Neural tissue unit and use of such a unit as a drug

Abstract

The present invention relates to a neural tissue unit comprising dopaminergic neurons and neural progenitor cells, said unit being organized in three dimensions (3D) and having a largest dimension less than 600 µm. The invention also relates to its use as a drug, particularly in the prevention and / or treatment of neurodegenerative diseases by implanting said unit alone or encapsulated in a cellular microcompartment in the nervous system of a mammal, especially a human.

Description

Title of the invention: Neural tissue unit and use of such a unit as a drug technical field

[0001] The invention relates to a neural tissue unit, comprising dopaminergic neurons and neural progenitor cells, said unit being organized in three dimensions (3D) and having a largest dimension of less than 600 µm. The invention also relates to its use as a drug, particularly in the prevention and / or treatment of neurodegenerative diseases by implanting said unit alone or encapsulated in a cellular microcompartment in the nervous system of a mammal, in particular a human. Prior art

[0002] Neurodegenerative diseases continue to pose a major challenge to society, both in terms of understanding the biological phenomena involved and in terms of treatment. Indeed, more and more people are affected by these diseases, which have significant consequences for both the patient and their family.

[0003] Neurodegenerative diseases encompass a very large number of diseases, characterized primarily by neuronal death that is more rapid than during normal aging, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc. The nervous system can be affected in various ways, leading to a variety of symptoms including motor, balance, behavioral, and / or cognitive disorders.

[0004] Currently, the causes and mechanisms of neurodegenerative diseases are often poorly understood, making their treatment all the more complex. There is currently no proven treatment that can cure these diseases.

[0005] In recent years, several trials of intracerebral cell therapy, particularly the transplantation of neurons into damaged areas of the brain of subjects with neurodegenerative diseases to permanently replace neurons destroyed by the disease, have been successfully performed in several patients with Parkinson's disease. However, this approach involves the use of tissues derived from aborted fetuses, which raises ethical and logistical problems. Therefore, many laboratories have turned to human induced pluripotent stem cells (hIPSCs).

[0006] The efficient differentiation of hIPSCs into neurons with the phenotype necessary to replace destroyed neurons in patients is a very active area of ​​research.

[0007] However, neurons are very fragile cells. Therefore, cellular microcompartments have been developed that allow the differentiation of human induced pluripotent stem cells (hIPSCs) into neurons in the form of a neural tissue unit. Neural tissue units suitable for implantation in a patient suffering from such a disease are thus already known.

[0008] Although this solution is satisfactory, there is a need for new neural tissue units to improve graft take, survival rate and functionality once the units are implanted, through improvement of cell organization within said neural tissue unit. Summary of the invention

[0009] To meet this need, the inventors have developed a new type of neural tissue unit or neural micro-tissue exhibiting improved cell organization in a biomimetic manner of in vivo structuring. In particular, the invention is a neural tissue unit or neural micro-tissue, that is to say, a cluster of cells exhibiting a biomimetic organization, comprising a plurality of neural cells, the plurality of neural cells including in particular progenitor cells but also dopaminergic neurons and possibly neural stem cells, in which said unit comprises at least one lumen bordered by a layer of cells comprising predominantly neural progenitor cells (also called neural progenitor cells), said neural progenitor cells expressing at least the SOX2 marker.Furthermore, this unit is organized in three dimensions and has a largest dimension of less than 600 pm.

[0010] Thus, the invention relates to a three-dimensionally organized neural tissue unit comprising at least dopaminergic neurons and neural progenitor cells, and optionally also neural stem cells, said unit having a largest dimension of less than 600 pm, and comprising at least one lumen bordered by a Cl layer of cells comprising predominantly progenitor cells expressing at least SOX2. Preferably, the dopaminergic progenitors express at least SOX2 and OTX2.

[0011] The neural tissue unit according to the invention thus comprises at least one lumen which is lined by the Cl layer of cells comprising predominantly neural progenitor cells expressing at least SOX2. In other words, the neural tissue unit includes a layer Cl of cells forming a border juxtaposed to said lumen. Said layer Cl of cells being a concentric layer around said lumen.

[0012] According to a preferred embodiment of the present invention, the Cl layer at the edge of the lumen is itself surrounded by a C2 cell layer comprising dopaminergic neurons, more preferably at least 3%, at least 5%, at least 10%, and even more preferably at least 15% of the cells in the C2 layer are dopaminergic neurons. The C2 cell layer thus forms a second concentric layer around the CL layer. The C2 cell layer may also include neural progenitors.

[0013] Advantageously, several concentric layers are thus formed around the light, namely: - a border layer of cells (the Cl cell layer), and - around said border at least one more concentric layer distinct from the Cl layer which includes dopaminergic neurons (the C2 layer).

[0014] According to one embodiment, the neural tissue unit according to the invention comprises one or more neural stem cells, preferably in the Cl layer and / or in the C2 layer.

[0015] According to one embodiment, the microtissue according to the invention may comprise several lumens, at least one lumen of which is surrounded by a border layer of cells (the cell layer C1), and around said border at least one further concentric layer distinct from the C1 layer that comprises dopaminergic neurons (the C2 layer). Preferably, if it comprises several lumens, each lumen is surrounded by a border layer of cells (the cell layer C1), and around said border at least one further concentric layer distinct from the C1 layer that comprises dopaminergic neurons (the C2 layer).

[0016] Very preferentially, dopaminergic neurons are substantially excluded from the periluminal zone, that is to say from the zone comprising the light and the CL layer. Indeed, dopaminergic neurons are essentially present in a second cortical zone, namely the C2 layer.

[0017] Preferably, layer C2 comprises at least 15% of cells positive for tyrosine hydroxylase (TH), namely at least 15% of dopaminergic neurons expressing the TH marker.

[0018] According to another object of the present invention, at most 50% of the cells included in the neural tissue unit express the Ki-67 marker, preferably between 5 and 50% of said cells express the Ki-67 marker. Neural progenitors expressing Ki-67.

[0019] According to another particularly preferred object, the three-dimensional organized neural tissue unit, comprising at least dopaminergic neurons and cells neural progenitor cells, said unit has a largest dimension less than 600 pm, and comprises at least one lumen bordered by a Cl layer of cells consisting essentially of progenitor cells expressing at least SOX2.

[0020] Preferably, the neural tissue unit according to the present invention is in the form of an ovoid, a sphere, a ball, or a teardrop, or in a substantially ovoid, substantially spheroid, substantially ball-shaped, or substantially teardrop-shaped form. Preferably, the neural tissue unit according to the present invention is in a substantially spheroid shape; in particular, said unit is a cluster of substantially spheroid-shaped cells, the cells of which are specifically organized, namely, a layer of C1 cells and a layer of C2 cells, which are successively organized around said lumen.

[0021] Preferably, the largest dimension of the neural tissue unit according to the invention is between 100 and 300 pm, more preferably 200 pm (+ or - 20 pm).

[0022] According to another preferred object of the invention, the neural tissue unit according to the invention comprises neural progenitor cells that express FOXA2 and / or OTX2 and / or LMX1A, in addition to the SOX2 marker. Most preferably, the cell layer Cl comprises dopaminergic progenitors that express FOXA2 and / or OTX2 and / or LMX1A, in addition to the SOX2 marker; even more preferably, the cell layer Cl comprises dopaminergic progenitors that express FOXA2 and / or OTX2 and / or LMX1A and SOX2.

[0023] According to another preferred object of the invention, the neural tissue unit according to the invention comprises neural cells expressing EN1 and / or GIRK2 or PAX6. Optionally, the neural tissue unit may contain neural cells that may also express SOX1.

[0024] According to another object, the neural tissue unit according to the invention may optionally include glial cells.

[0025] In the context of the invention, the neural tissue unit is advantageously obtained in a three-dimensional cellular microcompartment.

[0026] Thus, the neural tissue unit according to the invention is preferably obtained in a hollow hydrogel cell microcompartment. The hollow hydrogel cell microcompartment thus forms a hollow capsule having an outer layer (shell) of hydrogel. Such a cell microcompartment may comprise one or more neural tissue units according to the invention. Preferably, it comprises a single neural tissue unit according to the present invention.

[0027] According to another preferred object of the invention, the neural tissue unit according to the invention is obtained in a hollow hydrogel cell microcompartment, by neural differentiation from pluripotent cells.

