Neural tissue unit and use of such unit for implantation into nervous system of mammal

Abstract

To provide a neuronal cell implantation system to improve the survival of implanted cells, in particular to permanently replace altered neurons in a patient with neurodegenerative disease.SOLUTION: The invention provides a neural tissue unit for use in implantation into the nervous system of a human or non-human mammal, wherein the neural tissue unit contains differentiated post-mitotic neuronal cells in an extracellular matrix, the unit being obtained from a cellular microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, and the hydrogel capsule being at least partially removed before use of the neural tissue unit. The invention also provides a process for preparing such a neural tissue unit.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a neural tissue unit comprising a plurality of postmitotic neurons, and optionally glial cells, organized in a three-dimensional (3D) network in an extracellular matrix, and its use as a unit implanted into the nervous system of a mammal. Such a neural tissue unit may be particularly used to transplant neurons with a phenotype of interest into the nervous system of a subject to treat neurodegenerative diseases. [Background technology]

[0002] Over the past decades, neurodegenerative diseases have been a major social challenge, both in terms of understanding the physiological phenomena involved and in terms of treatment. More and more people are affected by these diseases, and the consequences for the patients, as well as their families and friends, are dramatic.

[0003] The collective term "neurodegenerative diseases" encompasses a large number of diseases that are primarily characterized by the death of neurons more rapid than normal aging, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc. The nervous system can be affected in a variety of ways, resulting in a variety of symptoms, including impairment of movement, balance, behavior and / or cognition.

[0004] Currently, the causes and mechanisms of neurodegenerative diseases are often poorly understood, making their treatment even more complicated. There are currently no effective treatments to cure these diseases.

[0005] In recent years, development priorities have been focused on intracerebral cell therapy, more specifically on grafting neurons into damaged areas of the brain of patients suffering from neurodegenerative diseases. The aim of this transplantation is to permanently replace the neurons destroyed by the disease. Thus, local transplantation of embryonic cells or tissues has been successfully performed in several patients with Parkinson's disease. However, this approach involves the use of tissue from aborted fetuses, which raises ethical and logistical issues. Also, many laboratories have turned to human induced pluripotent stem (hIPS) cells as a possible alternative source of high-quality human cells. Effective differentiation of hIPS into neurons with the necessary phenotype to replace neurons lost by patients is a very active area of ​​research. However, neurons are very fragile cells. Injection of neuronal suspensions generally results in the death of more than 98% of the neurons due to anoikis. Alternatively, it has been considered to transplant neuronal precursors, which are known to be less fragile than neurons. This solution is also insufficient. Indeed, cell differentiation of neuronal precursors after implantation is not controlled and there is no certainty that the implanted cells will differentiate appropriately and / or into the desired neuronal types.

[0006] Thus, there is a need for neuronal implantation systems to improve the survival rate of implanted cells, particularly to permanently replace altered neurons in patients with neurodegenerative diseases. Summary of the Invention

[0007] While working on three-dimensional (3D) culture of cells, the inventors discovered that when dealing with particularly fragile cells such as neurons, it is possible to improve their survival rate by culturing and treating them as a 3D cell network rather than as a cell suspension. They developed a neural tissue unit containing neurons with a desired phenotype that can be injected into the patient's nervous system in order to integrate the neurons into the nervous system with a much higher survival rate than that obtained when injecting a neuronal suspension. The developed neural cell unit contains neurons pre-organized in a 3D network and embedded in an extracellular matrix, which promotes the integration and survival of the cells in the nervous system after injection. In fact, the neural cells are transplanted into the host nervous system while maintaining the integrity of the local environment, in particular the interactions between the cells and with the extracellular matrix developed within the neural tissue unit. The neural tissue unit according to the invention also contains postmitotic neurons with a perfectly controlled degree of differentiation and limits the risk of introducing undesired cell types responsible for the growth of teratomas and tumors. Interestingly, it has been observed that the neural tissue units according to the invention are able to project axons into the host tissue, promoting good integration of the cells into said host tissue and resulting in clear functional benefits.

[0008] Thus, the present invention relates to a neural tissue unit for use in implantation into the nervous system of a human or non-human mammal, said neural tissue unit comprising differentiated post-mitotic neurons in an extracellular matrix, said unit being derived from an intracellular microcompartment comprising a hydrogel capsule surrounding a single neural tissue unit, said hydrogel capsule being at least partially removed prior to implantation of said neural tissue unit.

[0009] Thus, the present invention relates to a neural tissue unit for use in the treatment of a neurodegenerative disease by implanting at least one neural tissue unit comprising differentiated postmitotic neurons in an extracellular matrix into the nervous system of a human or non-human mammal having a neurodegenerative disease, the unit being obtained from an intracellular microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, the hydrogel capsule being at least partially removed prior to implantation of the neural tissue unit.

[0010] In particular, the present invention relates to a neural tissue unit for use in the treatment of Parkinson's disease by implanting at least one neural unit comprising postmitotic neurons differentiated into dopaminergic neurons in an extracellular matrix into the nervous system of a human or non-human mammal having Parkinson's disease, the unit being obtained from an intracellular microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, the hydrogel capsule being at least partially removed prior to implantation of the neural tissue unit.

[0011] The present invention also relates to a method for the treatment of Parkinson's disease by means of at least one neural tissue unit comprising postmitotic neurons differentiated into dopaminergic neurons in an extracellular matrix which is implanted into the nervous system of a patient with Parkinson's disease, said unit being obtained from an intracellular microcompartment comprising a hydrogel capsule surrounding said neural tissue unit, said hydrogel capsule being at least partially removed prior to implantation of the neural tissue unit.

[0012] Similarly, the present invention relates to a neural tissue unit for use in the treatment of Huntington's disease by implanting at least one neural tissue unit comprising postmitotic neurons differentiated into GABAergic neurons in an extracellular matrix into the nervous system of a human or non-human mammal having Huntington's disease, the unit being obtained from an intracellular microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, the hydrogel capsule being at least partially removed prior to implantation of the neural tissue unit.

