Neuronal differentiation from pluripotent cells

Abstract

This invention relates to the field of cell biology, more specifically to the field of cell culture. Accordingly, this invention relates to a method for differentiating pluripotent cells into nerve cells that is suitable for and optimized for three-dimensional cell culture. The nerve cells thus obtained can be used in cell therapy for treating neurodegenerative diseases, such as Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, more specifically to the field of cell culture. Thus, the present invention relates to a method for differentiating pluripotent cells into neural cells, which is suitable for and optimized for three-dimensional cell culture. In particular, a plurality of neural cells obtained in the form of microtissues can be used, in particular, in cell therapy for treating neurodegenerative diseases such as Parkinson's disease.

Background Art

[0002] Pluripotent embryonic stem (ES) cells have proliferative and differentiation properties that make them promising tools for cell therapy, but are associated with many ethical issues. In fact, these ES cells are mainly obtained from tissues of aborted fetuses. Therefore, their clinical use is difficult to tolerate, which is why many governments prohibit their use for clinical purposes.

[0003] The discovery of induced pluripotent stem cells (iPS or iPSC) by Professor Yamanaka in 2006 gave a new impetus to this field and made it possible to overcome most of the ethical problems associated with the use of ES cells. Clinical trials were immediately initiated for the purpose of treating diseases that could not be treated with conventional drugs such as neurodegenerative diseases. Transplantation of neural cells obtained from iPSCs, preferably in the form of microtissues, offers the possibility of developing new curative treatments.

[0004] For example, according to the World Health Organization (WHO), more than 8.5 million people worldwide are suffering from Parkinson's disease, and the cases are increasing more rapidly than other neurological disorders. However, with the aging of the world's population, the number of people suffering from Parkinson's disease is set to double between 2005 and 2030, and as a result, without a curative treatment, the cost to society will continue to rise worldwide.

[0005] In fact, to date, there are no curative treatments for these diseases; at best, only treatments that can slow their progression have been developed, and the results are very mixed. For Parkinson's disease, L-dopa and dopaminergic agonists can be mentioned.

[0006] Recent advances in cell culture technology appear to offer the most promising solutions for developing curative therapies, in contrast to conventional drug treatments that are potent and often induce unwanted side effects.

[0007] In this context, advances made in the field of cell culture of pluripotent cells for tissue regeneration offer true hope for repairing damaged nerve tissue and thereby maintaining or even restoring neural activity. Processes for differentiating pluripotent cells into nerve cells in two or three dimensions are already known. However, each of these has the following drawbacks: namely, very high cell mortality or low differentiation yield. An example is the differentiation process disclosed in U.S. Patent Application Publication No. 2017 / 0130199.

[0008] While these processes are adaptable to three-dimensional cell culture, they need to be fully optimized to overcome the aforementioned drawbacks, namely to reduce cell mortality, improve neuronal yield and differentiation rates, and enhance transplantation efficiency. Indeed, prior art has shown that the vulnerability of mature cells, particularly dopaminergic neurons, requires considerable attention in clinical settings. Furthermore, a simple replacement of two-dimensional differentiation processes with three-dimensional systems certainly overcomes the problem of high cell mortality, but at the expense of differentiation yield. On the other hand, known two-dimensional processes provide good differentiation yields, but at the expense of cell mortality.

[0009] As a result, no differentiation process specific to three-dimensional cell culture is known that overcomes all of these shortcomings, namely reducing cell mortality, minimizing the risk of graft mortality as much as possible, and providing better differentiation yields than those currently known in two-dimensional cell culture.

[0010] Therefore, there is a need for a novel process for differentiating pluripotent cells into neurons that avoids the drawbacks of two-dimensional (2D) differentiation processes known from background technologies, namely low production yield, high cell mortality, and low differentiation rates, and is particularly suitable and optimized for three-dimensional cell culture. [Overview of the Initiative]

[0011] Furthermore, to meet this need, the present invention proposes a novel process for differentiating pluripotent cells into nerve cells, which is carried out by at least one three-dimensional cellular microcompartment, and involves exposing the pluripotent cells to a mixture of several factors known to be involved in the differentiation of pluripotent cells into nerve cells, namely, at least one SMAD inhibitor, at least one SHH activator, an FGF activator, and a Wnt activator.

[0012] While these factors are known individually or in combination in the differentiation process of the background technology, the present invention solves the aforementioned drawbacks through the dynamics of cellular exposure to the factor in a three-dimensional cellular microcompartment.

[0013] In fact, the inventors have observed that specific exposure to pluripotent cells associated with a certain increase in SMAD-type factors for a predetermined period, prior to the addition of other differentiation factors and thus to cell exposure to them, can improve yield, differentiation rate, and in particular the proportion of dopaminergic neurons in the resulting mixture containing multiple neurons, reduce cell mortality, and improve the success rate of transplantation of these multiple neurons obtained by the process according to the present invention.

[0014] In contrast to the prior art teachings which aim to expose pluripotent cells to all target factors, particularly SMAD factors, at a given concentration and time, we have found that by continuously or discontinuously increasing the concentration of at least one SMAD inhibitor in the culture medium for at least three days from the initial exposure of pluripotent cells to the SMAD inhibitor, it is possible to induce a decrease in the amount of neural stem cells (a decrease in PAX6+ and / or SOX1+ markers) and an increase in the amount of differentiation-involved cells, such as dopaminergic progenitor cells (FOXA2 and / or OTX2), in the resulting mixture, thereby overcoming the aforementioned drawbacks.

[0015] Therefore, the present invention is an in vitro method for differentiating pluripotent cells into nerve cells, which is carried out by at least one three-dimensional cell microcompartment in a suitable culture medium, the microcompartment comprising the pluripotent cells, and the method comprises, - At least two SMAD inhibitors, - At least one SHH activator, - At least one FGF activator, and - At least one step of exposure to at least one Wnt activator, The present invention relates to a method comprising the step of increasing the concentration of at least one SMAD inhibitor in culture medium by at least 20%, preferredly at least 30%, more preferably at least 40%, from the initial concentration of the SMAD inhibitor for at least 3 days from the initial exposure of pluripotent cells to the SMAD inhibitor, either continuously or discontinuously, to a plurality of neurons, wherein at least 30%, preferredly at least 40%, of the cells express at least the FOXA2 and / or OTX2 markers. Thus, the resulting mixture of plurality of neurons at the end of the process contains a greater amount of FOXA2 marker-positive cells and a smaller amount of PAX6 and / or SOX1 marker-positive cells than the 3D culture process of the background technology.

[0016] According to a preferred object of the present invention, the continuous or discontinuous increase in at least one SMAD inhibitor is exponential. Furthermore, the concentration of at least one SMAD inhibitor increases exponentially with preference.

[0017] Advantageously, the process according to the present invention aims to expose pluripotent cells to at least two SMAD inhibitors, the concentrations of the two SMAD inhibitors increasing exponentially or logarithmically with respect to the initial exposure of the pluripotent cells to the SMAD inhibitors.

[0018] For another preferred purpose, the concentration of at least one SMAD inhibitor is increased for at least one day, at least three days, and up to four days, and preferably up to five days, after initial exposure of pluripotent cells to the SMAD inhibitor, more preferably two SMAD inhibitors.

[0019] According to another particularly advantageous objective, the method according to the present invention exposes pluripotent cells to two SMAD inhibitors, each of which can act on different cellular signaling pathways. Thus, the first SMAD inhibitor (i) can act on the BMP-2,4,7 pathway, particularly by preventing the interaction between SMAD-1,5,8 and the SMAD4 cofactor, and in particular by preventing the phosphorylation of SMAD-1,5,8, and thus preventing the activation of transcription by the SMAD-1,5,8 / SMAD-4 complex. The second SMAD inhibitor (ii) can act on the TGFβ,activin,Nodal pathway, particularly by preventing the interaction between SMAD-2,3 and the SMAD4 cofactor, and in particular by preventing the phosphorylation of SMAD-2,3, and thus preventing the activation of transcription by the SMAD-2,3 / SMAD-4 complex.

[0020] Favorably, 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% one day after the initial exposure of pluripotent cells to the SMAD inhibitor; 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 present invention, two days after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) is increased by at least 33%, the concentration of the second SMAD inhibitor (ii) is increased by at least 4950%, and 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 process according to the present invention comprises exposing pluripotent cells to two SMAD inhibitors, the first SMAD inhibitor (i) being selected from noggin factor, LDN193189, dolsomorphin, DMH1, A83-1, and combinations thereof, and the second SMAD inhibitor (ii) being selected from SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

[0023] For another purpose, the process according to the present invention also exposes pluripotent cells to at least one SHH factor. Preferably, at least one SHH activator is added at least 3 days and up to 5 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor. Also, in contrast to processes known in the background art, the exposure of cells to at least one SHH activator is not simultaneous with the initial exposure of pluripotent cells to the SMAD inhibitor. Very preferably, at least two SHH activators are added at least 3 days and up to 5 days after the initial exposure of pluripotent cells to at least one SMAD inhibitor.

[0024] According to another preferred object of the present invention, the FGF activator, particularly FGF-8b, is also preferably added at least 3 days and at most 5 days after the first exposure to at least one SMAD inhibitor and the pluripotent cells, and advantageously simultaneously with the SHH activator, that is, during the first exposure to at least one SHH activator.

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

[0026] Therefore, the method according to the present invention advantageously comprises exposing the pluripotent cells, - at least one SHH activator is added at least 3 days after the first exposure to at least one SMAD inhibitor and the pluripotent cells, and / or - the FGF activator, particularly FGF-8b, is added at least 3 days after the first exposure to at least one SMAD inhibitor and the pluripotent cells, and / or - at least one Wnt activator is added at least 6 days after the first exposure to at least one SMAD inhibitor and the pluripotent cells.

[0027] When adding at least one SMAD inhibitor to expose the pluripotent cells to the SMAD inhibitor, preferably it is in an isolated form or an aggregate form. Also, the cell culture is a three-dimensional cell culture, and the cells are encapsulated in three-dimensional cell microcompartments. Such microcompartments are well-known and are particularly described in International Publication No. WO2018 / 096277.

[0028] In the context of the present invention, the three-dimensional microcompartments, particularly those containing a layer of the cells, extracellular matrix or extracellular matrix substitute, are suitable for culturing pluripotent cells. As an example, the layer may be of Matrigel (registered trademark) or fibrin type, and the microcompartment includes an outer layer of a hydrogel, such as alginate.

[0029] Given that exposure of pluripotent cells to at least one SMAD inhibitor may occur simultaneously with or after the encapsulation of such pluripotent cells, the pluripotent cells may therefore be in the form of isolated cells, aggregates, or a mixture containing both isolated cells and aggregates.

[0030] According to another object of the present invention, the pluripotent cells may be of any type and can differentiate into cells of interest, particularly nerve cells, and most preferably, the pluripotent cells are induced pluripotent stem cells (iPSCs). Various cell culture methods for obtaining induced pluripotent stem cells (iPSCs) are well documented in the background, particularly in the papers by Yu et al. (Science 2007, 318(5858):1917-1920), Takahashi et al. (Cell, 207, 131(5):861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1):101-106).

