NL2029861B1 - A method for generating a human cortical spheroid from self-renewing stem cells capable of differentiation - Google Patents

A method for generating a human cortical spheroid from self-renewing stem cells capable of differentiation Download PDF

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NL2029861B1
NL2029861B1 NL2029861A NL2029861A NL2029861B1 NL 2029861 B1 NL2029861 B1 NL 2029861B1 NL 2029861 A NL2029861 A NL 2029861A NL 2029861 A NL2029861 A NL 2029861A NL 2029861 B1 NL2029861 B1 NL 2029861B1
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human cortical
cells
spheroid
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human
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Julianus Maria Martens Gerardus
Mary Anne De Kleijn Kimberly
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Neurodrug Res B V
Jan Kelders Beheer B V
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Abstract

The present invention relates to a method for generating a human cortical spheroid from self-renewing stem cells capable of differentiation wherein cells are cultured in culture medium comprising low amounts of BMP inhibitor and TGF-ß inhibitor during the first step of the method. The present invention further relates to a human cortical spheroid, comprising central nervous system (CNS) cell types obtained or obtainable by the method of the present invention, as well as the use of the human cortical spheroid or a plurality of human cortical spheroids of the present invention as a disease model, in particular wherein a neuroinflammatory response is induced by administering at least one pro-inflammatory factor, to screen pharmacological compounds, biomolecules or cells for their effect on the formation of the human cortical spheroid, or on the formed human cortical spheroid.

Description

Title: A method for generating a human cortical spheroid from self- renewing stem cells capable of differentiation
Technical field
The present invention relates to a method for generating a human cortical spheroid from self-renewing stem cells capable of differentiation. The present invention further relates to a human cortical spheroid, comprising central nervous system (CNS) cell types obtained or obtainable by the method of the present invention, as well as the use of the human cortical spheroid or a plurality of human cortical spheroids of the present invention as a disease model, in particular wherein a neuroinflammatory response is induced by administering at least one pro-inflammatory factor, to screen pharmacological compounds, biomolecules or cells for their effect on the formation of the human cortical spheroid, or on the formed human cortical spheroid.
Background
Stem cell-derived three-dimensional (3D) human cortical spheroids are in vitro culture systems used to study brain development and (dys)functioning of the CNS. Human cortical spheroids display complex developmental processes, including coordinated cell proliferation, migration, and organization. After sufficient maturation during which cells self-organize into functional neuronal networks and distinct cortical layers, human cortical spheroids further display characteristics of the developing brain such as human cerebral cortex patterning, and developing electrophysiological activity. Such characteristics are inaccessible in traditional in vitro cultures. The complexity and cellular diversity of these human cortical spheroids has increased such that now the major neuroectoderm-derived cell types can be produced in 3D. Current human cortical spheroid protocols result in the generation and maturation of various cortical cell types in 3D, including neural progenitors, mature (but not adult) neuron subtypes, and astrocytes. Recently, protocols were developed to induce neurons, astrocytes and myelinating oligodendrocytes in human cortical spheroids derived from induced
Pluripotent Stem cells (iPSCs) or Embryonic Stem Cells (ESCs) (Madhavan et al, 2018, Nat Methods 15, 700 — 706, Pamies et al., 2017, ALTEX 34, 362 — 376, Matsui et al., 2018, Neurosci Lett 670, 75-82 and Shaker et al., 2021, Front Cell Neurosci 15, 631548). Current human cortical spheroids lack microglia, the innate immune cells of the CNS, and endothelial cells, key elements of the microvasculature that forms the blood-brain-barrier (BBB). Some protocols do describe the incorporation of cells from a microglial cell line (Immortalized Human Microglia-SV40) or microglial cells differentiated from iPSCs into iPSC-derived human cortical spheroids already containing neuroectoderm-derived neurons, astrocytes and oligodendrocytes (Abud et al. 2017, Neuron 94, 278-293 e279, Abreu et al., 2018 Front Microbiol 9, 2766, Bejoy et al, 2019, Stem Cells Int 2019, 2382534, Song et al., 2019 Sci Rep 9, 11055).
However, an innate development of microglia in human cortical spheroids is more complex as these cells are not derived from neuroectoderm, but from mesodermal epithelium of the embryonic yolk sac. Mature and inducible microglial cells have been innately grown in an iPSC-derived human cortical spheroid model containing neuronal and astrocyte populations, but in these human cortical spheroids the presence of oligodendrocyte populations and myelination was not reported (Ormel et al., 2018, Nat
Commun 9, 4167). Recent in silico analyses of single-cell transcriptomes of eight human cortical spheroids each produced with a different protocol showed that the highest degree of cellular diversity was generated with a non-guided protocol (Quadrato et al., 2017, Nature 545, 48-53). These human cortical spheroids contained neural progenitors, neuronal populations, astrocytes, oligodendrocytes, proteoglycan- expressing endothelial precursors and mesoderm progenitor cells, but lacked mature microglia and endothelial cells (Tanaka et al., 2020, Cell Rep 30, 1682-1689, e1683).
Assembly of 3D human cortical spheroids from various 2D iPSC-derived human cortical spheroid cultures did result in a model comprising all major CNS cell types, including endothelial cells, microglia and pericytes (Nzou et al., 2018, Sci Rep 8, 7413). However, a disadvantage of such an assembly method is that it does not allow for analysis of the stepwise innate development of the various cell types into a complex system. Accordingly, there is a demand for the generation of a guided iPSC- or ESC- derived human cortical spheroid model which contains innately grown neurons, astrocytes, oligodendrocytes, and innately grown microglia and endothelial cells. The aforementioned drawbacks, among others, are overcome by the invention as defined inthe appended claims.
Summary of the invention
The present invention relates to a method for generating a human cortical spheroid from self-renewing stem cells capable of differentiation, the method comprising the steps of: a. a neurocortical patterning step, comprising culturing the self-renewing cells in a culture medium containing a ROCK inhibitor, a BMP inhibitor and a TGF-B inhibitor to form a spheroid from the self-renewing cells; b. a neural and mesodermal progenitor differentiation step, comprising culturing the spheroid in a culture medium containing growth factors to form a spheroid comprising neural and mesodermal progenitor cells;
C. a neuronal and mesodermal differentiation step, comprising culturing the spheroid comprising neural and mesodermal progenitor cells in a culture medium containing growth factors to form a spheroid comprising neuronal and mesodermal cells; d. a glial differentiation step, comprising culturing the spheroid comprising neuronal and mesodermal cells in a culture medium containing growth factors to form a spheroid comprising neuronal, glial and mesodermal cells; and e. a maturation step, culturing the spheroid comprising neuronal, glial and mesodermal cells in a culture medium containing a pro-myelinating factor to induce unguided maturation of central nervous system oligodendrocytes to form a human cortical spheroid; wherein the concentration of the BMP inhibitor C(BMP) in the culture medium is chosen such that:
C(BMP) / IC50(BMP) is between 1 and 50; and wherein the concentration of the TGF-B inhibitor C{TGFb) in the culture medium is chosen such that
C(TGFb) / IC50(TGFb) is between 5 and 150; and wherein IC50(BMP) is the IC50 value of the BMP inhibitor and IC50(TGFb) is the IC50 value of the TGF-B inhibitor.