[0013] The present invention also relates to a method for the treatment of Huntington's disease by means of at least one neural tissue unit comprising postmitotic neurons differentiated into GABAergic neurons in an extracellular matrix which is implanted into the nervous system of a patient with Huntington's disease, said unit being obtained from an intracellular microcompartment comprising a hydrogel capsule surrounding said neural tissue unit, said hydrogel capsule being at least partially removed prior to implantation of the neural tissue unit.

[0014] Similarly, the present invention relates to a neural tissue unit for use in implantation into the nervous system of a human or non-human mammal, said neural tissue unit comprising genetically modified neurons. Such a neural tissue unit may be used in particular to assist, improve or restore cognitive and / or behavioral functions in a human or non-human mammal.

[0015] The present invention also relates to a method for the preparation of such a neural tissue unit intended to be implanted into the nervous system of a human or non-human mammal, the method comprising the steps of: (1) producing within a hydrogel capsule an intracellular microcompartment containing extracellular matrix and human or non-human mammalian cells capable of differentiating into neural cells, and which is advantageously immunocompatible with the mammal intended to receive the neural tissue unit; (2) inducing cell differentiation within the intracellular microcompartment to obtain neurons with at least one desired postmitotic phenotype; (3) At least partially removing the hydrogel capsule to harvest the neurons as neural tissue units. The present invention relates to a method of manufacturing the same.

[0016] The present invention also relates to a neural tissue unit implantation kit comprising at least one intracellular microcompartment comprising at least one hydrogel capsule surrounding a neural tissue unit according to the present invention, and means for at least partially removing the hydrogel capsule.

[0017] The present invention also relates to a nervous tissue unit implantation kit comprising at least one, preferentially 10-100 and up to 1000 nervous tissue units according to the invention, wherein the nervous tissue units are loaded into a surgical implantation device capable of implanting the nervous tissue units into the nervous system of a mammal. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 is a schematic representation of the procedure for preparing different rats made to suffer from Parkinson's disease and treated or not by injection of neural tissue according to the invention: Sham: control rats; 6-OHDA: rats made to suffer from Parkinson's disease (negative control); 6-OHDA encapsulated precursors (or Pro encap): rats made to suffer from Parkinson's disease and given a neural tissue unit according to the invention from intracellular microcompartments in which commercially available neuronal precursor cells were encapsulated; 6-OHDA CNO (control nervous system organoid): rats made to suffer from Parkinson's disease and given a neural tissue unit according to the invention (corresponding to CNO in this example) from intracellular microcompartments in which pluripotent cells, including dopaminergic neurons, were encapsulated before their differentiation into neurons; [Diagram 2]2A shows the lesion (2A) and transplantation (2B) procedures applied to obtain Parkinson's disease rats and to transplant neuronal tissue according to the invention; [Diagram 3] Figure 3 is a photomicrograph showing the multi-millimeter axonal projections of human neurons 6 days after implantation of an implantable neural unit according to the invention (in this example, CNO type) into the central nervous system of a Parkinsonian rat (6-OHDA CNO). This is a confocal image of a section of the rat striatum in the area of ​​implantation that was fixed, frozen, cut on a cryostat, and revealed by histochemistry using an antibody against human cytoplasm (HCM); [Figure 4A] 4A-E show the results obtained for behavioral tests carried out on rats made Parkinson's disease and receiving or not receiving a transplant of a neural tissue unit according to the invention: (4A) cylinder test; (4B) stepping test; (4C) rotameter test; (4D) anxiety test. [Figure 4B] 4A-E show the results obtained for behavioral tests carried out on rats made Parkinson's disease and receiving or not receiving a transplant of a neural tissue unit according to the invention: (4A) cylinder test; (4B) stepping test; (4C) rotameter test; (4D) anxiety test. [Figure 4C] 4A-E show the results obtained for behavioral tests carried out on rats made Parkinson's disease and receiving or not receiving a transplant of a neural tissue unit according to the invention: (4A) cylinder test; (4B) stepping test; (4C) rotameter test; (4D) anxiety test. [Figure 4D] 4A-E show the results obtained for behavioral tests carried out on rats made Parkinson's disease and receiving or not receiving a transplant of a neural tissue unit according to the invention: (4A) cylinder test; (4B) stepping test; (4C) rotameter test; (4D) anxiety test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention discloses a method for implanting or transplanting a neural tissue unit, in which neural cells are organized into a 3D network, into the nervous system of a mammal. The transplantation of neural cells in the form of dense spheres facilitates their implantation or integration into the host nervous system. The implantation method according to the invention, and more generally the neural tissue unit developed according to the invention, is intended for use in human or non-human mammals, more preferentially in human subjects.

[0020] Neural Tissue Unit The present invention also proposes the use of neural tissue units for implantation into the nervous system of a subject, instead of neuronal suspensions, to treat all kinds of neurodegenerative diseases and / or to assist, improve or restore cognitive or behavioral functions in a subject.

[0021] According to the invention, the implanted neural tissue unit advantageously comprises postmitotic neurons and glial cells, such as astrocytes and / or oligodendrocytes, differentiated into an extracellular matrix.

[0022] "Postmitotic" neurons are neurons that have lost the ability to divide. The neurons contained in the microcompartments are distinct in the sense that they have a particular phenotype.

[0023] In accordance with the present invention, the hydrogel capsule surrounding the neural tissue unit is at least partially removed prior to implantation into the subject's nervous system.

[0024] In the context of the present invention, "hydrogel capsule" refers to a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferentially water. Advantageously, the hydrogel used is biocompatible in the sense that it is not toxic to the cells. The hydrogel capsule must also allow the diffusion of oxygen or nutrients to be supplied to the cells contained in the microcompartments, allowing them to survive. For example, the hydrogel capsule comprises alginic acid. Preferentially, the capsule comprises only alginic acid. In the context of the present invention, "alginic acid" refers to a linear polysaccharide formed from β-D-mannuronic acid and α-L-guluronic acid, their salts and derivatives. Advantageously, the alginic acid is sodium alginate, composed of 80% α-L-guluronic acid and 20% β-D-mannuronic acid, with an average molecular weight between 100 and 400 KDa (see, for example, PRONOVA). TM SLG100) and the total concentration is 0.5% by mass to 5% by mass.