[0031] According to another specific object of the present invention, the method according to the present invention may include an additional step of further maturing neurons by exposing them to factors selected from at least rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and combinations thereof, to obtain neurons expressing at least FOXA2 and / or OTX2, and particularly enabling the emergence of dopaminergic neurons expressing FOXA2 and TH+ markers.

[0032] Advantageously, the process according to the present invention allows for an increase in cells expressing FOXA2 and TH+ and a decrease in cells expressing PAX6 / SOX1, respectively, resulting in an increase in the number of mature neurons, i.e., at least dopaminergic cells (neurons and dopaminergic progenitor cells), and a decrease in neural stem cells, leading to a better commitment of pluripotent cells to differentiation into neurons. Finally, the absence of pluripotency markers, particularly TRA-1-60+ / OCT+, confirms the absence of pluripotent stem cells in the final product obtained at the end of the process, i.e., multiple neurons in a microtissue morphology. Such absence reduces the risk of toxic or uncontrolled differentiation.

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

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

[0035] According to another object of the present invention, the method for differentiating pluripotent cells into neurons includes an intermediate step of at least one day in which the medium does not contain a TGF-β pathway inhibitor, for removing differentiation factors from the medium, provided that the microcompartment contains at least 50% neurons expressing at least the FOXA2+ / OTX2+ factor.

[0036] Finally, according to another aspect, the present invention also relates to a plurality of nerve cells obtained by a process according to any one of the embodiments described above. Preferably, the final product obtained by this process is a mixture of nerve cells, particularly dopaminergic neurons, dopaminergic progenitor cells, and glial cells. Thus, the present invention also relates to a heterogeneous population of nerve cells of particular interest for nerve cell transplantation. This heterogeneous population, consisting of a plurality of nerve cell types, preferably striatal and / or cortical populations, is very preferably aggregate or microtissue morphology.

[0037] In another aspect, the present invention also relates to a plurality of nerve cells, preferably in the form of microtissue, for use as a pharmaceutical, more preferably for use in the prevention and / or treatment of Parkinson's disease.

[0038] Other features and advantages will become apparent from the following detailed description of the present invention, examples, and drawings. [Brief explanation of the drawing]

[0039] [Figure 1] This shows the steps of a differentiation process according to a specific embodiment of the present invention. [Figure 2] This report shows the dynamics of marker expression during the neural differentiation process according to the present invention in a bioreactor, as measured by flow cytometry. The selected differentiation days correspond to the iPSC encapsulation day (D0), as well as the end of the neural induction phase (D5), ventralization phase (after rinsing) (D12), maturation phase (D17), and late maturation phase (D24). The data are presented in histogram format, aggregating data from 16 different independent bioreactors (n=16). [Figure 3]The left panel shows the transcriptome expression of the tyrosine hydroxylase gene (TH) relative to the expression of the ACT-B housekeeping gene, and the transcriptome expression of the Engrailed gene (EN-1) relative to the expression of the ACT-B housekeeping gene, by RT-qPCR in a process outside the scope of the present invention, namely at the end of the neural induction phase (D5) of the differentiation process according to the present invention (D12), the end of the ventricular phase (after rinsing) (D17), and the end of the late maturation phase (D24), and in a process outside the scope of the present invention, which does not include the step of adding at least one SMAD inhibitor that has been continuously or discontinuously increased for at least 3 days (left panel), and the transcriptome expression of the Engrailed gene (EN-1) relative to the expression of the ACT-B housekeeping gene (right panel). [Figure 4] At the end of a differentiation process outside the scope of the present invention, a comparative flow cytometry analysis (FACS) of the neuronal cell mixture obtained by the differentiation process according to the present invention is performed (neuronal microtissue of D24). The results are shown in the form of a histogram. An increase in FOXA2-positive cells indicates an increase in the number of dopaminergic cells (neurons and dopaminergic precursors), while a decrease in PAX6+ / SOX1+-positive cells indicates a decrease in the number of neural stem cells in the resulting cell mixture. In D24, the neuronal microtissue obtained according to the process of the present invention contains less than 40%, more preferably less than 30%, of cells expressing FOXA2. [Figure 5] This shows the mRNA expression levels of cells obtained by the process according to the present invention, which constitutes the final product at D24 by RT-qPCR. The values ​​are expressed as a percentage of the iPSC(D0) value by comparing the mean Ct value with the values ​​of five different housekeeping genes (ACTB, PSMB4, NONO, C1orf43, and YWHAZ). The experiment was performed twice and is shown as a Tukey box plot (n=17). [Figure 6]Using an amphetamine-induced rotation test (rotometer), we describe the ability of multiple neurons with microtissue morphologies (modified forms without lumen) obtained by the differentiation process according to the present invention to restore motor symmetry in a unilateral Parkinson's disease rat model. Post-injury grafts were unilaterally transplanted into the rat striatum. Injury was pre-induced by injecting a neurotoxin (6-OHDA) into the medial forebrain bundle (MFB). This lesion replicates severe destruction of dopaminergic neurons and induces unilateral motor deficits, similar to the motor symptoms of Parkinson's disease. [Figure 7] These are cross-sectional images of postmortem grafts 20 weeks after transplantation of nerve cells obtained according to the process of the present invention. Sections of grafts stained with the dopaminergic neuron marker TH (B), stained with the human marker Stem121 (C), and containing both markers. Rectangles indicate the magnified zone shown in the right panel (E). Scale bars are 2.5 mm (B, C, and D) and 50 μm (E). Panel D shows nerve reinnervation from the graft. [Figure 8] A modified example of a nerve tissue unit obtained by the process according to the present invention is shown, having a lumen and whose cells are stained for the markers FOXA2 and OTX2, as well as Ki67. [Figure 9] This shows a modified example of a nerve tissue unit obtained by the process according to the present invention, in which the cells are stained for the markers SOX2 and nestin. [Figure 10] A and B) Hematoxylin-eosin-saffron (HES) staining of microtissues according to the present invention at D24. A) Lumen-containing microtissue obtained by carrying out the process according to the present invention, showing a high-density zone of the nucleus and a radially organized C1 layer (dotted line) around the lumen ("L"). B) Lumen-less microtissue obtained by carrying out the process according to the present invention. Scale bar: 50 μm. [Figure 11] Image of a modified solid nerve microtissue without a lumen in the microcompartment. [Figure 12]Immunofluorescence images of modified microtissues according to the present invention having a lumen, in D24. Neural microtissues were immunostained for the dopaminergic neuron marker TH (A) and the dopaminergic precursor marker FOXA2 (B), and counterstained with the nuclear dye DAPI (C). Scale bar: 50 μm. * indicates the presence of a lumen. Cells organized radially around the lumen are mainly FOXA2 positive and correspond to layer C1, while TH expression is localized around the cells surrounding the lumen, corresponding to layer C2. [Figure 13] Immunofluorescence images of microtissue modifications obtained by the present invention using a lumen, at D24. Nerve microtissue was immunostained for the proliferative cell marker Ki67 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 μm. * indicates the presence of a lumen. Cells organized radially around the lumen are positive for KI67 (less than 50% of cells). [Figure 14] Immunofluorescence images of microtissues obtained by the process according to the present invention in D24. Neural microtissues were immunostained for the dopaminergic precursor marker OTX2 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 μm. Asterisks (*) indicate the presence of a lumen. Cells organized radially around the lumen are mainly OTX2-positive, corresponding to layer C1. [Figure 15] Microtissue size distribution obtained by the method according to the present invention in D24 (modified example with lumen). Microtissues were imaged using a wide-field microscope, and their size was measured using image analysis software. The average diameter of the microtissues was 176.8 ± 60.48 μm. [Figure 16]Postmortem histological analysis of rats transplanted with microtissues according to the present invention, having a lumen (Batch 1) and not having a lumen (Batch 2). A and B) show animal brain sections 20 weeks after transplantation of Batch 1 (A) and Batch 2 (B), immunostained for TH and the human marker Stem121. Scale bar: 2.5 mm. C, D) Quantification of the number of TH-expressing cells per volume of injected microtissue (C) and the number of TH-expressing cells per number of injected microtissues (D). Nonparametric Mann-Whitney U test was performed. ns=p>0.05, *p<0.05. [Modes for carrying out the invention]

[0040] definition For the purposes of the present invention, “microcompartment” or “capsule” means a partially or completely closed, hollow, three-dimensional structure containing one or more cells. The structure comprises a preferentially cured hydrogel outer layer and a hollow interior containing at least one cell and / or at least one cell aggregate and / or cellular microtissue (or tissue unit), and optionally an extracellular matrix and / or extracellular matrix substitute suitable for cell culture and proliferation of said cells.

[0041] For the purposes of this invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even if not explicitly stated otherwise, differentiated cells, including cells, pluripotent cells, progenitor cells, and nerve cells, are obtained from or derived from human cells or immunologically humanized non-human mammalian cells.

[0042] For the purposes of this invention, "pluripotent" cells mean cells that have the ability to form all tissues present in the entire organism from which they originate, but cannot form the entire organism, because they have already undergone a first differentiation step and can only produce cells of the embryo, endoderm, mesoderm, and ectoderm layers, and can no longer produce cells of the trophectoderm. Human pluripotent cells may be referred to as hPSCs or ES cells in the context of this invention. In particular, these may be induced pluripotent stem cells (iPSCs, or hiPSCs for human induced pluripotent stem cells). The pluripotency of these cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG, and SOX2, as well as surface markers such as SSEA4 / 5, Tra-1-60, and Tra-1-81. In an optional and very specific embodiment, pluripotent cells obtained from embryonic stem cells can be obtained without destroying the embryo from which they originate, for example, using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)):113-117). Human-derived embryonic stem cells can be optionally excluded.

[0043] For the purposes of this invention, "induced pluripotent stem cells," "iPSCs," or "hiPSCs" mean pluripotent stem cells induced to become pluripotent through genetic reprogramming of differentiated somatic cells. These cells are particularly positive for staining with alkaline phosphatase and for pluripotency markers such as the expression of proteins NANOG, SOX2, OCT4, and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the papers by Yu et al. (Science 2007, 318(5858):1917-1920), Takahashi et al. (Cell, 207, 131(5):861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1):101-106).

[0044] For the purposes of this invention, “nerve” cells mean all cells of the nervous system. These may be mature cells, such as dopaminergic or GABAergic neurons, so-called glial supporting cells (astrocytes, oligodendrocytes, etc.), or progenitor cells (dopaminergic or GABAergic progenitor cells, etc.), or neural tissue stem cells (neural stem cells). They may also be a mixture of nerve-type cells, in particular a heterogeneous population of nerve cells including several cell types, such as neurons, dopaminergic neurons, precursors, neural stem cells, and optionally glial cells.

[0045] For the purposes of this invention, “progenitor” cells mean cells in the differentiation process, that is, cells that are already involved in the differentiation pathway but have not yet differentiated. In the context of this invention, progenitor cells are, for example, radial glial cells or radial glial progenitor cells (RGPCs), but are also dopaminergic progenitor cells that express at least the foxa2 factor.