The invention further relates to a human cortical spheroid, comprising central nervous system cell types obtained or obtainable by the method of the present invention.
The invention furthermore relates to the use of the human cortical spheroid, or a plurality of human cortical spheroids, according to the present invention as a disease model, preferably a model for a neuroinflammatory disease such as Multiple Sclerosis,
a neurodevelopmental disorder or a neurodegenerative disease, wherein the disease model is induced by a method comprising administration of a compound, genetic manipulation, up- or downregulation of gene expression (e.g., using antisense technology), or transient regulation of gene expression.
Brief description of drawings
The present invention is described hereinafter with reference to the accompanying drawings in which embodiments of the present invention are shown.
Figure 1: Schematic overview of the human cortical spheroid culturing protocol, depicting crucial proteins and small molecules that induce neural, mesodermal, neuronal, microglial, endothelial, and glial differentiation.
Figure 2: Human cortical spheroid culturing protocol of Madhavan et al. 2018 (Nat Methods 15, 700 — 706) and of the present invention.
Figure 3: Generation of mature neuronal subtypes in 150-days-old (d150) human cortical spheroids.
Figure 4: Generation of astrocytes and oligodendrocyte populations that develop in close vicinity of neuronal cells in d150 human cortical spheroids.
Figure 5: Generation of innately developing microglia subpopulations within neuron-, astrocyte-, and oligodendrocyte-containing d150 human cortical spheroids.
Figure 6: Generation of innately developing endothelial lineage cell populations in neuron-, astrocyte-, oligodendrocyte- and microglia-containing d150 human cortical spheroids.
Figure 7: Robust presence of both neuroectoderm-derived and mesoderm-derived cell types in two independently grown d150 human cortical spheroid batches, with slightly different ratios in cell type and subpopulation composition.
Figure 8: Relative distributions of distinct cell types based on transcriptomic comparisons of d150 human cortical spheroids generated with the protocol of the present invention and 94-days-old (d94) human cortical spheroids generated with the T3-induction protocol of Madhavan et al., 2018 (Nat Methods 15, 700 — 706).
Figure 9: Cell-type compositions of d150 human cortical spheroids show a high similarity to those of human cortical fetal brain regions and in particular to those of the frontal cortex at gestational week (GW) 19-26.
Figure 10: Stimulation of d150 human cortical spheroids with a 5 cocktail of pro-inflammatory factors (bacterial lipopolysaccharides (LPS), Tumour
Necrosis Factor alpha (TNFa) and Interleukin-1 beta (IL-1B)) induces a neuroinflammatory response.
Detailed description of the invention
The inventors have now developed a novel method of generating stem cell-derived three-dimensional (3D) human cortical spheroids comprising innately grown, neuroectoderm-derived neurons, astrocytes. oligodendrocytes, and innately grown mesoderm-derived microglia and endothelial cells, where mild SMAD inhibition during early neurocortical patterning allows the development of mesoderm-derived cell types in neuroectoderm-directed human cortical spheroids. The inventors found that the reduced presence of one of the dual-SMAD inhibitors generates not only innately grown neurons, astrocytes and oligodendrocytes, but also various microglial subtypes and endothelial lineage cells in stem-cell derived human cortical spheroids.
Furthermore, the inventors found that 150-day old human cortical spheroids display a significant similarity with frontal and dorsal, rather than more inferior, regions of midgestational (weeks 19-26) human fetal brain. Without wishing to be bound by theory, this may be a consequence of the occurrence, in the human cortical spheroid, of the appropriate type, sufficient numbers, and appropriate ratios of excitatory and inhibitory neurons, oligodendrocytes, astrocytes, and endothelial cells as well as a higher number of excitatory and inhibitory neurons and astrocytes in the frontal regions compared to inferior surface regions. In addition to this, it was further found that applying a pro-myelinating factor results in the development of the human cortical spheroid into a mature oligodendrocyte differentiation stage.
A first aspect of the present invention provides herewith a method of generating a human cortical spheroid, wherein the method comprises the subsequent steps of:
a) a neurocortical patterning step, comprising culturing the self- renewing stem cells in a culture medium containing a ROCK inhibitor, a BMP inhibitor and a TGF-B inhibitor to form a spheroid from the self-renewing cells; b) a neural and mesodermal progenitor differentiation step, comprising culturing the spheroid in a culture medium containing growth factors to form a spheroid comprising neural and mesodermal progenitor cells; c) a neuronal and mesodermal differentiation step, comprising culturing the spheroid comprising neural and mesodermal progenitor cells in a culture medium containing growth factors to form a spheroid comprising neuronal and mesodermal cells; d) a glial differentiation step, comprising culturing the spheroid comprising neuronal and mesodermal cells in a culture medium containing growth factors to form a spheroid comprising neuronal, glial and mesodermal cells; and e) a maturation step comprising culturing the spheroid comprising neuronal, glial and mesodermal cells in a culture medium containing a pro-myelinating factor to induce unguided maturation of CNS cell types oligodendrocytes to form a human cortical spheroid; wherein the concentration of the BMP inhibitor C(BMP) in the culture medium is chosen such that:
C(BMP) /IC50(BMP) is between 1 and 50; and wherein the concentration of the TGF-B inhibitor C(TGFb) in the culture medium is chosen such that
C(TGFb) / IC50(TGFb) is between 5 and 150; and wherein IC50(BMP) is the IC50 value of the BMP inhibitor and IC5O(TGFb) is the IC50 value of the TGF-B inhibitor.
Neurocortical patterning in step a) is understood to mean the generation of a highly organised spheroid with a well-defined morphology recapitulating the organization of the developing human brain. More specifically, during neurocortical patterning neuroectodermal and mesodermal lineages are formed from self-renewing stem cells. Self-renewing stem cells may be any type of self- renewing stem cells suitable for performing the method of the present invention. By way of example, any cell line as listed by the National Institutes of Health (NIH) in the
NIH Human Embryonic Stem Cell Registry would be appropriate (hips: //grents.nih.gov/siem cellsregisty/curent. htm). In a preferred embodiment,
the self-renewing cells are selected from but not restricted to the group consisting of embryonic stem cells, induced pluripotent stem cells, human embryonic stem cell line
H1, human embryonic stem cell line H7, and human embryonic stem cell line H9.