[0025] At the time the neural tissue unit is produced, the hydrogel capsule protects the cells from the extracellular environment while allowing controlled differentiation into one or more desired neuronal types. According to the present invention, the hydrogel capsule surrounds a single neural tissue unit. Thus, each neural tissue unit is individually surrounded by a hydrogel capsule. Once one or more desired cell types are obtained and the size of the neural tissue in the capsule is sufficient, the capsule is at least partially removed. By "at least partially removed" is meant that the hydrogel capsule is at least partially hydrolyzed, dissolved, perforated or ruptured to allow at least the axons of at least some of the neural cells of the unit to exit the capsule. Any biocompatible means, i.e., non-toxic to cells, that allows hydrolysis, dissolution, perforation and / or at least partial rupture of the hydrogel capsule can be used. For example, phosphate buffered saline, divalent ion chelating agents, enzymes such as alginate degrading enzymes, laser microdissection, soluble beads incorporated into the hydrogel, etc. can be used.

[0026] Advantageously, the capsule is completely removed so that the neural tissue unit intended to be implanted into the nervous system of a subject is free of hydrogel.

[0027] Preferentially, the extracellular matrix layer of the nervous tissue unit forms a gel. The extracellular matrix layer comprises a mixture of proteins and extracellular compounds necessary for cell culture, more particularly for nervous cell culture. Preferentially, the extracellular matrix comprises structural proteins, such as laminin with α1 or α4 or α5 and β1 or β2 and γ1 or γ3 subunits, entactin, vitronectin, collagen, and growth factors, such as TGF-β and / or EGF. In one embodiment, the extracellular matrix layer consists of or comprises Matrigel® and / or Geltrex®.

[0028] The nervous tissue unit according to the invention has the advantage that it comprises nervous system cells organized into a 3D network in which interactions between the cells and the extracellular matrix already exist.

[0029] Advantageously, the nervous system cells of the nervous tissue unit form a group of nervous system cells, in particular of ovoid, tubular or spherical shape, with a smallest dimension preferably comprised between 10 μm and 1000 μm ± 10%, preferentially between 150 μm and 400 μm ± 10%, even more preferentially between 200 μm and 300 μm ± 10%. These dimensions are particularly favorable for the survival of neurons within the nervous tissue unit and optimize the remodeling and vascularization of the graft after implantation. "Smallest dimension" means the smallest distance between two points on either side of the cell population.

[0030] Preferentially, the nervous tissue unit according to the invention comprises between 100 and 100,000 nervous system cells.

[0031] Within the nervous tissue, the neurons advantageously have one or more phenotypes of interest, selected according to the intended use of the nervous tissue unit. According to the invention, the implanted nervous tissue unit comprises neurons differentiated according to one or more selected and controlled phenotypes. Preferentially, the nervous tissue unit comprises 10-100%, preferentially 50-100%, more preferentially more than 90% of postmitotic neurons of the phenotype of interest. Other cells present in the nervous tissue unit may in particular be glial cells and / or postmitotic neurons whose phenotype differs from the phenotype of interest. Advantageously, the nervous tissue unit consists essentially of postmitotic neurons with the same phenotype.

[0032] The desired phenotype of the postmitotic neuron depends on the application of the unit. Thus, in the case of cell therapy applications, the phenotype of the neuron advantageously corresponds to that of the defective cell to be replaced.

[0033] Indeed, the nervous tissue units according to the invention may advantageously be used in cell therapy for the treatment of neurodegenerative diseases, such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease or neuronal ceroid lipofuscinoses, such as Batten disease.

[0034] Thus, for the treatment of Parkinson's disease, the nervous tissue unit advantageously comprises dopaminergic neurons. Preferentially, the post-mitotic neurons contained in such a nervous tissue unit are essentially dopaminergic neurons.

[0035] In the context of the present invention, "essentially" means at least 90%, preferentially at least 95% and even more preferentially at least 99% of the cells.

[0036] For the treatment of Huntington's disease, the neural tissue unit advantageously comprises GABAergic neurons. Preferentially, the post-mitotic neurons contained in such a neural tissue unit are essentially GABAergic neurons.

[0037] The nervous tissue unit according to the present invention may also include genetically modified nervous system cells. Thus, it is possible to implant nervous system cells in which the expression of one or more genes is suppressed or increased into the nervous system of a subject. It is also possible to implant nervous system cells into which a heterologous gene has been introduced in order to introduce a function that is not normally present in the nervous system.

[0038] Such neural tissue units comprising genetically modified cells are of particular interest for assisting, improving or repairing cognitive and / or behavioral functions, e.g. impaired or deficient cognitive and / or behavioral functions in the subject under consideration.

[0039] For example, in the treatment of Huntington's disease, which has a known genetic cause (expression of a CAG triplet repeat in the gene encoding huntingtin), it is possible to remove cells from the patient and produce a neural tissue unit according to the invention containing GABAergic neurons in which the defective gene has been corrected, before performing an autologous transplantation of the neural tissue unit.

[0040] For patients with Parkinson's disease, it is possible to produce neural tissue units containing modified dopaminergic neurons that express light-sensitive channels, such as rhodopsin channels, and control the activity of the transplanted neural tissue by simple illumination in a manner similar to deep brain stimulation, but less invasive. In general, controlling the activity of the transplanted neural tissue, especially by expression of light-sensitive channels, can be beneficial for all potential applications.

[0041] Neural Tissue Unit Embedding Kit The present invention also proposes a nervous tissue unit implantation kit comprising at least one intracellular microcompartment comprising a hydrogel capsule surrounding the nervous tissue unit according to the present invention, and means for at least partially removing the hydrogel capsule (hydrolysis, dissolution, perforation and / or rupture).

