

[0238] The study aims to demonstrate the restoration of motor asymmetry in a non-clinical efficacy study with neural microtissues according to the invention. The results are presented in Figure 6 and 7.

[0239] The inventors observed d-amphetamine-induced rotations measured preoperatively, at 4, 8, 12, 16, 20 weeks (n = 43) and 24, 28, 32 weeks (n = 2) after transplantation. Rotations were measured in a rotometer, 10 minutes after injection of d-amphetamine (2.5 mg / kg ip) for 40 minutes. Neural microtissue transplantation allowed complete functional recovery of motor deficits from 16 weeks for all Cryo 10K (p < 0.0001), Cryo 21K (p < 0.0001), Fresh 3K (p < 0.01) and Fresh 10K (p < 0.0001). A delay was observed for the Cryo 6K group, with complete functional recovery at 20 weeks (p < 0.0001). Long-term functional motor recovery was observed, with the number of amphetamine-induced rotations stabilizing between 16 and 32 weeks (n = 2).Cryo 6K, Cryo 10K, and Cryo 21K correspond, respectively, to a cryopreserved product with an estimated total of 6,000, 11,000, and 21,000 dopaminergic neurons (TH+ cells) initially grafted. Fresh 6K and Fresh 10K correspond, respectively, to a fresh product with an estimated total of 3,000 and 10,000 dopaminergic neurons initially grafted. Comparisons were made with the vehicle group using two-way ANOVA and Tuckey's multiple comparison test up to 20 weeks (**** p < 0.0001). From 24 to 32 weeks, two-way ANOVA and Sidak's multiple comparison test were used. Data are presented as mean ± SEM.

[0240] The results in Figure 7 are fluorescence microscopy images of a graft section 20 weeks after transplantation of a microtissue according to the invention, immunostained for the TH marker (B) and the human marker Steml21 (C). A fusion of the two labels is also shown confirming the presence of dopaminergic neurons from the microtissue (D). The rectangle indicates the magnification area shown in the right panel (E) indicating reinnervation of the host tissue from the graft as revealed by the labeling of axonal projections from the graft.

[0241] Example 6 - Preparation of a micro-tissue comprising a lumen.

[0242] The protocol is identical to that described in example 1 except that the cells encapsulated, namely hiPSCs, are 2 to 20 hiPSC cells per capsule (preferably 5) in order to limit internal pressure and facilitate better cellular organization during differentiation in the capsule. The final concentration of cells in the cell / matrix solution is then between 2.2 x 10 A 6 and 3.7 x 10 A 6 viable cells / mL, called encapsulation density.

[0243] An example of a obtained neural tissue unit is shown in Figure 10A.

[0244] Example 7 - Characterization of the micro-tissue consisting of a plurality of neural cells according to the invention also comprising a lumen (10A) and a unit of plurality of neural cells according to the invention without lumen (10B).

[0245] This study aims to characterize the micro-tissue on the present invention obtained after neurodifferentiation.

[0246] Hematoxylin-eosin-safron (HES) staining and feature scoring

[0247] Microtissues were fixed with 4% paraformaldehyde at room temperature for 1 hour, then washed in phosphate buffer. MTs were centrifuged (10 min, 1200 rpm) and pre-embedded in 3% agarose. After dehydration in successive baths of alcohol, acetone, and xylene, samples were embedded in paraffin. 5 μm slices were cut with a microtome and then glued with an albumin-glycerol mixture onto treated slides. After deparaffinization, sections were successively immersed in Harris hematoxylin, eosin, and saffron solutions. After dehydration, sections were mounted between slide and coverslip using Entellan®. Hematoxylin-eosin-saffron staining allows observation of tissue morphology and structure. The cytoplasm appears pink and the nuclei appear blue-violet. The extracellular matrix has been stained yellow to pink.

[0248] The stained microtissues were imaged using a slide scanner (NanoZoomer 2. ORS, Hamamatsu) and evaluated by a neuropathologist for the presence or absence of physiological features of the human embryonic midbrain: the ventricle (referred to as lumen in the invention), the ventricular zone (referred to as zone Cl in the invention), the intermediate zone, and the mantle zone (referred to as zone C2 in the invention) (Arenas et al. 2015, https: / / doi.org / 10.1242 / dev.097394). Abnormal histological features, including abnormal cytonuclear morphology and karyorrhexis, were also noted.

[0249] These microtissues are represented in Figure 10A (with light) and 10B (without light).

[0250] Immunofluorescence labeling, microscopy and image analysis

[0251] Encapsulated 3D neuronal microtissues with lumen were harvested for confocal microscopy at the end of the neurodifferentiation process (D24). The alginate capsule was removed by incubating the sample for 5 min in RelesR at room temperature with a final concentration of 20% capsules (V / V). The microtissues with lumen were fixed with 4% PFA for 1 h at room temperature in the dark. After fixation, the samples were washed 3 times with 0.1% Tween20 in PBS. A permeabilization step was performed in a PBS solution containing 5% Triton X-100 for 30 min under shaking (170 rpm) at room temperature. The samples were washed 3 times with 0.1% Tween20 in PBS. Samples were incubated in appropriate primary and secondary antibodies 0.1% Tween20 in PBS for 72 h at room temperature with shaking (170 rpm).Samples were rinsed five times with 0.1% Tween20 in PBS after each incubation, including two rinses with shaking for 30 min at 170 rpm. These microtissues are shown in Figs 12, 13 and 14 and were imaged with a fluorescence microscope with deconvolution or with a confocal microscope.