Typically this step is performed from day -1, 0 or 1 to day 5, 6, 7, 8 or 9, such as for example from day O to day 7.
It will be appreciated that the self-renewing stem cells to be cultured in step a) are obtained by dissociating a cell suspension from intact self-renewing stem cell-colonies into single cells, which in turn are obtained by plating self-renewing stem cells in medium, growing, either feeder independent or first feeder dependent (on e.g. mouse embryonic fibroblasts), then feeder independent, on coated surfaces (such as
Matrigel or Laminin). Any differentiated cells are marked with a cell-culture marker and removed. The undifferentiated stem-cell phenotype may be confirmed by the high expression of NANOG and OCT4, and the low expression of PAX8 and NEFL over several passages. The cells obtained through this procedure are then seeded in a culture medium containing a ROCK inhibitor, a BMP inhibitor and a TGF-6 inhibitor to form a spheroid from the self-renewing cells. The number of cells to be seeded may range from 10.000 to 60.000 cells. The culture medium may be, but is not limited to
Spheroid Starter Medium (SSM: DMEM-F12 (Gibco; 31331-028)). The cells are seeded on a plate suitable for performing the method of the present invention, which may be a low-adherence V-bottom 96-wells plate (S-Bio; #MS-9096VZ).
The Rock inhibitor may be a Rock inhibitor suitable for performing the method of the present invention. In a preferred embodiment, the Rock inhibitor is selected from but not restricted to the group comprising Thiazovivin, Y-27632 dihydrochloride, the proprietary ROCK inhibitor of RevitaCell'™ Supplement, Y-39983 dihydrochloride, GSK429286, GSK269962A HCI, ARKI-1447, Azaindole 1, AR-13324,
ZINC00881524, KD025, K-115 hydrochloride dihydrate, Hydroxyfasudil, and AT 13148.
In an embodiment the BMP inhibitor is selected from but not restricted to the group consisting of Dorsomorphin (ALK2 inhibitor, IC50: 50 nM), LDN-214117 (ALK2 inhibitor, IC50: 24 nM), DMH1 (ALK2 inhibitor, IC50: 107.9 nM), LDN-193189 /
LDN-193189 2HCI (ALK2 inhibitor, 1C50: 5 nM) or ML347 (ALK2 inhibitor, IC50: 32 nM).
In an embodiment the BMP inhibitor is Dorsomorphin, and the cells are cultured in culture medium comprising 0.05 to 2.5 uM Dorsomorphin in step a).
In an embodiment the TGF-B inhibitor is selected from but not restricted to the group consisting of SB431542, RepSox, A83-01 or Galunisertib.
In an embodiment the TGF-B inhibitor is SB431542, and the cells are cultured in culture medium comprising 0.5 to 15 pM SB431542 in step a.
The culture medium in step a) may be refreshed daily for seven days.
When used herein the term BMP inhibitor preferably refers to a BMP4 inhibitor. The term inhibitor refers to a signaling pathway inhibitor, meaning that the inhibitor may directly inhibit BMP (e.g. BMP4) by binding to it and e.g. preventing it from binding to the receptor, inhibit the receptor, a co-receptor such as ALK2, ALK3 or ALK6, or downstream signaling of the activated receptor complex.
When used herein the term TGF-B inhibitor preferably refers to a TGF-
B signaling pathway inhibitor, meaning that the inhibitor may directly inhibit TGF-B by binding to it and e.g. preventing it from binding to the receptor, inhibit the receptor, a co-receptor such as ALK5, or downstream signaling of the activated receptor complex.
Neural and mesodermal progenitor differentiation in step b) is understood to mean the differentiation from neuroectodermal lineages from step a to neural progenitors and the differentiation from mesodermal lineages from step a to mesodermal progenitors. Neural progenitor cells (NPCs) give rise to the glial and neuronal cell types that populate the CNS. Mesodermal progenitor cells (MPCs) give rise to, among other cell types, microglia and endothelial cells. Typically this step is performed from day 5, 6, 7, 8 or 9 to day 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, such as for example from day 7 to day 25.
The growth factors may be growth factors suitable for performing step b. of the method of the present invention. In a preferred embodiment the growth factors are basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF), of which 10 to 50 ng/ml FGF2 and 4 to 20 ng/ml EGF may be added to the culture medium used to change half of the culture medium daily from day 7 to day 17 and every other day from day 17 to day 25 in step b). In case of daily medium changes, medium change may occur between 16 — 36 hours. In case of every other day medium changes, medium change may occur between 36 — 60 hours. Culture medium in step b) may be switched to Neurobasal-A spheroid medium (Thermo Fisher; 10888-022).
Neuronal and mesodermal differentiation in step c) is understood to mean the differentiation of neural and mesodermal progenitors from step b into neuronal cells, more specifically, excitatory and inhibitory neurons, and mesodermal cells, such as microglial cells and endothelial cells. Typically this step is performed from day 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 to day 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, such as for example from day 25 to day 51.
The growth factors may be growth factors suitable for performing step c. of the method of the present invention. In a preferred embodiment the growth factors are Neurotrphin-3 (NT-3) and Brain-derived neurotrophic factor (BDNF), of which 15 — 30 ng/ml NT-3 and 15 — 30 ng/ml BDNF may be added to the culture medium used to change the culture medium every other day from day 27 to until day 43 in step c.
Glial differentiation in step d) is understood to mean the differentiation of oligodendrocyte precursor cells to mature oligodendrocytes. Figures 1 and 2 contain representative images of the whole-spheroid signal of P2RY12 (a microglial cell marker) and scans of PECAM1+ (an endothelial lineage cell marker) of d150 human cortical spheroids according to the invention. Typically this step is performed from day 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 to day 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 78, 77, 78, 79, 80 or 81, such as for example from day 51 to day 71.
The growth factors may be growth factors suitable for performing step d. of the method of the present invention. In a preferred embodiment, the growth factors are Platelet-derived growth factor AA (PDGF-AA) and Human insulin-like growth factor 1 (hIGF1), of which 5 — 25 ng/ml PDGF-AA and 5 — 25 ng/ml hIGF1 may be added to the culture medium used to change half of the culture medium every other day between day 51 until day 59 in step d.
Maturation in step e) is understood to mean the differentiation and diversification of any cell type occurring in the human cortical spheroids in the maturation phase, such as excitatory and inhibitory neurons, mesodermal cells such as microglial cells, glial cells such as mature oligodendrocytes as well as the formation of synaptic networks. Typically the step is performed from day 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 77, 78, 79, 80 or 81 until the cortical spheroid is harvested, such as for example from day 71 to day 150. The cortical spheroid may be harvested for example between day 100 and day 200, preferably between day 110 and day 190 more preferably between day 120 and day 180, between day 130 and day 170, between day 140 and day 160, between day 142 and day 158, between day 144 and day 156 or between day 148 and day 152.