[0028] According to one variant, the neural tissue unit according to the invention is obtained in a hollow hydrogel cell microcompartment, by neural differentiation from neural stem cells and / or progenitor cells.

[0029] According to another particularly preferred object, the neural tissue unit was obtained from at most 20 pluripotent stem cells and / or neural stem cells and / or progenitor cells, more preferably from at most 10 pluripotent stem cells and / or neural stem cells and / or progenitor cells, even more preferably from at most 5 pluripotent stem cells and / or neural stem cells and / or progenitor cells.

[0030] According to another preferred object, pluripotent cells are pluripotent stem cells, more preferably induced pluripotent stem cells (iPSC or hiPSC for human induced pluripotent stem cells).

[0031] In another aspect, the invention relates to a composition comprising at least two units of neural tissue according to any one of the embodiments described above. In a variant, the invention relates to a kit comprising at least two units of neural tissue according to any one of the embodiments described above.

[0032] According to another aspect, the invention relates to a neural tissue unit according to the present invention or a composition according to the invention or a kit according to the invention, for its use as a drug.

[0033] According to one embodiment, the invention also relates to a cellular microcompartment comprising at least one neural tissue unit according to the present invention, for use as a medicinal product. In this embodiment, said microcompartment comprising said neural tissue unit according to the invention is injected or administered into a subject in need.

[0034] Thus, the invention also relates to a three-dimensional, hollow cellular microcompartment comprising an outer hydrogel layer, said microcompartment comprising at least one neural tissue unit according to the invention. In certain embodiments, the cellular microcompartment may comprise extracellular matrix of natural origin (in particular naturally produced by the encapsulated cells themselves), and / or Matrigel®, and / or an extracellular matrix substitute, these extracellular matrices being present, in particular, in the intercellular spaces that may remain in the neural tissue unit.

[0035] Preferably, the neural tissue unit according to the present invention, or the composition according to the invention, or the kit according to the invention, is intended to be used in the prevention and / or treatment of a neurodegenerative disease, most preferably Parkinson's disease. Thus, the invention also relates to a neural tissue unit according to the present invention or a composition according to the invention or a kit according to the invention, for its use in preventing and / or treating a neurodegenerative disease, preferably Parkinson's disease.

[0036] According to another aspect, the invention also relates to an assembly of three-dimensional cellular microcompartments, said assembly comprising at least one cellular microcompartment as described above, each cellular microcompartment comprising at least one neural tissue unit according to the invention.

[0037] Finally, according to a last aspect, the invention also relates to a method for obtaining the neural tissue unit according to the invention, comprising the following steps: - encapsulation in a hollow hydrogel microcompartment of no more 20 pluripotent stem cells, preferably no more than 10, more preferably no more than 5, - induction of cell differentiation of pluripotent cells present in the cellular microcompartment, so as to obtain at least one unit of neural tissue according to the invention, - possibly remove at least partially the outer hydrogel layer of the microcompartment (i.e. the hydrogel capsule) to recover the neural tissue unit according to the invention.

[0038] The induction of cell differentiation of pluripotent cells will be initiated in particular by the addition of the corresponding differentiation factors which will be described below, in particular SMAD, SHH, FGF, and Wnt.

[0039] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures that will follow. Brief description of the Figures

[0040] [Fig. 1] is a representation of a neural tissue unit according to the invention, the cells of which are stained for the markers FOXA2 and OTX2, as well as Ki67

[0041] [Fig. 2] is a representation of a neural tissue unit according to the invention, the cells of which are stained for the markers SOX2 and NESTIN

[0042] [Fig. 3] A and B) Rhematoxylin-eosin-safranin (H&E) staining of microtissues according to the invention at D24. A) Microtissue according to the invention exhibiting a dense zone of nuclei, the Cl layer (dotted lines) organized radially around a lumen (“L”) Protocol 1. B) Microtissue without a lumen outside the scope of the invention; these figures notably show cells with abnormal cytonuclear morphology such as larger nuclei with decondensed chromatin (arrowheads) and cells with karyorrhexis (*). Scale bars: 50 pm.

[0043] [Fig.4] Hematoxylin-eosin-safranin (HES) staining of micro tissues according to the invention at D24 without progressive addition of SMAD factors during neurodifferentiation (Protocol 2).

[0044] [Fig. 5] Immunofluorescence images of microtissues according to the invention at D24. The neural microtissues were immunostained for the dopaminergic neuron marker TH (A), the dopaminergic progenitor marker FOXA2 (B), and counterstained with the nuclear dye DAPI (C). Scale bar: 50 µm. * indicates the presence of the lumen. Cells arranged radially around the lumen are predominantly positive for FOXA2 and correspond to layer C1, while TH expression is localized to the periphery of the cells surrounding the lumen, corresponding to layer C2.

[0045] [Fig. 6] Immunofluorescence images of microtissues according to the invention at D24. The neural microtissues were immunostained for the proliferative cell marker Ki67 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 µm. * indicates the presence of light. Cells arranged radially around the lumen are positive for Ki67 (approximately less than 50% of the cells).

[0046] [Fig. 7] Immunofluorescence images of microtissues according to the invention at D24. The neural microtissues were immunostained for the dopaminergic progenitor marker OTX2 (A) and counterstained with the nuclear dye DAPI (B). Scale bar: 50 µm. Asterisks (*) indicate the presence of lumen. Cells arranged radially around the lumen are predominantly positive for OTX2, corresponding to the CL layer

[0047] [Fig. 8]. Size distribution of the microtissues according to the invention at D24. The microtissues were imaged using a wide-field microscope and their size was measured using proprietary image analysis software. The average diameter size of the microtissues is 176.8 ± 60.48 pm.

[0048] [Fig. 9]. Post-mortem histological analysis of rats transplanted with microtissues according to the invention (Lot 1) and outside the invention (Lot 2). A and B) represent brain sections of animals 20 weeks after transplantation with Lot 1 (A) and Lot 2 (B), immunostained for TH and the human marker Steml21. Scale bars: 2.5 mm. C, D) Quantification of the number of TH-expressing cells per volume of injected microtissue (C) and the number of TH-expressing cells per number of injected microtissues (D). The non-parametric Mann-Whitney test was performed. ns = p > 0.05; * p < 0.05.

[0049] [Fig. 10] Post-mortem histological analysis of Macaca fascicularis transplanted with microtissues according to the invention. Panels A and B represent sections of animal brain (left hemisphere) 30 days after transplantation, immunostained for TH+ and the human marker Steml21. Detailed description of the invention

[0050] Definition

[0051] For the purposes of this invention, "microcompartment," "cellular microcompartment," or "capsule" refers to a partially or totally enclosed, hollow, three-dimensional structure containing one or more cells. The structure consists of an outer layer of hydrogel, preferably rigidified, and an inner hollow portion comprising at least one cell and / or at least one aggregate of cells and / or cellular microtissue (or tissue unit), and optionally an extracellular matrix and / or extracellular matrix substitute adapted for cell culture and the growth of said cells.

[0052] By "differentiated cells" within the meaning of the invention, we mean cells that exhibit a particular phenotype, as opposed to pluripotent cells, which are not differentiated, or progenitor cells, which are in the process of differentiation. In this context, differentiated cells are mature cells, for example, neuronal cells, i.e., neurons, for example, dopaminergic neurons, expressing at least the FOXA2 and TH factors.

[0053] For the purposes of this invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even where not specified, cells, pluripotent cells, progenitor cells, and differentiated cells, including neural cells, are obtained or derived from human cells or immunologically humanized non-human mammalian cells.

[0054] By "pluripotent" cells within the meaning of the invention, we mean cells that have the capacity to form all the tissues present in the entire organism of origin, without, however, being able to form an entire organism as such, since they have already undergone a first stage of differentiation; they can only generate the cells of the embryonic germ layers, endoderm, mesoderm, and ectoderm, but can no longer generate the cells of the trophectoderm. Human pluripotent stem cells may be called hPSCs or ES in the context of the present invention. These may, in particular, 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, and surface markers. such as SSEA4 / 5, Tra-1-60, and Tra-1-81. Optionally, according to a very specific embodiment, pluripotent cells obtained from embryonic stem cells are obtained without destruction of the embryo from which they originate, for example, using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, said human embryonic stem cells may be excluded.