[0046] For the purposes of this invention, “differentiated” cells mean cells having a specific phenotype, as opposed to undifferentiated pluripotent stem cells or progenitor cells that have undergone differentiation. In this context, differentiated cells are mature cells, such as neuronal cells, i.e., neurons, such as dopaminergic neurons expressing at least FOXA2 and TH factors.

[0047] For the purposes of this invention, “layer of cells” or “bed of cells” means several cells that form a layer or bed that can be structured around a lumen, which may be, for example, a group of cells that function in coordination with one another and are assembled three-dimensionally. The thickness of the layer of cells or bed may be variable. This layer or bed is organized three-dimensionally within a nerve tissue unit.

[0048] For the purposes of the present invention, “microtissue,” “neuronal tissue unit,” or “tissue unit” means at least one neural tissue unit comprising a plurality of nerve cells obtained by the differentiation process according to the present invention, wherein the plurality of nerve cells include, for example, dopaminergic neurons, precursors, particularly dopaminergic precursors, and glial cells, and the cells are optionally organized into a three-dimensional network in the extracellular matrix. The microtissue may be encapsulated in three-dimensional cellular microcompartments or deencapsulated, and is suitable for transplantation into the nervous system of mammals, preferably humans.

[0049] For the purposes of this invention, "exponential increase" or "exponentially increased" means an exponential increase in the amount of differentiation factors, such as SMAD inhibitors, i.e., in the context of this invention, their concentration. This is a mathematical definition known to those skilled in the art, characterized in that the increase follows the law of exponents over time.

[0050] For the purposes of this invention, "logarithmic increase" or "logarithmically increased" means a logarithmic increase in the amount of a differentiation factor, such as a SMAD inhibitor, i.e., in the context of this invention, its concentration. Therefore, it is a mathematical definition known to those skilled in the art, characterized in that the increase follows the logarithmic law over time.

[0051] For the purposes of this invention, "SMAD inhibitor" refers to a family of molecules involved in the signal transduction of transforming growth factor-β (TGF-β) and its analogs. Mammals have at least eight types of SMAD proteins, numbered from SMAD1 to SMAD8. When TGF-β binds to its cell surface receptor, it forms a complex with SMAD4 and then phosphorylates SMAD2 and SMAD3, which can then move to the nucleus. The SMAD complex thus formed binds to the promoter sequences of target genes, activating their transcription and thus mediating the biological effects of TGF-β. Conversely, proteins SMAD6 and SMAD7 inhibit TGF-β signal transduction. When BMP-2 binds to its cell surface receptor, it forms a complex with SMAD4 and then phosphorylates SMAD1, SMAD5, and SMAD8, which can then move to the nucleus. The SMAD complex thus formed binds to the promoter sequences of target genes, activating their transcription and thus mediating the biological effects of BMP-2. Conversely, proteins SMAD6 and SMAD7 inhibit BMP-2 signal transduction. For example, SMAD inhibitors can be selected from noggin, LDN193189, dolsomorphine, DMH1, and A83-1, SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

[0052] For the purposes of this invention, "SHH activator" means a molecule capable of activating the Sonic Hedgehog signaling pathway. The Sonic Hedgehog protein is one of three mammalian proteins involved in the Hedgehog signaling pathway, and the SHH protein is a ligand for the Hedgehog signaling pathway that plays a crucial role in regulating organogenesis in vertebrates, such as the growth of fingers in 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, SHH activators are selected from SHH, SHH C25II, SAG (Smoothened Agonist), and palmorfamine.

[0053] In the object of this invention, "FGF activator" means a molecule capable of activating FGF receptor signaling. FGF receptors belong to a family of transmembrane receptors in which the intracellular portion has protein tyrosine kinase activity. When a tyrosine kinase receptor binds to its extracellular ligand, it becomes possible to phosphorylate intracellular proteins or other transmembrane receptors on specific tyrosine amino acids, thus enabling signal transduction from the outside to the inside of the cell. Major tyrosine kinase receptors are receptors for polypeptide growth factors (EGF, FGF, PDGF, VEGF, etc.). Preferably, the FGF activator is rhFGF-8b.

[0054] For the purposes of this invention, "Wnt activator" or "Wnt signaling pathway activator" means a molecule capable of activating the Wnt signaling pathway, which plays various roles in animal development and stem cell maintenance. Preferably, the Wnt activator is selected from CHIR 99021, XAV939, and BIO.

[0055] For the purposes of this invention, "prevention" means reducing to a lower degree a certain phenomenon, for example, in the context of this invention, the risk or possibility of developing Parkinson's disease.

[0056] For the purposes of this invention, “treatment” means suppressing the progression of a disease, such as Parkinson’s disease, stabilizing, reversing, or even interfering with or inhibiting its progression.

[0057] For the purposes of this invention, "Ferré diameter" means the distance between two tangents, in particular "d" or "D", where these two tangents are parallel, and as a result, the entire projection is contained between these two parallel tangents.

[0058] For the purposes of this invention, the "maximum dimension" of X means the value of the maximum Ferret diameter of X.

[0059] For the purposes of the present invention, “lumen” means a substantially cell-free volume containing an aqueous solution, topologically surrounded by cells, in particular at least one layer of cells that form a barrier to fluid circulation, and characterized by the presence of ZONULA OCCLUDENS 1, i.e., ZO-1 marker-positive tight junctions adjacent to the lumen.

[0060] The process of differentiating pluripotent cells into nerve cells Therefore, the object of the present invention is an in vitro method for differentiating pluripotent cells into nerve cells, which is carried out by at least one three-dimensional cell microcompartment in a suitable culture medium, the microcompartment comprising the pluripotent cells, and the method comprises, - At least two SMAD inhibitors, - At least one SHH activator, - At least one FGF activator, and - comprising at least one step of exposure to at least one Wnt activator, The method involves increasing the concentration of at least one SMAD inhibitor in the culture medium by at least 20%, preferably at least 30%, and more preferably at least 40%, from the initial concentration of the SMAD inhibitor to at least 3 days from the initial exposure of pluripotent cells to the SMAD inhibitor, either continuously or discontinuously, to obtain multiple neurons in which at least 30%, preferably at least 40%, of the neurons express at least the following factor, namely FOXA2 / OTX2.

[0061] In the context of the present invention, pluripotent cells are encapsulated in three-dimensional microcompartments or capsules that enable three-dimensional cell culture under conditions as close as possible to in vivo physiological conditions. The advantages of three-dimensional cell culture are now well described and are well known to those skilled in the art.

[0062] This method may include a preliminary step of culturing pluripotent cells, followed by encapsulation of the pluripotent cells into three-dimensional cellular microcompartments. Three-dimensional cellular microcompartments are described in particular in International Publication No. WO2018 / 096277. Briefly speaking, encapsulation may advantageously include the following steps: a. A step of encapsulating a mixture comprising pluripotent cells, culture medium, and extracellular matrix or an extracellular matrix substitute in an outer hydrogel layer, wherein the encapsulation includes the following sub-steps. i. The step of bringing the mixture into contact with a hydrogel solution intended to form the outer layer to form at least one droplet, and ii. A step of curing the hydrogel solution and collecting the droplets obtained in a calcium bath capable of forming the outer layer of each micro-compartment. b. A step of culturing the capsules obtained in the preceding step in culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably 3 to 50 days, and, c. A step of collecting the obtained cellular microcompartments containing the target pluripotent cells.

[0063] Preferably, the encapsulation process is carried out by co-injection of a hydrogel solution intended for the formation of an outer layer, particularly a mixture containing pluripotent cells and optionally an extracellular matrix or extracellular matrix substitute, and optionally an intermediate solution. This co-injection is performed concentrically through a microfluidic or millifluidic injector, which forms a jet of the mixture of solutions at the injector outlet, and the jet is divided into droplets. The droplets are then collected in a calcium bath by gravity, curing the hydrogel solution to form an outer layer and, as a result, a cellular microcompartment containing the desired pluripotent cells. The pluripotent cells may be in isolated form, or in the form of cell aggregates or cell clusters. The encapsulated pluripotent cells are also suspended in the capsule in the form of single, i.e., isolated cells and / or cell clusters or aggregates. Preferably, isolated cells account for less than 50% of the total number of encapsulated cells, and more preferably, the isolated cells are iPSC cells.

[0064] Once the target cells are encapsulated, preferably in isolated and / or aggregated forms, they are exposed to several differentiation agents, such as at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator. These differentiation agents are preferentially added directly to the culture medium containing the microcompartments in a bioreactor. The differentiation agents diffuse through the outer layer of the cellular microcompartments that allow for their diffusion. They also diffuse within each cellular microcompartment and into the culture medium present within that microcompartment.

[0065] In this context, the present inventors have developed a method to improve yield in three-dimensional cell culture, increase differentiation rate, and thereby improve the success rate of transplantation of the neuronal cell mixture obtained by the process according to the present invention and reduce cell mortality, in relation to the teachings of the background technology.

[0066] To achieve this, the inventors observed that exposure of encapsulated pluripotent cells to at least one SMAD inhibitor, which is added stepwise, i.e., in a stepwise increasing manner, before the addition of other differentiation factors, namely SHH activator, FGF activator, and Wnt activator, overcomes the shortcomings of the background art.

[0067] Therefore, the present invention relates to the stepwise addition of at least one SMAD inhibitor, preferably two SMAD inhibitors, before the initiation of neuronal differentiation, which is initiated by the addition of other factors, namely SHH activator, FGF activator, and Wnt activator.

[0068] In contrast, background techniques instruct that after culturing pluripotent cells for amplification, the resulting pluripotent cells be exposed to high concentrations of differentiation factors, particularly a combination of SMAD inhibitors, SHH activators, and FGF activators. Exposure of pluripotent cells enables differentiation into neurons, but at the cost of high cell death. Indeed, sudden exposure of cells that do not yet express receptors for differentiation factor induction pathways to high concentrations induces significant cellular stress, resulting in a high rate of cell death. In addition, differentiation rates are low and production yields are poor. Conversely, a stepwise increase of these factors promotes the cellular adaptability and tolerance to the differentiation process, i.e., the ability of cells to respond to signals, thereby reducing cell death.

[0069] Accordingly, the present invention aims to expose encapsulated pluripotent cells expressing OCT / NANOG, SSEA5 / SSEA4, and TRA-1-60 / OCT to at least one SMAD inhibitor at T0. The SMAD inhibitor is added at a concentration lower than those known in the background art in order to avoid causing cellular stress and significant cell death. Advantageously, the concentration of the SMAD inhibitor is increased continuously or discontinuously, more preferably by at least 40% from the initial concentration of the SMAD inhibitor, for at least 3 days from the initial exposure of the pluripotent cells to the SMAD inhibitor.

[0070] In the object of the present invention, "continuous increase" means that the concentration of the target differentiation factor increases linearly over time, preferably over a given period, for example, three days.

[0071] Conversely, for the purposes of the present invention, "discontinuous increase" means that the concentration of the target differentiation factor increases stepwise over a predetermined period, for example, three days, until it reaches the target final concentration.