The pro-myelinating factor may be any pro-myelinating factor suitable for performing step e. of the method of the present invention. In a preferred embodiment, the pro-myelinating factor is selected from but not restricted to the group comprising Ketoconazole, Miconazole, and Clobetasol. In a preferred embodiment, the pro-myelinating factor is Ketoconazole, of which 1 — 10 uM is added to the culture medium used to change half of the culture medium every other day from day 61 until day 73 in step e.
It will be appreciated that microfluidic technology may be used for example for medium change, administration of cells or cell solutions, and administration of drugs, such as inhibitors, growth factors, and pro-myelinating factors.
A second aspect of the present invention provides herewith a human cortical spheroid, comprising CNS cell types obtained or obtainable by the method of the present invention.
A third aspect of the present invention provides herewith the use of the human cortical spheroid, or a plurality of human cortical spheroids according to the present invention as a disease model, preferably a model for a neuroinflammatory disease such as Multiple Sclerosis, a neurodevelopmental disorder or a neurodegenerative disease, such as Alzheimer’s disease and Parkinson's disease, wherein the disease model is induced by a method comprising administration of a compound, genetic manipulation, up- and downregulation of gene expression (e.g., using antisense technology), and transient regulation of gene expression. In a preferred embodiment the human cortical spheroid, or a plurality of human cortical spheroids, according to the present invention, is or are used as a model for neuroinflammatory disease, wherein a neuroinflammatory response is induced by a method comprising administration of at least one pro-inflammatory factor. Non-limiting examples of pro-inflammatory factors are bacterial lipopolysaccharides (LPS), Tumour
Necrosis Factor alpha (TNFa) and Interleukin-1 beta (IL-1B).
It is appreciated that the skilled person may select an appropriate genetic manipulation technique for direct manipulation of genes using biotechnology. Commonly used methods, known by the skilled person, include microinjection of DNA into the nucleus of cells, electroporation of cell membranes and polycationic neutralization of cell membranes. The skilled person may further select an appropriate technique for up- and downregulation of gene expression, allowing for selective manipulation of gene expression. Techniques known to the skilled person are for instance a) antisense oligonucleotides, synthetic DNA oligomers that hybridize to a target RNA in a sequence-specific manner, allowing for interference with several steps of RNA processing and message translation, degradation of RNA such as mRNA or pre-mRNA or non-coding RNAs, or targeting of aberrant splice junctions, b) RNA interference, in particular employing siRNA (small interfering RNA), a class of double-stranded RNA or non-coding RNA molecules, interfering with the expression of specific genes with complementary nucleotide sequences by degrading RNA such as mRNA after transcription, preventing translation, ¢) and designed transcription factors, typically modular proteins containing a DNA binding domain responsible for the specific recognition of base sequences and one or more effector domains that can activate or repress transcription. The skilled person may also select an appropriate technique for transient gene expression, that is the temporary expression of genes after introduction of a nucleic acid, most commonly through introduction of a DNA encading expression cassette in a viral or non-viral expression vector or select an appropriate compound for regulating expression.
An embodiment of the third aspect of the present invention relates to the use of the human cortical spheroid, or a plurality of human cortical spheroids, according to the present invention, to screen pharmacological compounds, biomolecules or cells for their effect on the human cortical spheroid formation, the use comprising: a. performing any one or several steps of the method of the present invention in the presence of pharmacological compounds, biomolecules or cells to be tested; b. monitoring the effect of the pharmacological compounds, biomolecules or cells an human cortical spheroid formation; c. monitoring the toxic effect of the pharmacological compounds, biomolecules or cells on human cortical spheroid formation; d. monitoring the intended clinical effect, side effect and adverse effect of potential drugs on human cortical spheroid formation.
It will be appreciated that monitoring the effect of the pharmacological compounds, biomolecules or cells, the toxic effect and/or the intended clinical effect of said compounds, biomolecules or cells, may be carried out using any suitable method known in the art. For example, any of cell survival, growth proliferation, differentiation, migration, morphology, electrophysiology, cell-cell communication, multicellular organization, and human cortical spheroid architecture may be assessed by microscopy and image analysis, genomic, transcriptomic, proteomic and metabolomic analysis, and recording and analysis of spontaneous and/or stimulated electrical activity via intracellular or extracellular methods. For example, level of presence of certain cell types can be assessed by determination of mRNA-expression levels of markers specific to said cell types, as described, for example, in Examples 4 — 9 and 11. Presence of certain cell types can also be assessed, for example, by quantification of immunocytochemistry data, as described, for example, in Examples 4 — 7 and 10. It is appreciated that the skilled person may select the appropriate technique to assess a given property.
It will be appreciated that use of the human cortical spheroid, or a plurality of human cortical spheroids according to the present invention allows the assessment of the effect of pharmacological compounds, biomolecules or cells on how a particular cell type affects the function of another cell type.
It will be appreciated that the use of the human cortical spheroid, or a plurality of human cortical spheroids according to the present invention allows the assessment of the function of a cellular gene or protein, for example by using a pharmaceutical compound that is an inhibitor of said gene or protein. For example, the pharmaceutical compound may be any of a chemical inhibitor, a peptide inhibitor, an siRNA molecule or an shRNA construct or any pharmaceutical compound capable of effecting a gene knockdown. It may also be desirable to use more than one inhibitor (e.g. with different selectivities) to provide further insight into the function of a cellular gene or protein. Similarly, by exposing multiple multicellular spheroids to a range of respective agents, the use can be scaled up to investigate the cellular functions, e.g. using genome or proteome arrays on a larger scale.
The pharmacological compounds, biomolecules or cells may be applied to the human cortical spheroid after formation, or may be present in the medium in which the human cortical spheroid is formed, or it may be administered to the culture medium along with the self-renewing stem cells.
Another embodiment of the third aspect of the present invention relates to the use of the human cortical spheroid, or a plurality of human cortical spheroids, according to the present invention, to screen pharmacological compounds, biomolecules or cells for their effect on the human cortical spheroid, the use comprising: e. administration of pharmacological compounds, biomolecules or cells to be tested to the human cortical spheroid obtained by the method according to the invention; f. monitoring the effect of the pharmacological compounds, biomolecules or cells on the human cortical spheroid; g. monitoring the toxic effect of the pharmacological compounds, biomolecules or cells on the human cortical spheroid; h. monitoring the intended clinical effect, side effect and adverse effect of potential drugs on the human cortical spheroid.