[0055] For the purposes of this invention, "induced pluripotent stem cell" or "iPSC" or "hiPSC" refers to a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for pluripotency markers, such as alkaline phosphatase staining and the expression of the proteins NANOG, SOX2, OCT4, and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 2007, 131(5): 861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0056] For the purposes of this invention, "neural cells" refers to all cells of the nervous system. These may be mature cells, for example, dopaminergic neurons, GABAergic neurons, etc., or supporting cells known as glial cells (astrocytes, oligodendrocytes, etc.); progenitor cells (dopaminergic progenitors, GABAergic progenitors, etc.); or stem cells that give rise to neural tissue (neural stem cells). They may also be a mixture of neural-type cells, in particular a heterogeneous population of neural cells comprising several cell types, for example, dopaminergic neurons, progenitors, neural stem cells, and possibly glial cells.

[0057] For the purposes of this invention, "progenitor cells" or "progenitor" are understood to be cells that are undergoing differentiation, that is, already committed to a differentiation pathway, but not yet differentiated. In the context of this invention, progenitor cells are neural progenitor cells or neural-type progenitor cells, for example, radial glial cells, or radial glial progenitor cells (RGPCs), but also dopaminergic progenitors. For the purposes of this invention, "cell layer" or "cell layer" refers to several cells forming a layer or layer structured around a lumen; this could be, for example, a group of cells cooperating with each other and grouped in three dimensions. The thickness of the cell layer or layer can be variable. This layer or layer is organized in three dimensions within the neural tissue unit.

[0058] For the purposes of this invention, "drop" also means a three-dimensional structure formed from at least one liquid solution comprising the constituents of an unrigidified hydrogel (polymerization precursors, chains of non- or partially cross-linked polymers), of hydrogel precursor elements. Also, the droplet constitutes a transitional state between the co-injection of the different constituents and the microcompartment according to the invention.

[0059] By "Feret diameter" in the sense of the invention, we mean the distance, in particular "d" or "D", between two tangents, these two tangents being parallel, such that the whole of the projection is contained between these two parallel tangents.

[0060] For the purposes of this invention, "micro-tissue" or "neural tissue unit" refers to a neural tissue unit comprising a plurality of neural cells, such as dopaminergic neurons, progenitor cells (particularly dopaminergic progenitor cells), and glial cells. These cells are organized in a three-dimensional network within an extracellular matrix. This micro-tissue may be encapsulated in a three-dimensional cellular microcompartment or uncapped and suitable for implantation in the nervous system of a mammal, preferably a human.

[0061] By "the largest dimension" of X in the sense of the invention, we mean the value of the largest diameter of Feret of X.

[0062] For the purposes of this invention, "light" or "lumen" means a substantially acellular volume containing an aqueous solution, topologically surrounded by cells, in particular by at least one layer of cells forming a barrier to the flow of fluids, characterized by the presence of tight junctions positive to the ZONULA OCCLUDENS 1 or ZO-1 marker bordering said light.

[0063] By "extracellular matrix substitute" is meant a substitute for the extracellular matrix naturally produced by cells, such as Matrigel® or one or more molecules or any substitute as described in particular in applications WO2024 / 033284, WO2024 / 105130.

[0064] For the purposes of this invention, "prevention" means reducing to a lesser degree the risk or probability of occurrence of a given phenomenon, for example, in the context of the present invention, Parkinson's disease.

[0065] For the purposes of this invention, "treatment" means a reduction in the progression of the disease, a stabilization, a reversal or regression, or even an interruption or inhibition of the progression of a disease, for example Parkinson's disease.

[0066] Neural tissue unit

[0067] The present invention therefore relates to a three-dimensionally organized neural tissue unit comprising at least dopaminergic neurons, neural progenitor cells, and optionally neural stem cells. This unit has a largest dimension of less than 600 µm and comprises at least one lumen bordered by a Cl layer of cells consisting predominantly of cells neural progenitor cells expressing at least SOX2. Preferably, the Cl layer comprises neural progenitor cells expressing OTX2. Preferably, neural progenitor cells in the Cl layer express at least SOX2 and OTX2.

[0068] The neural tissue unit according to the invention thus comprises at least one lumen which is bordered by the Cl layer of cells comprising predominantly neural progenitor cells expressing at least SOX2. In other words, the neural tissue unit comprises a Cl layer of cells forming a border juxtaposed to said lumen. Said Cl layer of cells being a concentric layer around said lumen.

[0069] The presence of light within the neural tissue unit according to the invention allows, in particular, for improved cell survival and preserves the specific physiological cytoarchitecture of such tissue. During transplantation, such micro-tissue helps to improve the differentiation potential and / or survival of TH+ cells, i.e., dopaminergic neurons, thus improving the survival rate and the grafting of the neural tissue unit according to the invention.

[0070] According to a preferred embodiment of the present invention, the Cl layer at the edge of the lumen is itself surrounded by a layer or stratum of C2 cells comprising dopaminergic neurons. The C2 cell layer or stratum advantageously comprises at least 3%, at least 5%, at least 10%, or at least 15% dopaminergic neurons. The C2 cell layer thus forms a second concentric layer around the CL layer. Very advantageously, the C2 cell layer comprises between 3% and 50%, or even more advantageously between 15% and 50%, dopaminergic neurons, these neurons expressing at least the marker tyrosine hydroxylase (TH).

[0071] Also, preferably, several concentric layers are therefore formed around the light, namely: - a border layer of cells (the Cl cell layer), and - around said border at least one more concentric layer distinct from the Cl layer which includes dopaminergic neurons (the C2 layer).

[0072] According to one embodiment, the microtissue according to the invention may comprise several lumens, at least one lumen of which is surrounded by a border layer of cells (the cell layer C1), and around said border at least one further concentric layer distinct from the C1 layer that comprises dopaminergic neurons (the C2 layer). Preferably, if it comprises several lumens, each lumen is surrounded by a border layer of cells (the cell layer C1), and around said border at least one further concentric layer distinct from the C1 layer that comprises dopaminergic neurons (the C2 layer).

[0074] Very preferentially, dopaminergic neurons are substantially excluded from the periluminal zone, that is to say from the zone comprising the light and the Cl layer. Indeed, dopaminergic neurons are present within a second layer, topologically more external, which can be described as a second layer cortical zone.

[0075] By "substantially excluded," for the purposes of the invention, we mean that the periluminal zone may contain some dopaminergic neurons. In other words, we cannot exclude the presence of at least one dopaminergic neuron, in particular between 1 and 50 dopaminergic neurons in the periluminal zone.

[0076] By "second layer cortical zone" in the meaning of the invention, we mean in particular the C2 cell layer, excluding any zone directly bordering the lumen.

[0077] Preferably, the C2 layer therefore comprises at least 15% of cells positive for tyrosine hydroxylase (TH). The TH gene is a marker of dopaminergic neurons. Thus, the C2 layer comprises at least 15% of cells expressing this marker, and therefore at least 15% of dopaminergic neurons are present in the C2 layer.

[0078] According to another object of the present invention, at most 50% of the cells included in the neural tissue unit express the Ki-67 marker, preferably between 5 and 50% of said cells express the Ki-67 marker. Advantageously, the Cl layer has a higher ratio of cells expressing Ki-67 than the C2 layer.

[0079] According to another particularly preferred object, the three-dimensional organized neural tissue unit, comprising at least dopaminergic neurons and neural progenitor cells, said unit has a largest dimension less than 600 pm, and in that it comprises at least one lumen bordered by a Cl layer of cells consisting essentially of neural progenitor cells expressing at least SOX2.

[0080] Preferably, the neural tissue unit according to the invention has a substantially spheroid shape, in particular said unit is a cluster of substantially spheroid-shaped cells, whose cells are specifically organized, namely a layer of Cl cells and a layer of C2 cells which are successively organized around said lumen.

[0081] Alternatively, the neural tissue unit according to the invention has an ovoid, spherical, ball or teardrop shape or a substantially ovoid, substantially ball or substantially teardrop shape.

[0082] Preferably, the neural tissue unit according to the invention has a larger dimension of between 10 pm and 600 pm plus or minus 10%, preferably between 150 pm and 400 pm plus or minus 10%, more preferably between 100 pm and 300 pm plus or minus 10%, and even more preferably 200 pm plus or minus 10%. These dimensions are particularly favorable to the survival of neurons within the neural tissue unit and optimize the reorganization and vascularization of the graft after implantation.