[0072] The increase in the concentration of SMAD inhibitors can take several forms. Therefore, the increase may follow an exponential or logarithmic scale. Most preferably, the increase in the concentration of at least one SMAD inhibitor is exponential. Furthermore, the concentration of at least one SMAD inhibitor preferentially increases exponentially, reducing cell death and improving differentiation rates in the target encapsulated cells.

[0073] Preferably, the concentration of at least one SMAD inhibitor should be increased for up to 4 days, and more preferably up to 5 days, after the initial exposure of pluripotent cells to the SMAD inhibitor.

[0074] According to a further object of the present invention, pluripotent cells are exposed to at least two SMAD inhibitors, wherein at least a first SMAD inhibitor (i) is increased sequentially or discontinuously, preferentially exponentially, and the pluripotent cells are exposed to a second SMAD inhibitor (ii), the concentration of which may be constant over time, or continuously or discontinuously increased. Thus, the concentrations of the at least two SMAD inhibitors are preferentially increased relative to the initial exposure concentration of the pluripotent cells to the SMAD inhibitors.

[0075] 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, which has the effect of maintaining pluripotency and preventing pluripotency from committing to undesirable pathways, such as pathways that allow pluripotency to commit to the mesoendoderm. To a particularly preferred purpose, the increase in the concentration of the first SMAD inhibitor (i) is exponential, and the increase in the concentration of the second SMAD inhibitor (ii) is logarithmic.

[0076] Preferably, the concentrations of both SMAD inhibitors should be increased for up to 4 days, and more preferably up to 5 days, after the initial exposure of pluripotent cells to the SMAD inhibitor in question.

[0077] Furthermore, the present invention aims at a differentiation method comprising the step of exposing encapsulated pluripotent cells to at least one, preferably at least two, SMAD inhibitors, wherein the concentration of each SMAD inhibitor is increased over a given period, preferably 3 to 5 days. During this period, the concentration of the first SMAD inhibitor (i) is preferably increased exponentially, while the concentration of the second SMAD inhibitor (ii) is constant or increased, preferably on a logarithmic scale.

[0078] According to another particularly interesting objective, one day after the initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 40% and the concentration of the second SMAD inhibitor (ii) increases by at least 900% compared to the initial concentration of the SMAD inhibitor, i.e., compared to day 0, which is the initial exposure of the SMAD inhibitor to the target encapsulated pluripotent cells.

[0079] Preferably, two days after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 67% and the concentration of the second SMAD inhibitor (ii) increases by at least 9900% compared to the initial concentration.

[0080] According to a preferred objective of the present invention, the SMAD inhibitor can be selected from (i) a SMAD inhibitor capable of acting on the BMP-2, 4, and 7 pathways, and (ii) a SMAD inhibitor capable of acting on the TGFβ, activin, and Nodal pathways.

[0081] SMAD inhibitors (i) capable of acting on the BMP-2, 4, and 7 pathways are advantageous in that they prevent transcriptional activation by the SMAD-1, 5, 8 / SMAD-4 complex, particularly by preventing the interaction between SMAD-1, 5, 8 and the SMAD4 cofactor, and especially by preventing the phosphorylation of SMAD-1, 5, 8.

[0082] SMAD inhibitors (ii) capable of acting on the TGFβ, activin, and Nodal pathways are advantageous in that they prevent transcriptional activation by the SMAD-2,3 / SMAD-4 complex, particularly by preventing the interaction between SMAD-2,3 and SMAD4 cofactors, and especially by preventing the phosphorylation of SMAD-2,3.

[0083] Preferredly, SMAD inhibitors can be selected from noggin, LDN193189, dorsomorphine, DMH1, and A83-1, SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

[0084] In a particularly preferred method, the first SMAD inhibitor (i) is selected from noggin, LDN193189, dolsomorphine, DMH1, factor A83-1, and combinations thereof. In a particularly preferred method, the second SMAD inhibitor (ii) is selected from SB431542, SB505124, LY2157299, LY550410, these factors, and combinations thereof.

[0085] For the purposes of this invention, "noggin" refers to a secreted homodimeric glycoprotein that binds to and inactivates members of the transforming growth factor β (TGF-β) signaling protein superfamily, such as bone morphogenetic protein 4 (BMP4). Noggin proteins are generally 65 kDa proteins expressed in human cells as glycosylated, disulfide-linked dimers. (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).

[0086] For the purposes of this invention, "LDN193189" or "LDN-193189" refers to a compound analogous to noggin protein, a bone formation pathway (BMP) inhibitor that inhibits ALK1, ALK2, ALK3, and ALK6. This is a derivative of dorsomorphine, which is generally used at concentrations about 100 times lower (Sanvitale et al., Vogt et al.). This compound significantly 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 embryonic endoderm derived from human and mouse pluripotent stem cells (Kearns et al.), and promotes the differentiation of inner ear sensory epithelial cells from mouse embryonic stem cells (Koehler et al.). This compound can be accessed in particular via its CAS number 1062368-24-4.

[0087] For the purposes of this invention, "dorsomorphine" refers to the AMPK inhibitor of formula C24H25N5O, named 6-[4-[2-(1-piperidyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride. Dorsomorphine inhibits the BMP pathway by targeting type I receptors such as ALK2, ALK3, and ALK6. It is a compound analogous to the Noggin protein and compound LDN193189. It can be accessed via CAS number 866405-64-3.

[0088] For the purposes of this invention, "DMH1" refers to a highly selective small molecule inhibitor of the BMP pathway that promotes hiPSC neurogenesis, accessible via CAS number 1206711-16-1.

[0089] In the object of this invention, "A83-1" refers to a selective inhibitor of the TGF-β1 type ALK receptor of formula 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide. In particular, it inhibits TGF-β-induced epithelial-mesenchymal transition (EMT) by inhibiting SMAD2 phosphorylation. Previous studies have shown that A83-1 significantly promotes somatic cell reprogramming.

[0090] For the purposes of this invention, "SB431542" or "SB-431542" refers to activin receptor-type receptor kinase inhibitors of ALK5, ALK4, and ALK7. In particular, it is used in combination with LDN193189, CHIR99021, and DAPT to transform astrocytes into neurons. It can be accessed via CAS number 301836-41-9.

[0091] For the purposes of this invention, "SB505124" or "SB-505124" also refers to a selective inhibitor of the transforming growth factor-βI type ALK4, ALK5, and ALK7 receptors. It selectively and concentration-dependently inhibits the ALK4, ALK5, and ALK7-dependent activation of the downstream cytoplasmic signaling transducers Smad2 and Smad3, as well as components of the TGF-β activated protein kinase pathway, but does not alter Smad signaling induced by ALK1, ALK2, ALK3, or ALK6. SB-505124 is 3 to 5 times potent than SB-431542, a previously described related ALK5 inhibitor.

[0092] In the object of the present invention, "LY2157299" or "garnicertib" refers to a small molecule inhibitor of the TGF-β signaling pathway, particularly the TGF-β receptor I, which inhibits the activation of the normal pathway by specifically regulating SMAD2 phosphorylation.

[0093] For the purposes of this invention, "LY550410" is also used herein to refer to a small molecule inhibitor of the TGF-β signaling pathway, particularly the TGF-β receptor I.

[0094] Therefore, with the utmost preference, the concentration of a first SMAD inhibitor (i), selected from noggin, LDN193189, dorsomorphin, DMH1, factor A83-1, and combinations thereof, increases exponentially over a period of 3–5 days after the initial exposure of encapsulated pluripotent cells to the SMAD inhibitor, i.e., T0.

[0095] According to another highly preferred objective, the concentration of a second SMAD inhibitor (ii) selected from factors SB431542, SB505124, LY2157299, LY550410, and combinations thereof increases logarithmically over a period of 3–5 days after initial exposure of encapsulated pluripotent cells to the SMAD inhibitor.

[0096] According to another preferred object of the present invention, the first SMAD inhibitor(i) is noggin factor, and the concentration at initial exposure to encapsulated pluripotent cells is 50–70 ng / mL. Advantageously, the noggin concentration is then exponentially increased for at least 3 days after initial exposure to encapsulated pluripotent cells at a concentration of 50–70 ng / mL. Preferredly, the maximum concentration of noggin factor added is 100 ng / mL.

[0097] According to another preferred object of the present invention, the second SMAD inhibitor (ii) is factor SB431542, the concentration of which is 0.1–0.3 μM at initial exposure to encapsulated pluripotent cells. Advantageously, the concentration of SB431542 is then increased exponentially or logarithmically for at least 3 days after initial exposure to encapsulated pluripotent cells at a concentration of 50–70 ng / mL, until a maximum additive concentration of factor SB431542 of 20 μM is reached.

[0098] Therefore, unlike known 2D or 3D processes, exposing encapsulated pluripotent cells to at least one SMAD inhibitor, preferably two, in 3D culture, and gradually increasing the concentration of said inhibitors before adding other differentiation factors, particularly SHH activator and FGF activator, overcomes the shortcomings of the background technology, namely reducing cell death and simultaneously improving differentiation yield. For the purposes of the present invention, "before adding other differentiation factors" means exposing the cells to at least one SMAD inhibitor at least three days before adding other differentiation factors, particularly SHH activator and FGF activator.

[0099] This time-delayed exposure of pluripotent cells to various differentiation factors, combined with increasing concentrations of at least one SMAD inhibitor, preferably at least two SMAD inhibitors, for at least three and up to five days, results in improved yields, achieved by both reduced cell death and better differentiation rates of the target cells, and further facilitates the synchronization of the various cell populations obtained in capsules. In fact, pluripotent cells are always in the cell cycle; however, the cycle influences the cellular response to a given signal. Consequently, exposure to increasing concentrations over a characteristic cell cycle time (24 hours) optimizes the probability that cells receive the optimized signal at the point of their peak capability.

[0100] The differentiation method includes a step of exposing pluripotent cells involved in the differentiation process to at least one SHH activator and at least one FGF activator at least 3 days and up to 5 days after initial exposure of pluripotent cells to at least one SMAD inhibitor. Preferably, this process includes exposure to at least two SHH activators. Finally, exposure of pluripotent cells to the SMAD inhibitor is maintained for the duration of exposure to at least one SHH activator and at least one FGF activator.

[0101] Therefore, at least 3 days and up to 5 days after the initial exposure of pluripotent cells to at least two SMAD inhibitors, the pluripotent cells are exposed to the maximum concentration of SMAD inhibitors supplied to D3-D5 cells.

[0102] When the SMAD inhibitors are noggin and SB431542, respectively, the concentrations to which cells are exposed 3–5 days after differentiation are 100 ng / mL and 20 μM, respectively, over a period of 9–12 days.

[0103] SHH activators significantly activate the Sonic Hedgehog signaling pathway. The normal SHH protein signaling traverses multicomponent receptors, including Patched (PTCH1, PTCH2) and Smoothed (SMO). Binding of the SHH protein to PTCH inactivates the basal inhibition of SMO by PTCH. This pathway can be activated using the SHH protein, SHH-C25II, SAG (Smoothed Agonist), and palmorfamine. This pathway is involved in the construction of the developing central nervous system. The SHH protein regulates the fate of neural stem cells through tissue morphogenesis.