[0042] Thus, a practitioner who must use the nerve tissue unit according to the present invention will at least partially remove the hydrogel capsule, if necessary at the time of use, to obtain a nerve tissue unit ready to be implanted into the nervous system of a subject.

[0043] An implantation kit according to the present invention may also include a surgical implantation device capable of implanting the neural tissue unit into the nervous system of a mammal.

[0044] The present invention also proposes a nervous tissue unit implantation kit comprising at least one nervous tissue unit according to the invention, preferentially 1 to 10,000, more preferentially 10 to 1000 nervous tissue units, loaded into a surgical implantation device capable of implanting the nervous tissue unit into the nervous system of a mammal. Advantageously, the amount of nervous tissue units implanted depends on the size of the host brain and the application.

[0045] The nervous tissue unit may optionally be frozen prior to introduction into the surgical implantation device and / or may be frozen simultaneously with the surgical implantation device, although of course a subsequent thawing step will be required before the nervous tissue unit can be implanted into the nervous system of a subject.

[0046] Manufacturing method The invention also relates to a method for producing a neural tissue unit that can be implanted into the nervous system of a mammal. More specifically, the invention proposes to produce a neural tissue unit that comprises postmitotic neurons, whose phenotype depends on the intended use of the neural tissue unit. According to the invention, it is possible in particular to produce intracellular microcompartments comprising hydrogel capsules surrounding nervous system cells from differentiated mammalian cells that are reprogrammed before or after encapsulation and then differentiated into one or more neural types within the hydrogel capsule. The capsule is then at least partially removed, allowing the embedding of the nervous system cells into the host tissue after implantation.

[0047] Generally, the method for producing such a nervous tissue unit according to the invention, intended to be implanted in the nervous system of a human or non-human mammal, comprises the following steps: (1) producing within a hydrogel capsule an intracellular microcompartment containing extracellular matrix and human or non-human mammalian cells capable of differentiating into neural cells, and which is advantageously immunocompatible with the mammal intended to receive the neural tissue unit; (2) inducing cell differentiation within the intracellular microcompartment to obtain postmitotic neurons with at least one desired phenotype; (3) At least partially removing the hydrogel capsule to harvest the neural cells as neural tissue units. Includes.

[0048] Any method for producing intracellular microcompartments containing extracellular matrix and cells capable of giving rise to neural cells within a hydrogel capsule may be used. In particular, it is possible to produce the microcompartments using a microfluidic device as described in Alessandri et al. 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604).

[0049] In certain embodiments, the cell to be encapsulated is pluripotent stem cell, for example induced pluripotent stem cell (IPS) cell or embryonic stem cell (ES).In the context of the present invention, "induced pluripotent stem cell" (IPS cell) is defined as pluripotent stem cell obtained by genetic reprogramming of differentiated somatic cell, and has morphology partially similar to embryonic stem cell, and self-renewal ability and pluripotency.These cells are particularly positive for pluripotent markers, for example alkaline phosphatase staining and expression of protein NANOG, SOX2, OCT4 and SSEA3 / 4. Methods for obtaining induced pluripotent stem cells are well known to those skilled in the art and are described in particular in the publications of 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). In the case of embryonic stem cells, the pluripotent stem cells are cells that originate from the inner cell mass of the blastocyst and have the ability to cause the formation of all tissues of an organism. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4 and NANOG and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. Embryonic stem cells can be obtained without destroying the embryo from which they are derived, for example by using the technique described by Chung et al. (Cell Stem Cell, 2008, 2(2): 113-117). In certain embodiments, for legal or ethical reasons, stem cells are defined as excluding human embryonic stem cells.

[0050] Alternatively, it is possible to encapsulate neuronal precursor cells, i.e. stem cells that have already committed to cell differentiation into nervous system cells. In another embodiment, it is possible to encapsulate differentiated cells that will be differentiated into nervous system cells by transdifferentiation. It is also possible to encapsulate glial cells, e.g. cells that can form somatic cells.

[0051] In another embodiment, it is possible to use differentiated cells that are reprogrammed into pluripotent stem cells before or after encapsulation. Since the neural tissue unit must be able to be implanted in the patient's nervous system for a long period of time, it is preferable to start with cells that are immunocompatible with the subject, to avoid the risk of rejection. In a particular embodiment, the cells used to produce the neural tissue unit have been previously harvested from the human or non-human mammal in which the neural tissue unit will be implanted.

[0052] In all cases, once the subcellular microcompartments are obtained, the cells contained therein must undergo cytodifferentiation in order to differentiate them into neurons according to one or more desired phenotypes.

[0053] Any method conventionally used in 2D culture (such as petri dishes) for cell differentiation can be used, such as those described by 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 Lippmann et al. ("Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors", Stem Cells 2014). In certain embodiments, the differentiation medium contains 24(S),25-epoxycholesterol to enhance dopaminergic differentiation.

[0054] Preferentially, the intracellular microcompartments are cultured in differentiation medium for at least 3 weeks and prior to removal of the hydrogel capsule.

[0055] Generally, the pluripotent intracellular microcompartments may be frozen prior to use and thawed as needed.

[0056] In one exemplary embodiment, cell differentiation can be induced to differentiate into dopaminergic cells within intracellular microcompartments ("Generating regionalized neuronal cells from pluripotency, a step-by-step protocol" Kirkeby et al. Frontiers in Cellular Neuroscience 2012). Neural tissue units derived from such intracellular microcompartments can then be implanted into the nervous system of a subject with Parkinson's disease to at least partially replace the subject's failing dopaminergic neurons.

[0057] In another exemplary embodiment, cell differentiation can be induced to differentiate into GABAergic cells within the subcellular microcompartments ("Derivation of striatal neurons from human stem cells" Viegas et al. Prog Brain Res 2012). Neural tissue units derived from such subcellular microcompartments can then be implanted into the nervous system of a subject with Huntington's disease to at least partially replace the subject's impaired GABAergic neurons.

