Means and methods for generating midbrain organoids

The present invention provides means and methods for the generation of midbrain organoids which are useful for studying neurodevelopmental and neurodegenerative diseases. Neuroepithelial stem cells serve as a starting population for the generation of midbrain organoids by contacting them with differentiation medium under agitating conditions in three-dimensional cell culture comprising a matrix.

1. A method of generating a midbrain organoid, comprising the steps of

(i) culturing neuroepithelial stem cells for a first period of time with differentiation medium (I) comprising

(a) at least two different neurotrophins, and

(b) an antioxidant, and

(c) a SHH-pathway activator,

(ii) culturing neuroepithelial stem cells obtained from step (i) for a second period of time with differentiation medium (II) comprising

(a) said at least two different neurotrophins, and

(b) said antioxidant, and

(iii) culturing neuroepithelial stem cells obtained from step (ii) for a third period of time

with differentiation medium (II) which is further supplemented with FGF8, wherein in steps (i), (ii) and (iii) the neuroepithelial stem cells are cultured in a three-dimensional cell culture comprising a matrix, wherein at least a part of the-culturing in steps (i)-(iii) is performed under agitating conditions, thereby obtaining a midbrain organoid, wherein said midbrain organoid comprises one or more of astrocytes, oligodendrocytes, and oligodendrocyte progenitors, wherein said midbrain organoid comprises an asymmetric polar organization of dopaminergic neurons, and wherein said asymmetric polar organization of dopaminergic neurons within the midbrain organoid is characterized by the localization of

(a) dopaminergic neurons expressing TUJ1 and TH in the outermost part of the midbrain organoid, and/or

(b) dopaminergic neurons expressing MAP2 and TH in the inner part of the midbrain organoid.

2. The method of claim 1, wherein said neuroepithelial stem cells are human neuroepithelial stem cells.

3. The method of claim 1, wherein said neuroepithelial stem cells are genetically modified or obtained from a patient suffering from a neurological disease.

4. The method of claim 1, wherein said neuroepithelial stem cells have been produced from induced pluripotent stem cells (iPSCs).

5. The method of claim 1, wherein the matrix comprises one or more of polymers, metals, ceramics and/or carbon nanotubes.

6. The method of claim 1, wherein FGF8 is added to the differentiation medium (II) after 8 days of culturing in the differentiation medium (II).

7. The method of claim 1, wherein the neuroepithelial stem cells are cultured in a maintenance medium before they are cultured in step (i) with differentiation medium (I), wherein the maintenance medium comprises (i) a SHE-pathway activator; (ii) a canonical WNT-signaling activator; and (iii) an antioxidant.

8. The method of claim 1, wherein the neuroepithelial stem cells are present in a colony.

9. The method of claim 1, wherein said agitating conditions comprise shaking, spinning, stirring, moving and/or mixing of the three-dimensional cell culture.

10. The method of claim 1,

wherein agitating is started after starting culturing of the neuroepithelial stem cells in step (i) and continued in steps (ii) and (iii).

11. The method of claim 1, wherein the midbrain organoid is obtained after 1 or more weeks of differentiation.

12. The method of claim 1, wherein said midbrain organoid is an early midbrain organoid or a late midbrain organoid.

13. The method of claim 1, wherein said midbrain organoid further comprises one or more of:

(a) neural progenitor cells;

(b) young neurons;

(c) young dopaminergic neurons;

(d) mature neurons;

(e) mature dopaminergic neurons;

and (f) processes that expand from the midbrain organoid through the matrix.

14. The method of claim 1, wherein the at least two different neurotrophins in the differentiation media are selected from the group consisting of NGF, BDNF, NT-3, NT-4, CNTF and GDNF.

The midbrain or mesencephalon is a portion of the central nervous system associated with vision, hearing, motor control, sleep/wake, arousal (alertness), and temperature regulation. During embryonic development, the midbrain arises from the second vesicle, also known as the mesencephalon, of the neural tube. Unlike the other two vesicles, the forebrain and hindbrain, the midbrain remains undivided for the remainder of neural development.

The midbrain is also that region of the brain, where the majority of the neurotransmitter dopamine (DA) is produced. Dopamine plays, inter alia, a major role in motivation and habituation of species from humans to the most elementary animals such as insects. The regions of DA producing neurons are derived from the tegmentum and are called the substantia nigra pars compacta (SNc, A9 group), and the ventral tegmental area (VTA, A10 group). Especially the mesencephalic dopaminergic (mDA) neurons of the SNc play an important role in the control of multiple brain functions. Their axons ascend rostrally into the dorsolateral striatum of the cortex, where they release the neurotransmitter dopamine (Abeliovich and Hammond, 2007; Gale and Li, 2008). The SNc is of particular interest since mDA neurons of this region selectively undergo degeneration in Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra The loss of mDA neurons leads to a lack of DA in the striatum, which controls voluntary body movements under physiological conditions (Abeliovich and Hammond, 2007). In PD, the degeneration of DA neurons and the subsequent lack of DA in the striatum are associated to motor symptoms including tremor, bradykinesia, and rigidity. Mutation or deletions of several genes have been identified to predispose to the pathology.

Neuronal differentiation towards the dopaminergic lineage is a complex process, which highly relies on the sequential activation of specific transcription factors. In mammals, mDA progenitors start to develop at E7.5. LIM homeobox transcription factor 1a (LMX1A) and forkhead box protein A2 (FOXA2), both induced by SHH signaling, are important determination factors of mDA differentiation. Expression of LMX1A triggers dopaminergic differentiation and recruits MSX1/2, an inhibitor of negative regulators of neurogenesis. It further induces the expression of proneural factors such as Neurogenin 2 (NGN2), which are necessary for the proper development of mDA neurons (Gale and Li, 2008). Upon maturation, mDA progenitos migrate to exit the proliferative zone at E10.5-E11.5. At this stage, the phenotypic marker of mDA neuons, tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis is induced and mDA progenitors start to express the early neuronal marker βIII Tubulin (TUJ1) (Abeliovich and Hammond, 2007).

Many of the underlying studies of CNS development and specification have been carried out in mouse and chick embryos. Even though it is likely that many of the mechanisms are conserved between mammalian species, there are strong differences between murine and human neurodevelopment. For instance, the size of the cortex is remarkably increased in humans. Some regions, such as the outer subventricular zone or the inner fibre layer are completely absent in mice but present in humans. Notably, rodents are naturally not susceptible to neurodegenerative disorders such as PD and Alzheimer's disease (AD). In addition to differences in neurodevelopment, another obvious but striking difference is the ability to speak.

Parkinson's disease is characterized by the progressive loss of midbrain dopaminergic neurons in the substantia nigra pars compacta. The underlying mechanisms that lead to severe cell death remain poorly understood. Mouse models often cannot recapitulate the phenotype seen in vivo, especially in the case of complex pathologies such as neurodegenerative disorders, which do not affect animals. For this reason, given the divergences between humans and mice, which are most frequently used as in vivo model systems, as well as the shortcomings of 2D cell cultures, there is an obvious need for robust in vitro models of human brain development, especially in terms of studying neurodevelopmental and neurodegenerative diseases.

The technical problem underlying the present application is to comply with this need. The solution to said technical problem is the provision of means and methods for generating midbrain organoids as reflected in the claims, described herein, illustrated in the Figures and exemplified in the Examples of the present application.

Much to their surprise the present inventors observed that they were able to obtain midbrain organoids by contacting neuroepithelial stem cells with differentiation medium, when they cultured said neuroepithelial stem cells in a three-dimensional cell culture comprising a matrix under agitating conditions. Specifically, the present inventors used a human neural precursor cell line, called human neuroepithelial stem cells (hNESCs), which served as a starting population for the generation of midbrain organoids. A single colony was embedded into a matrix allowing three-dimensional cell culture, said matrix being e.g. a droplet of Matrigel. Matrigel droplets are described, for example, in Lancaster and Knoblich (2014a). A single colony was cultured under agitating conditions, such as continuous spinning. The medium used for differentiation contained, inter alia, signaling molecules for the induction of midbrain development as described herein. At several time points, midbrain organoids were characterized using immunohistochemical stainings in order to determine whether indeed a differentiation into midbrain-specific structures was detectable. After about 3 weeks of differentiation, midbrain organoids developed several neural cell types, including dopaminergic neurons, and an asymmetric organization, consistent with brain development in vivo. Such midbrain organoids may serve as 3D models which can, for example, be used to study neurodevelopmental and/or neurodegenerative diseases, such as Parkinson's disease (PD), Multiple sclerosis, Batten's disease or Alzheimer's disease.

During their studies, the present inventors observed that NESCs seeded on round-bottom plates with low attachment surfaces formed globular colonies that expanded quickly (FIG. 4). However, this involved increasing cell death in the centre of the colonies due to a limited nutrient exchange. Sectioning of early organoids between day 6 and 10 revealed that the majority of the cells in the core were dead. However, surprisingly, cell death significantly decreased when the 3D structures were kept under agitating conditions.

NESCs embedded in a matrix allowing three-dimensional cell culture such as MATRIGEL and kept under agitating conditions grew further and quickly started to develop long processes that migrated through the entire ECM (FIG. 4d-f). These processes expressed the neuronal markers TUJ1 and partially MAP2, indicative of axon formation (FIG. 6 and FIG. 7). Upon neurogenesis, young neurons adopt bipolar morphology with leading and trailing processes, which eventually become axons between embryonic day 11 to 18 (Lewis et al., 2013). Cell bodies were mainly located adjacent to the core. Under maintenance conditions, hNESCs stably express the neural progenitor markers SOX1, SOX2, and NESTIN (FIG. 3). This expression pattern is retained in early midbrain organoids that were seeded on ultralow attachment plates (FIG. 5). Some SOX2 positive cells remained in the inner part of the organoid after 24 days of differentiation, abutting to TH positive DA neurons (FIG. 7). During the course of midbrain development in vivo, the neuroepithelium in the midbrain regions thickens and becomes layered. Some neural stem cells remain in the inner layer and retain their proliferative properties, while other cells migrate to an intermediate zone and start to differentiate into mDA neurons. Immature neurons continue to migrate to reach the marginal zone where they become mature neurons. The mDA neuron progenitors express LMX1, FOXA2, TH, and the neuronal marker TUJ1 (Ang, 2009; Gale and Li, 2008). In vitro, midbrain organoids seem to develop a similar layered organisation with neural stem cells remaining adjacent to the inner core and mDA neurons migrating basally. DA neurons started to develop after 6 days under differentiation conditions. Interestingly, DA neurons and neural precursors accumulated in a particular area and were not equally distributed across the tissue at day 30 and 44, suggesting self-patterning of midbrain organoids along the D-V axis (FIG. 6 and FIG. 7). However, not all TH positive cells are necessarily DA neurons. All catecholamines, including dopamine, noradrenaline, and adrenaline are synthesised from tyrosine. TH is the rate-limiting enzyme that produces L-DOPA from tyrosine (Sharples et al., 2014). To confirm that the TH⁺ cells are indeed DA neurons, further markers such as DAT are to be considered. Importantly, midbrain organoids developed asymmetric polar structures, similar to brain development in vivo. To this end, the present inventors successfully generated midbrain organoids by using neuroepithelial stem cells as starting population for the generation said midbrain organoids by contacting them with differentiation medium under agitating conditions in three-dimensional cell culture comprising a matrix.

Accordingly, the present invention provides in one aspect a method of generating a midbrain organoid, comprising contacting neuroepithelial stem cells, which are cultured in a three-dimensional cell culture comprising a matrix, with differentiation medium, wherein the culturing is performed under agitating conditions, thereby obtaining a midbrain organoid.

The present invention also provides in another aspect midbrain organoid obtainable by the method of the present invention as well as in yet another aspect uses and methods applying the midbrain organoids of the present invention for testing compounds for their ability to elicit a cellular response on said midbrain organoid.

Furthermore, the present invention provides in a further aspect midbrain organoids as described herein for use in transplantation.

Having recognized that neuroepithelial stem cells can successfully be used for the generation of midbrain organoids as described herein, the present invention also provides in a yet further aspect the use of neuroepithelial stem cells, which are cultured in a three-dimensional cell culture comprising a matrix under agitating conditions, for generating a midbrain organoid unit.