[0104] FGF-8b is a member of the fibroblast growth factor family. FGF-8b is widely expressed during embryogenesis and regulates epithelial-mesenchymal transition. FGF-8b plays a role in systematizing and inducing gastrulation and is also involved in the structuring of the midbrain / lobe.

[0105] Unlike SMAD inhibitors, the concentrations of SHH activators and FGF activators remain constant over time. Therefore, the preferred exposure period for SHH activators and FGF activators is 9–12 days, starting with exposure to cells already involved in differentiation that express particularly low levels of the markers OCT / NANOG and TRA-1-60 / OCT compared to pluripotent cells.

[0106] Therefore, at least one SHH activator is advantageously added at least 3 days and up to 5 days after initial exposure to encapsulated pluripotent cells and at least one SMAD inhibitor. Preferably, at least two SHH activators are added.

[0107] According to one object of the present invention, at least one FGF activator is advantageously added 3 to 5 days after the initial exposure of cells to at least one SMAD inhibitor and encapsulated pluripotent cells, and very preferentially in parallel with, i.e., simultaneously with, the exposure of cells to at least one SHH activator, more preferably two SHH activators. Therefore, the FGF activator is preferentially added 3 to 5 days after the initial exposure of cells to at least one SMAD inhibitor and pluripotent cells.

[0108] According to a particularly preferred object of the present invention, at least one SHH activator is SHH factor C25II at a target concentration of 100 ng / mL.

[0109] According to another particularly preferred object of the present invention, at least one SHH activator is a purmorphamine factor at a target concentration of 2 μM.

[0110] According to a preferred objective of the present invention, the FGF activator is rhFGF-8b at a concentration of 100 ng / mL.

[0111] The process according to the present invention also includes the step of exposing differentiated cells to a Wnt activator. This is preferably added for the exposure of differentiated cells, at least cells expressing factor PAX6 / SOX1, i.e., radial glial cells or radial glial progenitor cells (RGPCs), which are bipolar progenitor cells involved in the production of all neurons in the cerebral cortex and also produce specific glial cell lineages, particularly astrocytes and oligodendrocytes.

[0112] In preference, the Wnt activator is factor CHIR 99021 at a target concentration of 3 μM.

[0113] For another purpose, the concentration of at least one of the factors selected from SHH activator, FGF activator, and Wnt activator is 30–300% of the target concentration, preferably 50–200%, and more preferably 90–110%.

[0114] According to a preferred object of the present invention, the Wnt activator is added at least 6 days after initial exposure to at least one SMAD inhibitor and pluripotent cells. According to one modification, the Wnt activator is added at least 3 days after exposure to at least one SHH activator and one FGF activator to pluripotent cells committed to differentiation that express at least factor PAX6 / SOX1. According to another modification, if the cells are radial glial progenitor cells, at least one Wnt activator is added.

[0115] After exposing encapsulated pluripotent cells to at least two SMAD inhibitors, at least one SHH activator, at least one FGF activator, and at least one Wnt activator for 12–17 days, a heterogeneous population of neurons is obtained. This population contains multiple neuronal-type cells, of which at least 30%, preferably 40%, express the FOXA2 and / or OTX2 markers.

[0116] Preferably, the method may include an intermediate step for removing differentiation factors from the mixture. Advantageously, the intermediate step lasts 12 to 48 hours, and very advantageously, 24 hours.

[0117] According to a further object of the present invention, the differentiation process may include an additional maturation step of exposing the resulting cells to differentiation factors selected from at least rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and combinations thereof.

[0118] Therefore, a further object of the present invention is an in vitro method for differentiating pluripotent cells, further comprising exposing neurons obtained after exposing encapsulated pluripotent cells to at least two SMAD inhibitors for 12 to 17 days to at least rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, compound E, trichostatin A, and combinations thereof.

[0119] Furthermore, after exposing encapsulated pluripotent cells to at least one SMAD inhibitor for 12–17 days, the resulting neurons are exposed to differentiation factors (so-called mature differentiation factors or mature factors) selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and combinations thereof. These mature factors are added at a constant concentration for at least 5 days during the exposure period of at least 5 days.

[0120] For another purpose, the maturation factor is added at a constant concentration for a maximum of 30 days, preferably for a maximum of 11 days, and even more preferably for a maximum of 5 days.

[0121] Advantageously, the rhBDNF factor is added at one concentration for at least 12 days. According to a particularly preferred embodiment, rhBDNF is added at a target concentration of 10 ng / mL.

[0122] For particularly preferred purposes, L-ascorbic acid is added at a target concentration of 200 μM.

[0123] In a particularly preferred embodiment, rhGDNF is added at a target concentration of 10 ng / mL.

[0124] For particularly preferred purposes, rhTGF-β3 is added at a target concentration of 1 ng / mL.

[0125] In a particularly preferred embodiment, rhFGF-20 is added at a target concentration of 5 ng / mL.

[0126] In a particularly preferred embodiment, dbcAMP is added at a target concentration of 0.5 mM.

[0127] In a particularly preferred embodiment, DAPT is added at a target concentration of 10 μM.

[0128] For particularly preferred purposes, compound E is added at a target concentration of 1 μM.

[0129] For particularly preferred purposes, trichostatin A is added at a target concentration of 10 nM.

[0130] According to another objective, the concentration of at least one factor selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, and Trichostatin A is 30–300% of the target concentration, preferably 50–200% of the target concentration, and more preferably 90–110% of the target concentration.

[0131] Preferably, at the end of exposure of neurons to maturation factors selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and combinations thereof, the method includes an additional rinsing step. This step is designed to avoid simultaneous activation and inhibition of the TGF-β pathway.

[0132] In fact, according to another purpose, the method preferentially includes an additional rinsing step of at least one day if the microcompartment contains at least 50% of neurons expressing at least the FOXA2 and / or OTX2 markers.

[0133] Microtissue obtained by the method of the present invention At the end of the differentiation process according to any one of the embodiments described above, a plurality of nerve cells are obtained, and these nerve cells are organized in three dimensions to form a microtissue or neural tissue unit. Thus, according to another embodiment, the present invention also relates to a microtissue comprising a plurality of three-dimensionally organized nerve cells and extracellular matrix, the nerve cells which can be obtained by the differentiation process of the present invention. This plurality of nerve cells advantageously includes at least dopaminergic neurons, progenitor cells, in particular dopaminergic progenitor cells, and glial cells.

[0134] These multiple neurons express several cell type-specific markers obtained through the differentiation process. Preferably, the multiple neurons constituting the microtissue display cells positive for large amounts of the FOXA2 marker and cells positive for small amounts of the PAX6 / SOX1 marker, indicating an increase in the number of dopaminergic cells and a decrease in neural stem cells within the microtissue, reflecting a greater commitment to differentiation into the dopaminergic pathway, i.e., a better differentiation rate. Furthermore, high expression of the EN1 marker, a population shown to play a crucial role in transplantation efficiency (Kirkeby et al., 2017), leads to the acquisition of microtissue with improved functionality. Additionally, higher expression of the TH marker, a marker of dopaminergic neurons, reflects a greater commitment to differentiation into the dopaminergic pathway and therefore a better differentiation rate.

[0135] According to one embodiment, the nerve tissue unit may be solid without a lumen. This variation is preferably obtained when more than 20 cells are encapsulated per capsule at the time of pre-differentiation cell encapsulation.

[0136] In another modification, the nerve tissue unit may have at least one lumen. This modification is preferably obtained when, at the time of pre-differentiation cell encapsulation, no more than 20 cells are encapsulated per capsule.

[0137] In certain modifications, the object of the present invention is a three-dimensionally organized neural tissue unit comprising at least dopaminergic neurons, neural progenitor cells, and optionally neural stem cells, the unit having a maximum dimension of less than 600 μm and comprising at least one lumen surrounded by a C1 cell layer mainly comprising neural progenitor cells expressing at least SOX2. Preferably, the C1 layer comprises neural progenitor cells expressing OTX2. Preferably, the neural progenitor cells of the C1 layer express at least SOX2 and OTX2.

[0138] Therefore, the neural tissue unit comprises at least one lumen surrounded by a C1 cell layer primarily composed of neural progenitor cells expressing at least SOX2. In other words, the neural tissue unit comprises a C1 cell layer that forms a boundary juxtaposed within the lumen. The C1 cell layer is a concentric layer around the lumen.

[0139] The presence of a lumen within the nerve tissue unit significantly enhances cell survival and preserves specific physiological cellular structures in such tissues. During transplantation, such microtissues contribute to improved differentiation and / or survival of TH+ cells (i.e., dopaminergic neurons), thereby improving the viability and transplantation of nerve tissue units according to the present invention.

[0140] According to preferred purposes, the C1 layer at the rim of the lumen is itself surrounded by a C2 layer or cell bed containing dopaminergic neurons. The C2 layer or cell bed advantageously contains at least 3%, at least 5%, at least 10%, or at least 15% of dopaminergic neurons. Thus, the C2 cell layer forms a second concentric layer around the C1 layer. Most advantageously, the C2 cell layer contains at least 3%–50%, and more advantageously 15%–50%, of dopaminergic neurons expressing a tyrosine hydroxylase (TH) marker.

[0141] Furthermore, preferentially, several concentric layers are formed around the lumen, which are as follows: - Marginal cell layer (C1 cell layer), and - Around the edge, there is at least one concentric layer (C2 layer) distinct from layer C1, which contains dopaminergic neurons.

[0142] In one modification, the microtissue according to the present invention may include several lumens, of which at least one lumen is surrounded by a marginal cell layer (C1 cell layer), and around the margin there is further at least one concentric layer (C2 layer) distinct from the C1 layer, containing dopaminergic neurons. Preferably, if it includes several lumens, each lumen is surrounded by a marginal cell layer (C1 cell layer), and around the margin there is further at least one concentric layer (C2 layer) distinct from the C1 layer, containing dopaminergic neurons.

[0143] With the utmost priority, dopaminergic neurons are substantially excluded from the periluminal region, i.e., the region encompassing the lumen and layer C1. In fact, dopaminergic neurons are located in a second, more externally lateral bed and can be described as a cortical region of the second bed.

[0144] For the purposes of this invention, "substantially excluded" means that the periluminal region may contain a small number of dopaminergic neurons. In other words, the presence of at least one dopaminergic neuron, particularly 1 to 50 dopaminergic neurons, in the periluminal region cannot be excluded.

[0145] In the object of the present invention, the "cortical region of the second bed" refers in particular to the C2 cell layer, excluding any region directly adjacent to the lumen.

[0146] Therefore, preferentially, layer C2 contains at least 15% tyrosine hydroxylase (TH)-positive cells. The TH gene is a marker of dopaminergic neurons. Thus, layer C2 contains at least 15% of cells expressing this marker, i.e., at least 15% of dopaminergic neurons are present in layer C2.

[0147] According to a further object of the present invention, up to 50% of the cells contained in a nerve tissue unit express the Ki-67 marker, and preferentially 5-50% of such cells express the Ki-67 marker. Advantageously, layer C1 has a higher proportion of cells expressing KI67 than layer C2.