[0058] The method for producing a nerve tissue unit according to the invention also comprises the further steps of: - loading the surgical implantation device with at least one nerve tissue unit, preferentially 10-1000, more preferentially 10-100 nerve tissue units; may include.

[0059] The implantation device is then ready to be used to transplant neural cells into the nervous system of a subject.

[0060] According to the invention, it is possible to freeze the nervous system cells as 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 manufacturing process without first being frozen.

[0061] Advantageously, the process according to the invention comprises the following intermediate steps: - To confirm the phenotype of neural cells contained in hydrogel capsules after the cell differentiation process. Includes.

[0062] For example, in the case of one or more neural tissue units containing dopaminergic neurons, it is possible to non-invasively measure the activity of the implantable neural units prior to implantation by removing the supernatant from the culture medium and performing microdialysis analysis coupled to HPLC.

[0063] More generally, it may be advantageous to characterize the electrical activity of neurons representative of a sample by the so-called "patch clamp" technique, which is based on the recording of ionic currents across the cell membrane.

[0064] The neural tissue units are advantageously cultured in batches under identical conditions and a more detailed analysis can also be carried out on a representative sample of the considered neural tissue unit population, in particular by immunocytochemistry, RNA and / or proteomic sequencing, to ensure the exact quality of the differentiation. For example, in the case of neural tissue units containing dopaminergic neurons, an immunocytochemistry analysis targeting the tyrosine hydroxylases FOXA2 and / or NURR1 can be carried out ("Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease" Kriks et al. 2012).

[0065] Thus, it is possible to verify that the post-mitotic neurons and / or neural cells indeed have the desired phenotype prior to implantation into the subject's nervous system.

[0066] Implantation of Neural Tissue Units into the Nervous System of Humans or Non-Human Mammals The present invention also proposes a method for implanting or transplanting the neural tissue unit into the nervous system of a subject in need, in particular a human. Indeed, the neural tissue unit according to the present invention may advantageously be used for the treatment of neurodegenerative diseases. In the context of the present invention, the term "treatment" refers to a therapeutic, symptomatic or preventive treatment. The term "treatment" of a disease as used herein refers to any action intended to extend the quality and / or lifespan of the patient. The treatment may be designed to eradicate the disease, to stop or slow the progression of the disease and / or to promote the regression of the disease. The term "treatment" of a disease also refers to any action intended to alleviate the symptoms associated with the disease. The patient to be treated may be any mammal, preferably a human.

[0067] Generally, any method for implanting neural tissue cells can be used to implant the neural tissue unit into the nervous system of a subject. In particular, it is possible to use a cannula, such as a glass cannula, that is preloaded with a neural tissue unit.

[0068] In certain embodiments, the embedding comprises the steps of: - anesthetizing and / or immobilizing the subject that will receive the nerve tissue unit; - Opening the subject's skull; - positioning the implantation device comprising the nerve tissue unit on the implantation area, for example by means of a so-called "stereotactic" device; - Inserting the implantation device into the implantation area; - Involves injecting a nerve tissue unit while gradually removing the implanted device from the implantation area.

[0069] Advantageously, the injection process is controlled by a microfluidic pump and motorization system to manage the insertion depth of the implanted device into the subject's nervous system and provide the necessary space for neural tissue units in said nervous system, thereby depositing in the withdrawal path of the implanted device.

[0070] In a particular embodiment, the implantation device consists of a glass cannula with an inner diameter of 0.3-2 mm, preferentially 0.4 mm, and an outer diameter of 0.4-3 mm, preferentially 0.55 mm. To reduce the risk of tissue damage when inserting the cannula and to reduce the risk of occlusion by thrombus, the cannula advantageously has a rounded or beveled tip, the surface of which is silanized with a fluorinated compound. The nerve tissue unit is loaded into the cannula, for example from the front. The cannula is inserted into the subject's cranial position.

[0071] Advantageously, 10-40 neural tissue units are implanted during the same transplantation, where each neural tissue unit contains preferentially 1000-10,000 neural cells. Naturally, the number of neural tissue units implanted is adapted according to the subject to be treated and the disease and / or cognitive or behavioral function to be modified. Several transplantations more or less separated in time can also be considered. Similarly, transplantation can be performed simultaneously or in a short time in different areas of the subject's nervous system. EXAMPLES

[0072] 1. Fabrication of implantable neural tissue units for the treatment of Parkinson's disease: Precursors were obtained in cultures derived from human induced pluripotent stem cells using 24(S),25-epoxycholesterol (at concentrations between 1 and 10 μM) to optimize differentiation into dopaminergic neurons, according to a method published by Kriks et al. ("Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease" Kriks et al. 2012).

[0073] The precursors obtained are then encapsulated according to the method published by Alessandri et al. ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016).

[0074] Once intracellular microcompartments containing the precursors are obtained, so-called "terminal" differentiation into dopaminergic neurons is performed according to the method published by Kriks et al. ("Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease" Kriks et al. 2012), again using 24(S),25-epoxycholesterol (at concentrations of 1–10 μM).

[0075] Particular attention is paid to the exchange of the culture medium, since the proliferation of cells in the intracellular microcompartments increases the rate at which medium is consumed by the implantable neural unit during its fabrication.

[0076] Although all differentiation occurs in intracellular microcompartments, it is possible to deencapsulate and reencapsulate cells as long as the cells support dissociation of a single cell suspension (up to 1 week after addition of γ-secretase inhibitors and / or Notch signaling, e.g., DAPT, CoumpoundE or other molecules that promote terminal differentiation).

[0077] Therefore, the intracellular microcompartments obtained after 3 weeks of differentiation are treated by successive rinsing in PBS solutions to dissolve the hydrogel shell and retrieve the neural tissue units before injection.

[0078] Fabrication of the infusion cannula: A borosilicate capillary (inner diameter: 0.4 mm, outer diameter: 0.55 mm) was cut to a length of 5 cm using a ceramic blade.

[0079] The front face of the capillary was rounded with 12 μm aluminum oxide abrasive paper and then rounded with 1 μm optical fiber.