The above aspects of present invention as well as preferred aspects thereof may also be summarized in the following items:

• • (1) A method of generating a midbrain organoid, comprising: contacting (only) neuroepithelial stem cells, which are cultured in a three-dimensional cell culture comprising a matrix, with differentiation medium, wherein the culturing is performed under agitating conditions, thereby obtaining a midbrain organoid.
  • (2) The method of item 1, wherein said neuroepithelial stem cells are human neuroepithelial stem cells, preferably said neuroepthelial cells express SOX1, SOX2, PAX6 and NESTIN.
  • (3) The method of item 2, wherein said neuroepithelial stem cells are hNESC-K7 or smNPCs.
  • (4) The method of any one of items 1-3, wherein said neuroepithelial stem cells are genetically modified or obtained from a patient suffering from a neurological disease.
  • (5) The method of item 4, wherein the genetic modification comprises a mutation, a knock-out or a knock-in.
  • (6) The method of item 4, wherein the neurological disease is a neurodegenerative disease such as Parkinson's disease, Multiple sclerosis, Batten's disease or Alzheimer's disease.
  • (7) The method of any one of items 1-6, wherein said neuroepithelial stem cells have been produced from induced pluripotent stem cells (iPSCs).
  • (8) The method of item 7, wherein the iPSCs are produced from fibroblasts or peripheral blood mononuclear cells (PBMCs), wherein the fibroblasts or PBMC have preferably been obtained from a patient.
  • (9) The method of any one of items 1-8, wherein the three-dimensional cell culture is performed in a gel, a bioreactor in ultra-low adhesion conditions or a microchip, preferably a hydrogel and/or a hydrogel droplet such as a Matrigel droplet.
  • (10) The method of any one of items 1-9, wherein the matrix is an extracellular matrix and/or wherein the matrix comprises one or more of natural molecules, synthetic polymers, biological-synthetic hybrids, metals, ceramics, bioactive glasses and/or carbon nanotubes.
  • (11) The method of any one of items 1-10, wherein the matrix comprises collagen, Matrigel, fibrin, hyaluronic acid, chitosan, alginate, silk fibrils, ethylene glycol such as PEG, poly(vinyl alcohol) and/or poly(2-hydroxy ethyl methacrylate), preferably the matrix comprises Matrigel.
  • (12) The method of any one of items 1-11, wherein said differentiation medium (differentiation medium I) comprises
    • (i) a SHH-pathway activator;
    • (ii) at least two different neurotrophins; and
    • (iii) an antioxidant.
  • (13) The method of any one of items 1-12, wherein said differentiation medium (differentiation medium II) comprises
    • (i) at least two different neurotrophins; and
    • (ii) an antioxidant.
  • (14) The method of item 12, wherein the SHH-pathway activator is selected from the group consisting of purmorphamine, SHH, smoothened agonist (SAG), Hh-Ag 1.5 and Gli-2, preferably the SHH-pathway activator is purmorphamine.
  • (15) The method of any one of items 12-14, wherein the at least two neurotrophins are selected form the group consisting of NGF, BDNF, NT-3, NT-4, CNTF and GDNF, preferably the at least two neurotrophins are GDNF and BDNF.
  • (16) The method of any one of items 12-15, wherein the antioxidant is selected from the group consisting of ascorbic acid, superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, glutathione, lipoic acid, epigallocatechin gallate, curcumine, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E and uric acid, preferably the antioxidant is ascorbic acid.
  • (17) The method of any one of items 12-16, wherein the differentiation medium (differentiation medium I and/or II) further comprises an activator of activin/transforming growth factor-β (TGF-β) signaling pathway and/or wherein the differentiation medium II does not comprise a SHH-pathway activator.
  • (18) The method of item 17, wherein the activator of activin/TGF-β signaling pathway is selected from the group consisting of TGβ1, TGβ2, TGβ3, activin A, activin B, activin AB and nodal, preferably the activator of activin/TGF-β signaling pathway is TGFβ3.
  • (19) The method of any one of items 12-18, wherein the differentiation medium (differentiation medium I and/or II) further comprises a cAMP analogue.
  • (20) The method of item 19, wherein the cAMP analogue is selected from the group consisting of forskolin, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8CPT-2Me-cAMP), 8-Chloro-cAMP (8-Cl-cAMP), Bucladesine, Rp-adenosine, 3′, 5′-cyclic monophosphorothioate sodium salt (Rp-cAMPS), Sp-8-hydroxyadenosine, 3′, 5′-cyclic monophosphorothioate sodium salt (Sp-80H-cAMPS) and Rp8-hydroxyadenosine, 3′, 5′-cyclic monophosphorothioate sodium salt (Rp-80H-cAMPS) and dbcAMP, preferably the cAMP analogue is dbcAMP.
  • (21) The method of any one of items 12-20, wherein the differentiation medium (differentiation medium I and/or II) is a N2B27 medium.
  • (22) The method of item 21, wherein the N2B27 medium comprises equal amounts of Neurobasal medium and DMEM/F12 medium.
  • (23) The method of any one of items 12-22, wherein the differentiation medium (differentiation medium I and/or II) further comprises penicillin and streptomycin.
  • (24) The method of any one of items 12-23, wherein the differentiation medium (differentiation medium I and/or II) further comprises glutamine, preferably L-glutamine, more preferably L-glutamine at a concentration of 2 mM.
  • (25) The method of any one of items 12-24, wherein the differentiation medium further comprises B27 supplement without vitamin A, preferably at a concentration of 1:100 (supplement:medium).
  • (26) The method of any one of items 12-25, wherein the differentiation medium ((differentiation medium I and/or II) further comprises N2 supplement, preferably at a concentration of 1:200 (supplement:medium).
  • (27) The method of any one of items 12-26, wherein the differentiation medium (differentiation medium I and/or differentiation medium II) does not comprise FGF8.
  • (28) The method of any one of items 12-27, wherein the cells are kept in the differentiation medium I for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more days, preferably the cells are kept in the differentiation medium I for 6 days and/or wherein the cells are kept in differentiation medium II for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, 38, 40, 50 or more days, preferably the cells are kept in the differentiation medium II for 1, 24 or 38 days.
  • (29) The method of item 28, wherein differentiation medium I is replaced by differentiation medium II after 6 days of differentiation by omitting the SHH-pathway activator from the differentiation medium.
  • (30) The method of any one of items 1-29, wherein FGF8 is added to the differentiation medium (differentiation medium I and/or II) after 8 days of differentiation.
  • (31) The method of any one of items 1-30, wherein the neuroepithelial stem cells are contacted by a maintenance medium before they are contacted with the differentiation medium.
  • (32) The method of item 31, wherein the maintenance medium comprises
    • (i) a SHH-pathway activator;
    • (ii) canonical WNT-signaling activator; and
    • (iii) an antioxidant.
  • (33) The method of item 31 or 32, wherein the maintenance medium comprises N2B27 medium as defined in any one of items 21-27.
  • (34) The method of item 32 or 33, wherein the canonical WNT-signaling activator is selected from the group consisting of Norrin, R-spondin 2 or WNT protein.
  • (35) The method of any one of items 32-34, wherein canonical WNT-signaling activator blocks Axin or APC e.g. via siRNA.
  • (36) The method of any one of items 32-34, wherein canonical WNT-signaling activator is a GSK-3 inhibitor.
  • (37) The method of item 36, wherein the GSK-3 inhibitor is selected from the group consisting of CHIR 99021, SB-216763, 6-bromoindirubin-3′-oxime, Tideglusib, GSK-3 inhibitor 1, AZD1080, TDZD-8, TWS119, CHIR-99021 HCl, CHIR-98014, SB 415286, SB 216763, LY2090314, AR-A014418 and IM-12, preferably the GSK-3 inhibitor is CHIR 99021.
  • (38) The method of item 31-37, wherein the maintenance of the neuroepithelial stem cells takes place in a two-dimensional and/or three-dimensional cell culture.
  • (39) The method of item 38, wherein the maintenance of the neuroepithelial stem cells is performed in a three-dimensional cell culture for 1, 2, 3, 4, 5, 6 or more days, preferably wherein the maintenance of the neuroepithelial stem cells is performed in a three-dimensional cell culture for 2 days.
  • (40) The method of any one of items 1-39, wherein the neuroepithelial stem cells are present in a colony.
  • (41) The method of item 40, wherein the colony is a cluster of cell clones.
  • (42) The method of item 40 or 41, wherein said colony of neuroepithelial stem cells is obtainable by culturing said neuroepithelial stem cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more days in the maintenance medium as defined in any one of items 31-39, preferably said neuroepithelial stem cells are cultured for 10 days in the maintenance medium as defined in any one of items 31-39.
  • (43) The method of item 42, wherein at least 50, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000 or more neuroepithelial stem cells, preferably about 9000 neuroepithelial stem cells are used as a starting cell population.
  • (44) The method of any one of items 40-43, wherein the colony of neuroepithelial stem cells is obtainable by culturing neuroepithelial cells in round bottom ultralow attachment 96-well plates and/or ultralow attachment 24-well-plates and/or in a three-dimensional cell culture.
  • (45) The method of any one of items 40-44, wherein the colony of neuroepithelial stem cells is cultured in a Matrigel droplet for at least 1 day.
  • (46) The method of any one of items 9-45, wherein said Matrigel droplet is cultured under agitating conditions after at least 1, 2, 3, 4, 5, 6, 7 or more days after initiation of differentiation, preferably the Matrigel droplet is cultured under agitating conditions after 2 or after 4 days after initiation of differentiation.
  • (47) The method of any one of items 1-46, wherein said agitating conditions comprise shaking, spinning, stirring, moving and/or mixing of the three-dimensional cell culture.
  • (48) The method of item 47, wherein the spinning is performed with a spinning bioreactor and/or the shaking is performed with an orbital shaker.
  • (49) The method of item 47 or 48, wherein said orbital shaker is shaking at least at 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, or more, preferably said orbital shaker is shaking at 80 rpm.
  • (50) The method of any one of items 1-49, comprising:
    • (i) contacting neuroepithelial stem cells with the maintenance medium as defined in any one of items 32-39;
    • (ii) contacting neuroepithelial stem cells, which are cultured in a three-dimensional cell culture comprising a matrix, with the differentiation medium (I) as defined in any one of items 12, 14-30, wherein the culturing is performed under agitating conditions, wherein the agitating is started after 0, 1, 2, 3, 4, 5, 6, 7, 8 or more days, preferably 2 days or 4 days, after starting culturing of the neuroepithelial stem cells in the differentiation medium I;
    • (iii) contacting neuroepithelial stem cells with the differentiation medium (II) as defined in any one of items 13-30 under agitating conditions.
      thereby obtaining a midbrain organoid.
  • (51) The method of any one of items 1-50, comprising:
    • (i) contacting neuroepithelial stem cells with the maintenance medium as defined in any one of items 32-39;
    • (ii) culturing the neuroepithelial stem cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more days in the maintenance medium;
    • (iii) contacting neuroepithelial stem cells with the differentiation medium (I) as defined in any one of items 12, 14-30,
    • (iv) culturing the cells of step (iii) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more days in the differentiation medium I, wherein the culturing is performed under agitating conditions, wherein the agitating is started after 0, 1, 2, 3, 4, 5, 6, 7, 8 or more days, preferably 2 days, after starting culturing of the neuroepithelial stem cells in the differentiation medium I;
    • (v) contacting the cells obtained in step (iv) with the differentiation medium II as defined in any one of items 13-30; and
    • (vi) culturing the cells of step (v) for 1, 2, 3, 4, 5, 6, 7, 8, 9, or more weeks in the differentiation medium II under agitating conditions;
      thereby obtaining a midbrain organoid.
  • (52) The method of item 51, wherein the culturing of the neuroepithelial stem cells in step (ii) is performed for 9 or 10 days.
  • (53) The method of item 51 or 52, wherein the culturing of the cells of step (iv) is performed for 6 days.
  • (54) The method of any one of items 51-53, wherein the culturing the cells of step (vi) is performed for 1, 24 or 38 days.
  • (55) The method of any one of items 1-54, wherein the midbrain organoid is obtainable after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more weeks of differentiation, preferably the differentiation is performed for 6, 7, 16, 30 or 44 days.
  • (56) The method of any one of items 1-55, wherein said midbrain organoid is an early midbrain organoid or a late midbrain organoid.
  • (57) The method of item 56, wherein the early midbrain organoid is a midbrain organoid, which has been differentiated for 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days, preferably the early midbrain organoid has been differentiated for 6, 7 or 16 days.
  • (58) The method of item 56, wherein the late midbrain organoid is a midbrain organoid, which has been differentiated for at least 25, 30, 35, 40, 50, 60 or more days, preferably the late midbrain organoid has been differentiated for 30, 44 or 51 days.
  • (59) The method of any one of items 1-58, wherein said midbrain organoid comprises
    • (a) neural progenitor cells;
    • (b) young neurons;
    • (c) young dopaminergic neurons;
    • (d) mature neurons;
    • (e) mature dopaminergic neurons;
    • (f) an asymmetric organization of the midbrain organoid;
    • (g) oligodendrocytes;
    • (h) oligodendrocyte progenitors;
    • (i) astrocytes; and/or
    • (j) processes that expand from the midbrain organoid through the matrix.
  • (60) The method of any one of items 56, 57 or 59, wherein the early midbrain organoid comprises
    • (a) neural progenitor cells;
    • (b) young neurons; and/or
    • (c) Ki67-positive cells and/or
    • (d) young dopaminergic neurons.
  • (61) The method of any one of items 56, 58 or 59, wherein the late midbrain organoid comprises
    • (a) neural progenitor cells;
    • (b) young neurons;
    • (c) young dopaminergic neurons;
    • (d) mature neurons;
    • (e) mature dopaminergic neurons, preferably comprising or producing dopamine;
    • (f) an asymmetric organization of the midbrain organoid;
    • (g) oligodendrocytes, preferably expressing O4 and/or CNPase;
    • (h) oligodendrocyte progenitors, preferably expressing NG2;
    • (i) astrocytes, preferably expressing S100b and/or GFAP;
    • (j) clustering of dopaminergic neurons within the organoid; and/or
    • (k) processes that expand from the midbrain organoid through the matrix.
  • (62) The method of any one of items 59-61, wherein said neural progenitor cells are characterized by the expression of the markers SOX2 and/or nestin.
  • (63) The method of any one of items 59-62, wherein said young neurons are characterized by the expression of the marker TUJ1.
  • (64) The method of any one of items 59-63, wherein said young dopaminergic neurons are characterized by the expression of the markers TUJ1 and tyrosine hydroxylase (TH).
  • (65) The method of any one of items 59-64, wherein said mature neurons are characterized by the expression of the marker MAP2.
  • (66) The method of any one of items 59-65, wherein said mature dopaminergic neurons are characterized by the expression of the markers MAP2, TH, FOXA2 and/or the expression of the marker LMX1A and/or said mature dopaminergic neurons are characterized by the expression of the mRNA encoding for LMX1A, LMX1B, EN1, NURR1, AADC, and/or TH.
  • (67) The method of any one of items 59-66, wherein the asymmetric organization of the midbrain organoid is an asymmetric polar organization of dopaminergic neurons and/or an asymmetric organization of neuronal progenitor cells within the midbrain organoid.
  • (68) The method of any one of items 59-67, wherein said asymmetric polar organization of dopaminergic neurons within the midbrain organoid is characterized by the localization of
    • (a) mature dopaminergic neurons in the outermost part of the midbrain organoid;
    • (b) young dopaminergic neurons in the inner parts of the midbrain organoid.
  • (69) The method of item 68, wherein young dopaminergic neurons migrate towards the outermost part of the midbrain organoid upon maturation.
  • (70) The method of any one of items 59-69, wherein the asymmetric organization of neuronal progenitor cells within the midbrain organoid is characterized by the localization of neuronal progenitors in a ring-like structure surrounding the inner core of the midbrain organoid.
  • (71) The method of any one of items 59-70, wherein said oligodendrocytes are characterized by the expression of the marker O4 and/or expression of CNPase.
  • (72) The method of any one of items 59-71, wherein said oligodendrocyte progenitors are characterized by the expression of the marker NG2.
  • (73) The method of any one of items 59-72, wherein said astrocytes are characterized by the expression of the markers GFAP and/or S100b.
  • (74) The method of any one of items 59-73, wherein said clustering of dopaminergic neurons within the organoid is characterized by the accumulation of more than 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dopaminergic neurons in a specific region of the midbrain organoid.
  • (75) The method of any one of items 59-74, wherein said processes that expand from the midbrain organoid through the matrix have a length of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm or more.
  • (76) The method of any one of items 1-75, wherein the neuroepithelial stem cell is obtainable by a method comprising
    • a) optionally obtaining/providing induced pluripotent stem cells (iPSCs);
    • b) cultivating said iPSCs in a medium comprising
      • (i) an activin/transforming growth factor-β (TGF-β) signaling inhibitor;
      • (ii) a canonical WNT-signaling activator;
      • (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and
      • (iv) a SHH-pathway activator; and
    • c) cultivating the cells obtained in b) in a medium comprising
      • (i) an activin/TGF-β signaling inhibitor;
      • (ii) a canonical WNT-signaling activator;
      • (iii) a BMP signaling inhibitor; and
      • (iv) a SHH-pathway activator; and
    • d) further cultivating the cells obtained in c) in a medium comprising
      • (i) a canonical WNT-signaling activator;
      • (ii) SHH-pathway activator; and
      • (iii) an antioxidant; and
        thereby obtaining a neuroepithelial stem cell.
  • (77) The method of item 76, wherein the method further comprises
    • e) maintaining the cells obtained in d) in a medium comprising
      • (i) a FGF signaling activator;
      • (ii) an EGF signaling activator; and
      • (iii) a LIF signaling activator.
  • (78) Midbrain organoid obtainable by the method of any one of items 1-77.
  • (79) Use of the midbrain organoid of item 78 for testing compounds for their ability to elicit a cellular response on said midbrain organoid.
  • (80) Use of item 79, wherein said compound is a drug, small molecule, hormone, growth factor, binding protein, nucleic acid molecule, peptide protein or (co-cultured) cell.
  • (81) Use of item 79 or 80, wherein said cellular response is the frequency or survival of a certain type of cell.
  • (82) Use of item 81, wherein said type of cell is a dopaminergic neuron.
  • (83) Method for testing a compound of interest for its ability to elicit a cellular response, comprising:
    • (a) contacting the midbrain organoid of item 78 with said compound of interest; and
    • (b) determining whether said compound of interest elicits a cellular response.
  • (84) The method of item 83, wherein said compound is a drug, small molecule, hormone, growth factor, binding protein, nucleic acid molecule, peptide protein or (co-cultured) cell.
  • (85) The method of item 83 or 84, wherein said cellular response is the frequency or survival of a certain type of cell.
  • (86) The method of item 85, wherein said type of cell is a dopaminergic neuron.
  • (87) The method for identifying molecules promoting or inhibiting dopaminergic neuronal differentiation and/or death of dopaminergic neurons in a midbrain organoid as defined in item 78, the method comprising contacting the midbrain organoid with a molecule of interest, wherein an increase of the differentiation into dopaminergic neurons compared to a control indicates that the molecule of interest promotes dopaminergic neuronal differentiation and/or inhibits death of dopaminergic neurons and wherein a decrease of the differentiation into dopaminergic neurons compared to a control indicates that the molecule of interest inhibits dopaminergic neuronal differentiation and/or induces death of dopaminergic neurons.
  • (88) The method of item 87, wherein the differentiation into dopaminergic neurons is measured by comparing neurite outgrowth.
  • (89) The method of item 87 or 88, wherein the differentiation into dopaminergic neurons is measured by comparing the expression of TH.
  • (90) The method of any one of items 87-89, wherein the control is a midbrain organoid which is not contacted with the molecule of interest.
  • (91) Composition comprising a midbrain organoid unit of item 78.
  • (92) The composition of item 91, which is a pharmaceutical composition.
  • (93) Use of neuroepithelial stem cells, which are cultured in a three-dimensional cell culture comprising a matrix under agitating conditions, for generating a midbrain organoid unit.
  • (94) Midbrain organoid of item 78 for use in transplantation.