[0148] According to another particularly preferred objective, a three-dimensionally organized neural tissue unit comprises at least dopaminergic neurons and neural progenitor cells, has a maximum dimension of less than 600 μm, and includes at least one lumen surrounded by a C1 cell layer substantially composed of neural progenitor cells expressing at least SOX2.

[0149] Preferably, the microstructure obtained by the present invention has a substantially spherical shape.

[0150] Alternatively, the nerve tissue units obtained by the present invention may be oval, spherical, ball-shaped, or teardrop-shaped, or substantially oval, substantially ball-shaped, or substantially teardrop-shaped.

[0151] Preferably, the nerve tissue units obtained by the present invention have a maximum dimension of 10 μm to 600 μm ± 10%, preferably 150 μm to 400 μm ± 10%, more preferably 100 μm to 300 μm ± 10%, and even more preferably 200 μm ± 10%. These dimensions are particularly favorable for neuronal survival within the nerve tissue units and optimize graft reconstruction and angiogenesis after transplantation.

[0152] According to another preferred object of the present invention, the neural tissue unit obtained by the present invention comprises neural progenitor cells expressing FOXA2 and / or OTX2 and / or LMX1A in addition to the SOX2 marker, particularly dopaminergic progenitor cells. In very preferred terms, the C1 cell layer comprises neural progenitor cells expressing FOXA2 and / or OTX2 and / or LMX1A in addition to the SOX2 marker, and even more preferred terms, in the case of a tissue unit comprising a lumen and C1 and C2 layers, the C1 cell layer comprises neural progenitor cells expressing FOXA2 and / or OTX2 and / or LMX1A as well as SOX2.

[0153] According to one object of the present invention, in the case of a tissue unit comprising a lumen and C1 and C2 layers, the C1 cell layer comprises neural progenitor cells expressing SOX2 and FOXA2, or SOX2 and OTX2, or SOX2 and LMX1A. According to one modification, the C1 cell layer comprises neural progenitor cells expressing SOX2 and FOXA2 and OTX2, or SOX2 and FOXA2 and LMX1A, or SOX2 and OTX2 and LMX1A. According to one modification, the C1 cell layer comprises neural progenitor cells expressing SOX2 and FOXA2 and OTX2 and LMX1A.

[0154] According to another preferred object of the present invention, the neural tissue units obtained by the present invention include nerve cells expressing EN1 and / or GIRK2 or PAX6, particularly dopaminergic neurons. Optionally, the nerve cells may also express SOX1.

[0155] According to one modification, the neural tissue unit according to the present invention includes nerve cells expressing EN1 and GIRK2. In fact, dopaminergic neurons express TH and / or GIRK2 in particular, while dopaminergic progenitor cells express LMX1A and / or OTX2 and / or FOXA2 and / or EN1 and / or CORIN in particular.

[0156] In fact, high expression of the EN1 marker, which has been shown to play an important role in transplant efficiency (Kirkeby et al., 2017), leads to the acquisition of neural tissue units with improved functionality.

[0157] For another purpose, the nerve tissue unit according to the present invention may include glial cells.

[0158] In the context of the present invention, nerve tissue units are obtained within three-dimensional cellular microcompartments, particularly within cellular microcompartments.

[0159] Therefore, the nerve tissue units according to the present invention are preferably obtained in hollow hydrogel cell microcompartments (or capsules). Such cell microcompartments may contain one or more units of nerve tissue according to the present invention. Preferably, they contain a single nerve tissue unit according to the present invention.

[0160] According to a preferred object of the present invention, the neural tissue unit is obtained in a hollow hydrogel cell microcompartment by neural differentiation from cells capable of differentiating into nerve cells. The stem cells are preferably pluripotent stem cells. This may be any neural differentiation or neural differentiation process known to those skilled in the art, or one of the processes described in this application. Cells capable of differentiating into nerve cells include stem cells and / or neural 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 nerve cells are immunocompatible with humans, to whom the neural tissue unit is intended to be received.

[0161] According to a preferred embodiment, the stem cells are pluripotent stem cells, more preferably induced pluripotent stem cells, and even more preferably induced human pluripotent stem cells. According to another embodiment, the pluripotent cells are pluripotent stem cells other than human or animal embryonic stem cells. Regardless of embryonic stem cells, the embryo is not destroyed.

[0162] In one modification, the neural tissue unit is obtained within a microcompartment from up to 20 cells capable of differentiating into nerve cells, i.e., stem cells and / or progenitor cells, more preferably from up to 10 cells capable of differentiating, and even more preferably from up to 5 cells capable of differentiating, all containing a lumen. This amount of differentiateable cells encapsulated within such a cellular microcompartment is particularly advantageous in reducing the internal pressure of the microcompartment, which promotes the formation of the neural tissue unit according to the present invention, i.e., a unit containing a lumen and at least one cell layer adjacent to the lumen. Conversely, if the internal pressure is too high, a neural tissue unit is formed that lacks a lumen and biomimetic cellular tissue, i.e., advantageously, a C1 cell layer adjacent to the lumen and a C2 cell layer surrounding the C1 cell layer.

[0163] The present invention also relates to a method for preparing and obtaining modified nerve tissue units including a lumen, intended for transplantation into the nervous system of human mammals, the method comprising the following steps: a. A step of encapsulating up to 20 cells, preferably up to 10, and more preferably up to 5, that are capable of differentiating into nerve cells, in a hollow hydrogel microcompartment. b. A step of inducing cell differentiation of cells capable of differentiating into nerve cells present in a cellular microcompartment to obtain at least one nerve tissue unit having at least one lumen, c. Optionally, a step of at least partially removing the outer hydrogel layer from the microcompartment and recovering the nerve tissue unit according to the present invention.

[0164] Microtissue as a pharmaceutical product In its final form, the present invention relates to microtissue or nerve tissue units obtained by carrying out a process according to the present invention for use as a pharmaceutical, preferably for preventing and / or treating neurodegenerative diseases, and more preferably for preventing and / or treating Parkinson's disease.

[0165] In fact, the inventors have demonstrated that transplantation is possible by injecting the nerve tissue units according to the present invention, particularly as shown in Figure 7 (using microtissue without a lumen) and Figure 16 (using microtissue with a lumen).

[0166] In a modified form, the present invention also relates to a cellular microcompartment comprising at least one microtissue according to the present invention for use as a pharmaceutical agent.

[0167] The nerve tissue units according to the present invention can subsequently be implanted into the nervous system of a subject suffering from a neurodegenerative disease, particularly Parkinson's disease, to at least partially replace the defective neurons of the subject.

[0168] Herein, the present invention is demonstrated by non-limiting examples of the process according to the present invention, the resulting microstructures, and the results. [Examples]

[0169] Example 1: Differentiation protocol according to the present invention Manufacturing of nerve microtissue 2D culture of hiPSCs All hiPSC strains were maintained on vitronectin and cultured in mTeSR1 medium. The cultures were changed daily and subcultured every 3-4 days at 37°C for 6 minutes using the enzyme-free reagent ReLeSR (culture density approximately 80%). The cells were then reseeded as small clusters (100-200 μm) at a density of approximately 20,000-40,000 cells / cm2. The cells were then incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0170] 3D encapsulation of hiPSC Prior to encapsulation, 2D stem cell colonies were detached. HiPSCs were resuspended in mTeSR1 medium supplemented with 10 μM Y-27632. The cells were then mixed with human fibrinogen and Y-27632 in a 1:1 volume ratio to achieve final fibrinogen and Y-27632 concentrations of 14 mg / mL and 10 μM, respectively. Thus, the final cell concentration in the cell / matrix solution was 7.1 × 10^6 to 7.4 × 10^6 live cells / mL, which was referred to as the encapsulation density. The tube was connected to the three inlets of a 3D glass-printed microfluidic colaminar flow device. The tip of the 3D glass-printed microcapillary was coupled to the nozzle outlet for better flow control. The cell / matrix suspension was filled into the internal channel of the three-way device. Sodium alginate SDS solution was injected into the outer channel. To prevent the gelation of the alginate inside the microfluidic device due to calcium release by cells in suspension, a calcium-free solution (sorbitol) is used in the intermediate channel of the co-extruded tip to act as a barrier against calcium diffusion. This solution is also supplemented with thrombin at a final concentration of 0.02 U / mL to enable fibrin crosslinking within the capsule. Typical flow rates for the three solutions were approximately 80 mL / h for the three channels (alginate solution, sorbitol solution, and cell + matrix suspension). At these rates, the combined solution forms a liquid jet that breaks down into droplets. When the droplets come into contact with a 100 mM calcium bath, the outer layer of alginate readily gels. As a result, the inner cell / matrix solution remains trapped within a closed, spherical, permeable microcompartment. More than 20 cells were encapsulated per capsule. Within 5 minutes of encapsulation, the capsules are rinsed with DMEM / F-12 supplemented with 2.9 mM CaCl2 and 15 mM HEPES to reduce the base calcium concentration. Finally, they are transferred to mTeSR1 medium supplemented with 10 μM Y-27632 as an initial suspension culture medium for neural differentiation.

[0171] 3D neural differentiation of hiPSCs in a static T-flask or bioreactor Further neural differentiation can be achieved in several culture systems, including static suspension culture using T flasks or well plates, and stirred suspension culture using 30 mL or 500 mL bioreactors.

[0172] Under static conditions, encapsulated hiPSCs were cultured in suspension using T flasks (5-30 mL) maintained in a cell culture incubator at 37°C and 5% CO2, with the culture medium being changed daily.

[0173] Stirred suspension culture was performed in various bioreactors under stirring conditions. We used benchtop rBSTs containing 30 mL or 500 mL bioreactors. The stirring speed was set to 150 rpm for D0-D7, 200 rpm for D7-D18, 250 rpm for D18-D24, or 55 rpm for D0-D24 in the 500 mL and 30 mL rBSTs, respectively. In both cases, the bioreactor was inoculated with capsules at a ratio of 25% (V / V) of the culture medium volume. In the 500 mL bioreactor, the culture volume was maintained at 300 mL for the duration of the culture. On day 1, the medium was completely replaced with fresh medium supplemented with the ROCK inhibitor. On day 2, no medium change was performed. On day 3, the medium was completely replaced with new medium without the ROCK inhibitor. From that day onward, the medium was changed by injection. On days 12, 13, and 18, the culture medium was completely replaced with fresh medium. The final volume of capsules relative to the medium was 22%–25%, and the pH was maintained at 7.2 ± 0.2. Dissolved oxygen (DO) levels were calibrated to 100% before and after autoclaving in an empty bioreactor, and under initial conditions (filled with medium). Oxygen levels were monitored and controlled throughout the experiment. Oxygen levels were adjusted by controlling them to 50%.