As well as being useful in screening pharmacological compounds, the third aspect of the present invention may also be useful in personalized medicine regimes. For example, using the human cortical spheroid, or a plurality of human cortical spheroids, according to the present invention, which may be generated from iPSCs derived from a person, such as the patient to be treated. Furthermore, pharmacological compounds may be screened for the efficacy of potential drug treatments, and so may aid the customization of treatments of individual patients.
It will be appreciated that the third aspect of the present invention may also be useful in monitoring disease progress wherein human cortical spheroids of increasing age represent the progressive stages of the disease.
Examples
Example 1 3D human cortical spheroid formation
In order to allow for mesoderm development alongside neuroectoderm development in the human cortical spheroids of the present invention, intact H9 ESC-colonies were detached with ReLESR (STEMcell technologies; 05872). A small volume of cell suspension was dissociated to single cells and counted. Low-adherence V-bottom 96-
wells (S-Bio; #MS-9096VZ) were seeded with 1.25*10% cells per well in 190 ul Spheroid
Starter Medium (SSM: DMEM-F12 (Gibco; 31331-028) with 20% knock-out serum (Gibco; 10828-028), 1% non-essential amino acids (Gibco; 11140050), 1% pen-strep and 0.1% 2-mercaptoethanol (Thermo Fisher; 31350-010)) supplemented with 10 uM $SB-431542 (Sigma-Aldrich; S4317), 1 HM dorsomorphin (Sigma-Aldrich; P5499) and 2 uM ROCK inhibitor thiazovivin (Sigma-Aldrich; SML1045). SSM (190 ul) supplemented with 10 uM SB-431542 and 1 uM dorsomorphin, and was refreshed daily for seven days. Subsequently, medium was switched to Neurobasal-A spheroid medium (Thermo fisher; 10888-022) with 2% B27 supplement minus vitamin A (Gibco; 12587-010), 1% GlutaMAX (Gibco; 35050-061) and 1% pen-strep (NSMnogel).
NSMnogel supplemented with 25 pg/ml FGF2 (Sigma-Aldrich; F0291) and 10 pg/ml
EGF (Gibco; PHG0311L) was refreshed daily until day 17 and half of the medium refreshed every other day from day 17 until day 25. Then, spheroids were transferred to low-attachment 60 cm dishes (~24 spheroids in 6 ml per dish) with a cut-open P1000 tip in NSM (NSMnogel containing 15 pg/ml Geltrex (Thermo Fisher; A1413302)) supplemented with 25 pg/ml FGF2 and 10 pg/ml EGF. Half of the medium was refreshed with NSM medium supplemented with 20 ng/ml NT-3 (Sigma-Aldrich;
SRP3128) and 20 ng/ml BDNF (Sigma-Aldrich; SRP3014) every other day from day 27 until day 43. Between days 43 and 51 half of the NSM was refreshed every other day with NSM (no supplements). Half of the medium was then refreshed with NSM supplemented with 10 ng/ml PDGF-AA (Sigma-Aldrich; H8291) and 10 ng/ml IGF1 (R&D systems; 291-G1-200) every other day from day 51 until day 59. Subsequently, half of the medium was refreshed with NSM medium containing ketoconazole (4 uM;
Sigma-Aldrich; K1003) every other day from day 61 until 73. Finally, from day 73 onwards, half of the medium was refreshed with NSM medium (no supplements) every other day until the spheroids were harvested. During the entire growth period and all experiments, human cortical spheroids were kept at 37 °C and 5% CO..
Example 2
Schematic overview of the human cortical spheroid culturing protocol, depicting crucial proteins and small molecules that induce neural, mesodermal, neuronal, microglial, endothelial, and glial differentiation (figure 1).
Example 3
Human cortical spheroid culturing protocol of Madhavan et al., 2018 (Nat Methods 15, 700 — 706) and of the present invention. The protocol of the present invention uses reduced concentration of one of the dual-SMAD inhibitors (Dorsomorphin), reduced concentration of ROCK-inhibitor (Thiazovivin) and every-other day half-media changes instead of daily half-media changes during days 15 — 25 compared to Madhavan et al., 2018 (figure 2).
Example 4
Generation of mature neuronal subtypes in d150 human cortical spheroids. a)
Representative ICC images of cells positive for MAP2 or the neuronal microtubule- associated protein marker TAU in d33 and d150 human cortical spheroids. b) Levels of normalized mRNA expression of the mature neuronal markers NEFL and MAP2 in
HO and d150 human cortical spheroids as determined by qPCR analysis. c) Levels of normalized mRNA expression of the synapse-associated transcripts SYP and SNAP25 in H9 and d150 human cortical spheroids. d) Representative ultrastructural transmission electron microscopy (TEM) image of a synapse (S) in d150 human cortical spheroids . e) Levels of normalized mRNA expression of the excitatory neuron markers SLC17A7, GRIN1 and NEURODB8, and the inhibitory neuron markers GAD1,
NPY and DLX1,2 in H9 and d150 human cortical spheroids . f) Representative ICC images of cells positive for the excitatory neuronal marker vGLUT1 and the inhibitory neuron markers GAD1, GAD2 and GAT1 in early (d27/d33) and late (d150) human cortical spheroids DAPI was used to visualize nuclei. *** p < 0.001, ** p < 0.01, * p < 0.05 and # p < 0.1. Scale bar light microscopy images: 75 um (figure 3).
Example 5
Generation of astrocytes and oligodendrocyte populations that develop in close vicinity of neuronal cells in d150 human cortical spheroids. a) Levels of normalized mRNA expression of the astrocyte-related transcripts VIM, GFAP and AQP4 in HS and d150 human cortical spheroids. b) Representative images of GFAP-positive cells in d33- and d150 human cortical spheroids. c) Ultrastructural transmission electron microscopy (TEM) image of an astrocyte cell (A) in d150 human cortical spheroid. d)
Double immunocytochemistry for neuronal marker MAP2 and GFAP does not show overlapping positive cells for these two cell-type markers. e) Levels of normalized
MRNA expression of oligodendrocyte precursor (OPC) marker NDRG1 and mature oligodendrocyte markers MBP and MAL in H9 and d150 human cortical spheroids. f)
Representative images of cells positive for oligodendrocyte marker CC1- and OPC- transforming marker O1 in d33- and d150 human cortical spheroids. g) Double immunocytochemistry for MAP2 and O1 does not show overlapping positive cells for these two cell-type markers. h) Representative images of MBP- and PLP1-positive cells in d33- and d150 human cortical spheroids. i) Ultrastructural TEM images of OPC and mature oligodendrocyte (mOL) in d150 human cortical spheroids. *** p < 0.001, ** p < 0.01 and * p < 0.05. Scale bar light microscopy images = 75 um (figure 4).