Notably, the method as described herein can also comprise:

• • (i) contacting neuroepithelial stem cells with the maintenance medium as defined herein;
  • (ii) culturing the neuroepithelial stem cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more days in the maintenance medium, preferably, the neuroepithelial stem cells are cultured for 10 days in the maintenance medium wherein optionally the neurepithelilal stem cells are transferred into a MATRIGEL droplet after 8 days;
  • (iii) contacting neuroepithelial stem cells with the differentiation medium (I) as described herein,
  • (iv) culturing the cells of step (iii) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more days, preferably 6 days in the differentiation medium I, wherein the culturing is performed under agitating conditions,

wherein the agitating is started after 0, 1, 2, 3, 4, 5, 6, 7, 8 or more days, preferably 4 days, after starting culturing of the neuroepithelial stem cells in the differentiation medium I;

• • (v) contacting the cells obtained in step (iv) with the differentiation medium II as described herein; and
  • (vi) culturing the cells of step (v) for 1, 2, 3, 4, 5, 6, 7, 8, 9, or more weeks in the differentiation medium II under agitating conditions;

thereby obtaining a midbrain organoid.

An “organoid” resembles a whole organ. Organoids exhibit an intrinsic potential to self-organise, forming the cellular organisation of an organ. Organoids hold great promise for diagnostic and therapeutic applications. The organoid of the present invention is preferably a midbrain organoid. Accordingly, a midbrain organoid of the present invention resembles the midbrain. The midbrain is the region of the brain, where the majority of the neurotransmitter dopamine (DA) is produced. A midbrain organoid of the present invention is preferably from a single colony of a NESC, preferably a hNESC. A midbrain organoid of the present invention has preferably the phenotype of a midbrain. A midbrain organoid of the present invention is either an early midbrain organoid or a late midbrain organoid. A midbrain organoid of the present invention has preferably the phenotype of a midbrain. As such, it comprises typical cells/cell types of a midbrain. Accordingly, a midbrain organoid of the present invention comprises neural progenitor cells, young neurons, young dopaminergic neurons, mature neurons, mature dopaminergic neurons, an asymmetric organization, oligodendrocytes, oligodendrocyte progenitors, astrocytes, and/or processes that expand from the midbrain organoid through the matrix. These cell types are preferably characterized by the markers as described herein. Likewise, the asymmetric organization is preferably characterized as described herein and as well as the clustering of dopaminergic neurons and the processes that expand from the midbrain organoid through the matrix. Presence of the cells/cell types in a midbrain organoid of the present invention can be tested by means and methods known in the art and as described herein. Likewise, expression of the markers as described herein by said cells/cell types or the asymmetric organization can be tested as is known in the art and as described herein.

Organoids have the potential to model degenerative and developmental diseases and/or cancer, and represent a valuable tool to study genetic disorders and to identify subtle phenotypes. Somatic cells derived from patients can be reprogrammed into iPSCs and thereof-derived organoids can be used as a patient-specific model for drug tests or in regenerative medicine for organ replacement therapies. Insertion and correction of mutations in hiPSC-derived organoids might help to understand disease mechanisms. This is advantageously envisioned by the present invention, i.e. midbrain organoids of the present invention may serve as models which can, for example, be used to study neurodevelopmental and/or neurodegenerative diseases, such as Parkinson's disease (PD), Multiple sclerosis, Batten's disease or Alzheimer's disease.

The methods of the present invention can be carried out in any cell culture, while, however, three-dimensional cell culture is preferred. Culture conditions may vary, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel comprising one or more of the following: a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, gases (O₂, CO₂) and/or regulated physico-chemical environment (pH, osmotic pressure, temperature). Cell culture as described herein refers to the maintenance and growth of cells in a controlled laboratory environment. Such in vitro cell culture models are well-known in experimental cell biological research. For example, cells can be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture). Cells can also be grown floating in the culture medium (suspension culture). However, it is preferred that cells of the present invention are cultured under agitating conditions.

Medium for cell culture, such as maintenance medium or differentiation medium is described herein elsewhere, e.g. in the items above.

Differentiation media as applied in the methods of the present invention comprise at least two different neurotrophins. The term “neurotrophins”, as used herein, relates to a family of proteins that regulate the survival, development, and function of neurons. Exemplary neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) as well as GDNF family of ligands and ciliary neurotrophic factor (CNTF). The GDNF family of ligands includes glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN), and persephin (PSPN).

Accordingly, the term “at least two different neurotrophins” refers to two or more of the recited molecules. Preferably, the at least two different neurotrophins are BDNF and GDNF (Gene Symbols: BDNF and GDNF, respectively). BDNF can e.g. be the human BDNF protein of Uniprot/Swissprot accession no. P23560 (version 1 as of Oct. 31, 1991). GDNF can e.g. be the human GDNF protein of Uniprot/Swissprot accession no. P39905 (version 1 as of Jan. 31, 1995).

BDNF and GDNF can both independently from each other be employed in a concentration of between about 0.0001 and about 50 ng/μl each, more preferably between about 0.001 and about 25 ng/μl each, and most preferably the amount is about 0.001 ng/μl each. BDNF and GDNF may for example be obtained from Peprotech.

The differentiation medium as applied in the methods of the present invention may further comprise an antioxidant. An antioxidant is a molecule that inhibits the oxidation of other molecules. The terms “oxidation” and “antioxidant” are well known in the art and have been described, for example, in Nordberg J, Amer E S. (2001) “Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.” Free Radic Biol Med. 31(11):1287-312. In short, oxidation is a chemical reaction involving the loss of electrons or an increase in oxidation state. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. Accordingly, an antioxidant refers to an inhibitor of a molecule involved in cellular oxidative processes.

Exemplary antioxidants include ascorbic acid, superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, glutathione, lipoic acid, epigallocatechin gallate, curcumine, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E or uric acid. Thus, the antioxidant can be ascorbic acid.

Any medium for cell culture as described herein may contain a ROCK inhibitor. A “ROCK inhibitor” as used herein is compound that acts as an inhibitor of Rho-associated protein kinase, i.e. reduces or even abolishes ROCK functionality. The capability of a compound to act as a ROCK inhibitor can be assessed by various means, e.g. by determining its ability to compete with ATP for binding to ROCK and/or by assessing its effects on cell morphology, G1-S Transition and cytokinesis as described in Ishizaki T Mol Pharmacol. 2000 May; 57(5):976-83. The inhibitor may be either unspecific or specific for either of the ROCK isoforms ROCK1 and/or ROCK2. ROCK inhibitors known in the art have been reviewed in Liao et al. J Cardiovasc Pharmacol. 2007 July; 50(1): 17-24 and include Fasudil, Y-27632, Thiazovivin, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, ROKa inhibitor, XD-4000, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, HMN-1152, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, with Y-27632 or Thiazovivin being particularly envisaged for use in the method of the invention.

Differentiation medium as applied in the methods of the present invention can further comprise an activator of activin/transforming growth factor-β (TGF-β) signaling pathway. The activin/TGF-β signaling pathway is known in the art and for example described in Heldin, Miyazono and ten Dijke (1997) “TGF-bold beta signaling from cell membrane to nucleus through SMAD proteins.” Nature 390, 465-471. In short, Receptor ligands, including, for example, TGFB1, TGFB2, TGFB3, ACTIVIN A, ACTIVIN B, ACTIVIN AB, and/or NODAL, bind to a heterotetrameric receptor complex consisting of two type I receptor kinases, including, for example, TGFBR2, ACVR2A, and/or ACVR2B, and two type II receptor kinases, including, for example, TGFBR1, ACVR1 B, and/or ACVR1C. This binding triggers phosphorylation and activation of a heteromeric complex consisting of an R-smad, including, for example, SMAD2, and/or SMAD3, and a Co-smad, including, for example, SMAD4. Accordingly, the term “activator of the activin/TGF-β signaling pathway” refers to an activator of any one of the above recited molecules that form part of this signaling pathway.