[0080] The glass is activated by plasma treatment for 5 minutes and then silanized in the gas phase by deposition of 20 μl of 1H,1H,2H,2H-perfluorooctyltrichlorosilane adjacent to the chip placed in a closed Petri dish.

[0081] After 30 minutes of reaction, the injection cannula is prepared and connected to a 10 μl syringe (Hamilton) and attached to a standard stereotaxic apparatus powered by a syringe pump (Harvard Apparatus). The injection arm of the stereotaxic apparatus is also motorized (PI).

[0082] Surgery: The rats then underwent standard nerve implantation surgery, which included the following steps: 1) Induce medicated (ketamine) sleep and anesthesia in animals. 2) Accurately position the animal using a stereotaxic device. 3) The animal's head is shaved and then treated with betadine, then opened with a scalpel to remove the skin from the skull, and then a microdrill is used to create a window in the skull to allow passage of the cannula. The meninges are carefully spread apart without damaging the exposed cortex underneath. 4) A cannula attached to a syringe is positioned over the implantation area using a stereotactic device. 5) The nerve tissue unit is loaded into the cannula from the anterior side (approximately 80 capsules or 10,000-800,000 cells). The implantation corresponds to a total of 4 μl or a 4 cm height of the cannula. In rats, unilateral injections are made into the striatum at two sites with a distance of approximately 1 mm. 6) The cannula is inserted into the animal's skull at a first site within the ventral striatum. During the 2 μl injection, the cannula is removed simultaneously with the injection using a microfluidic pump system and motorized control of the cannula depth, freeing up space required for the implantation, which is deposited approximately 2 mm into the retrieval track. 7) The cannula is left in place for 1 minute before being gently removed. 8) The second site is then implanted using the same procedure. 9) The rat's skin is sutured (3-5 stitches) after rinsing the wound with Betadine.

[0083] Porting results: To demonstrate survival of the implanted mature human neurons, animals are sacrificed one week after surgery to test survival and embedding of the grafts by immunocytochemistry of brain slices cut with a vibratome in the transplantation pathway.

[0084] Estimated survival based on graft size (known before implantation) is >84% ± 33%, compared to a 2% survival rate obtained with transplantation of mature dopaminergic neurons in suspension (DM Marchionini et al., "Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease." J Comp Neurol 464, 172-179 (2003)). Neuronal phenotype is maintained (tyrosine hydroxylase positive), and axonal processes of several millimeters are observed, indicating that the neurons immediately begin to integrate into the rat brain.

[0085] 2. Implantation of neuron-containing neural tissue units for the treatment of Parkinson's disease 2.1- Surgical procedure Rats (7 / 8 weeks old) are housed in a temperature-controlled room with a 12-h light / dark cycle with free access to food and water.

[0086] FIG. 1 summarizes the different procedures carried out on Parkinson's disease rats to obtain these rats to which the transplantation of neural tissue according to the invention can then be applied.

[0087] Preparation of Parkinson's disease rats Animals are fasted prior to surgery. Food and water, including nutritional supplements, are maintained.

[0088] Place each animal in an anesthesia chamber supplied with a continuous flow of air (0.3 L / min) + oxygen (0.3 L / min) and 2% isoflurane. Inject xylocaine (7 mg / kg, sc), bolgar (7.5%, im) and buprenorphine (0.1 mg / kg, sc). Then, keep the animal under anesthesia with an isoflurane-based anesthetic administered by a volume-controlled ventilator. Core temperature is recorded through the rectum during the surgery. Use a heating pad to maintain body temperature if necessary. Administer ophthalmic ointment (Lipo ophthalmic gel) to each eye. Cut the fur from the skull to the neck. After loss of consciousness, place the animal in a stereotaxic frame (Kopf) and fix its head in position using ear bars.

[0089] Rats were made to mimic Parkinson's disease by unilateral stereotactic injection of 2.5 μl of 6-hydroxydopamine (6-OHDA) (Sigma, 5 mg / ml in sterile 0.9% NaCl) with 0.1% ascorbic acid at the coordinates of -2.8 mm anterior-posterior, 2 mm lateral, and 8.4 mm dorsal in the medial forebrain bundle (MFB) at a rate of 1 μl / min using a Hamilton 1702N syringe (Ga22s / 51 mm / PST3) connected to a Legato 101 autoinjector (KD scientific) (Figure 2A).

[0090] After injection, the needle is left in place for 5 min before being slowly removed from the brain. 30 min before surgery, animals are given an intraperitoneal (ip) injection of desipramine (25 mg / kg, 5 ml / kg) dissolved in 0.9% NaCl to protect the noradrenergic system.

[0091] When the injection is complete, the skin is sutured closed and the animals are then allowed to recover from anesthesia.

[0092] transplant Maintaining the mature neuronal microenvironment throughout the transplantation procedure allows for survival and integration into the host brain.

[0093] A group of rats (6-OHDA encapsulated precursors) are given transplants of neural tissue units according to the invention containing commercially available neuronal precursor cells (Cellular Dynamics iCell DopaNeurons), which are encapsulated and matured in vitro for 2 weeks prior to use.

[0094] A second group of rats (6-OHDA CNO) will receive transplants of neural tissue units according to the invention containing control nervous system organoids (CNO), produced and preserved in this laboratory.

[0095] Neural tissue units containing CNO cells were obtained by encapsulating pluripotent or untreated pluripotent cells in alginate and differentiating them into neuronal precursor cells according to the following procedure:

[0096] All incubations are carried out at 37° C., 5% CO 2 and >95% humidity.