[0174] Under static and agitated conditions, the molecules were added as follows. From day 0, 10 μM Y-27632 was added to the mTeSR1 medium for ROCK inhibition for the first 72 hours of culture. The mTeSR1 medium was stored until day 3. GMP recombinant human nogin protein was initially added at a concentration of 60 ng / mL on day 0 (encapsulation day), and gradually increased to 84 ng / mL and 100 ng / mL on days 1 and 2, respectively. Similarly, SB431542 GMP was initially added at a concentration of 0.2 μM on day 0, and gradually increased to 2 μM and 20 μM on days 1 and 2, respectively. On day 3, the medium was changed to Neurobasal® CTS® and DMEM / F-12, supplemented with GlutaMAX® in a 1:1 ratio, and supplemented with N-2 CTS® and B-27® GMP supplements. From day 3 to day 11, 100 ng / mL of GMP human nogin recombinant protein, 20 μM of SB431542 GMP, 100 ng / mL of Sonic Hedgehog / Shh(C24II)N-Term GMP human recombinant protein, 100 ng / mL of HumanKine® FGF-8b human recombinant protein, and 2 μM of StemMACS palmorfamine were added. CHIR99021 GMP was added from day 6 to day 12. Next, from D13 to D17, 200 μM ascorbic acid, 10 ng / mL GMP recombinant human GDNF protein, 1 ng / mL recombinant human TGF-β3 protein HumanKine®, 5 ng / mL recombinant human FGF-20 protein, 0.5 mM dibutyryl cAMP, 10 μM DAPT RMU, 1 μM Compound E, and 10 nM trichostatin A were added. Finally, 20 ng / mL HumanKine recombinant BDNF protein was added from D13 to D24. This differentiation protocol is shown in Figure 1.

[0175] Images of nerve microtissue obtained on D24 are shown in Figures 11 and 10B.

[0176] Example 2 - Dynamics of the emergence of different cell types during the differentiation process according to the present invention. protocol The protocol for preparing the microtissue is the same as the protocol in Example 1.

[0177] Flow cytometry analysis Nerve microtissue was collected at various stages of neuronal differentiation. The alginate capsule was removed by incubating the sample in a 20% final concentration capsule (V / V) in Reles® at room temperature for 5 minutes. After washing in Reles®, the nerve microtissue was dissociated using a neurosphere dissociation kit at 37°C for 40 minutes with agitation (150 rpm), and manually resuspended every 10 minutes using a micropipette. The cells were then fixed at a maximum cell density of 5.10^6 per mL using a 1:4 ratio of Fixation / Permeabilization concentrate and Fixation / Permeabilization diluent, and permeabilized. The cells were then resuspended in flow cytometry staining buffer at a cell density of 833,333 / mL. The cells were centrifuged at 500 g for 5 minutes at room temperature, and the supernatant was removed. The samples were then incubated with a specific antibody diluted 1:50 in 1× permeabilization buffer (Invitrogen) at room temperature in the dark for 30 minutes. Finally, the samples were washed twice, centrifuged at 500g for 5 minutes at room temperature, and the cells were resuspended in staining buffer. The samples were analyzed using MACSQuant® Analyzer 10 (Miltenyi Biotec). Isotype control experiments were performed to determine the positive limit beyond which the sample was considered positive, and antibody specificity was verified. Spectral overlap was eliminated using corrected controls for each fluorescent dye. Data were post-processed with FlowJo analysis software. Unstained cells were used as negative controls to distinguish between hepatocyte or neuron-specific antigen staining and nonspecific background signals. Therefore, significant signals from the NANOG+ / OCT4+, SSEA5+ / SSEA4+, and TRA-1-60+ / OCT+ populations were used to identify pluripotent cells and calculate their proportions. Signals from the PAX6+ / SOX1+ or FOXA2+ / OTX2+ populations were used to identify neural stem cells and dopaminergic progenitor cells in the samples and calculate their proportions.

[0178] RNA extraction and RT-PCR analysis Samples were homogenized in Tri-reagent (Euromedex), and RNA was isolated using a standard chloroform / isopropanol protocol. RNA was processed and analyzed according to modified known methods. cDNA was synthesized from 2 μg of total RNA using Maxima reverse transcriptase (Fisher Scientific). qPCR was performed using the LightCycler® 480 Real-Time PCR system (Roche). qPCR reactions were performed twice 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. Reference genes were determined using the RefFinder method. Where no specific reference gene was mentioned, relative expression analysis was corrected for five reference genes. The following genes were used: Proteasome subunit β4 (Psmb4), non-POU domain-containing octamer binding (Nono), chromosome 1 open reading frame 43 (C1orf43), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activated protein zeta (Ywhaz), and actin β (Actb). PCR data were exported and analyzed using software tools developed at NeuroCentre Magendie. Relative expression levels were assessed by calculating 2-ΔCt = 2-(Ct(target gene)-Ct(average of 5 reference genes)).

[0179] statistical analysis All statistical analyses were performed using GraphPad Prism 8. Behavioral analysis (in vivo) was performed using two-way ANOVA up to 20 weeks and Tukey's multiple comparison test to compare with the vehicle group. **** p<0.0001). Two-way ANOVA and Sidak's multiple comparison test were used from 24 to 32 weeks. * p<0.05).

[0180] result The results are shown in Figure 2. In this example, the selected differentiation days correspond to the iPSC encapsulation day (D0), as well as the end of the neural induction phase (D5), ventralization phase (after rinsing) (D12), maturation phase (D17), and late maturation phase (D24). At D0, almost all encapsulated cells express the markers OCT / NANOG, SSEA5 / SSEA4, and TRA-1-60 / OCT, which are characteristic of pluripotent iPSC-type cells. At D5, there is a significant decrease in the OCT / NANOG and TRA-1-60 / OCT markers, as well as an increase in the FOXA2 / OTX2 marker, thus indicating the initiation of cell differentiation. At D12, almost all cells express FOXA2 / OTX2, a marker characteristic of dopaminergic progenitor cells.

[0181] Example 3 - Comparison of the method according to the present invention and a method outside the scope of the present invention. protocol The test protocol is the same as that disclosed in Examples 1 and 2.

[0182] result The results shown in Figure 3 illustrate the transcriptome 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 to the expression of ACT-B housekeeping genes. The TH gene is a marker of dopaminergic neurons, and therefore, a higher proportion of dopaminergic neurons is demonstrated in the microtissues obtained using the process according to the present invention, due to the greater number of cells expressing this marker.

[0183] The results shown in Figure 3 illustrate the transcriptome 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 to the expression of ACT-B housekeeping genes. The EN-1 gene is a marker for dopaminergic progenitor cells, and therefore, higher expression of this marker in the microtissues obtained using the process of the present invention demonstrates a higher proportion of dopaminergic progenitor cells. Furthermore, this cell type is particularly desirable and interesting because EN-1 gene expression has been shown to be a predictive marker for transplantation success when using dopaminergic progenitor cells (Kirkeby et al., 2017). By using the process according to the present invention, EN-1 gene expression in the microtissues is increased compared to microtissues obtained using processes outside the scope of the present invention.

[0184] The results shown in Figure 4 describe the flow cytometry (FACS) analysis of dissociated microtissue obtained using the process according to the present invention, compared with microtissue obtained using a standard process outside the scope of the present invention.

[0185] The decrease in the number of neural stem cells (PAX6+ / SOX1+) and the increase in the number of associated dopaminergic cells (FOXA2+) reflect a greater commitment to differentiation into the dopaminergic pathway. The absence of the pluripotency marker (TRA-1-60+ / OCT+) confirms the absence of pluripotent stem cells in the microtissue obtained by the process according to the present invention. These results demonstrate a better commitment to differentiation and a resulting improvement in differentiation yield using the process according to the present invention.

[0186] Example 4 - Characterization of a microtissue composed of multiple nerve cells according to the present invention. protocol The test protocol is the same as that disclosed in Examples 1 and 2.

[0187] result Characterization of multiple cells constituting the microtissue by qPCR revealed the presence of numerous mature or differentiated cells. The results are shown in Figure 5. In particular, it was possible to observe the presence of cells such as neurons (MAP2, TUBB3), dopaminergic neurons (TH, GIRK2), dopaminergic precursors (LMX1A, OTX2, FOXA2, EN1, CORIN), astrocytes (GFAP), GABAergic neurons (GAD1), and oligodendrocytes (OLIG2).

[0188] Therefore, the microtissue contains multiple neural cells, including more mature cells or cells at the end of differentiation, confirming a better commitment to differentiation of neurons obtained using the process according to the present invention.

[0189] Example 5 - Injection of microtissue according to the present invention for the treatment of Parkinson's disease. protocol Induction of a rat model of unilateral Parkinson's disease Rowett nude rats (Rnu+) were first anesthetized using an induction chamber containing 4% isoflurane (induction chamber parameters: i4 Oxy1 Air1). Then, the rats were fitted with an anesthetic mask using approximately 2.5% isoflurane (induction chamber parameters: i2.5 Oxy0.4 Air0.4). The concentration could be slightly adjusted by manually checking the foot temperature and body temperature periodically. 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 infusion of 6-hydroxydopamine hydrobromide (6-OHDA / HBr). 2.5 μL of freshly prepared 0.5% (w / v) 6-OHDA / HBr was perfused into the medial forebrain bundle (MFB) using a stereotactic frame, according to the coordinate system (referenced to bregma): AP: -3.8, ML (right): 1.6, DV: -8 to -7. Three weeks after lesion stabilization, behavior was assessed using an amphetamine-induced rotation test.

[0190] Amphetamine-induced rotation test (rotometer) Amphetamine-induced rotational behavior was evaluated pre-transplant (3 weeks after 6-OHDA infusion) and every 4 weeks post-transplant. Rotation was recorded 10 minutes after intraperitoneal injection of amphetamine (2.5 mg / kg mg / kg-1) over 40 minutes. Results are shown in rotations / minute. Only rats that showed more than 3 rotations per minute after lesion stabilization were included in the behavioral analysis.

[0191] Injection of neuronal microtissue in a rat model of unilateral Parkinson's disease Rowett nude rats (Rnu+) were first anesthetized using an induction chamber containing 4% isoflurane (induction chamber parameters: i4 Oxy1 Air1). Then, the rats were fitted with an anesthetic mask using approximately 2.5% isoflurane (induction chamber parameters: i2.5 Oxy0.4 Air0.4). The concentration could be slightly adjusted by manually monitoring foot temperature and body temperature periodically. Analgesia was achieved by subcutaneous injection of buprenorphine and lidocaine at concentrations of 0.05 mg / kg and 5 mg / kg, respectively. Next, the rats were injected with nerve microtissue into the right striatum using a custom administration medium consisting of 25 μL of Hamilton (Hamilton, ref. 1702 CX SYR) fitted with a custom needle (glass cannula) and carboxymethylcellulose and dextran 70 kDa (CMC-DA70). Injection was performed in two trajectories using a stereotactic frame. At the end of the surgery, Metacam was administered subcutaneously at a concentration of 1 mg / kg for postoperative pain management.