[0083] According to another preferred object of the invention, the neural tissue unit according to the invention comprises neural progenitor cells, in particular dopaminergic progenitors, which express FOXA2 and / or OTX2 and / or LMX1A, in addition to the marker SOX2. Most preferably, the cell layer Cl comprises neural progenitor cells which express FOXA2 and / or OTX2 and / or LMX1A, in addition to the marker SOX2; even more preferably, the cell layer Cl comprises neural progenitor cells which express FOXA2 and / or OTX2 and / or LMX1A and SOX2.

[0084] According to one object of the invention, the cell layer Cl comprises neural progenitor cells that express SOX2 and FOXA2, or SOX2 and OTX2, or SOX2 and LMX1A. According to one embodiment, the cell layer Cl comprises neural progenitor cells that express SOX2 and FOXA2 and OTX2, or SOX2 and FOXA2 and LMX1A, or SOX2 and OTX2 and LMX1A. According to one embodiment, the cell layer Cl comprises neural progenitor cells that express SOX2 and FOXA2 and OTX2 and LMX1A.

[0085] According to another preferred object of the invention, the neural tissue unit according to the invention comprises neural cells, in particular dopaminergic neurons, expressing EN1 and / or GIRK2 or PAX6. Optionally, the neural cells may also express SOX1.

[0086] According to one embodiment, the neural tissue unit according to the invention comprises neural cells expressing EN 1 and GIRK2. Indeed, dopaminergic neurons express in particular TH and / or GIRK2, dopaminergic progenitors express in particular LMX1A and / or OTX2 and / or FOXA2 and / or EN1 and / or CORIN.

[0087] Indeed, greater expression of the EN1 marker, a population that has been shown to play a key role in the effectiveness of the transplant (Kirkeby et al., 2017), reflects the obtaining of a neural tissue unit with improved functionality.

[0088] According to another object, the neural tissue unit according to the invention may include glial cells.

[0089] In the context of the invention, the neural tissue unit is obtained in a three-dimensional cellular microcompartment, in particular a cellular microcompartment.

[0090] Thus, the neural tissue unit according to the invention is preferably obtained in a hollow hydrogel cell microcompartment (or capsule). Such a cell microcompartment may comprise one or more tissue units neural according to the invention. Preferably it comprises a single unit of neural tissue according to the present invention.

[0091] According to a preferred embodiment of the invention, the neural tissue unit is obtained in a hollow hydrogel cell microcompartment by neural differentiation from cells capable of differentiating into neural cells. The stem cells are preferably pluripotent stem cells. This may be any neural differentiation or neurodifferentiation process known to those skilled in the art or one of the processes described in this application. Cells capable of differentiating into neural cells are understood to mean neural 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 neural cells are immunocompatible with the human being intended to receive the neural tissue unit;

[0092] In 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. In another embodiment, the pluripotent cells are pluripotent stem cells, excluding human or animal embryonic stem cells. Regardless of the embryonic stem cells used, no destruction of the embryo is performed.

[0093] Preferably, the neural tissue unit was obtained from at most 20 cells capable of differentiating into neural cells, i.e., stem and / or progenitor cells, more preferably from at most 10 cells capable of differentiating, and even more preferably from at most 5 cells capable of differentiating. Such a quantity of cells capable of differentiating encapsulated in such a cellular microcompartment as described above is particularly advantageous for reducing the internal pressure in said microcompartment, which improves the formation of a neural tissue unit according to the present invention, namely a unit comprising a lumen and at least one cell layer bordering said lumen.Conversely, excessively high internal pressure leads to the formation of neural tissue units lacking a lumen and biomimetic cellular organization, namely advantageously a layer of Cl cells bordering said lumen and a layer of C2 cells surrounding said layer of CL cells.

[0094] Method for obtaining the neural tissue unit.

[0095] The invention also relates to a method for preparing and obtaining such a unit of neural tissue according to the invention, intended to be implanted in the nervous system of a human mammal, said method comprising the following steps: - encapsulation in a hollow hydrogel microcompartment of at most 20 cells capable of differentiating into neural cells, preferably at most 10, more preferably at most 5, - induction of cell differentiation of cells capable of differentiating into neural cells present in the cellular microcompartment, so as to obtain at least one unit of neural tissue according to the invention, - possibly remove at least partially the outer hydrogel layer of the microcompartment to recover the neural tissue unit according to the invention.

[0096] The encapsulation step in a hollow hydrogel capsule of cells capable of differentiating into neural cells consists of encapsulating at most 20 cells capable of differentiating into neural cells, preferably at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5. Preferably this step consists of encapsulating in a hollow hydrogel capsule at least 2 cells capable of differentiating, preferably between 2 and 20.

[0097] The induction of cell differentiation of pluripotent cells can be initiated in particular by the addition of differentiation factors in particular SMAD, SHH, FGF, and Wnt.

[0098] Advantageously, the process comprises the following steps: - to produce cellular microcompartments comprising, within an outer hydrogel layer, cells capable of differentiating into neural cells, and possibly extracellular matrix, - induce cell differentiation within the cell microcompartment, so as to obtain neural cells exhibiting at least one phenotype of interest; - possibly remove at least partially the outer hydrogel layer of the microcompartment to recover the neuronal cells as a unit of neural tissue.

[0099] Any method for producing hollow cellular microcompartments containing cells capable of differentiating into neural cells within an outer hydrogel layer may be used. In particular, the microcompartments may be obtained by encapsulation using concentric co-injection via a microfluidic injector. The injector produces a jet consisting of a mixture of the various useful solutions, which then breaks into droplets. These droplets are then collected in a bath, in particular a calcium bath, capable of stiffening the hydrogel solution to form the outer layer of each microcompartment.

[0100] According to an embodiment enabling the formation of a tube, the co-injection is also carried out concentrically via a microfluidic injector, said injector comprising a tip, said tip being in contact with a calcium solution, forming a jet at the injector outlet consisting of the mixture of said solutions, said jet forming the tube in the calcium solution.

[0101] The microcompartment can be any three-dimensional shape, that is, it can be any three-dimensional object. The microcompartment can have any shape compatible with cell encapsulation. Preferably, the microcompartment according to the invention is spherical or elongated. It can be ovoid, cylindrical, spheroidal, or spherical. In particular, it can be hollow spheroidal, ovoidal, cylindrical, or spherical.

[0102] According to one embodiment, the microcompartment can be obtained by a process comprising the following steps: - prepare a solution containing cells capable of differentiating into neural cells, - prepare at least one aqueous solution and / or at least one hydrogel, - encapsulate the solutions and / or hydrogels from the previous steps by collinear flow in an external hydrogel layer, so that the microcompartments comprise at most 20 cells capable of differentiating into neural cells, preferably at most 10, preferably at most 5, - to cultivate the microcompartments obtained in the previous step in a suitable culture medium with differentiation factors capable of inducing neural cell differentiation, preferably in a bioreactor, so as to obtain a unit of neural tissue according to the invention and - optionally recover the resulting cellular microcompartments.

[0103] In a particular embodiment, the encapsulated cells are pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), or possibly embryonic stem cells (ESCs). Preferably, the encapsulated cells number at most 20 pluripotent stem cells, more preferably at most 10 pluripotent stem cells, and even more preferably at most 5 pluripotent stem cells, in order to facilitate the structuring of the neural tissue unit in a biomimetic manner, including in particular at least one lumen.

[0104] Alternatively, it is also possible to encapsulate neural progenitor cells, i.e. stem cells already committed to cell differentiation into neural cells.

[0105] Any known differentiation method classically implemented in 2D culture (petri dish and other) to force cell differentiation may be used, such as the method described in Chambers et al. (“Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling”, Nature Biotechnology 27, 275 - 280 (2009)), or in Lippmann et al., (“Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors”, Stem Cells 2014).

[0106] According to one variant, the differentiation method is as follows, namely an in vitro method for differentiating pluripotent cells into neural cells, implemented by means of at least one three-dimensional cell microcompartment in a suitable culture medium, said microcompartment comprising said pluripotent cells, said method comprising at least one step of exposing said pluripotent cells to: - at least two SMAD inhibitors, - at least one SHH activator, - at least one FGF activator, and - at least one Wnt activator, wherein the concentration in the culture medium of at least one SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of pluripotent cells to said SMAD inhibitor, by at least 20%, preferably at least 40%, relative to the initial concentration of said SMAD inhibitor.