[0097] D0-D3 (day 0 to day 3): N2B27 (For 500 mL of medium: 250 mL of Neurobasal medium + 250 mL of glutamax-supplemented Dmem F12 medium + 1 N2 supplement + 1 B27 supplement + 0.5 μM LDN-193189 + 10 μM SB431542 + 100 ng / mL Shh (Sonic Hedgehog) + 100 ng / mL FGF-8 (Fibroblast Growth Factor 8) + 2 μM Parmorphamine + 10 μM 24(S),25-epoxycholesterol) D3-D9: N2B27 + LDN + SB + Shh + FGF-8 + Parmorphamine + CHIR + 24(S),25-epoxycholesterol D9-D10: N2B27+LDN+CHIR+24(S),25-epoxycholesterol D10-D15 onwards: N2B27 + cAMP + AA + GDNF + BDNF + FGF-20 + TGFβ + trichostatin + CpE + 24(S),25-epoxycholesterol

[0098] [Table 1]

[0099] [Table 2]

[0100] Prior to injection of the neural tissue units (6-OHDA encapsulated precursors and 6-OHDA CNO), the alginate capsules of the intracellular microcompartments are dissolved by simply replacing the medium with PBS.

[0101] The injection setup is a syringe or cannula attached to a stereotactic device. The nerve tissue unit is loaded into the injection cannula by drawing the injection volume (approximately 1 μL of 25 CNO) into the body of the cannula.

[0102] For injection into four injection sites on two orbits (Figure 2B): 4 μl containing precursor (group 3), encapsulated precursor (6-OHDA precursor) or CNO (6-OHDA CNO) is injected into the striatum at the following coordinates: A / P+1.2; M / L-2.6; D / V-5.0 (3 μl) and -4.0 (3 μl); and A / P+0.5; M / L-3.0; D / V-5.0 (3 μl) and -4.0 (3 μl); tooth bar-2.4 (Grealish et al., 2014).

[0103] After the surgical procedure Four groups of rats are formed from immunodeficient rats (NR or nude rats): - "Sham": non-lesioned rats - "6-OHDA": Rats in which dopaminergic neurons in the right hemisphere were chemically killed but not implanted (control experiment) "CNO" or "6-OHDA CNO": rats in which the dopaminergic neurons of the right hemisphere have been chemically killed and in which a neural tissue unit according to the invention containing approximately 250,000 cells has been transplanted into the right striatum - "Pro encap" or "6-OHDA encapsulated precursor": rats in which dopaminergic neurons of the right hemisphere are chemically killed and matured in a capsule for 2 weeks, and a neural tissue unit according to the invention containing approximately 250,000 Cellular Dynamics iDopaneurons cells is transplanted into the right striatum.

[0104] Each animal is closely observed and kept warm until righting and swallowing reflexes are restored. Animals are cared for by experienced technicians with readily available experienced veterinary support.

[0105] Other medications for the clinical management of the animals are used as necessary. Each medication, dose, route, and site of administration will be recorded in the surgical record.

[0106] The surgical incision will be monitored at least daily (until the incision is healed) for signs of infection, inflammation and general integrity. All observations made and results will be recorded in the surgical record. The study coordinator will be informed of any abnormalities. The study monitor will be informed of any abnormalities as soon as reasonably possible.

[0107] Checks on the animals' condition will be conducted twice a day (11am to 10pm).

[0108] Animals are evaluated according to standards for fair and humane treatment. Appropriate veterinary support is provided as necessary. Any animals showing signs of severe pain and distress that may persist are promptly euthanized. They are then subjected to a gross necropsy. In addition to the necropsy, the brains are removed for histological examination and processed as indicated in the euthanasia section. All reasonable efforts are made to apply standards for fair and humane treatment and to prevent animals from being found dead.

[0109] 2.2- Behavioral research Amphetamine-induced rotation (modified from Grealish et al., 2014 ). Only animals with more than 5 rotations per minute after injection of 6-hydroxydopamine (6-OHDA) into the central telencephalic bundle were considered successfully lesioned. Rotational bias after systemic injection of amphetamine (2.5 mg / kg, i.p., Apoteksbolaget) was recorded using an automated system (Omnitech Electronics). Animals' rotations were recorded for 90 min and only complete body rotations were counted and then expressed as net rotations per minute, with rotations towards the lesioned side (clockwise) counted as positive.

[0110] Stepping Test Each rat is placed on a flat surface; its hind paws are raised by gently holding the tail so that only the front paws touch the platform. The experimenter pulls the rat 1 m backwards at a constant speed. The contralateral and ipsilateral forepaw adjustment movements are counted independently for each path. Data are expressed as the percentage of adjustment movements with the paw by calculating the ratio contralateral / (contralateral + ipsilateral).

[0111] Cylinder Test Each animal was placed in a glass cylinder and the cylinder test was performed by counting the number of contacts of the ipsilateral and contralateral paws to the wall of the cylinder during a 5 min period, and the results were expressed by calculating the ratio contralateral / (contralateral + ipsilateral), with a maximum of 20 contacts.

[0112] "Open Field" Activity Meter Voluntary locomotor activity is measured using a photoelectron actimeter (Actitrack, Panlab, SL, Barcelona, ​​Spain). The apparatus consists of a transparent cage connected to a photocell. A light beam detects movement and the total number of horizontal movement detections for each rat is recorded daily over two consecutive 10-minute sections. All actimeter tests are performed in an isolated room between 8:00 am and 1:00 pm. The first 3-day phase is for habituation. The fourth day is considered as the test day. Only locomotor activity recorded during the second 10-minute section is used for data analysis.

[0113] 2.3- Post-mortem research evaluation – histological analysis At the end of the experiment, the rats are sacrificed by intracardiac injection of 4% paraformaldehyde and the brains are harvested, frozen in isopentane at −45° C. and stored at −80° C. The injected brains are cut into 50 μm coronal sections on a cryostat.

[0114] Confirmation of dopaminergic (DA) lesions Loss of DA cells in the central nervous system and loss of DA fibers in the striatum are confirmed by immunohistochemistry for tyrosine hydroxylase (TH) as described previously (Bouali-Benazzouz et al., 2009). The number of tyrosine hydroxylase (TH) immunoreactive cells is obtained by applying the optical sectioning method using a Leica DM6000B microscope with Mercator Pro software (ExploraNova, version 7.9.8).