Example 6
Generation of innately developing microglia subpopulations within neuron-, astrocyte — and oligodendrocyte-containing d150 human cortical spheroids. a) Levels of normalized mRNA expression of (resting-state) MO microglia markers AIF1, CX3CR1,
TGF-B2, TMEM119 and PTPRC in H9 and d150 human cortical spheroids. b)
Representative images of TMEM119-, P2RY12-, AIF1- and CD68-positive cells in d33 human cortical spheroids and d150 human cortical spheroids. ¢) Levels of normalized
MRNA expression of M1/M2 microglia marker HLA-DRA, M1 microglia marker CD14 and M2 phagocytosis-related markers ITGAM, TREM2 and ARG1 in H9 and d150 human cortical spheroids. d) Double immunohistochemistry for TMEM119 and
P2RY12 shows both TMEM119-positive/P2RY 12-positive microglia and TMEM112- positive/P2RY 12-negative cells. e) Ultrastructural transmission electron microscopy image of a microglia cell (M) in d150 human cortical spheroid. f) Quantification of immunocytochemistry data for P2RY12 in five independently cultured human cortical spheroid batches. *** p <0.001, ** p < 0.01, * p < 0.05 and # p < 0.1. Scale bar light microscopy images=75 um (figure 5).
Example 7
Generation of innately developing endothelial lineage cell populations in neuron-, astrocyte-, oligodendrocyte- and microglia-containing d150 human cortical spheroids. a) Levels of normalized mRNA expression of endothelial lineage transcripts SPARC,
COL4A2, VWF and CD143 in H9 and d150 human cortical spheroids. b)
Representative images of PECAM1-positive cells in d17 and d150 human cortical spheroids. Scale bar: 75 um. ¢) Ultrastructural transmission electron microscopy images of endothelial-like cells in d150 human cortical spheroids. * p < 0.05 (figure 8).
Example 8
Robust presence of both neuroectoderm-derived and mesoderm-derived cell types in two independently grown d150 human cortical spheroid batches, with slightly different ratios in cell type and subpopulation composition. a) Log10-converted fold change of median GAPDH-normalized cell-type-specific marker expression in batch-1 relative to batch-2 d150 human cortical spheroids (see table 1 for markers used). The bar represents the median of the expression of all cell type markers. b) Log10-converted fold change of median GAPDH-normalized cell-type-specific marker expression in batch-1 relative to batch-2 d150 human cortical spheroids, data per individual marker (see table 1 for markers used). SC = stem cell; NPC = neural progenitor; EXC = excitatory neuron; INH = inhibitory neuron; ASTR = astrocyte; OPC = oligodendrocyte precursor cell, pOL = pre-myelinating oligodendrocyte; MG = microglia, ENDO = endothelial cell (figure 7).
Table 1. Cell-type-specific mRNA expression of brain cell-type markers in the single- cell RNA-sequencing dataset from human fetal brain (Fan et al, 2018, Cell
Res 28, 730-745). These markers were selected on the basis of extensive literature searches and showed cell-specific mRNA expression (with a median fold change, FC, larger than 3.5) in neural progenitor cells (NPCs), excitatory neurons, inhibitory neurons, astrocytes, endothelial cells, oligodendrocyte precursor cells (OPCs), (pre- myelinating) oligodendrocytes ((p)OLs) and microglia.
Cell type Marker Median FC | Low discriminative value expression over | for (FC < 2.5) other CNS cell types
AE
Mes reeme
So
SLC17A7 [6.639055 - emer oer
DLX2 | 36.75871 -
ERE [we
EErmEe me meme]
Aar mes
SLCO1CI [20.2895 -
Endothelial cell
ADRESS]
Rae [me com ees 10.70552 -
Fo [aen a
COON mes 6.122841 Astrocyte ee
RE mee —
ew TEmes a
Microglia 41.4727 -
FTC jews
FRE wwe
Pe [wmw ef ic ae aas 48.38399 -
Example 9
Relative distributions of distinct cell types based on transcriptomic comparisons of d150 human cortical spheroids generated with the protocol of the present invention and d94 human cortical spheroids generated with the T3-induction protocol of
Madhavan et al., 2018 (Nat Methods 15, 700 — 706). a) Log10-converted fold-change
MRNA expression of cell-type-specific markers normalized by GAPDH in batch-1 d150 human cortical spheroids (present invention) relative to that in d94 human cortical spheroids (Madhavan et al., 2018, Nat Methods 15, 700 — 706). b) Log10-converted fold-change mRNA expression of cell-type-specific markers normalized by GAPDH in batch-2 d150 human cortical spheroids (present invention) relative to that in d94 human cortical spheroids (Madhavan et al., 2018, Nat Methods 15, 700 — 706) (figure 8).
Example 10
Cell-type compositions of d150 human cortical spheroids of the present invention show a high similarity to those of human cortical fetal brain regions and in particular to those of the frontal cortex at gestational week (GW) 19-26. a) A priori determined cell-type composition of human cortical spheroids of the present invention at day 150 in culture using immunocytochemistry (ICC) signals for the markers PAX6 (NPC), vGLUT1 (EXC), GAD1 (INH), GFAP (ASTR), O1 (OPC), CC1 (OPC), MBP (pOL),
TMEM119 (MG) and PECAM1 (ENDO) in multiple independently grown human cortical spheroid batches. b) Distance scores of estimated cell-type compositions to ICC established cell-type compositions per cell type, per gestational week. c) Distance scores of estimated cell-type compositions to ICC established cell-type compositions per cell type, per fetal brain region. NPC = neural progenitor cell; EXC = excitatory neuron; INH = inhibitory neuron; ASTR = astrocyte; OPC = oligodendrocyte precursor cell; pOL = pre-myelinating oligodendrocyte; MG = microglia; ENDO = endothelial cell;
FC = frontal cortex; PC = parietal cortex; TC = temporal cortex; OC = occipital cortex,
IS = inferior surface (figure 9).
Example 11
Stimulation of d150 human cortical spheroids with a cocktail of pro-inflammatory factors (LPS, TNFa and IL-1B) induces a neuroinflammatory response. a) Levels of normalized mRNA expression (as determined by qPCR) of microglial, astrocyte, neuron, oligodendrocyte and endothelial cell markers in unstimulated human cortical spheroids (U) and human cortical spheroids stimulated with a cocktail of LPS 100 ng/ml (24 hours) followed by TNFa and IL1B (5 ng/ml each, 24 hours) (S). b) Levels of normalized mRNA expression of the neuroinflammatory cytokines IL-6, CXCLS,
CCL20, CCL5 in unstimulated human cortical spheroids (U) and stimulated human cortical spheroids (S) . *** p < 0.001, ** p < 0.01, * p < 0.05 (figure 10).