Exemplary activators of the activin/TGF-β signaling pathway include TGβ1, TGβ2, TGFβ3, activin A, activin B, activin AB or nodal. Thus, the activator of activin/TGF-β signaling pathway can be TGFβ3. The activator of the activin/TGF-β signaling pathway such as TGFβ3 can be utilized in an amount of 0.0001 ng/μl to 0.1 ng/μl such as e.g. in an amount of 0.001 ng/μl.

Differentiation medium as applied in the methods of the present invention can further comprise a cAMP analogue. Such cAMP analogs are compounds that have similar physical, chemical, biochemical, or pharmacological properties as the cyclic adenosine monophosphate (cAMP). cAMP is known to the skilled artesian and described in e.g. Fimia G M, Sassone-Corsi P. (2001) “Cyclic AMP signalling.” J Cell Sci; 114 (Pt 11):1971-2.

Differentiation medium as applied in the methods of the present invention can further be a N2B27 medium (into which the different compounds are diluted). This means that the medium comprises a N2 supplement and a B27 supplement. Both supplements are well known to the person skilled in the art and freely available. The B27 supplement can be a B27 supplement without vitamin A. This B27 can be used at a concentration of 1:10-1:1000, such as 1:100 (supplement:medium). The B27 supplement can for example be obtained from Life technologies. Likewise, also the N2 supplement can for example be obtained from Life technologies. The N2 supplement may be used at a concentration of 1:20 to 1:2000, such as 1:200 (supplement:medium).

Differentiation medium may also be a Neurobasal medium and/or a DMEM-F12 medium. Both media can for example be obtained from Life technologies. The N2B27 medium can for example comprise equal amounts of Neurobasal medium and DMEM/F12 medium.

Differentiation medium as applied in the methods of the present invention can further comprise an antibiotic.

Differentiation medium as applied in the methods of the present invention can further comprise glutamine.

Differentiation medium as applied in the methods of the present invention can comprise N2B27 medium comprising about 50% DMEM-F12 (e.g. from Life technologies)/about 50% Neurobasal (e.g. from Life technologies), about 1:200 N2 supplement (e.g. from Life technologies), about 1:100 B27 supplement lacking vitamin A (e.g. from Life technologies), 1% Penicillin/Streptomycin (e.g. from Life technologies) and 2 mM L-glutamine (e.g. from Life technologies).

In addition, cells may be cultured in a two-dimensional cell culture. This type of cell culture is well-known to the person skilled in the art. In two-dimensional (2D) cell culture cells are grown on flat plastic dishes such as Petri dish, flasks and multi-well plates. However, biologically derived matrices (e.g. fibrin, collagen and as further described herein) and synthetic hydrogels (e.g. PAA, PEG and as further described herein) can be used to elicit specific cellular phenotypes that are not expressed on rigid surfaces.

The methods of the present invention are, however, preferably be carried out in a three dimensional cell culture. A “three-dimensional cell culture” or “3D cell culture” as used herein means that cells are grown in an artificially-created environment in which cells are permitted to grow or interact with its surroundings in all three dimensions. For example, in order to achieve the three dimensional property of the cell culture, cells are grown or differentiated in matrices or scaffolds. In principle, suitable matrices or scaffolds, which can be used in three dimensional cell cultures are known to the skilled artesian. Such matrices or scaffolds can therefore be any matrix or scaffold. For example, the matrix or scaffold can be an extracellular matrix comprising either natural molecules or synthetic polymers, a biological and synthetic hybrid, metals, ceramic and bioactive glass or carbon nanotubes.

Exemplary natural extracellular matrix molecules include collagen, basement membranes such as laminin or fibrin, alginates, chitosan, hyaluronic acid, silk fibroin, cellulose actetate, casein, chitin, fibrinogen, gelatine, elastin or poly-(hydroxyalkanoate). Synthetic extracellular matrix polymers include Hyaluronic acid (HA) modified forms, Poly-ethylen glycol (PEG) modified forms, Self-assembling protein hydrogels, Poly(lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL), Polyurethane or PGS. Biological and synthetic hybrids can for example include Polycaprolactone-chitosan, PLLA-Hydroxyapatite, Hydroxyapatite-bioglass-ceramic, Poly-(hydroxylalkanoate)-bioglass, Hydroxyapatite-collagen, PCL-gelatin or PCL-collagen. Exemplary metals include Tantalam, Magnesium and its alloys, Titanium and its alloys or Nitinol (nickel and titanium alloys). Examples of Ceramics and bioactive glass matrices/scaffolds include Titanium and tri calcium phosphate, Hydroxyapatite and Tricalcium phosphate, Bioactive silicate glass (SiO2-Na2O—CaO—P2O5), Hydroxyapatite and bioglass, Calcium phosphate glass or Phosphate glass. Carbon nanotubes can be constructed using graphite ranging from 0.4 to 2 nm. Carbon nanotubes can comprise CNT-polycaprolactone, CNT-ceramic matrix, 45S5 bioglass-CNT, CNT studded with gelatine hydrogel, CNT-TiO2, CNT-laminin, CNT grafted with polyacrylic acid or CNT-TGF-β.

The matrix or scaffold can also be a hydrogel such as Matrigel, fibrin gel or alginate gel. Matrigels can be a reconstituted basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins. Matrigel can be constituted of 60% laminin, 30% type IV collagen and 8% entactin. Optionally growth factors and other molecules can be added to the Matrigel. The Matrigel can also be BD Matrigel™ (obtainable from BD Biosciences).

Neuroepithelial stem cells (NESCs) when referred to herein can be derived from actual stem cells in several different stages of neural development. Neuroepithelial cells are a class of stem cell and have similar characteristics as stem cells. For example, these cells are able to self-renew. Self-renewal is the ability to go through numerous cell cycles of cell division while maintaining the undifferentiated state. In addition, neuroepithelial stem cell cells have the capacity to differentiate further into multiple types of cells, such as neurons, astrocytes and other glial cells. Thus, these cells are also multipotent. They are restricted to the neural lineage and can differentiate into neurons, astrocytes, and oligodendrocytes (Gage, 2000). Methods for testing if a cell has the capacity to self-renew and if a cell is multipotent are known to the skilled artesian. Self-renewal may be tested by passaging the cells over more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more passages. Passaging includes splitting of the cells before re-plaiting them as a single cell suspension. Multipotency can be tested by differentiating said cells into different lineages such as astrocytes, oligodendroctyes and neurons.

Furthermore, a neuroepithelial stem cell can express markers such as PAX6, Notch 1, Nestin, PCNA, Hes5 and Sox1. In particular, the neuroepithelial stem cells used in the methods of the present invention can be mammalian neural plate border stem cells (NPBSC) as described in WO2013104752. Furthermore, the neuroepithelial stem cells used in the methods of the present invention can also be NPBSCs as described in WO2013104752, which are also obtained by the method as described in WO2013104752. These NPBSC can be characterized by the expression of at least three markers selected from the group consisting of FORSE1, MSX1, PHOX2B, PAX3, PAX6, SOX1, SOX2, NESTIN, IRX3, HOXA2, HOXB2, HESS, DACH1, PLZF, LM03, EVI1 and ASCL1. Furthermore, these cells can be characterized by a lack of expression of at least one of the markers OCT4, NANOG, AFP, T, SOX17, EOMES, GSH2, OLIG2, CK8, CK18, NKX2.2, NKX6.1, HOXB8, HOXAS, FOXA2 and VCAM-1.

It is further envisioned that the neuroepithelial stem cells (N ESC) used in the present invention express the markers SOX1, SOX2, PAX6 and/or NESTIN. Additionally or alternatively, the NESCs used in the present invention have been passaged for more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20 or more times. It is also envisioned that the NESCs used in the present invention have been passaged less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 times. It is also envisioned that the NESCs used in the present invention have been passaged less than 20 and more than 2 times.

Notably, NESCs differ from cells of the embryoid bodies, which are three-dimensional aggregates of pluripotent stem cells. These embryoid bodies can be neurally induced. However, such induced cells may not be patterned well towards midbrain/hindbrain identity. In particular, compared to iPSCs as a starting population, NESCs are already patterned towards midbrain/hindbrain identity. This makes NESCs particularly suitable for use in the present invention. It is also envisioned that FGF8 is firstly added after 8 days of differentiation to the mediums (e.g. differentiation medium I and/or differentiation medium II) as described herein. The reason for this is that in this way the efficiency of the generation of dopaminergic neurons can be increased.

The neuroepithelial stem cell can be a mammalian NESC. It is also encompassed by the present invention that the NESC is a human NESC (hNESC), such as hNESC-K7. A neuroepithelial stem cell may be obtained by different means and methods known to the skilled artesian. For example, a neuroepithelial stem cell may be derived or obtained from pluripotent cells. NESC of the present invention may be genetically modified or be obtained from a patient suffering from a neurological disease, such as PD. Also, NESC may be produced from iPSCs, fibroblasts or PBMCs as described herein.

Totipotent stem cells (lat. “capable of everything”) can give rise to all cell types of the body, including the germ line of the trophectoderm (Weissman, 2000).

A “pluripotent stem cell” when referred to herein relates to a cell type having the capacity for self-renewal, and the potential of differentiation into different cell types. Pluripotent stem cells can differentiate into nearly all cells, i.e. cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm. The term pluripotent stem cells also encompasses stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst.

Multipotent stem cells are already restricted to a tissue or an organ and can give rise to all the tissue-specific cells.

Notably, recent advances in embryonic stem cell research have led to the possibility of creating new embryonic stem cell lines without destroying embryos, for example by using a blastomere biopsy-based technique, which does not interfere with the embryo's developmental potential (Klimanskaya (2006) “Embryonic stem cells from blastomeres maintaining embryo viability.” Semin Reprod Med. 2013 January; 31(1):49-55). Furthermore, a large number of established embryonic stem cell lines are available in the art. Thus, it is possible to work with embryonic stem cells without the necessity to destroy an embryo. Takahashi and Yamanaka addressed these concerns and published a method to generate induced pluripotent stem cells (iPSCs) from somatic cells (Takahashi and Yamanaka, 2006). Skin fibroblasts were induced with four defined factors to reprogram the cells back to the pluripotency state. Those stem cells have the same essential characteristics as ESCs (FIG. 1) (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007). In one embodiment, the pluripotent stem cells are embryonic stem cells, which have not been obtained via the destruction of a human embryo. Thus, the pluripotent stem cells are embryonic stem cells obtained from an embryo, without the destruction of the embryo.

A neuroepithelial stem cell can also be derived or obtained from another pluripotent cell, namely an induced pluripotent stem cell (iPSC). “Induced pluripotent stem cells”, as used herein, refers to adult somatic cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Thus, induced pluripotent stem cells derived from a non-pluripotent cell.

Induced pluripotent stem cells are an important advancement in stem cell research, as they allow obtaining pluripotent stem cells without the use of embryos. Mouse iPSCs were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126 (4): 663-76), and human iPSCs were first reported in 2007 (Takahashi et al. (2007) “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell; 131(5):861-72). Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expression of stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. Such stem cell markers can include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) as well as stem cell-specific antigen 3 and 4 (SSEA3/4). Also the chromatin methylation patterns of iPSC are also similar to that of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014) “Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90).

In addition, iPSCs are able to self-renew in vitro and differentiate into cells of all three germ layers. The pluripotency or the potential to differentiate into different cell types of iPSC can tested, e.g., by in vitro differentiation into neural or glia cells or the production of germline chimaeric animals through blastocyst injection.

Methods for the generation of human induced pluripotent stem cells are well known to the skilled person. Usually forced expression of Oct3/4, Sox2 and Klf4 (as well as OCT3/4, SOX2 and KLF4) is sufficient to generate an induced pluripotent stem cell out of an adult somatic cell, such as a fibroblast. However, also the combination of Oct3/4, Sox2, c-Myc and Klf4 (as well as OCT3/4, SOX2, C-MYC) and KLF4 is sufficient for the generation of an iPSC from an adult somatic cell. In addition, also the combination of OCT3/4, SOX2, NANOG and LIN28 was efficient for reprogramming (Tanabe, Takahashi, Yamanaka (2014) “Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90). For this, these genes are usually cloned into a retroviral vector and transgene-expressing viral particles or vectors, with which the somatic cell is co-transduced. However, also other techniques known to the skilled artesian can be used for that purpose. Human skin fibroblasts can also be co-transduced with all four vectors e.g. via protein transduction or naked DNA.

Further methods for obtaining iPSCs are also known to the skilled artesian and for example described in WO2009115295, WO2009144008 or EP2218778. Thus, the skilled artesian can obtain an iPSC by any method.

In principle, induced pluripotent stem cells may be obtained from any adult somatic cell (of a subject). Exemplary somatic cells include peripheral blood Mononuclear Cells (PBMCs) from blood or fibroblasts, such as for example fibroblasts obtained from skin tissue biopsies.