[0192] Postmortem Examination Postmortem histological analysis The animals were first anesthetized by intraperitoneal injection of ketamine and xylazine. Next, the animals were intracardiac perfused with 150 mL of 0.9% NaCl, followed by 200 mL of 10% phosphate-buffered formalin. After extraction, the brains were fixed in 10% phosphate-buffered formalin at 4°C for 24 hours and finally stored in PBS at 4°C. 40 μm sections were collected using a vibratome and stored in PBS containing 0.01% azide. The sections were washed three times with PBS. The sections were permeabilized by incubation in PBS containing 0.1% Triton and 2% BSA at room temperature for 1 hour, and then washed three times with PBS. The sections were stained with tyrosine hydroxylase (TH, 1:1000) and human antigen (Stem121, 1:1000) by incubation of primary antibodies in PBS containing 0.05% Triton and 0.5% BSA overnight at 4°C. The sections were washed three times with PBS. Then, the sections were incubated for 2 hours at room temperature with 0.05% Triton and fluorescent conjugate secondary antibodies (Alexa 488, 568) (1:1000) in PBS containing BSA. After rinsing three times with PBS, the sections were mounted on slides using DAPI Fluoromount-G for nuclear counterstaining. The slides were imaged using a nanozoomer.

[0193] result The objective of the study was to demonstrate the restoration of motor asymmetry in a nonclinical efficacy study using the nerve microtissue according to the present invention. The results are shown in Figures 6 and 7.

[0194] The inventors observed d-amphetamine-induced rotation measured preoperatively and at 4, 8, 12, 16, and 20 weeks post-transplant (n=43) and 24, 28, and 32 weeks post-transplant (n=2). Rotation was measured using a rotometer 10 minutes after injecting d-amphetamine (2.5 mg / kg ip) for 40 minutes. Nerve microtissue transplantation resulted in complete functional recovery of motor impairment from week 16 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 in the Cryo 6K group, with complete functional recovery at week 20 (p<0.0001). Long-term functional motor recovery was observed, and the number of amphetamine-induced rotations remained stable between weeks 16 and 32 (n=2). Cryo 6K, Cryo 10K, and Cryo 21K correspond to cryopreserved products with an estimated total of 6,000, 11,000, and 21,000 dopaminergic neurons (TH+ cells) initially implanted, respectively. Fresh 6K and Fresh 10K correspond to newly manufactured products with an estimated total of 3,000 and 10,000 dopaminergic neurons initially implanted, respectively. Comparisons with the vehicle group were performed using two-way ANOVA and Tukey's multiple comparison test up to week 20. **** (p<0.0001). Two-way ANOVA and Sidak's multiple comparison test were used from 24 to 32 weeks. Data are presented in mean ± SEM format.

[0195] The results in Figure 7 are fluorescence microscopy images of graft sections 20 weeks after transplantation of microtissue according to the present invention, immunostained for the TH marker (B) and the human marker Stem121 (C). Fusion of the two markers is also shown, confirming the presence of dopaminergic neurons derived from the microtissue (D). The rectangles indicate the enlarged region shown in the right panel (E), showing reinnervation of host tissue from the graft, revealed by the labeling of axonal projections derived from the graft.

[0196] Example 6 - Preparation of microtissue including the lumen. The protocol is identical to that described in Example 1, except that the encapsulated cells, i.e., HiPSCs, are 2 to 20 (preferably 5) HiPSC cells per capsule to limit internal pressure during differentiation within the capsule and promote better cell organization. The final cell concentration in the cell / matrix solution is then 2.2 × 10^6 to 3.7 × 10^6 live cells / mL, which is referred to as the encapsulation density.

[0197] An example of the obtained nerve tissue units is shown in Figure 10A.

[0198] Example 7 - Characterization of a microtissue (10A) consisting of multiple nerve cells including the lumen according to the present invention, and a unit of multiple nerve cells (10B) according to the present invention that does not include the lumen. The purpose of this study is to characterize the microtissue of the present invention obtained after neural differentiation.

[0199] Hematoxylin-eosin-saffron (HES) staining and histological feature scoring Microtissue was fixed with 4% paraformaldehyde at room temperature for 1 hour, then washed with phosphate buffer. The microtissue was centrifuged (10 minutes, 1200 rpm) and pre-coated with 3% agarose. After dehydration in a continuous bath of ethanol, acetone, and xylene, the samples were embedded in paraffin. 5 μm sections were prepared using a microtome and then attached to a processing slide using an albumin-glycerol mixture. After deparaffinization, the sections were continuously immersed in Harris hematoxylin, eosin, and saffron solutions. After dehydration, the sections were placed between the slide and a coverslip using Entellan®. Hematoxylin-eosin-saffron staining allowed observation of the tissue's morphology and structure. The cytoplasm appeared pink, and the nucleus appeared bluish-purple. The extracellular matrix was stained yellow to pink.

[0200] Stained microtissues were imaged using a slide scanner (NanoZoomer 2.0RS, Hamamatsu), and neuropathologists evaluated the physiological features of the human fetal midbrain: the presence or absence of ventricles (referred to as lumen in this invention), ventricular zone (referred to as zone C1 in this invention), intermediate zone, and mantle zone (referred to as zone C2 in this invention) (Arenas et al. 2015, https: / / doi.org / 10.1242 / dev.097394). Abnormal histological features, including abnormal cell nuclear morphology and nuclear disintegration, were also observed.

[0201] These microtissues are shown in Figure 10A (with lumen) and Figure 10B (without lumen).

[0202] Immunofluorescence labeling, microscopy, and image analysis Encapsulated 3D neural microtissues showing the lumen were collected for confocal microscopy at the end of the neural differentiation process (D24). The alginate capsules were removed by incubating the samples in ReleaseR at room temperature for 5 minutes with a final concentration of 20% capsules (V / V). The microtissues with lumen were fixed with 4% PFA in the dark at room temperature for 1 hour. After fixation, the samples were washed three times with 0.1% Tween20 in PBS. The permeabilization process was performed in a PBS solution containing 5% Triton X-100 with stirring (170 rpm) at room temperature for 30 minutes. The samples were washed three times with 0.1% Tween20 in PBS. The samples were incubated with appropriate primary and secondary antibodies and 0.1% Tween20 in PBS with stirring (170 rpm) at room temperature for 72 hours. After each incubation, the samples were rinsed five times with 0.1% Tween20 in PBS, two of which were rinses with stirring at 170 rpm for 30 minutes. These microtissues are shown in Figures 12, 13, and 14 and were imaged using a fluorescence microscope or confocal microscope with deconvolution.

[0203] The results are shown in Figures 12-14, where we confirm the presence of dopaminergic neurons that are not radially arranged around the lumen. In contrast, dopaminergic precursors (precursor cells) are organized radially around the lumen [Figure 12].

[0204] Figure 13 confirms the presence of KI67-labeled cells in the nerve tissue unit.

[0205] Figure 14 confirms the presence of dopaminergic precursors (precursor cells) surrounding the lumen in the C1 cell layer adjacent to the lumen.

[0206] Figure 15 shows the size distribution of microtissues according to the present invention in D24. Microtissues were imaged using a wide-field microscope, and their size was measured using proprietary image analysis software. The average diameter of the microtissues was 176.8 ± 60.48 μm.

[0207] Example 8 - Injection of a lumen-containing microtissue for the treatment of Parkinson's disease. The protocol is identical to that of Example 5, except that the injected microtissue contains a lumen.

[0208] result The results are shown in Figure 16, which are fluorescence microscopy images of graft sections 20 weeks after microtissue transplantation, immunostained for the TH marker, the human marker Stem121, and the nuclear dye DAPI. Compared to rats transplanted with Batch 2 (microtissue without lumen according to the present invention), rats transplanted with Batch 1 (microtissue with lumen according to the present invention) showed a greater abundance of microtissue-derived TH+ dopaminergic neurons, which were more uniformly distributed in the graft.

[0209] Panel C represents the steological quantification of the total number of TH-expressing cells in the graft at 20 weeks, corrected for the volume of injected microtissue with or without a lumen.

[0210] Panel D shows the steological quantification of the total number of TH-expressing cells, corrected for the number of luminal or non-luminal microtissues injected at 20 weeks post-transplantation.

[0211] Each dot corresponds to a rat. A non-parametric Mann-Whitney test was performed. ns=p>0.05. * p<0.05(C, D).

Claims

1. An in vitro method for differentiating pluripotent cells into nerve cells, the method being carried out in at least one three-dimensional cell microcompartment in a suitable culture medium, the microcompartment comprising the pluripotent cells, the method comprising, - At least two SMAD inhibitors, - At least one SHH activator, - At least one FGF activator, and - At least one step of exposure to at least one Wnt activator, A method comprising the step of increasing the concentration of at least one SMAD inhibitor in the culture medium by at least 30% from the initial concentration of the SMAD inhibitor, continuously or discontinuously, for at least 3 days from the initial exposure of the pluripotent cells to the SMAD inhibitor, thereby obtaining neurons expressing at least FOXA2.

2. The method according to claim 1, wherein the concentration of at least one SMAD inhibitor, preferably at least two SMAD inhibitors, increases exponentially.

3. The method according to claim 1 or 2, wherein the concentration of the at least one SMAD inhibitor increases up to four days after the initial exposure of the pluripotent cells to the SMAD inhibitor.

4. The method according to claim 3, wherein, one day after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 40%, and the concentration of the second SMAD inhibitor (ii) increases by at least 900%.

5. The method according to claim 4, wherein two days after initial exposure of pluripotent cells to the SMAD inhibitor, the concentration of the first SMAD inhibitor (i) increases by at least 67%, and the concentration of the second SMAD inhibitor (ii) increases by at least 9,900%.

6. The method according to any one of claims 1 to 5, wherein the first SMAD inhibitor (i) is capable of acting on the BMP-2, 4, and 7 pathways, and the second SMAD inhibitor (ii) is capable of acting on the TGFβ, activin, and Nodal pathways.

7. The method according to claim 6, wherein the first SMAD inhibitor (i) is selected from noggin factor, LDN193189, dorsomorphine, DMH1, A-83-01, and combinations thereof, and the second SMAD inhibitor (ii) is selected from SB431542, SB505124, LY2157299, LY550410, and combinations thereof.

8. The method according to claim 7, wherein the concentration of the noggin factor at the time of initial exposure is 50 to 70 ng / mL.

9. The method according to claim 7 or 8, wherein the concentration of factor SB431542 at the time of initial exposure is 0.1 to 0.3 μM.

10. a. The at least one SHH activator is added at least three days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and / or b. The FGF activator is added at least three days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and / or c. The method according to any one of claims 1 to 9, wherein the at least one Wnt activator is added at least 6 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells.

11. The method according to any one of claims 1 to 10, wherein the pluripotent cells are in an isolated form or an aggregated form.

12. The method according to any one of claims 1 to 11, wherein the pluripotent cells are induced pluripotent stem cells (iPSCs).

13. The method according to any one of claims 1 to 12, further comprising exposing the obtained nerve cells to at least one factor selected from rhBDNF, L-ascorbic acid, rhGDNF, rhTGF-β3, rhFGF-20, dbcAMP, DAPT, Compound E, Trichostatin A, and combinations thereof to obtain nerve cells expressing at least the FOXA2 / OTX2 marker.

14. The method according to claim 13, wherein the microcompartment comprises at least 50% of nerve cells expressing at least the FOXA2 / OTX2 marker, and further comprises an additional intermediate step of at least one day in which the culture medium does not contain a TGF-β pathway inhibitor.

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