[0107] In this context, pluripotent cells are thus encapsulated in three-dimensional microcompartments or capsules allowing three-dimensional cell culture, making it possible to approach physiological conditions in vivo as closely as possible.

[0108] The method may include a preliminary step of cell culture of pluripotent cells, followed by the encapsulation of said pluripotent cells in three-dimensional cellular microcompartments.

[0109] Briefly, the encapsulation may advantageously comprise the following steps: - encapsulating the mixture comprising the pluripotent cells, a culture medium and an extracellular matrix, in an outer layer of hydrogel, the encapsulation comprising the following substeps: i. bringing said mixture into contact with a hydrogel solution intended to form said outer layer to form at least one droplet, and ii. collect the drop obtained in a calcium bath suitable for stiffening said hydrogel solution to form the outer layer of each microcompartment. - cultivate the capsules obtained in the previous step in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days, and - to recover the resulting cellular microcompartments, including the pluripotent cells of interest.

[0110] Preferably, at most 20 pluripotent cells are encapsulated, more preferably at most 10 pluripotent cells, even more preferably at most 10 pluripotent cells.

[0111] Preferably, the encapsulation step is carried out by the simultaneous co-injection of the hydrogel solution intended to form the outer layer, the mixture comprising, in particular, the pluripotent cells and optionally the extracellular matrix or extracellular matrix substitute, and optionally an intermediate solution. This co-injection is performed concentrically via a microfluidic or millifluidic injector, which produces a jet at the injector outlet consisting of the mixture of said solutions. This jet breaks into droplets. The droplets are then collected by gravity in the calcium bath, allowing the hydrogel solution to stiffen and form the outer layer and, consequently, the cellular microcompartment containing the pluripotent cells of interest. These pluripotent cells may be in isolated form or optionally in the form of cell aggregates or clusters.Also, the encapsulated pluripotent cells are suspended within the capsule, either as single or isolated cells and / or as clusters or aggregates of cells. Preferably, isolated cells represent less than 50% by number of all encapsulated cells, and more preferably, the isolated cells are iPSCs.

[0112] Once the cells of interest are encapsulated, advantageously in single and / or aggregate form, said cells are exposed to several differentiation agents to induce differentiation, 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 added directly to the culture medium comprising said microcompartments, preferably in a bioreactor. The differentiation agents diffuse through the outer layer of the cell microcompartments, which is permissive to their diffusion. Thus, the differentiation agents diffuse within each cell microcompartment, into the culture medium present within said cell microcompartment.

[0113] In this context, the exposure of encapsulated pluripotent cells with at least one SMAD inhibitor added gradually, i.e., its concentration is increased gradually before the addition of the other differentiation factors, to Knowing about SHH activators, FGF activator and Wnt activator is of particular interest.

[0114] Thus, said pluripotent cells are encapsulated with at least one SMAD inhibitor at T0. Advantageously, the concentration of said SMAD inhibitor is increased continuously or discontinuously for at least 3 days from the initial exposure of the pluripotent cells to said SMAD inhibitor, more preferably by at least 40% compared to the initial concentration of said SMAD inhibitor.

[0115] Advantageously, pluripotent cells are exposed to at least two SMAD inhibitors, at least one first SMAD inhibitor (i) is increased continuously or discontinuously, preferably exponentially, and the pluripotent cells are exposed to a second SMAD inhibitor (ii), the concentration of said SMAD inhibitor (ii) being either constant over time or increased continuously or discontinuously. Thus, the concentration of at least two SMAD inhibitors is preferentially increased, relative to the initial exposure concentration of the pluripotent cells to said SMAD inhibitors.

[0116] Advantageously, 1 day after the initial exposure of pluripotent cells to said SMAD inhibitors, the concentration of a first SMAD inhibitor (i) is increased by at least 40% and the concentration of the second SMAD inhibitor (ii) is increased by at least 900%, compared to the initial concentration of said SMAD inhibitors, i.e. at 0 day, i.e. the initial exposure of the SMAD inhibitors with the encapsulated pluripotent cells of interest.

[0117] Advantageously, 2 days after the initial exposure of pluripotent cells to said SMAD inhibitors, the concentration of the first SMAD inhibitor (i) is increased by at least 67% and the concentration of the second SMAD inhibitor (ii) is increased by at least 9,900%, compared to the initial concentration.

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

[0119] Most advantageously, at least one SHH activator is added at least 3 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and the FGF-8b activator is added at least 3 days after the initial exposure of at least one SMAD inhibitor and pluripotent cells, and at least one Wnt activator is added at least 6 days after initial exposure to at least one SMAD inhibitor and pluripotent cells.

[0120] Said differentiation process thus enabling the formation of a neural tissue unit, in particular a neural tissue unit according to the invention, especially when at most 20 pluripotent cells are encapsulated, allowing the reduction of internal pressure, facilitating the formation of a neural tissue unit according to the invention comprising at least one lumen.

[0121] At the end of any of the differentiation processes known or described above, a plurality of neural cells is thus obtained, this plurality of neural cells is organized in three dimensions and thus forms the micro-tissue or neural tissue unit.

[0122] The invention thus relates to a micro-tissue consisting essentially of a plurality of neural cells organized in three dimensions and optionally of extracellular matrix. Said neural cells can be obtained by any of the differentiation processes known or described above. This plurality of neural cells advantageously comprises at least dopaminergic neurons, progenitor cells, in particular dopaminergic progenitors, and optionally glial cells.

[0123] This plurality of neural cells expresses several specific markers. Preferably, the plurality of neural cells thus constituting the micro-tissue presents a greater quantity of cells positive for the FOXA2 marker and a lower quantity of cells positive for the PAX6 and / or SOX1 markers.

[0124] Furthermore, greater expression of the EN1 marker indicates the formation of a microtissue with improved functionality. In addition, greater expression of the TH marker, a marker of dopaminergic neurons, in the C2 layer indicates better commitment of differentiation to the dopaminergic pathway and therefore a higher differentiation rate.

[0125] Composition or Kit

[0126] According to another aspect, the invention also relates to a composition or kit comprising one or more unit(s) of neural tissue according to the present invention, preferably at least two units of neural tissue according to the present invention.

[0127] The invention therefore also proposes a composition or a kit for implanting the neural tissue unit(s), the said neural tissue unit(s) being able to be encapsulated in one or more cellular microcompartment(s) comprising a hydrogel capsule enveloping a neural tissue unit according to the invention or possibly several but very preferably a single neural tissue unit according to the invention.

[0128] The practitioner who is to use the neural tissue unit according to the invention can thus, at the time of use and as needed, remove at least partially the hydrogel capsule to obtain the neural tissue unit(s) ready to be implanted in the nervous system of a subject.

[0129] When it is an implantation kit, it may also contain a surgical implantation device capable of implanting a unit of neural tissue into the nervous system of a mammal.

[0130] Thus, the invention proposes a neural tissue unit implantation kit comprising at least one neural tissue unit according to the invention (encapsulated or not in a cellular microcompartment), preferably between 1 and 10,000, more preferably between 10 and 1,000 neural tissue units. Advantageously, the quantity of neural tissue units implanted depends on the size of the host brain and the application.

[0131] Alternatively, the invention proposes a composition comprising at least one unit of neural tissue according to the invention (encapsulated or not in a cellular microcompartment), preferably between 1 and 10,000, more preferably between 10 and 1,000 units of neural tissue.

[0132] When neural tissue units are frozen, of course, a thawing step is then necessary before the implantation of the neural tissue units into the subject's nervous system.

[0133] Microcompartment comprising the neural tissue unit or set of microcompartments

[0134] The invention also proposes a three-dimensional, hollow cellular microcompartment comprising an outer hydrogel layer, said microcompartment comprising at least one neural tissue unit according to the invention. In certain embodiments, the cellular microcompartment may comprise extracellular matrix of natural origin, and / or Matrigel®, and / or an extracellular matrix substitute, the latter being present in particular in the spaces that may remain within the neural tissue unit.

[0135] Examples of such microcompartments are particularly well described in applications WO2024 / 033284, WO2024 / 105130.

[0136] According to another object, the invention also relates to a set of microcompartments, in which said set comprises at least one cellular microcompartment according to any of the preceding embodiments.