Therefore, it is envisioned by the present invention that the NESC is produced or derived or obtained from an induced pluripotent stem cell (iPSC). Different ways how to differentiate iPCSs into neuroepithelial stem cells are known to the skilled artesian and for example described in WO2013/104752. In addition, it is envisioned by the present invention that the iPSCs can be produced from somatic cells such as fibroblasts. Furthermore, the iPSC can be a human iPSC (hiPSC).

It is further encompassed by the present invention that the somatic cells such as fibroblasts have been obtained from a subject. The term “subject” can also mean human or an animal. The subject can also be a subject suffering from a neurodegenerative disease such as Parkinson's disease. In particular, the subject may be a subject comprising the LRRK2-G2019S mutation, which is associated with familial Parkinson's disease. The subject can also be a subject not suffering from a neurodegenerative disease such as Parkinson's disease. Also encompassed by the present invention is that the subject is a healthy subject. The subject can be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, dogs, horses, mice and rats. A mammal can be a human, dog, cat, cow, pig, mouse, rat etc. Thus, in one embodiment, the subject is a vertebrate. The subject can also be a human subject.

“Agitation” or “agitating” when used herein encompasses any technique that keeps cells in motion, i.e. cells are essentially not allowed to adhere to surfaces. Agitation can be achieved in a number of ways, including shaking, spinning, stirring, moving and/or mixing. Spinning can, for example, be achieved by the use of spinner flasks containing a magnetic paddle or impeller. Alternatively, the methods of the invention can be carried out in a bioreactor. A number of types of bioreactor are available, including bioreactors in which agitation of the medium is achieved using a paddle or impeller and rotary wall bioreactors. Rotary wall bioreactors can additionally be used to simulate conditions of reduced gravity (microgravity). It is also desirable to monitor and/or control the shear forces experienced by cells in operation of the methods of the present invention. For example, optimal conditions in cultures subjected to agitation require balancing the requirement for even distribution of oxygen and nutrients throughout the culture against the need to avoid cell damage due to excessive shear forces.

The term “activator”, as used herein, is defined as a compound/molecule enhancing or achieving the activity of a target molecule or pathway. The activator may achieve this effect by enhancing or inducing the transcription of the gene encoding the protein to be activated and/or enhancing the translation of the mRNA encoding the protein to be activated. It can also be that the protein to be activated performs its biochemical function with enhanced efficiency in the presence of the activator or that the protein to be activated performs its cellular function with enhanced efficiency in the presence of the activator. Accordingly, the term “activator” encompasses both molecules/compounds that have a directly activating effect on the specific pathway but also molecules that are indirectly activating, e.g. by interacting for example with molecules that negatively regulate (e.g. suppress) said pathway. The activator can also be an agonist of the pathway to be activated. Methods for testing if a compound/molecule is capable to induce or enhance the activity of a target molecule or pathway are known to the skilled artesian. For example, an activator of a SHH, WNT or other activator as described herein can be tested by performing Western Blot analysis of the amount of e.g. pathway effector proteins such as Gli proteins, LEF1 or TCF1 protein, respectively.

The compound/molecule that can be used as an activator can be any compound/molecule, which can activate the respective pathway or which inhibits a suppressor of the pathway to be activated. Exemplary activators can include suitable binding proteins directed e.g. against suppressors of a certain pathway.

An activator may enhance or increase the pathway to be activated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more when compared to the activity of the pathway without or before the addition of the activator.

The “Hedgehog signaling pathway” or “SHH pathway” is well known in the art and has been described, for example, in et al. (2014) “Sonic hedgehog signalling pathway: a complex network.” Ann Neurosci. 21(1):28-31. Hedgehog ligands, including, for example, Sonic hedgehog, Indian hedgehog, and/or Desert hedgehog, bind to the receptor, including, for example, Patched or the patched-smoothened receptor complex, which induces a downstream signaling cascade. Downstream target genes of SHH signaling include GLI1, GLI2 and/or GLI3. Accordingly, the term “activator of the Hedgehog signaling pathway” also refers to an activator of any one of the above recited molecules that form part of this signaling pathway.

Exemplary activators of the Hedgehog signaling (SHH) include purmorphamine (PMA; 2-(1-Naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine 9-Cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy); CAS No.: 483367-10-8), SHH, smoothened agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-benzo[b]thiophene-2-carboxamide; CAS No.: 912545-86-9) and Hh-Ag 1.5 (3-chloro-4,7-difluoro-N-(4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide; CAS No.: 612542-14-0) as well as Gli-2. The SHH-pathway activator can also be selected from the group consisting of purmorphamine, SHH, SAG Analog and Gli-2. The SHH-pathway activator can therefore be purmorphamine. The SHH pathway activator can also be a recombinant or truncated form of SHH, which retains SHH pathway activating functions such as e.g. SHH 02411.

The SHH signaling pathway activator such as purmorphamine can be employed in a concentration of between about 0.25 μM and about 1 M, more preferably between about 0.4 μM and about 0.5 μM, and most preferably the amount is about 0.5 μM.

The SHH signaling pathway activator such as SHH can also be employed between about 50 and about 1000 ng/ml. The SHH signaling pathway activator such as SHH 02411 can also be employed in a concentration of about 10 and about 500 ng/ml. The SHH signaling pathway activator such as SAG can be employed in a concentration of about 1 and about 100 nM. The SHH signaling pathway activator such as Hh-Ag1.5 can also be employed in a concentration of about 1 and about 50 nM.

The media as used in the methods of the present invention can additionally or alternatively comprise a canonical WNT-signaling activator. The canonical Wnt signaling pathway is known to the skilled artesian and for example described in Logan and Nusse (Annu. Rev. Cell Dev. Biol. (2004) 20:781-810). In short, a Wnt ligand binds to Frizzled receptors, which triggers displacement of the multifunctional kinase GSK-3β from a regulatory APC/Axin/GSK-3β-complex. In the absence of Wnt-signal (Off-state), β-catenin, is targeted by coordinated phosphorylation by CK1 and the APC/Axin/GSK-3β-complex leading to its ubiquitination and proteasomal degradation through the β-TrCP/SKP pathway. In the presence of Wnt ligand (On-state), the co-receptor LRP5/6 is brought in complex with Wnt-bound Frizzled. This leads to activation of Dishevelled (Dvl), which displaces GSK-3β from APC/Axin. The transcriptional effects of Wnt ligand is mediated via Rac1-dependent nuclear translocation of β-catenin and the subsequent recruitment of LEF/TCF DNA-binding factors as co-activators for transcription. Exemplary Wnt ligands include for example Wnt1, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt7b, and/or Wnt11.

Accordingly, the term “canonical WNT-signaling activator” as described herein refers to an activator of any one of the above recited molecules that form part of this signaling pathway.

Exemplary canonical WNT-signaling activators include Norrin, R-spondin 2 or WNT protein. However, the canonical WNT-signaling activator can also block Axin or APC. This can be achieved for example via siRNA or miRNA technology.

Exemplary canonical WNT-signaling activators can thus include Norrin, R-spondin 2 or WNT protein. However, the canonical WNT-signaling activator can also block Axin or APC. This can be achieved for example via siRNA or miRNA technology. It is also encompassed by the present invention that the canonical WNT-signaling activator is a GSK-3 inhibitor. Exemplary GSK-3 inhibitors include CHIR 99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS No.: 252917-06-9), SB-216763 (3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS No.: 280744-09-4), 6-bromoindirubin-3′-oxime (CAS No.: CAS 667463-62-9), Tideglusib (4-Benzyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione), GSK-3 inhibitor 1 (CAS No.: 603272-51-1), AZD1080 (CAS No.: 612487-72-6), TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione; CAS No.: 327036-89-5), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol; CAS No.: 601514-19-6), CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-Pyridinediamine; CAS No.: 252935-94-7), SB 415286 (3-[(3-Chloro-4-hydroxyphenyl)-amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione; CAS No.: 264218-23-7), LY2090314 (3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione; CAS No.: 603288-22-8), AR-A014418 (N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-Aurea; CAS No.: 487021-52-3 and/or IM-12 (3-(4-Fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS No.: 1129669-05-1). Thus, the GSK-3 inhibitor can also be CHIR 99021.

The canonical WNT-signaling activator such as CHIR 99021 can be employed in a concentration of between about 0.01 μM and about 1 M, more preferably between about 0.1 μM and about 5 μM, and most preferably the amount is about 3 μM. CHIR 99021 can for example be obtained from Axon Medchem.

The media as used in the methods of the present invention can for example comprise an activin/TGF-β inhibitor.

The activin/TGF-β signaling pathway is known in the art and for example described in Heldin, Miyazono and ten Dijke (1997) “TGF-bold beta signaling from cell membrane to nucleus through SMAD proteins.” Nature 390, 465-471. In short, receptor ligands, including, for example, TGFB1, TGFB2, TGFB3, ACTIVIN A, ACTIVIN B, ACTIVIN AB and/or NODAL, bind to a heterotetrameric receptor complex comprising two type I receptor kinases, including, for example, TGFBR2, ACVR2A, and/or ACVR2B, and two type II receptor kinases, including, for example, TGFBR1 (ALK5), ACVR1B (ALK4) and/or ACVR1C (ALK7). This binding triggers phosphorylation and activation of a heteromeric complex consisting of an R-smad, including, for example, SMAD2, and/or SMAD3, and a Co-smad, including, for example, SMAD4. Accordingly, the term “activator of the activin/TGF-β signaling pathway” refers to an activator of any one of the above recited molecules that form part of this signaling pathway, while the term “inhibitor of the activin/TGF-β signaling pathway” refers to inhibitors of any one of the above recited molecules that form part of this signaling pathway. In addition, such an activator can be an agonist of the ACVR2A and/or ACVR1B (ALK4) receptor or an agonist of the TGFβR11 receptor and/or ALK5 receptor. Such an inhibitor can be an antagonist of the ACVR2A and/or ACVR1B (ALK4) receptor or an antagonist of the TGFβR11 receptor and/or ALK5 receptor. In principle such inhibitors/activators of the activin/TGF-β signaling pathway are known to the skilled artesian and are commercially available.

The invention contemplates that the activin/TGF-β inhibitor is an inhibitor of the TGF-[3 type I receptor activin receptor-like kinase(s). Further envisioned by the present invention is that the activin/TGF-β inhibitor inhibits ALK5, ALK4 and/or ALK7. Exemplary but non-limiting examples of an activin/TGF-β inhibitor are A-83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; CAS No.: 909910-43-6), D4476 (44442,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide; CAS No.: 301836-43-1), GW788388 (4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide; CAS No.: 452342-67-5), LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline; CAS No.: 396129-53-6), R268712 (4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol; CAS No.: 879487-87-3), SB-431542 (4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate; CAS No.: CAS Number 301836-41-9), SB-505124 (2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate; CAS No.: 694433-59-5), SD208 (2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine; CAS No.: 627536-09-8), SB-525334 (6-[2-tert-Butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline; CAS No.: 356559-20-1) and ALK5 Inhibitor II (CAS: 446859-33-2). The activin/TGF-β inhibitor can thus be SB-431542.

The activin/TGF-β inhibitor such as SB-431542 can be employed in a concentration of between about 0.01 μM and about 1 M, more preferably between about 5 μM and about 15 μM, and most preferably the amount is about 10 μM. For example, SB-431542 can be obtained from Ascent Scientific.

The media as used in the methods of the present invention can additionally or alternatively comprise a BMP signaling inhibitor. The BMP signaling pathway is known to the skilled artesian and for example described in Jiwang Zhanga, Linheng Lia (2005) BMP signaling and stem cell regulation Developmental Biology Volume 284, Issue 1, 1 Aug. 2005, Pages 1-11.

In short, BMP functions through receptor-mediated intracellular signaling and subsequently influences target gene transcription. Two types of receptors are required in this process, which are referred to as type I and type II. While there is only one type II BMP receptor (BmprII), there are three type I receptors: Alk2, Alk3 (Bmprla), and Alk6 (Bmprlb). BMP signal transduction can take place over at least two signaling pathways. The canonical BMP pathway is mediated by receptor I mediated phosphorylation of Smad1, Smad5, or Smad8 (R-Smad). Two phosphorylated R-Smads form a heterotrimeric complex coaggregate with a common Smad4 (co-Smad). The Smad heterotrimeric complex can translocate into the nucleus and can cooperate with other transcription factors to modulate target gene expression. A parallel pathway for the BMP signal is mediated by TGFβ1 activated tyrosine kinase 1 (TAK1, a MAPKKK) and through mitogen activated protein kinase (MAPK), which also involves cross-talk between the BMP and Wnt pathways.

It is envisioned by the present invention that the inhibitors of BMP signaling can only block/reduce the canonical BMP pathway. Thus, the BMP signaling inhibitor can be a cancoical BMP signaling inhibitor. One such inhibitor selective for canocial BMP signaling pathway is dorsomorphin. Exemplary, but non-limiting, examples of BMP signaling inhibitors include chordin, noggin, DMH1 (CAS 1206711-16-1), K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol; CAS No.: 1431985-92-0), dorsomorphin (6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4-yl pyrazolo[1,5-a]pyrimidine; CAS No.: 866405-64-3) and LDN 193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride, CAS No.: 1062368-24-4). The BMP signaling inhibitor can also be dorsomorphin.