[0115] Verifying the transplant Rat brain sections and CNOs were subjected to immunohistochemistry assays using antibodies to analyze cell survival, proliferative potential and phenotypic maturation of the transplants (identified using human cytoplasmic markers (HCM)) and characterized with traditional DA markers (TH, DAT, FOXA2), total neuronal markers (MAP2, NeuN), serotonin (FOXG1), proliferation marker Ki-67 / PCNA and glial marker (human specific GFAP).

[0116] Neuronal viability assay 6 CNO rats are sacrificed 6 days after implantation to assess neuronal survival after implantation.

[0117] result Behavioral test results Behavioral tests show extremely positive effects of the transplantation according to the invention in transplanted rats (6Pro-encaps and CNO), since behavioral modifications are obtained for 4-6 weeks for the stepping test (Figure 4B) and cylinder test (Figure 4A) and for 2 weeks for the rotameter test (Figure 4C), whereas only modest effects shown in the literature of precursor transplantation show behavioral improvements after about 20 weeks (Kriks et al. 2011, Grealish et al. 2014, Kirkeby et al. 2017, Doi et al. 2014).

[0118] As expected, implantation according to the invention, on the other hand, has no effect on anxiety outcomes (FIG. 4D).

[0119] Histological results Histological analysis of the transplants performed in the two CNO and Pro-encap groups indicates the presence of 10-15% TH neurons in the transplants. In comparison, previously published studies have described TH neuron levels after progenitor implantation as around 5% (Kirkeby et al. 2017) or 6% (Kriks et al. 2011).

[0120] Analysis of neuronal survival showed that 6 days after implantation, multi-millimeter axonal projections were observed in the brains of CNO rats (Figure 3), confirming the remarkable survival of neurons after implantation according to the invention.

Claims

1. A neural tissue unit for use in implantation into the nervous system of a human or non-human mammal, the neural tissue unit comprising differentiated postmitotic neurons in an extracellular matrix, the unit being derived from an intracellular microcompartment comprising a hydrogel capsule surrounding a single neural tissue unit, the hydrogel capsule being at least partially removed prior to use of the neural tissue unit.

2. The nerve tissue unit for use according to claim 1, wherein the nerve tissue unit does not contain a hydrogel.

3. 3. A nervous tissue unit for use according to claim 1 or 2, wherein the nervous system cells form a group of nervous system cells, preferably having a smallest dimension of between 50 μm and 500 μm ± 10%, preferentially between 150 μm and 400 μm ± 10%, even more preferentially between 200 μm and 300 μm ± 10%.

4. The nervous tissue unit for use according to any one of claims 1 to 3, wherein said nervous cell unit comprises nervous cells and glial cells.

5. A nervous tissue unit for use according to any one of claims 1 to 4, wherein said unit comprises 10 to 100%, preferentially 50 to 100%, more preferentially more than 90% of post-mitotic neurons of the phenotype of interest.

6. A nervous tissue unit for use according to any one of claims 1 to 5, wherein the unit comprises between 100 and 100,000 nervous system cells.

7. 7. A nervous tissue unit for use according to any one of claims 1 to 6 for the treatment of a neurodegenerative disease, such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease or a neuronal ceroid lipofuscinosis, such as Batten disease.

8. A nervous tissue unit for use according to claim 7, wherein said nervous tissue unit advantageously comprises dopaminergic neurons for the treatment of Parkinson's disease.

9. 8. A neural tissue unit for use according to claim 7, wherein said neural tissue unit comprises GABAergic neurons for the treatment of Huntington's disease.

10. A nervous tissue unit for use according to any one of claims 1 to 9, wherein said nervous tissue unit comprises genetically modified nervous system cells.

11. A neural tissue unit for use according to claim 10 to assist, improve or restore cognitive and / or behavioral functions.

12. A method for producing a nerve tissue unit according to any one of claims 1 to 11, intended to be implanted into the nervous system of a subject, said method comprising the following steps: (1) producing, within a hydrogel capsule, an intracellular microcompartment comprising an extracellular matrix and human or non-human mammalian cells capable of differentiating into neural cells, and which is advantageously immunocompatible with the mammalian host intended to receive the neural tissue unit; (2) inducing cell differentiation within the intracellular microcompartment to obtain postmitotic neurons having at least one desired phenotype; (3) At least partially removing the hydrogel capsule to harvest the neurons as neural tissue units. A manufacturing method comprising:

13. Further steps as follows: (4) Loading the surgical implant device with at least one nerve tissue unit, preferentially at least 10-1000 nerve tissue units, and more preferentially 10-100 units. A method for producing a nerve tissue unit according to claim 12, comprising:

14. Further steps as follows: - freezing the nervous system cells as tissue units, either before or after at least partial removal of the hydrogel capsule; A method for producing a nerve tissue unit according to claim 12 or 13, comprising:

15. The method for producing a nervous tissue unit according to any one of claims 12 to 14, wherein the cells used in step (1) have been previously collected from a human or a non-human mammal into which the nervous tissue unit is to be implanted.

16. The following intermediate steps: - Confirming the phenotype of the neural cells contained in the hydrogel capsules after the cell differentiation in step (2). A method for producing a nerve tissue unit according to any one of claims 12 to 15, comprising:

17. A method for producing a neural tissue unit according to any one of claims 12 to 16, wherein the cells are differentiated into dopaminergic cells or GABAergic cells.

18. A neural tissue unit implantation kit comprising at least one intracellular microcompartment comprising at least one hydrogel capsule encapsulating a neural tissue unit according to any one of claims 1 to 11, and means for at least partially removing the hydrogel capsule.

19. 20. The nervous tissue unit implantation kit of claim 18, further comprising a surgical implantation device capable of implanting the nervous tissue unit into the nervous system of a mammal.

20. A nervous tissue unit implantation kit comprising at least one nervous tissue unit according to any one of claims 1 to 11, preferentially 1 to 10,000, more preferentially 10 to 1,000 nervous tissue units, in a surgical implantation device adapted for implanting the nervous tissue unit into the nervous system of a mammal.

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