[0137] The microcompartment according to the invention, or the assembly of microcompartments according to the invention, is particularly well suited for use in cell culture, especially in three-dimensional cell culture, allowing the production of large quantities of cells of interest, microtissues, or even organoids of interest, which can be used, for example, in cell therapy. In particular, these allow the production of neural tissue units according to the invention in large quantities, which is of particular interest in cell therapy. Also, the invention also relates to a set of microcompartments for its use as a medicine.

[0138] According to one embodiment, the invention also relates to using the microcompartment or a set of microcompartments to fabricate neural tissue units. It is understood that in a very particular embodiment, the neural tissue units are not implanted in a human being or an animal. By way of example, they can be used as an ex-vivo model.

[0139] Use as a medicinal product

[0140] According to another aspect, the invention relates to the micro-tissue according to the invention for its use as a medicinal product, preferably for the prevention and / or treatment of neurodegenerative disease, even more preferably for the prevention and / or treatment of Parkinson's disease.

[0141] Indeed, the inventors have demonstrated that it is possible to inject and therefore graft such a unit of neural tissue according to the present invention, in particular described in [Fig.9].

[0142] According to one variant, the invention also relates to a cellular microcompartment comprising at least one micro-tissue according to the invention, for its use as a drug.

[0143] The neural tissue unit according to the present invention can then be implanted in the nervous system of a subject suffering from a neurodegenerative disease, in particular Parkinson's disease, in order to at least partially replace the failing neurons of said subject.

[0144] Thus, the invention possibly relates to a method for preparing neural tissue units according to the invention also comprising at least one additional step consisting of loading a surgical implantation device with at least one neural tissue unit, preferably between 10 and 1000, more preferably between 10 and 100 neural tissue units.

[0145] Thus, the implantation device is ready to be used to graft at least one unit of neural tissue according to the invention into the nervous system of a subject.

[0146] According to the invention, it is also possible to freeze neural cells in the form of neural tissue units, before or after at least partial removal of the hydrogel capsule. Of course, the neural tissue units can be used directly after the preparation process, without having been frozen beforehand.

[0147] Advantageously, the preparation process according to the invention also includes an intermediate step consisting of checking the phenotype of the neural cells contained in the hydrogel capsule, after the cell differentiation step.

[0148] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples

[0149] Example 1 - Preparation of neural tissue units (invention and non-invention)

[0150] Production of neuronal microtissues

[0151] 2D Culture of hiPSCs

[0152] All hiPSC cell lines were maintained on Vitronectin and cultured in mTeSRl medium. The cultures were fed daily, passed with an enzyme-free reagent, ReLeSR, for 6 min at 37 °C every 3–4 days (around 80% confluence), and replanted as small clusters (between 100 and 200 µm) at a density of approximately 20,000–40,000 cells / cm². The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.

[0153] 3D encapsulation of hiPSCs

[0154] Prior to encapsulation, the 2D stem cell colonies were detached. The HiPSCs were resuspended in mTeSRl medium supplemented with 10 pM of Y-27632.

[0155] To obtain neural tissue units with lumens according to the invention, five HiPSC cells were then mixed with human fibrinogen and Y-27632 to achieve final concentrations of fibrinogen and Y-27632 of 14 mg / mL and 10 pM, respectively. The final cell concentration in the cell / matrix solution was therefore between 2.2 x 10⁶ and 3.7 x 10⁶ viable cells / mL, referred to as the encapsulation density.

[0156] To obtain neural tissue units outside the invention, the final concentration of cells in the cell / matrix solution is then between 7.1 x 10A6 and 7.4 x 10A6 viable cells / mL, called encapsulation density.

[0157] Tubes are connected to the three inlets of a 3D-printed glass colaminal flow microfluidic device. A 3D-printed glass microcapillary tip is glued to the nozzle outlet for improved flow control. The cell / matrix suspension is loaded into the inner channel of the three-way device. An SDS sodium alginate solution is injected into the outer channel. To prevent gelation of the alginate inside the microfluidic device due to calcium release from the suspended cells, a calcium-free solution (Sorbitol) is used in the intermediate channel of the co-extrusion chip and acts as a barrier against calcium diffusion. This solution is also supplemented with Thrombin was added to a final concentration of 0.02 U / mL to allow for fibrin cross-linking within the capsules. Typical flow rates for the three solutions were approximately 80 mL / h for the three channels: the alginate solution, the sorbitol solution, and the cell / matrix suspension. At these rates, the composite solution formed a liquid jet that fragmented into droplets. When the droplets came into contact with the 100 mM calcium bath, the outer alginate layer readily gelled. Consequently, the internal cell / matrix solution remained trapped in a closed, spherical, and permeable microcompartment.

[0158] Neural tissue units as shown in Figures 1, 2 or 3A, are obtained by encapsulating 2 to 20 cells per capsule.

[0159] The neural tissue units outside the invention, as shown in Figure 3B, are obtained by encapsulating more than 20 cells per capsule.

[0160] Within 5 minutes of encapsulation, the capsules are rinsed with DMEM / F-12, HEPES 15 mM supplemented with 2.9 mM CaC12, to reduce the baseline calcium concentration. Finally, they are transferred to mTeSRl medium supplemented with 10 µM Y-27632 as the initial suspension culture medium for neurodifferentiation.

[0161] Protocol 1: 3D neurodifferentiation of hiPSCs in static T flasks or bioreactors with progressive addition of the two SMAD inhibitors for at least 3 days.

[0162] Further neurodifferentiation can be achieved in several culture systems, including static suspension culture using T-flasks or well plates and agitated suspension cultures using 30 ml or 500 ml bioreactors.

[0163] Under static conditions, suspension cultures of encapsulated hiPSCs were carried out using T-flasks (from 5 to 30 mL) maintained in a cell culture incubator at 37 °C and 5% CO2, with daily medium changes.

[0164] Under agitated conditions, stirred suspension cultures were performed in various bioreactors. We used benchtop STBRs, including 30 mL and 500 mL bioreactors. The stirring speed was set at 150 rpm from day 0 to day 7, 200 rpm from day 7 to day 18, and 250 rpm from day 18 to day 24, or 55 rpm from day 0 to day 24 in the 500 mL and 30 mL STBRs, respectively. In both cases, the bioreactors were inoculated with 25% (v / v) capsules for an average volume. In the 500 mL bioreactor, the culture volume was maintained at 300 mL throughout the culture. On day 1, the medium was completely renewed with fresh medium supplemented with ROCK inhibitor. On day 2, no medium change was performed. On day 3, the medium was completely renewed with fresh ROCK inhibitor-free medium. From this day onward, the medium change was performed in Infusion. On days 12, 13, and 18, the medium was completely renewed with fresh medium. The final capsule volume relative to the medium was between 22% and 25%, and the pH was maintained at 7.2 ± 0.2. The dissolved oxygen (DO) level was calibrated to 100% before and after autoclaving in the empty bioreactor and under the starting conditions (filled with medium), respectively. During the experiment, the oxygen level was monitored and controlled. Oxygen was controlled at 50%, and the oxygen level was regulated.

[0165] Under static and agitated conditions, the molecules were added as follows. From day 0, the mTeSRl medium was supplemented with 10 pM Y-27632 for ROCK inhibition during the first 72 hours of culture. The mTeSRl medium was stored until day 3. Recombinant human protein Noggin GMP was initially added on day 0 (encapsulation day) at a concentration of 60 ng / mL and progressively increased on days 1 and 2 to 84 ng / mL and 100 ng / mL, respectively. Similarly, SB431542 GMP was initially added at a concentration of 0.2 pM on day 0 and progressively increased to 2 pM and 20 pM on days 1 and 2, respectively. On day 3, the medium was changed to Neurobasal™ CTS™ and DMEM / F-12, GlutaMAX™ supplement in a 1:1 ratio, supplemented with N-2 CTS™ supplement and B-27™ GMP supplement.From day 3 to day 11, 100 ng / mL of recombinant human protein Noggin GMP, 20 pM of SB431542 GMP, 100 ng / mL of recombinant human protein Sonic Hedgehog / Shh (C24II) N-Term GMP, 100 ng / mL of recombinant human protein FGF-8b HumanKine® and 2 pM StemMACS Purmorphamine were added. CHIR99021 GMP was added from day 6 to day 12. Then, 200 pM of ascorbic acid, 10 ng / mL of recombinant human GDNF GMP protein, 1 ng / mL of recombinant human TGF-beta 3 protein HumanKine®, 5 ng / mL of recombinant human FGF-20 protein, 0.5 mM Dibutyryl cAMP, 10 pM DAPT RMU, 1 pM Compound E and 10 nM Trichostatin A were added from day 13 to day 17. Finally, 20 ng / mL of recombinant human BDNF protein HumanKine was added from day 13 to day 24.