The BMP signaling inhibitor such as dorsomorphin can be employed in a concentration of between about 0.01 μM and about 1 M, more preferably between about 0.1 μM and about 5 μM, and most preferably the amount is about 0.1 μM. Dorsomorphin can for example be obtained from Tocris.

In accordance with the present invention, “Map2” refers to Microtubule-associated protein 2, a protein that in humans is encoded by the MAP2 gene. Map2 belongs to the microtubule-associated protein family. The proteins of this family are thought to be involved in microtubule assembly, which is an essential step in neuritogenesis. Human MAP2 mRNA can comprise sequence as shown in the NCBI reference NM_001039538 (SEQ ID NO: 1) and/or the protein can comprise the sequence as obtained from Uniprot No. P11137 (SEQ ID NO: 2). The term Map2 embraces any Map2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect Map2. Such methods are also described in the Examples.

In accordance with the present invention, “TH” refers to Tyrosine hydroxylase/tyrosine 3-monooxygenase/tyrosinase, a protein that in humans is encoded by the TH gene. TH is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). Human TH mRNA can e.g. comprise a sequence as shown in NCBI reference NM_000360 (SEQ ID NO: 3) and/or the protein sequence of Uniprot No. P07101 (SEQ ID NO: 4). The term TH embraces any TH nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect TH. Such methods are also described in the Examples.

In accordance with the present invention, “Tuj1” also known as “13111 Tubulin” is a protein that in humans is encoded by the TUBB3 gene. The protein 13111 Tubulin (TuJ1) is present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal percursors. Human Tuj mRNA can comprise sequence as shown in the NCBI reference NM_006086.3 (SEQ ID NO: 5) and/or the protein can comprise sequence as shown in the Uniprot No. Q13509 (SEQ ID NO: 6). The term Tuj1 embraces any Tuj1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect Tuj1. Such methods are also described in the Examples.

In accordance with the present invention, “GFAP” also known as Glial fibrillary acidic protein is a protein that in humans is encoded by the GFAP gene. Glial fibrillary acidic protein is an intermediate filament (IF) protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes. Human GFAP mRNA can comprise sequence as shown in the the NCBI reference NM_001131019 (SEQ ID NO: 7) and/or the protein can comprise sequence as shown in the Uniprot No. P14136 (SEQ ID NO: 8). The term GFAP embraces any GFAP nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect GFAP. Such methods are also described in the Examples.

In accordance with the present invention, “S100b” or “S10013” also known as S100 calcium-binding protein B is a protein that in humans is encoded by the S100 gene. S100 proteins are localized in the cytoplasm and nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Human S100b mRNA can comprise sequence as shown in the NCBI reference NM 006272 (SEQ ID NO: 9) and/or the protein can comprise sequence as shown in the Uniprot No. P04271 (SEQ ID NO: 10). The term S100b embraces any S100b nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect S100b. Such methods are also described in the Examples.

In accordance with the present invention, “PAX6” also referred to as Paired box protein Pax-6 also known as aniridia type II protein (AN2) or oculorhombin is a protein that in humans is encoded by the PAX6 gene. Pax6 is a transcription factor present during embryonic development. The encoded protein contains two different binding sites that are known to bind DNA and function as regulators of gene transcription. It is a key regulatory gene of eye and brain development. Human PAX6 mRNA can comprise any sequence encoding for SEQ ID NO: 11. Human PAX6 can also comprise the protein sequence as shown by Uniprot No. P26367 (SEQ ID NO: 11). The term PAX6 embraces any PAX6 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect PAX6. Such methods are also described in the Examples.

In accordance with the present invention, “SOX1” also referred to as “SOX1 Sex determining region Y-box 1” is a protein that in humans is encoded by the SOX1 gene. SOX1 (for Sex determining region Y-box 1) is a transcription factor in the Sox protein family. Human SOX1 mRNA can comprise any sequence encoding for SEQ ID NO:12. SOX1 can also comprise the protein sequence of Uniprot No. 000570 (SEQ ID NO: 12). The term SOX1 embraces any SOX1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect SOX1. Such methods are also described in the Examples.

In accordance with the present invention, “SOX2” also known as sex determining region Y-box 2, is a protein that in humans is encoded by the SOX2 gene. SOX2 is a transcription factor that is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic stem cells. Sox2 has a critical role in maintenance of embryonic and neural stem cells. Human SOX2 mRNA can comprise any sequence encoding for SEQ ID NO: 13. SOX2 can also comprise the protein sequence of Uniprot No. P48431 (SEQ ID NO: 13). The term SOX2 embraces any SOX2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect SOX2. Such methods are also described in the Examples.

In accordance with the present invention, “nestin” is a protein that in humans is encoded by the NES gene. Nestin is a type VI intermediate filament (IF) protein. Human nestin mRNA can comprise any sequence encoding for SEQ ID NO: 14. Nestin can also comprise a protein sequence such as depicted by Uniprot No. P48681 (SEQ ID NO: 14). The term nestin embraces any nestin nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect nestin. Such methods are also described in the Examples.

In accordance with the present invention, “LMX1A” (LIM homeobox transcription factor 1, alpha) is a protein that in humans is encoded by the LMX1A gene. LMX1 is a LIM homeobox transcription factor that binds an A/T-rich sequence in the insulin promoter and stimulates transcription of insulin. Human LMX1A mRNA can comprise any sequence encoding for SEQ ID NO: 15. LMX1A can also comprise a protein sequence such as depicted by Uniprot No. Q8TE12 (SEQ ID NO: 15). The term LMX1A embraces any LMX1A nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect LMX1A. Such methods are also described in the Examples.

In accordance with the present invention, “FOXA2” is a protein that in humans is encoded by the FOXA2 gene. Forkhead box protein A2 is a member of the forkhead class of DNA-binding proteins. Human FOXA2 mRNA can comprise any sequence encoding for SEQ ID NO: 16. FOXA2 can also comprise a protein sequence such as depicted by Uniprot No. Q9Y261 (SEQ ID NO: 16). The term FOXA2 embraces any FOXA2 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect FOXA2. Such methods are also described in the Examples.

In accordance with the present invention, “LMX1B” is a protein that in humans is encoded by the LMX1B gene. LMX1B is a LIM homeobox transcription factor which plays a central role in dorso-ventral patterning of the vertebrate limb. Human FOXA2 mRNA can comprise any sequence encoding for SEQ ID NO: 17. LMX1B can also comprise a protein sequence such as depicted by Uniprot No. 060663 (SEQ ID NO: 17). The term LMX1B embraces any LMX1B nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect LMX1B. Such methods are also described in the Examples.

In accordance with the present invention, “NURR1” is a protein that in humans is encoded by the NR4A2 gene. NURR1 is a member of the nuclear receptor family of intracellular transcription factors. NURR1 plays a key role in the maintenance of the dopaminergic system of the brain. Human NURR1 mRNA can comprise any sequence encoding for SEQ ID NO: 18. NURR1 can also comprise a protein sequence such as depicted by Uniprot No. P43354 (SEQ ID NO: 18). The term NURR1 embraces any NURR1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect NURR1. Such methods are also described in the Examples.

In accordance with the present invention, “AADC” is an Aromatic L-amino acid decarboxylase. Human AADC mRNA can comprise any sequence encoding for SEQ ID NO: 19. AADC can also comprise a protein sequence such as depicted by Uniprot No. P20711 (SEQ ID NO: 19). The term AADC embraces any AADC nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect AADC. Such methods are also described in the Examples.

In accordance with the present invention, “EN1” is a protein that in humans is encoded by the EN1 gene. Engrailed (EN) is a homeodomain transcription factor involved in many aspects of multicellular development. Human EN1 mRNA can comprise any sequence encoding for SEQ ID NO: 20. EN1 can also comprise a protein sequence such as depicted by Uniprot No. Q05925 (SEQ ID NO: 20). The term EN1 embraces any EN1 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof. The skilled person knows how to detect EN1. Such methods are also described in the Examples.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms “about” and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

The following sequences are mentioned herein:

I. Material and Methods

1. Cell Culture

Cell culture work was performed under sterile conditions using a laminar flow hood. All cells were cultured in an incubator at constant 37° C. and 5% CO₂. For cells that were grown under 2D conditions, cell culture plates (NUNC™, THERMO SCIENTIFIC™) were coated with MATRIGEL® (CORNING) resuspended in cold Knockout DMEM (LIFE TECHNOLOGIES). 1.5 ml of diluted MATRIGEL was added to each well of a 6-well-plate. The plates were kept at room temperature (RT) overnight. Coated plates were stored at 4° C. for up to one month.

2. Generation of Cerebral Organoids

Cerebral organoids were generated according to the established protocol by Lancaster et al. (Lancaster and Knoblich, 2014a; Lancaster et al., 2013) with slight modifications. Feeder-independent human iPS cells were used as a starting population instead of feeder-dependent iPSCs. As an initial step, embryoid bodies (EBs) were generated. These 3D aggregates undergo cell specification through differentiation into the three germ lineages endoderm, mesoderm, and ectoderm, similar to the embryonic development described in Section 1.1 of the Introduction. Next, the formation of neuroepithelial tissue was initiated, restricting the cell fate to the neural lineage. Once the neuroepithelial tissue developed, the 3D structures were transferred to droplets of MATRIGEL, which gives structural support and helps the tissue to maintain its 3D shape. This matrix, derived from the Engelbroth-Holm-Swarm mouse sarcoma, is composed of laminin, collagen IV, nidogen/enactin, and proteoglycan, thus resembling the extracellular matrix (ECM). The MATRIGEL-embedded tissue was cultured in medium that favoured neuronal differentiation by using Neurobasal medium supplemented with N2 and B27 containing vitamin A. Non-essential amino acids and GlutaMax™ were added in order to increase cell growth and viability. The reducing agent 2-Mercaptoethanol prevented the formation of toxic oxygen radical levels. The tissue was kept under dynamic conditions to prevent the tissue from attaching to the bottom. It further increases the nutrient exchange and controls waste product exchange. For this purpose, Lancaster et al. used a spinning bioreactor. In this project, the tissue was kept in a non-treated 10 cm cell culture petri dish (GREINER) on an orbital shaker (IKA®), spinning at around 80 rpm.

2.1 Maintenance of Human Induced Pluripotent Stem Cells

A wild type human induced pluripotent stem cell line from CORIELL was seeded on a cell culture 6-well-plate and cultured in Essential8™-medium (LIFE TECHNOLOGIES). At 70-80% confluence and within one week after the last seeding, cells were passaged. To detach the cells from the surface, the medium was aspirated and 0.5 mM EDTA (INVITROGEN) was added. After an incubation time of 4 min at 37° C. and 5% CO₂, EDTA was aspirated. Cells were washed with PBS, and resuspended in Essential8™-medium. Cells were seeded into a new coated 6-well-plate in a ratio of 1:3-1:6. Medium was changed every day.

2.2 Immunocytochemical Characterisation of hiPSCs

Human iPS cells were seeded on cover slips in a 24-well-plate and cultured under the conditions described in section 2.1. After three days, cells were fixed with 4% paraformaldehyde (PFA) for 40 min at 4° C. and washed three times with PBS for 5 min. The cell membrane was permeabilised with 0.2% Triton-X-100 at RT for 10 min. After three washing steps with 0.05% Triton-X-100 in PBS, cells were blocked with 2% NGS+2% BSA in 0.05% Triton-X-100 in PBS at RT for 60 min. Primary antibodies were diluted in the blocking buffer (see Table 1). 35 μl drops of primary antibody solution were prepared on Parafilm in a wet chamber. Cover slips were placed upside down on the drops and incubated at 4° C. overnight.

The next day, the cover slips were washed with three changes of PBS for 5 min. Secondary antibodies raised against primary antibodies (Table 2) were diluted together with Hoechst dye (INVITROGEN, 1:10000) and drops on Parafilm were prepared as before. The cover slips were incubated for 1 h at RT and then washed three times with PBS, once with H₂O, and mounted on glass slides using fluorescence mounting medium. The stainings were analysed using a confocal laser scanning microscope (ZEISS LSM 710).

2.3 Generation of EBs

For the generation of EBs, colonies were washed once with PBS and detached using 0.5 mM EDTA (INVITROGEN). After an incubation period of 4 min at 37° C., EDTA was replaced by 1 ml pre-warmed StemPro® Accutase® (LIFE TECHNOLOGIES) and cells were incubated for another 4 min at 37° C. The colonies were detached from the dish using a 1 ml pipette tip with 1 ml of Essential8™-medium (LIFE TECHNOLOGIES). The cell suspension was transferred to a Falcon tube and triturated in order to create a single cell suspension. Two repetitions of 5 μl were collected for cell counting. While the cells were centrifuged at 270 g for 5 min at RT, cells were counted using an automated cell counter (Countess II FL LIFE TECHNOLOGIES) with trypan blue. After centrifugation, the supernatant was aspirated and cells were resuspended in 1 ml low-FGF2 hESC medium containing ROCK inhibitor (Table 3). Next, low-FGF2 hESC medium was added in order to obtain 9000 live cells per 150 μl. Finally, 150 μl were plated to each well of a round-bottom ultralow attachment 96-well-plate (CORNING), which allows the cells to settle down and favours the formation of EBs. Cells were cultured in an incubator at 37° C. and 5% CO₂. Medium was changed every other day by replacing half of the medium with fresh low-FGF2 hESC medium. ROCK inhibitor and low-FGF2 were added only for four days.