Claims (15)

CONCLUSIESCONCLUSIONS 1. Werkwijze voor het genereren van een menselijke corticale sferoide uit zelfvernieuwende stamcellen die tot differentiatie in staat zijn, waarbij de werkwijze de stappen omvat van:A method of generating a human cortical spheroid from self-renewing stem cells capable of differentiation, the method comprising the steps of: A. een stap van neurocorticale patroonvorming, omvattende het kweken van de zelfvernieuwende cellen in een kweekmedium dat een ROCK-remmer, een BMP- remmer en een TGF-B-remmer bevat om een sferoide te vormen van de zelfvernieuwende cellen;A. a neurocortical patterning step comprising culturing the self-renewing cells in a culture medium containing a ROCK inhibitor, a BMP inhibitor, and a TGF-β inhibitor to form a spheroid of the self-renewing cells; B. een differentiatiestap van neurale en mesodermale voorlopercellen, omvattende het kweken van de sferoide in een kweekmedium dat groeifactoren bevat om een sferoide te vormen die neurale en mesodermale voorlopercellen omvat;B. a differentiation step of neural and mesodermal progenitor cells comprising culturing the spheroid in a culture medium containing growth factors to form a spheroid comprising neural and mesodermal progenitor cells; C. een neuronale en mesodermale differentiatiestap, omvattende het kweken van de steroïde die neurale en mesodermale voorlopercellen omvat in een kweekmedium dat groeifactoren bevat om een sferoïde te vormen die neuronale en mesodermale cellen omvat;C. a neuronal and mesodermal differentiation step comprising culturing the steroid comprising neural and mesodermal progenitor cells in a culture medium containing growth factors to form a spheroid comprising neuronal and mesodermal cells; D. een gliale differentiatiestap, omvattende het kweken van de sferoide die neuronale en mesodermale cellen omvat in een kweekmedium dat groeifactoren bevat om een steroïde te vormen die neuronale, gliale en mesodermale cellen omvat; enD. a glial differentiation step comprising culturing the spheroid comprising neuronal and mesodermal cells in a culture medium containing growth factors to form a steroid comprising neuronal, glial and mesodermal cells; and E. een rijpingsstap omvattende het kweken van de sferoide die neuronale, gliale en mesodermale cellen omvat in een kweekmedium dat een pro-myeliniserende factor bevat om ongeleide rijping van oligodendrocyten van het centrale zenuwstelsel te induceren om een menselijke corticale sferoïde te vormen; waarbij de concentratie van de BMP-remmer C(BMP) in het kweekmedium zodanig wordt gekozen dat: C(BMP) / IC5O(BMP) ligt tussen 1 en 50; en waarbij de concentratie van de TGF-B-remmer C{TGFb) in het kweekmedium zodanig wordt gekozen dat C(TGFb)/IC50(TGFDb) ligt tussen 5 en 150; en waarbij IC50(BMP) de IC50-waarde van de BMP-remmer is en IC50(TGFb) de IC50- waarde van de TGF-B-remmer is.E. a maturation step comprising culturing the spheroid comprising neuronal, glial and mesodermal cells in a culture medium containing a pro-myelinating factor to induce unguided maturation of central nervous system oligodendrocytes to form a human cortical spheroid; wherein the concentration of the BMP inhibitor C(BMP) in the culture medium is selected such that: C(BMP) / IC50(BMP) is between 1 and 50; and wherein the concentration of the TGF-β inhibitor C(TGFb) in the culture medium is selected such that C(TGFb)/IC50(TGFDb) is between 5 and 150; and wherein IC50(BMP) is the IC50 value of the BMP inhibitor and IC50(TGFb) is the IC50 value of the TGF-B inhibitor. 2. Werkwijze volgens conclusie 1, waarbij de ROCK-remmer in stap A. is geselecteerd uit de groep bestaande uit Thiazovivine, Y-27832 dihydrochloride, de gepatenteerde ROCK-remmer van RevitaCelltm Supplement, Y-39983 dihydrochloride, GSK429288, GSK269962A HCI, ARKI-1447, Azaindole 1, AR-13324, ZINC00881524, KD025, K-115 hydrochloridedihydraat, Hydroxyfasudil en AT13148.The method of claim 1, wherein the ROCK inhibitor in step A is selected from the group consisting of Thiazovivine, Y-27832 dihydrochloride, RevitaCell™ Supplement's proprietary ROCK inhibitor, Y-39983 dihydrochloride, GSK429288, GSK269962A HCI, ARKI -1447, Azaindole 1, AR-13324, ZINC00881524, KD025, K-115 hydrochloride dihydrate, Hydroxyfasudil and AT13148. 3. Werkwijze volgens een van de voorgaande conclusies, waarbij de ROCK- remmer Thiazovivine is en de cellen tijdens stap A. worden gekweekt in een kweekmedium dat 0,2 — 5 uM Thiazovivine bevat.A method according to any one of the preceding claims, wherein the ROCK inhibitor is Thiazovivine and the cells are cultured during step A. in a culture medium containing 0.2-5 µM Thiazovivine. 4. Werkwijze volgens een van de voorgaande conclusies, waarbij de groeifactoren in stap B. basale fibroblastgroeifactor (FGF2) en epidermale groeifactor (EGF) zijn waarbij de helft van het kweekmedium dagelijks wordt ververst van dag 7 tot dag 17 en om de andere dag wordt ververst van dag 17 tot dag 25 met kweekmedium dat 10 tot 50 ng/ ml FGF2 en 4 tot 20 ng/ml EGF omvat.The method according to any of the preceding claims, wherein the growth factors in step B are basal fibroblast growth factor (FGF2) and epidermal growth factor (EGF) with half of the culture medium being changed daily from day 7 to day 17 and every other day. changed from day 17 to day 25 with culture medium comprising 10 to 50 ng/ml FGF2 and 4 to 20 ng/ml EGF. 5. Werkwijze volgens een van de voorgaande conclusies, waarbij de groeifactoren in stap C. Neurotrphin-3 (NT-3) en Brain-derived neurotrophic factor (BDNF) zijn, waarbij de helft van het kweekmedium om de dag wordt ververst met kweekmedium dat 15 — 30 ng/ml NT-3 en 15 — 30 ng/ml BDNF omvat.The method according to any of the preceding claims, wherein the growth factors in step C are Neurotrophin-3 (NT-3) and Brain-derived neurotrophic factor (BDNF), with half of the culture medium being refreshed every other day with culture medium containing 15 — 30 ng/ml NT-3 and 15 — 30 ng/ml BDNF included. 6. Werkwijze volgens een van de voorgaande conclusies, waarbij de groeifactoren in stap D. van bloedplaatjes afgeleide groeifactor AA (PDGF-AA) en humaan insuline- achtige groeifactor 1 (hIGF1) zijn, waarbij de helft van het kweekmedium om de dag wordt ververst met kweekmedium dat 5 — 25 ng/ml PDGF-AA en 5 — 25 ng/ml hIGF1 omvat.The method according to any of the preceding claims, wherein the growth factors in step D. are platelet-derived growth factor AA (PDGF-AA) and human insulin-like growth factor 1 (hIGF1), with half of the culture medium being refreshed every other day with culture medium comprising 5-25 ng/ml PDGF-AA and 5-25 ng/ml hIGF1. 