[0252] The results are presented in Figure 12 to 14 and thus confirm the presence of dopaminergic neurons, these not being present radially around the lumen. Conversely, dopaminergic progenitors (progenitor cells) are organized radially around the lumen [Fig. 12],

[0253] Figure 13 confirms the presence of KI67-labeled cells in the neural tissue unit.

[0254] Figure 14 confirms the presence of dopaminergic progenitors (progenitor cells) around the lumen, in a layer of Cl cells bordering the lumen.

[0255] Figure 15 shows the size distribution of the microtissues according to the invention at D24. The microtissues were imaged using a wide-field microscope and their size was measured using proprietary image analysis software. The average diameter size of the microtissues is 176.8 ± 60.48 pm.

[0256] Example 8 - Micro-tissue injection including light for the treatment of Parkinson's disease.

[0257] The protocol is identical to that of example 5, except that the micro-tissues are covered with light.

[0258] Results

[0259] The results are presented in Figure 16 and are fluorescence microscopy images of a graft section 20 weeks after microtissue transplantation, immunostained for the TH marker, the human marker Steml21 and the nuclear dye DAPI. Microtissue-derived TH+ dopaminergic neurons are more abundant and uniformly distributed in the graft in rats transplanted with batch 1 (Light -Microtissue according to the invention) compared to rats transplanted with batch 2 (Microtissue according to the invention - No light).

[0260] Panel C represents the quantification by sterology of the total number of TH-expressing cells in the graft at 20 weeks normalized by the volume of microtissues according to with or without light injected.

[0261] Panel D represents the stereology quantification of the total number of TH-expressing cells normalized by the number of lumen- or non-lumen-injected microtissues at 20 weeks post-transplantation.

[0262] Each point corresponds to one rat. Non-parametric Mann-Whitney tests were performed; ns= p>0.05; * p<0.05 (C, D).

Claims

Claims

1. In vitro method for differentiating pluripotent cells into neural cells, carried out by means of at least one three-dimensional cellular microcompartment in a suitable culture medium, said microcompartment comprising said pluripotent cells, said method comprising at least one step of exposing said pluripotent cells to: at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator, in which the concentration in the medium of at least one SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells to said SMAD inhibitor, by at least 30% relative to the initial concentration of said SMAD inhibitor, to obtain neural cells expressing at least FOXA2.

2. Method according to the preceding claim, in which the concentration of at least one SMAD inhibitor is increased exponentially, preferably at least two SMAD inhibitors.

3. Method according to one of the preceding claims, wherein the concentration of at least one SMAD inhibitor is increased up to 4 days after the initial exposure of the pluripotent cells to said SMAD inhibitor.

4. A method according to the preceding claim, wherein 1 day after initial exposure of the pluripotent cells to said SMAD inhibitors, the concentration of a first SMAD inhibitor (i) is increased by at least 40% and the concentration of the second SMAD inhibitor (ii) is increased by at least 900%.

5. Method according to the preceding claim, wherein 2 days after the initial exposure of the pluripotent cells to said SMAD inhibitors the concentration of a first SMAD inhibitor (i) is increased by at least 67% and the concentration of the second SMAD inhibitor (ii) is increased by at least 9900%.

6. Method according to one of the preceding claims, in which 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 TGFbeta, Activin, Nodal pathway.

7. Method according to the preceding claim, wherein the first SMAD inhibitor (i) is selected from Noggin factor, LDN193189, Dorsomorphin, DMH1, A-83-01, and their combination; and the second SMAD inhibitor (ii) is selected from SB431542 factor, SB505124, LY2157299, LY550410, and their combination.

8. Method according to the preceding claim, in which the concentration of the Noggin factor during the initial exposure is between 50 and 70 ng / mL.

9. Method according to one of claims 7 or 8, in which the concentration of the factor SB431542 during the initial exposure is between 0.1 and 0.3 pM.

10. A method according to any preceding claim, wherein a. the at least one SHH activator is added at least 3 days after the initial exposure of at least one SMAD inhibitor and the pluripotent cells, and / or b. the FGF activator is added at least 3 days after the initial exposure of at least one SMAD inhibitor and the pluripotent cells, and / or c. the at least one Wnt activator is added at least 6 days after the initial exposure of at least one SMAD inhibitor and the pluripotent cells.

11. Method according to one of the preceding claims, in which the pluripotent cells are in isolated form or in aggregate form.

12. A method according to any preceding claim, wherein the pluripotent cells are induced pluripotent stem cells (iPSCs).

13. Method according to one of the preceding claims, further comprising exposing the obtained neural cells with at least one factor selected from rhBDNF, L-Ascorbic acid, rhGDNF, rhTGF-Beta3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and their combination, to obtain neural cells expressing at least the FOXA2 / OTX2 markers.

14. Method according to the preceding claim, wherein the method comprises an additional intermediate step for at least 1 day during which the medium does not comprise inhibitors of the TGF-Beta pathway, when the microcompartment comprises at least 50% of neural cells expressing at least the FOXA2 / OTX2 markers.

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