[0166] Protocol 2: 3D neurodifferentiation of hiPSCs in static T flasks or bioreactors without progressive addition of the two SMAD inhibitors.

[0167] Tests were carried out using an alternative protocol. This protocol is identical to that described above, except that recombinant human protein Noggin GMP was added directly on day 0 (encapsulation day) at a concentration of 100 ng / mL. Similarly, SB431542 GMP was added directly at a concentration of 20 pM on day 0.

[0168] Example 2 - Characterization of a micro-tissue according to the invention

[0169] This study aims to characterize the tissue units according to invention and outside of invention obtained after neurodifferentiation.

[0170] The neural tissue unit in Figure 3A was obtained using Protocol 1 and the encapsulation of 2 to 20 cells in the capsule. The neural tissue unit in [Fig. 4] represents a microtissue according to the invention with a lumen obtained after Protocol 2 and the encapsulation of 2 to 20 cells in the capsule.

[0171] The neural tissue unit in Figure 3B was obtained with Protocol 1 and encapsulation of more than 20 cells in the capsule.

[0172] Hematoxylin-eosin-saffron (HES) staining and notation of histological characteristics

[0173] Microtissues were fixed with 4% paraformaldehyde at room temperature for 1 hour, then washed in phosphate buffer. The microtissues were centrifuged (10 min, 1200 rpm) and pre-embedded in 3% agarose. After dehydration in successive baths of alcohol, acetone, and xylene, the samples were embedded in paraffin. 5 µm slices were prepared using a microtome and then glued with an albumin-glycerol mixture onto treated slides. After deparaffinization, the sections were successively immersed in solutions of Harris's hematoxylin, eosin, and saffron. After dehydration, the sections were mounted between a slide and a coverslip using Entellan®. Hematoxylin-eosin-saffron staining allows observation of the tissue morphology and structure. The cytoplasm appears pink and the nuclei blue-violet. The extracellular matrix has been stained yellow to pink.

[0174] The stained microtissues were imaged using a slide scanner (NanoZoomer 2.0RS, Hamamatsu) and evaluated by a neuropathologist for the presence or absence of physiological features of the human embryonic midbrain: the ventricle (referred to as the lumen in the invention), the ventricular zone (referred to as zone Cl in the invention), the intermediate zone, and the mantle zone (referred to as zone C2 in the invention) (Arenas et al. 2015, https: / / doi.org / 10.1242 / dev.097394). Abnormal histological features, including abnormal cytonuclear morphology and karyorrhexis, were also noted.

[0175] These micro-tissues are represented: - in Figure 3A and 4 for the tissue units according to the invention, - in Figure 3B for the tissue units outside the invention.

[0176] The results are presented in [Fig.3]:

[0177] Figures 3A and 4 show the specific organization of micro-tissues according to the present invention, with the presence of the light and cells organized radially around this light.

[0178] Conversely, in the absence of light (Figure 3B), the inventors observed a disorganization of the cells.

[0179] Immunofluorescence labeling, microscopy and image analysis of units fabrics according to the invention

[0180] The encapsulated 3D neuronal microtissues according to the invention (with light) were harvested for confocal microscopy at the end of the neurodifferentiation process (D24). The alginate capsule was removed by incubating the sample for 5 min in RelesR at room temperature with a final capsule concentration of 20% (V / V). The microtissues were fixed with 4% PFA for 1 h at room temperature in the dark. After fixation, the samples were washed 3 times with 0.1% Tween20 in PBS. A permeabilization step was performed in a PBS solution containing 5% Triton X-100 for 30 min under stirring (170 rpm) at room temperature. The samples were washed 3 times with 0.1% Tween20 in PBS. The samples were incubated in suitable primary and secondary antibodies 0.1% Tween20 in PBS for 72 h at room temperature under stirring (170 rpm).The samples were rinsed five times with 0.1% Tween20 in PBS after each incubation, including two rinses under shaking for 30 min at 170 rpm. These microtissues are shown in [Fig. 5], 6 and 7 and were imaged with a fluorescence microscope with deconvolution or with a confocal microscope.

[0181] The results are presented in [Fig. 5] to [Fig. 7] and thus confirm the presence of dopaminergic neurons, which are not radially arranged around the lumen. Conversely, dopaminergic progenitor cells are radially arranged around the lumen ([Fig. 5]).

[0182] Fig. 6 confirms the presence of KI67-labeled cells in the neural tissue unit.

[0183] Figure 7 confirms the presence of dopaminergic progenitors (cells progenitors) around the lumen, in a layer of Cl cells bordering said lumen.

[0184] Figure 8 shows the size distribution of the microtissues according to the invention at D24. The microtissues were imaged using a wide-field microscope and their size was measured using proprietary image analysis software. The average diameter size of the microtissues is 176.8 ± 60.48 pm.

[0185] Example 3 - Injection of micro-tissue according to the invention for the treatment of Parkinson's disease.

[0186] Protocol

[0187] Induction of the hemi-parkinsonian rat model

[0188] Rowett (Rnu+) hairless rats were first anesthetized using a 4% isoflurane induction chamber (induction chamber parameters: i4 Oxyl Airl). The rats were then placed on an anesthesia mask, using approximately 2.5% isoflurane (induction chamber parameters: i2.5 OxyO.4 Air0.4). The concentration could be slightly adjusted by regularly checking paw temperature and body temperature manually. Analgesia was achieved by subcutaneous injection of buprenorphine and lidocaine at concentrations of 0.05 mg / kg and 5 mg / kg, respectively. Rats received an additional intraperitoneal injection of desipramine hydrochloride at a concentration of 25 mg / kg at least 20 minutes before the injection of 6-hydroxydopamine hydrobromide (6-OHDA / HBr). 2.5 µl of freshly prepared 6-OHDA / HBr at a concentration of 0.5% (w / v) were infused into the medial anterior cerebral bundle (MFB) using a stereotaxic frame with the coordinates (Bregma as reference): AP: -3.8; ML (right): 1.6; DV: from -8 to -7.

[0189] Injection of neuronal microtissues into the hemiparkinsonian rat model

[0190] Rowett hairless rats (Rnu+) were first anesthetized using an induction chamber with 4% isoflurane (induction chamber parameters: 14 Oxyl Airl). The rats were then placed on an anesthesia mask using approximately 2.5% isoflurane (induction chamber parameters: 2.5 Oxyl Airl). The concentration could be slightly adjusted by regularly checking paw temperature and body temperature manually. Analgesia was achieved by subcutaneous injection of buprenorphine and lidocaine at concentrations of 0.05 mg / kg and 5 mg / kg, respectively. The rats were then injected with neural microtissues according to the present invention into the right striatum using a 25pL Hamilton (Hamilton, ref.1702 CX SYR) with custom-made needles (glass cannula) and a custom-made administration medium composed of Carboxymethylcellulose and Dextran 70kDA (CMC-DA70). The injection was performed in two trajectories using a stereotaxic frame. At the end of surgery, Metacam was injected subcutaneously at a concentration of 1 mg / kg for postoperative pain management.

[0191] Post-mortem study

[0192] Post-mortem histological analysis

[0193] The animals were first anesthetized by intraperitoneal injection of ketamine and xylazine. They were then perfused intracardiacly with 150 mL of 0.9% NaCl, followed by 200 mL of 10% phosphate-buffered formalin. After extraction, the brains were post-fixed in 10% phosphate-buffered formalin for 24 hours at 4°C and then stored in PBS at 4°C. 40 µm sections were taken using a vibratome and stored in 0.01% azide PBS. The sections were washed three times with PBS. The sections were permeabilized by incubation for 1 hour at room temperature in 0.1% Triton and 2% BSA PBS, and then washed three times with PBS. The cups were colored for the Tyrosine hydroxylase (TH; 1:1000) and human antigen (Steml21 1:1000) were used to incubate the primary antibodies in 0.05% Triton 0.5% BSA PBS overnight at 4°C. The sections were washed three times with PBS. They were then incubated with fluorescently conjugated secondary antibodies (Alexa 488, 568) (1:1000) in 0.05% Triton 0.05% BSA PBS for 2 hours at room temperature. After being rinsed three times with PBS, the sections were mounted on slides with DAPI Fluoromount-G for nuclear counterstaining. The slides were imaged using a nanozoomer.