2.4 Neural induction

After six days, EBs with smooth edges that brightened at the border were transferred with a cut pipette tip to an ultralow attachment 24-well-plate (CORNING) containing neural induction medium (Table 4) and cultured for four days.

2.5 Transferring Neuroepithelial Tissue to MATRIGEL Droplets

To prepare a substrate that helps to generate MATRIGEL droplets, a square of Parafilm was placed over an empty tip tray for 200 μl pipette tips and small dimples were created by pressing a gloved finger into the Parafilm over the holes. The dimpled Parafilm was sprayed once again with ethanol and dried in a petri dish under the laminar flow hood. Once the Parafilm was dry, Neuroepithelial tissue was transferred into each dimple and the medium was gently removed. Next, 30 μl of MATRIGEL was added carefully and the tissue was placed in the centre of the droplet (FIG. 2.1). To allow the MATRIGEL to polymerise, it was incubated at 37° C. for 30 min. After the incubation time, the droplets were collected in 15 ml cerebral organoid differentiation medium without vitamin A (Table 5). To remove the MATRIGEL droplets from Parafilm, sterile forceps were used to hold the sheet while shaking the dish gently until the drops fell off. The dish was kept in an incubator under static conditions and the medium was changed after 48 h. After four days in static culture, the medium was changed again with fresh differentiation medium containing vitamin A. Finally, the dishes were placed on an orbital shaker installed in the incubator, shaking at approximately 80 rpm, and medium was changed every third or fourth day.

3. Generation of Midbrain Organoids

3.1 Human Neuroepithelial Stem Cells

To generate midbrain organoids, human neuroepithelial stem cells (hNESCs) served as a starting population. These neural progenitor cells have properties of stem cells, as they are capable of a robust, immortal expansion and can differentiate into cells of the CNS, including neurons, astrocytes, oligodendrocytes, and also into neural crest lineages. They solely require small molecules for self-renewal and expansion. Neural induction was initiated through the inhibition of non-neural BMP and dorsalising TGF-β signalling. To maintain the immortal self-renewal state, WNT and SHH signals were triggered. WNT signalling induces the formation of cells at the lateral border of the neural plate, while its antagonist SHH specifies ventral neural tube fates. CHIR99021 was used to stimulate the canonical WNT signalling pathway, and purmorphamine (PMA) was added to stimulate the SHH pathway. hNESCs can be efficiently differentiated into motor neurons and mDAs, designating them as a powerful tool to study early human development and neurodegenerative diseases.

3.2 Maintenance of Human Neuroepithelial Stem Cells

A wildtype human neuroepithelial stem cell line (hNESC-K7) that was derived from human induced pluripotent stem cells served as a starting population (Reinhardt et al., 2013). Cells were cultured in freshly supplemented N2B27 maintenance medium (Table 6 and Table 7). Cells were passaged at 80-90% confluence and within one week after the last seeding. To detach the cells from the surface, the medium was replaced by 700 μl warm StemPro® Accutase® (LIFE TECHNOLOGIES) and cells were incubated at 37° C. and 5% CO₂for 4-6 min. Cells were resuspended in 5 ml warm DMEM/F12 (LIFE TECHNOLOGIES) and centrifuged at 200 g for 3 min. After aspirating the supernatant, the pellet was resuspended in supplemented N2B27-maintenance medium and cells were seeded into a new plate in a ratio of 1:10-1:20, depending on the confluence. Medium was changed every other day.

3.3 Immunocytochemical Characterisation of hNESCs

hNESCs were seeded on cover slips in a 24-well-plate and cultured under the conditions described in Section 3.2. After 3 days, cells were fixed with 4% PFA for 40 min at 4° C. and washed three times with PBS for 5 min. ICC was performed according to the protocol described in Section 2.2 with different primary antibodies (Table 8) and the according secondary antibodies (Table 2).

3.4 Generation and Expansion of Three-Dimensional hNESC Colonies

In order to generate single 3D hNESC colonies, hNESCs were passaged as described in section 3.2. After centrifugation and resuspension in 1 ml N2B27 maintenance medium, cells were counted using an automated cell counter (Countess II FL LIFE TECHNOLOGIES) with trypan blue. N2B27 maintenance medium was added in order to obtain 9000 live cells per 150 μl. 150 μl were plated to each well of a round-bottom ultralow attachment 96-well-plate (CORNING), which allows the cells to form one single colony per well. Medium was changed every other day by replacing half of the medium with fresh N2B27 maintenance medium. After six days, the colonies were transferred to an ultralow attachment 24-well-plate (CORNING) in order to remove side colonies and to allow the tissue to expand. After two days, the colonies were embedded into MATRIGEL droplets in the same manner as described in Section 2.5. The MATRIGEL-embedded colonies were cultured for two more days under static maintenance conditions in 10 cm petri-dishes, before differentiation was initiated at day 10 with N2B27 differentiation medium containing PMA (Table 9). The differentiation protocol was adapted from Reinhardt et al. 2013 with a slight modification. FGF8 was not added for the first eight days, since this decreases the differentiation efficiency into mDA neurons. After two days under differentiation conditions, the dishes were placed on an orbital shaker and kept under dynamic conditions at approximately 80 rpm. Medium was changed every third or fourth day. PMA was only added the first six days of differentiation.

4. Immunohistochemical Characterisation of Human Cerebral and Midbrain Organoids

During brain development, cells rearrange themselves and form different functional and interdependent regions. Simultaneously, neuroepithelial cells differentiate into the various neurons and supportive glial cells of the CNS. In order to analyse whether cerebral and midbrain organoids develop different cell types and brain regions characteristic of the developing human brain, immunohistochemical (IHC) stainings were performed. The presence of neural progenitor cells, young and mature neurons, astrocytes, oligodendrocytes, dopaminergic neurons was analysed. Moreover, immunofluorescence stainings were performed to verify whether cerebral and midbrain organoids established midbrain identity.

4.1 Sectioning

At day 6 (n=3), 7 (n=3), 16 (n=3), 30 (n=3), and 44 (n=8), cerebral and midbrain organoids were fixed in 4% PFA at RT on a shaker overnight. Bigger organoids with a diameter >900 μm were sectioned prior to IHC. The fixed tissue was washed three times in PBS for 15 min and embedded into warm 3% low-melting point agarose and allowed to cool until solid. The block of agarose was then trimmed and glued on a metal block holder, and sectioned using a vibratome (LEICA, VT100 S) to 150 μm thickness at a speed of 0.5 mm/s and a frequency of 70 Hz.

4.2 IHC

The free floating agarose sections were collected in 24-well-plates containing TBS+++(lx Tris-buffered saline with 0.5% Triton-X-100, 0.1% sodium azide, 0.1% sodium citrate and 5% fetal bovine serum or normal goat serum) and blocked/permeabilised for at least 1 h at RT on a shaker. Primary antibodies (see Table 10) were diluted in TBS+++ and 300 μl of the antibody solution were added to each well. The sections were incubated for 48 h at 4° C. on a shaker and afterwards washed with three changes of TBS for 15 min. The sections were incubated in secondary antibodies (see Table 11) diluted in TBS+++ for 2 h at RT on a shaker, which was followed by three washing steps in TBS for 15 min. Finally, the sections were rinsed with H₂O and mounted with mounting medium on a glass slide. The stainings were analysed using a confocal laser scanning microscope (ZEISS LSM 710).

II. Results

1. Generation of Midbrain Organoids

1.1 Immunocytochemical Characterisation of hNESCs

ICC of human neuroepithelial stem cells revealed a stable expression of the neural progenitor markers SOX2 and NESTIN under maintenance conditions. Another neural progenitor marker, SOX1, was also expressed in hNESCs, though fluorescence intensities varied. FOXA2, a marker of the ventral neural tube, was slightly expressed in hNESCs. Upon early differentiation, few hNESCs differentiate into neurons that express neuron-specific class III beta-tubulin (TUJ1) and doublecortin (DCX), which mark early neurons (FIG. 3).

1.2 Development of Midbrain Organoids

For the generation of midbrain organoids, the wild type hNESC line K7 was used as a starting population. Human NESCs seeded on round-bottom ultralow attachment plates formed dense globular colonies with only few dead cells around. In some wells, small side colonies developed, which then merged with the main colony after some days. The colonies began to brighten within the first three days and showed smooth edges under maintenance conditions, indicating healthy tissue. However, early organoids at day 8 developed a dark core in the centre, indicative of cell death. (FIG. 4a, b). After two days of differentiation, the organoids developed small processes along the surface of the tissue (FIG. 4c). Within three weeks, the processes expanded and reached a length of approximately 1 mm with only few cell bodies outside the colony. This was not observed in colonies without MATRIGEL support (FIG. 4d-f).

1.3 Immunohistochemical Characterisation of Human Midbrain Organoids

Early midbrain organoids at day 6 and day 7 under maintenance conditions were stained without sectioning. The developing three dimensional human NESC colonies showed a stable expression of the neural progenitor markers SOX2 and NESTIN, as well as early differentiated young neurons (FIG. 5). Throughout the colonies, small cavities enclosed by radially organised progenitor cells developed.

Midbrain organoids at later stages under differentiation conditions developed a dense neuronal network, which gradually increased in complexity, consistent with gross morphological changes. Immunofluorescence staining with markers of dopaminergic neurons (TUJ1/TH) revealed that already at day 16 some DA neurons developed, increasing in density with later stages. Notably, DA neurons were located in distinct areas, which is suggestive of an asymmetric polar organisation of the 3D structures, consistent with brain development in vivo (FIG. 6, FIG. 7). To further analyse DA neurons in midbrain organoids, staining for the mature neuronal marker MAP2 together with TH was performed. A staining of different sections of the same organoid revealed that mature neurons were located at the outermost part of the organoid, while young TUJ1 positive neurons were more abundant in the inner core (FIG. 7a, c). Interestingly, some of the mature neurons also expressed TH, revealing the presence of mature DA neurons in midbrain organoids. Additionally, staining for neural progenitor marker SOX2 showed that some neural progenitor cells surrounded the inner core of the organoid at later stages. TH positive DA neurons were located adjacent to the layer of neural precursor cells (FIG. 7).

To further examine the cellular organisation of midbrain organoids, stainings of glial cell markers including GFAP (astrocytes) and O4 (oligodendrocytes) were performed. In some regions of midbrain organoids, O4 positive and TUJ1 negative cells were identified, indicating the development of oligodendrocytes in late-stage midbrain organoids (FIG. 8a-c). Only few astrocytes were found in midbrain organoids. However, some GFAP positive processes without associated cell bodies were observed across the tissue (FIG. 8c), as well as few GFAP positive cells (FIG. 8d)

Finally, midbrain organoids at later stages were stained for midbrain markers LMX1A, FOXA2, and EN1. Within the analysed sections and organoids, no cells expressing these transcription factors were found. Some cells showed evidence of LMX1A expression, though it was not located in the nucleus and diffuse, potentially revealing non-specific binding. Altogether the data indicate that midbrain organoids develop various neural cell types and exhibit an asymmetric polarisation of the tissue.

2. Further Generation of Human Midbrain-Specific Organoids

2.1 Materials and Methods

2.1.1 Midbrain Organoid Culture

The hiPSC-derived hNESCs were cultured as previously described (Reinhardt, et al. “Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.” PloS one 8, e59252). On day 0 of the organoid culture, hNESCs at passage <20 were treated with accutase for 5 min at 37° C., followed by gentle pipetting to generate single cells. A total of 9000 cells were seeded into each well of an ultra-low attachment 96-well round bottom plate (Corning) and cultured in N2B27 media supplemented with 3 μM CHIR-99021 (Axon Medchem), 0.75 μM purmorphamine (Enzo Life Science) and 150 μM ascorbic acid (Sigma) (referred to as N2B27 maintenance media). N2B27 medium consists of DMEM-F12 (Invitrogen)/Neurobasal (Invitrogen) 50:50 with 1:200 N2 supplement (Invitrogen), 1:100 B27 supplement lacking Vitamin A (Invitrogen), 1% L-glutamine and 1% penicillin/streptomycin (Invitrogen). The medium was changed every other day for 6 days, and 3D colonies were then transferred to ultra-low attachment 24-well plates (Corning) and cultured in N2B27 maintenance media.

On day 8 of the organoid culture, the 3D colonies were transferred to droplets of hESC-qualified Matrigel (BD Bioscience) as previously described (Lancaster and Knoblich (2014) “Generation of cerebral organoids from human pluripotent stem cells.” Nature protocols 9, 2329-2340). Droplets were cultured in N2B27 maintenance media either in 10-cm Petri dishes for short-term cultures or in ultra-low attachment 24-well plates (Corning) with one droplet per well for long-term cultures. On day 10, differentiation was initiated with N2B27 media supplemented with 10 ng/ml hBDNF (Peprotech), 10 ng/ml hGDNF (Peprotech), 500 μM dbcAMP (Peprotech), 200 μM ascorbic acid (Sigma), and 1 ng/ml TGF-83 (Peprotech). Additionally, 1 μM purmorphamine (Enzo Life Science) was added to this medium for an additional 6 days. On day 14 of the organoid culture, the plates were placed on an orbital shaker (IKA), rotating at 80 rpm, in an incubator (5% CO₂, 37° C.) and the organoids were kept in culture with media changes every second or third day.