7. Werkwijze volgens een van de voorgaande conclusies, waarbij de zelfvernieuwende cellen worden gekozen uit de groep omvattende embryonale stamcellen, geïnduceerde pluripotente stamcellen, menselijke embryonale stamcellijn H1, menselijke embryonale stamcellijn H7, menselijke embryonale stam cellijn HO.A method according to any one of the preceding claims, wherein the self-renewing cells are selected from the group comprising embryonic stem cells, induced pluripotent stem cells, human embryonic stem cell line H1, human embryonic stem cell line H7, human embryonic stem cell line HO. 8. Werkwijze volgens een van de voorgaande conclusies, waarbij de pro- myeliniserende factor wordt gekozen uit de groep bestaande uit ketoconazol, miconazol en clobetasol.A method according to any one of the preceding claims, wherein the promyelinating factor is selected from the group consisting of ketoconazole, miconazole and clobetasol. 9. Werkwijze volgens een van de voorgaande conclusies, waarbij de pro- myeliniserende factor ketoconazol is, waarbij in stap E. de helft van het kweekmedium om de dag wordt ververst met kweekmedium dat 1-10 uM ketoconazol bevat.A method according to any one of the preceding claims, wherein the promyelinating factor is ketoconazole, wherein in step E. half of the culture medium is refreshed every other day with culture medium containing 1-10 µM ketoconazole. 10. Menselijke corticale sferoïde, omvattende celtypen van het centrale zenuwstelsel verkregen of verkrijgbaar met de werkwijze volgens conclusies 1-9.A human cortical spheroid comprising central nervous system cell types obtained or obtainable by the method of claims 1-9. 11. Gebruik van een humane corticale sferoid, of meerdere humane corticale sferoiden, volgens conclusie 10 als ziektemodel, bij voorkeur een model voor een neuro-inflammatoire ziekte zoals multiple sclerose, een neurologische ontwikkelingsstoornis of een neurodegeneratieve ziekte, waarbij het ziektemodel wordt geïnduceerd door een werkwijze die toediening van ten minste één verbinding, genetische manipulatie, op- en neerwaartse regulatie van genexpressie en/of tijdelijke regulatie van genexpressie omvat.Use of a human cortical spheroid, or multiple human cortical spheroids, according to claim 10 as a disease model, preferably a model for a neuroinflammatory disease such as multiple sclerosis, a neurodevelopmental disorder or a neurodegenerative disease, wherein the disease model is induced by a method comprising administration of at least one compound, genetic engineering, up- and down-regulation of gene expression, and/or temporal regulation of gene expression. 12. Gebruik van een humane corticale sferoïd, of meerdere humane corticale steroïden, volgens conclusie 11, waarbij het gebruik omvat het induceren van een neuro-inflammatoire respons door toediening van ten minste één pro-inflammatoire factor.The use of a human cortical spheroid, or multiple human cortical steroids, according to claim 11, wherein the use comprises inducing a neuroinflammatory response by administering at least one proinflammatory factor. 13. Gebruik van de humane corticale sferoïden, of meerdere humane corticale steroïden, volgens conclusie 11, waarbij de neurodegeneratieve ziekte wordt gekozen uit de groep die de ziekte van Alzheimer en de ziekte van Parkinson omvat.Use of the human cortical spheroids, or multiple human cortical steroids, according to claim 11, wherein the neurodegenerative disease is selected from the group comprising Alzheimer's disease and Parkinson's disease. 14. Gebruik van de menselijke corticale sferoïd, of meerdere menselijke corticale sferoiden, volgens een van de conclusies 11-13, voor het screenen van farmacologische verbindingen, biomoleculen of cellen op hun effect op de vorming van de menselijke corticale sferoïden, waarbij het gebruik omvat:Use of the human cortical spheroid, or multiple human cortical spheroids, according to any one of claims 11-13, for screening pharmacological compounds, biomolecules or cells for their effect on the formation of the human cortical spheroids, the use comprising : A. het uitvoeren van één of meer stappen van conclusie 1 in aanwezigheid van farmacologische verbindingen, biomoleculen of te testen cellen;A. performing one or more steps of claim 1 in the presence of pharmacological compounds, biomolecules or cells to be tested; B. het volgen van het effect van de farmacologische verbindingen, biomoleculen of cellen op de vorming van menselijke corticale sferoiden;B. monitoring the effect of the pharmacological compounds, biomolecules or cells on the formation of human cortical spheroids; C. het monitoren van het toxische effect van de farmacologische verbindingen, biomoleculen of cellen op de vorming van menselijke corticale sferoïden;C. monitoring the toxic effect of the pharmacological compounds, biomolecules or cells on the formation of human cortical spheroids; D. het monitoren van het beoogde klinische effect, de bijwerking en het nadelige effect van potentiële geneesmiddelen op de vorming van menselijke corticale sferoïden.D. monitoring the intended clinical effect, side effect and adverse effect of potential drugs on human cortical spheroid formation. 15. Gebruik van de menselijke corticale sferoïd of meerdere menselijke corticale steroïden, volgens een van de conclusies 11-14, om farmacologische verbindingen, biomoleculen of cellen te screenen op hun effect op de menselijke corticale sferoïden, waarbij het gebruik omvat:Use of the human cortical spheroid or multiple human cortical steroids, according to any one of claims 11-14, to screen pharmacological compounds, biomolecules or cells for their effect on the human cortical spheroids, the use comprising: A. het toedienen van farmacologische verbindingen, biomoleculen of te testen cellen aan de menselijke corticale sferoïde verkregen door de werkwijze volgens conclusies 1-9;A. administering pharmacological compounds, biomolecules or cells to be tested to the human cortical spheroid obtained by the method of claims 1-9; B. het volgen van het effect van de farmacologische verbindingen, biomoleculen of cellen op de menselijke corticale sferoide;B. monitoring the effect of the pharmacological compounds, biomolecules or cells on the human cortical spheroid; C. het monitoring van het toxische effect van de farmacologische verbindingen, biomoleculen of cellen op de menselijke corticale sferoide;C. monitoring the toxic effect of the pharmacological compounds, biomolecules or cells on the human cortical spheroid; D. het monitoren van het beoogde klinische effect, de bijwerking en het nadelige effect van potentiële geneesmiddelen op de menselijke corticale sferoide.D. monitoring the intended clinical effect, side effect and adverse effect of potential drugs on the human cortical spheroid.
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