[0194] Results

[0195] The results are presented in [Fig. 9] and are fluorescence microscopy images of a section of a graft 20 weeks after microtissue transplantation, immunostained for the TH marker, the human marker Steml21, and the nuclear dye DAPI. Microtissue-derived TH+ dopaminergic neurons are more abundant and uniformly distributed in the graft in rats transplanted with lot 1 (Light - Microtissue according to the invention) compared to rats transplanted with lot 2 (Microtissue not according to the invention - No light).

[0196] Panel C represents the sterological quantification of the total number of TH-expressing cells in the graft at 20 weeks normalized by the volume of microtissues according to or outside the invention injected.

[0197] Panel D represents the stereological quantification of the total number of cells expressing TH normalized by the number of microtissues according to the invention or outside the invention injected 20 weeks after transplantation.

[0198] Each point corresponds to a rat. Non-parametric Mann-Whitney tests were performed; ns= p>0.05 ; * p<0.05 (C, D).

[0199] Example 4 - Intrastriatal injection of micro-tissue according to the invention for the treatment of Parkinson's disease in non-human primates

[0200] The objective of this study is to confirm the grafting of microtissues according to the invention following their intra-striatal injection in healthy non-human primates.

[0201] Protocol

[0202] The protocol is as follows. On day -30, immunosuppressive treatment is initiated until the animal is euthanized. The immunosuppressive treatment administered to the animal is as follows: - Prednisolone 5 mg / kg, once a day - Cyclosporine 3 mg / kg, twice a day - Mycophenolate mofetil lg / 5ml powder for oral suspension twice a day

[0203] At JO, the microtissues are injected intra-striatally. The injection site is determined according to the knowledge of the person skilled in the art. Then, the craniotomy area is cleaned with a sterile saline solution.

[0204] The injections were then performed using an Elekta injection / aspiration needle. The injected volume was 18 pL in the left hemisphere at a concentration of 14% (V / V) neural microtissue. The cannulas were coupled to an automatic injector, administering the test element at a rate of 2 pL / min. The cannula was then gradually withdrawn.

[0205] Once the injection was completed, the wound was closed. The animal was able to recover from the anesthesia. The animal's condition was monitored daily. Surgical incisions were examined at least once a day (until healing) to detect any signs of infection, inflammation, and overall integrity. No infection or inflammation was observed. The animals were assessed daily according to established end-of-life assessment criteria.

[0206] On the thirtieth day after the operation, the animals are euthanized by barbiturate overdose, in accordance with the guidelines of the European Association of Veterinary Medicine.

[0207] The brain was carefully removed and separated into two halves, left and right, and each hemisphere was cut into three slices. One slice corresponding to the mid-brain was fixed in paraformaldehyde (PFA; 4%) at 4°C, then cryoprotected in a 20% sucrose solution (in 0.1 M PBS) at 4°C, and then transferred to a 30% sucrose solution (in 0.1 M PBS) at 4°C. Finally, they were frozen at -45°C and stored at -80°C until they were cut.

[0208] The median slice of the left hemisphere was cut coronally on a cryostat, then the slices were stored at 4°C in PBS containing 0.2% azide before being used for immunohistochemistry.

[0209] Tyrosine hydroxylase (TH) immunohistochemistry was performed on a series of 40 µm thick sections with a primary antibody (monoclonal rabbit anti-TH) and an anti-rabbit horseradish peroxidase (HRP) labeled polymer. Then, the TH staining was revealed with 3,3'-diaminobenzidine (DAB).

[0210] STEM 121 immunohistochemistry was performed on a series of 40 µm thick sections with a primary antibody (anti-STEM121 mouse monoclonal antibody) and a horseradish peroxidase (HRP)-labeled anti-mouse polymer. Then, STEM121 staining was revealed with 3,3'-diaminobenzidine (DAB).

[0211] Hematoxylin and eosin (H&E) staining was also performed on a series of 16 µm thick sections with incubation in Mayer's hematoxylin solution, followed by incubation in hydrochloric acid and of an incubation in an eosin solution. The slides are rinsed between each incubation step.

[0212] Results

[0213] The results are presented in [Fig. 10] which represents the grafts in the left hemisphere of the animal.

[0214] The presence of human stem cells was assessed by anti-Steml21 immunohistochemistry. In the left hemisphere, very intense staining of cell bodies and neurites was observed. These results show that the grafts survived and that there was no rejection of them in subsequent anatomical injections.

[0215] The presence of dopaminergic neurons was assessed by anti-TH immunohistochemistry. In the left hemisphere, the staining was strong and localized to the same areas as the Steml21+ staining, demonstrating that the injected human cells, organized into organoids, survived and enabled the transplantation of TH+ cells of interest into the grafts. TH+ cell bodies and TH+ neurites were located 100–500 pm from the grafts, demonstrating migration of these cells and reinnervation of the surrounding tissue (arrows on panel B of [Fig. 10]), and thus the differentiation of the cells injected into the graft. Finally, hematoxylin and eosin (H&E) staining demonstrates that the tissue formed after injection is healthy.

Claims

Demands

1. A three-dimensional organized neural tissue unit comprising at least dopaminergic neurons and neural progenitor cells, characterized in that it has a largest dimension less than 600 pm, and in that it comprises at least one lumen lined by a Cl layer of cells comprising predominantly neural progenitor cells expressing at least SOX2, said unit does not comprise human embryonic stem cells.

2. Neural tissue unit according to the preceding claim, characterized in that the Cl layer at the edge of the lumen is surrounded by a layer of C2 cells which includes dopaminergic neurons.

3. Neural tissue unit according to any one of the preceding claims, wherein the largest dimension is between 100 and 300 pm.

4. A neural tissue unit according to any one of the preceding claims, characterized in that it has a substantially spheroid shape.

5. Neural tissue unit according to any one of the preceding claims, characterized in that it comprises neural progenitor cells that also express FOXA2 and / or OTX2 and / or LMX1A.

6. Neural tissue unit according to any one of the preceding claims, characterized in that the Cl layer of cells bordering the lumen comprises neural progenitor cells that express FOXA2 and / or OTX2 and / or LMX1A.

7. Neural tissue unit according to any one of the preceding claims, characterized in that it comprises neural cells expressing EN 1 and / or GIRK2 or neural cells expressing PAX6.

8. Neural tissue unit according to any one of the preceding claims, characterized in that it comprises glial cells.

9. Neural tissue unit according to any one of the preceding claims, characterized in that said unit was obtained in a hollow hydrogel cell microcompartment.

10. Neural tissue unit according to any one of the preceding claims, characterized in that said unit was obtained in a hollow hydrogel cell microcompartment, by neural differentiation of cells capable of differentiating into neural cells, excluding embryonic stem cells.

11. Neural tissue unit according to the preceding claim, characterized in that it was obtained from at most 20 cells capable of differentiating into neural cells, preferably at most 10 cells capable of differentiating into neural cells, more preferably at most 5 cells capable of differentiating into neural cells.

12. Neural tissue unit according to any one of claims 10 or 11, characterized in that the cells capable of differentiating into neural cells are induced pluripotent stem cells.

13. Composition comprising at least two units of neural tissue according to any one of the preceding claims.

14. Neural tissue unit according to any one of claims 1 to 12 or composition according to claim 13, for use as a medicinal product.

15. Neural tissue unit according to any one of claims 1 to 12 or composition according to claim 13, for its use in the prevention and / or treatment of a neurodegenerative disease.

16. Neural tissue unit according to any one of claims 1 to 12 or composition according to claim 13, for its use in the prevention and / or treatment of Parkinson's disease.

17. Three-dimensional, hollow, cellular microcompartment comprising an outer hydrogel layer, comprising at least one neural tissue unit according to any one of claims 1 to 12.

18. A set of three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the preceding claim.

19. A method for obtaining the neural tissue unit according to any one of claims 1 to 12, comprising the following steps: a. encapsulation in a hollow hydrogel microcompartment of at most 20 cells capable of differentiating into neural cells, b. induction of cell differentiation of cells capable of differentiating into neural cells present in the cellular microcompartment, so as to obtain at least one unit of neural tissue according to any one of claims 1 to 14, c. possibly remove at least partially the outer hydrogel layer of the microcompartment to recover the neural tissue unit.

Images