2.1.2 Immunohistochemical Analysis

Organoids were fixed with 4% paraformaldehyde overnight at RT and washed 3× with PBS for 1 h. Afterwards, they were embedded in 3% low-melting point agarose in PBS and incubated for 15 min at 37° C., followed by 30 minutes incubation at RT. The solid agarose block was covered with PBS and kept overnight at 4° C. If not indicated otherwise, 50 μm sections were cut using a vibratome (Leica VT1000s), and sections were permeabilized with 0.5% Triton X-100 in PBS and blocked in 2.5% normal goat or donkey serum with 2.5% BSA, 0.1% Triton X-100, and 0.1% sodium azide. Sections were incubated on a shaker for 48-72 h at 4° C. with primary antibodies in the blocking buffer at the following dilutions: rabbit anti-TH (1:1000, Abcam), chicken anti-TH (1:1000, Abcam), rabbit anti-TH (1:1000, Santa Cruz Biotechnology), goat anti-SOX2 (1:200, R&D Systems), rabbit anti-SOX2 (1:100, Abcam), goat anti-SOX1 (1:100, R&D Systems), mouse anti-nestin (1:200, BD), mouse anti-Ki67 (1:200, BD), rabbit anti-CC3 (1:200, Cell Signalling), mouse anti-FOXA2 (1:250, Santa Cruz Biotechnology), rabbit anti-LMX1A (1:200, Abcam), chicken anti-GFAP (1:1000, Millipore), mouse anti-S100β (1:1000, Abcam), mouse anti-TUJ1 (1:600, Covance), rabbit anti-TUJ1 (1:600, Covance), chicken anti-TUJ1 (1:600 Millipore), rabbit anti-PAX6 (1:300, Covance), mouse anti-synaptophysin (1:50 Abcam), rabbit anti PSD-95 (1:300, Invitrogen), mouse anti-MAP2 (1:200, Millipore), mouse anti-CNPase (1:200, Abcam), and mouse anti-O4 (1:400, Sigma). After incubation with the primary antibodies, sections were washed three times in 0.05% Triton X-100 and blocked for 30 min at RT on a shaker, followed by incubation with the secondary antibodies in 0.1% Triton X-100 (1:1000). All secondary antibodies (Invitrogen) were conjugated to Alexa Fluor fluorochromes. Dopamine was detected using a STAINperfect Immunostaining Kit (ImmuSmol) according to manufacturer's protocol. Sections were co-stained with chicken anti-TH primary antibody (Abcam), and nuclei were counterstained with Hoechst 33342 (Invitrogen). Sections were mounted in Fluoromount-G mounting medium (Southern Biotech) and analyzed with a confocal laser scanning microscope (Zeiss LSM 710). Images were further processed with Zen Software (Zeiss) and ImageJ. Three-dimensional surface reconstructions of confocal z-stacks were created using Imaris software (Bitplane). The asymmetric distribution of DNs was assessed based on fluorescence intensities with the ImageJ Interactive 3D surface plot plugin.

2.1.3 Quantitative Real-Time PCR

Total RNA was isolated from 48 days-old organoids. Typically, five organoids were pooled for one isolation. For dissociation, the organoids were washed once with PBS and lysed with QIAzol lysis reagent (Qiagen), passed through a needle three times and homogenized with QIAshredder columns (Qiagen). RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Subsequently, isolated RNA was reverse-transcribed following the protocol of the High Capacity RNA to DNA Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reactions (qRT-PCRs) were conducted with the Maxima SYBR Green qPCR Master Mix (Thermo Scientific). Amplification of 1 μg cDNA was performed in an AriaMx Real-time PCR System (Agilent Technologies) as follows: An initial denaturing step, 10 min at 95° C., 40 cycles of denaturation for 15 s at 95° C., annealing for 30 s at 60° C., and elongation for 30 s at 72° C. The expression levels were normalized relative to the expression of the housekeeping gene RPL37A using the comparative Ct-method 2-ΔΔCt. To evaluate the expression patterns of midbrain organoids, the values were compared to the expression levels of hNESCs, which were set to 1. The quality of the PCR products was assessed by melting curve analysis.

2.1.4 Evaluation of Electrophysiological Activity

Calcium imaging and multielectrode array (MEA) recording was used to analyze the spontaneous activity of organoids at day 50-52 and day 60-70, respectively. A concentration of 5 μM cell permeant Fluo-4 AM (Life Technologies) in a neurobasal medium was added to the well and incubated for 45 min at 37° C. on an orbital shaker. Fluorescent images were acquired using a live cell spinning disk confocal microscope (Zeiss) equipped with a CMOS camera (Orca Flash 4.0, Hamamatsu). Calcium time-series were acquired at 5 Hz for approximately 2 min and stored as single images. These images were analyzed using the ADINA toolbox (Diego et al. (2013) “Automated identification of neuronal activity from calcium imaging by sparse dictionary learning.” Proceedings of the IEEE 10th International Symposium on Biomedical Imaging, pp. 1058-1061), which is publicly available software that has been developed to automatically segment individual cell bodies and separate the overlapping ones. Fluorescent traces, expressed as relative changes in the fluorescence intensity (AF/F), were then measured for segmented cell bodies.

MEA recording was conducted using the Maestro system from Axion BioSystems. A 48-well MEA plate containing a 16-electrode array per well was precoated with 0.1 mg/ml poly-D-lysine hydrobromide (Sigma-Aldrich) and subsequently coated with 10 μg/ml laminin (Sigma-Aldrich) for 1 h at room temperature (RT). Midbrain organoids were placed onto the array after day 60-70. A coverslip was placed on top to ensure the contact of the free floating organoid with the electrodes. Spontaneous activity was recorded at a sampling rate of 12.5 kHz for 5 min for up to five days at 37° C. in neuronal maturation media. Using Axion Integrated Studio (AxIS 2.1), a Butterworth band pass filter with 200-3000 Hz cutoff frequency and a threshold of 6×SD were set to minimize both false-positives and missed detections. The Neural Metric Tool (Axion BioSystems) was used to analyze the spike raster plots. Electrodes with an average of spikes/min were defined as active. The spike count files generated from the recordings were used to calculate the number of spikes/active electrode/measurement. Further details regarding the MEA system were previously described (Bardy et al. (2015) “Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.” Proceedings of the National Academy of Sciences of the United States of America 112, E2725-2734).

2.2 Results

Previously described neuroepithelial stem cells (Reinhardt et al. “Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.” PloS one 8, e59252) were used as the starting population for the generation of human midbrain organoids. Compared to iPSCs as a starting population, NESCs are already patterned towards midbrain/hindbrain identity.

Typically, NESCs express the neural progenitor markers SOX1, SOX2, PAX6, and NESTIN prior to organoid generation. Cells were seeded on round-bottom ultralow adhesion 96-well plates enabling the cells to form three-dimensional colonies. They were cultured in the presence of the GSK3b inhibitor CHIR99021 to stimulate the canonical WNT signaling pathway, and the SHH pathway was activated using purmorphamine (PMA). On day 8, the three-dimensional NESC colonies were embedded into droplets of Matrigel for structural support, and two days after, specification into midbrain organoids was initiated by changing the media to differentiation media. We kept the organoids in 10 cm Petri dishes for short-term cultures or in ultralow adhesion 24-well plates for long-term cultures and placed them on an orbital shaker rotating at approximately 80 rpm (FIG. 9A).

After about 27 days of the whole procedure (after about 16 days of differentiation into midbrain organoids), the early midbrain organoids widely expressed the cell proliferation marker Ki67, which decreased upon maturation (FIG. 9B). Furthermore, these organoids expressed the neural progenitor marker SOX2, which also decreases during maturation and becomes more regionally restricted, resembling the formation of a stem cell niche (FIG. 9C). Importantly, the generation of midbrain organoids from NESCs could be reproduced without significant variation.

2.1 Neuronal Differentiation and Self-Organization of Human Neuroepithelial Stem Cell Derived Midbrain Organoids

After showing a decrease of proliferation and stem cell identity, neuronal differentiation and specification of midbrain dopaminergic neurons (mDNs) was assessed. Robust differentiation into TUJ1 positive neurons and TH positive DNs could be observed. These stainings revealed the formation of a complex neuronal network (FIG. 10A, 2B). Double-positive staining of the mature neuronal marker MAP2/TH demonstrated the maturation of DNs within the organoids. It was further examined whether NESC-derived organoids undergo differentiation into DNs with midbrain identity. In late-stage organoids, a large population of TH-, LMX1A-, and FOXA2-positive neurons was observed (FIG. 10C-E). qRT-PCR further revealed the upregulation of mDN differentiation markers, including LMX1A, LMX1B, EN1, NURR1, AADC, and TH (FIG. 10F, 10G). These data indicate that the obtained dopaminergic neurons indeed have midbrain identity.

To examine the degree of spatial organization in NESC-derived midbrain organoids, we evaluated the distribution pattern DA neuronal markers TUJ1/TH and depicted the results using surface plots. Strikingly, we found that DA neurons form clearly specified clusters within midbrain organoids (FIG. 10H, 10I).

To further demonstrate the identity of TH positive neurons as dopaminergic, we analyzed their ability to produce the neurotransmitter dopamine. Immunostainings of mature organoids demonstrated the presence of dopamine and TH-double positive cells (FIG. 10J). These results indicated that mDNs of NESC-derived organoids self-organize into a complex, spatially patterned and functional neuronal tissue.

3. Glial Differentiation in Midbrain Organoids

During the development of the fetal human brain, neural tube-derived cells not only differentiate into neurons but also into glia cells, including astrocytes and oligodendrocytes. Therefore, the presence of these glia cells in the midbrain organoids was investigated. In good agreement with brain development, where glia differentiation temporally follows neuronal differentiation, no significant amounts of glia cells in organoids (day 27 of the whole procedure, about 16 days after starting differentiation into midbrain organoid/early organoids) were detected. However, in more mature organoids (day 61 of the whole procedure, after about 51 days of differentiation into midbrain organoids), we observed astrocytes positive for the markers S10013 and GFAP. Interestingly, populations of astrocytes in both a quiescent state (negative for GFAP) and a reactive state characterized by GFAP expression were obtained (FIG. 11A, B).

Moreover, a fraction of cells that differentiated into O4-positive oligodendrocytes at day 44 of the whole procedure (at about 34 days of differentiation into midbrain organoids) was detected. Furthermore, interestingly, these oligodendrocytes typically showed a spatially asymmetric distribution within the organoids (not shown). In the central nervous system, mature oligodendrocytes form myelin sheaths that enwrap axons to accelerate the transmission of action potentials along axons. To analyze if the oligodendrocytes within the midbrain organoids are able to execute their actual function, i.e., formation of myelin sheets, we performed immunofluorescence staining against 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), a myelin-associated enzyme together with the neuronal marker TUJ1. A 3D surface reconstruction of these stainings revealed numerous TUJ1-positive neurites that were ensheathed by myelin sheets of CNPase-positive oligodendrocytes (FIG. 11C). Interestingly, these neurites often showed gaps of ensheathment, resembling the formation of nodes of Ranvier (FIG. 11C) that allow for saltatory fast neuronal transmission.

4. Functionality of Midbrain Organoids

One important requirement for neuronal transmission is the development of a mature neuronal network via the formation of synaptic connections. Therefore, synaptic connectivity was investigated using immunohistological staining against the presynaptic marker synaptophysin and the postsynaptic marker PSD95 at day 61 (51 days of differentiation into midbrain organoids). A subsequent 3D surface reconstruction demonstrated not only the formation of numerous pre- and postsynaptic puncta but also multiple synaptic connections (FIG. 11D). Synaptic connections have been developed between different neurites, indicated by the direct contact of synaptophysin-positive presynapses with PSD95-positive postsynapses (FIG. 11E). Accordingly, midbrain organoids exhibit the ability to forward signals via synaptic connections and thus fulfill the prerequisite for being electrophysiologically functional.

To further confirm their functionality and neuronal network activity, we performed Fluo-4AM calcium imaging on whole organoids. We measured the spontaneous neuronal activity based on calcium transients evoked by action potentials (FIG. 12A, B). Notably, some of the fluorescent traces showed regular firing patterns, which were indicative of tonic electrophysiological activity and resembled the pacemaker activity of dopaminergic neurons (Moreno et al. (2015), “Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.” Lab on a chip 15, 2419-2428; Cummings et al. (2014)” Alzheimer's disease drug-development pipeline: few candidates, frequent failures.” Alzheimer's research & therapy 6, 37). In addition to calcium imaging, a multielectrode array (MEA) system was used to examine the electrophysiological activity. This methodology allows non-invasive recordings of extracellular field potentials generated by action potentials. At day 60-70 (50-60 of differentiation), the midbrain organoids were placed on a grid of 16 electrodes in a 48-well tissue culture plate (FIG. 12C). Spontaneous activity was detected over several days by individual electrodes in the form of mono- and biphasic spikes (26.12±5.1 spikes/active electrode (n3), FIG. 12D,E). Furthermore, spikes occurred close in time on multiple electrodes, which represents neuronal network synchronicity (FIG. 12F). These findings indicate that midbrain organoids develop functional synaptic connections and show spontaneous neuronal activity.

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