Dopaminergic neural progenitor cells or cell derivatives thereof obtained from lineage-restricted pluripotent stem cells

Abstract

The present invention relates to pluripotent stem cells that have been restricted in their ability to differentiate into cell lineages distinct from dopaminergic neural progenitor cells and their derivatives by specific gene knockouts. In particular, the present invention relates to dopaminergic neural progenitor cells or cell derivatives thereof obtained from these lineage-restricted pluripotent stem cells, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to pluripotent stem cells that have been restricted in their ability to differentiate into cell lineages different from dopaminergic neural progenitor cells or their cellular derivatives by specific gene knockouts. In particular, the present invention relates to dopaminergic neural progenitor cells or their cellular derivatives obtained from these lineage-restricted pluripotent stem cells, and uses thereof. [Background technology]

[0002] The generation of midbrain dopaminergic (mDA) neurons has been an active area of ​​research in recent years. This is in part due to growing interest in regenerative medicine and the hope that a better understanding of how mDA neurons specialize, differentiate, and are maintained in vivo may facilitate the treatment of diseases that affect them, such as Parkinson's disease (PD). Insights can help instruct efforts to generate mDA neurons in vitro, which holds promise not only for cell replacement therapy but also for disease modeling and drug discovery.

[0003] Human pluripotent stem cells can differentiate into any cell type in the body. Therefore, pluripotent stem cells are actively investigated as a way to generate specific cell types for cell replacement therapy or to model diseases in vitro. Therefore, an unmet need in the field is to be able to reliably and efficiently induce pluripotent stem cells into a cell type of interest. To date, many protocols have been published for differentiating pluripotent stem cells into various cell types, but none of them can reliably generate a pure population of one cell type.

[0004] Mesencephalic dopaminergic (mesDA) neurons arise from the ventral midbrain of the neural tube. Morphogen SHH and members of the WNT family play key roles in specification and are essential for establishing the dorsal-ventral and anterior-posterior axes of the embryo, respectively (Castelo-Branco et al., 2003; Hynes et al., 1995). For mesencephalic dopaminergic neurons, high SHH signaling from the notochord is required to specify ventral neuroepithelial cells of the neural plate to a floor plate identity, while graded WNT signaling emanating from posterior regions of the embryo generates posterior neural precursors. Neural precursors in the midbrain region receive WNT1 and FGF8 from the isthmus organizer, which are involved in the patterning of cells to a midbrain identity. The isthmus organizer is essential for refining and directing cell identity to the ventral midbrain and caudal regions of the anterior rhombencephalon. Recapitulating these developmental steps in vitro using human pluripotent stem cells (hPSCs) has become a focus of cell transplantation therapy for Parkinson's disease.

[0005] In stem cell differentiation protocols, early and high SHH concentrations are sufficient to specify neural progenitor cells to floor plate identity (Fasano et al., 2010). However, for the AP (also called rostral-caudal) axis, tuned WNT concentrations within a precise concentration range are required to specify cells to the caudal midbrain (Kirkeby et al., 2012). Too high a concentration results in rhombencephalic cell types, while low concentrations result in anterior midbrain. Also, timed delivery of FGF8 has been shown to improve caudal midbrain specification (Kirkeby et al., 2017). Despite these advances in stem cell differentiation protocols, variability in cell lines and overall yield of mesDA neurons still hinders the clinical use of these cells (Nolbrant et al., 2017).

[0006] During the complex development from pluripotency to mesDA neurons, a succession of transcription factors are expressed that change as cells mature, and distinct transcriptomic profiles define specific maturation stages ( La Manno et al., 2016 ).

[0007] WO 2016 / 162747A1 discloses a method for generating stem cell-derived dopaminergic cells for use in treating neurodegenerative diseases. The cells are directed to the desired lineage by plating on a substrate coated with laminin-111 / 121 / 521 / 421 or 511.

[0008] Thus, improved methods for generating stem cell-derived dopaminergic cells would be advantageous, and in particular, more efficient and / or reliable methods for generating large quantities or purer populations of stem cell-derived dopaminergic cells would be advantageous. Summary of the Invention

[0009] Control of transcription factor expression at alternate lineage points can manipulate cell fate choices.

[0010] In this study, we chose to exploit the effects of gene knockout approaches as a way to restrict cell fate and block non-dopaminergic cell lineages with the aim of enhancing differentiation towards the midbrain dopaminergic (mesDA) fate. Specifically, we investigated the key points at which alternate cell fate choices are made early in development and explored which transcription factor determinants are important for those lineages but not required for the mesDA fate. By deleting genes for lineage determinants expressed in the alternate lineages, we were able to bias the differentiation of pluripotent stem cells towards a dopaminergic identity. We generated pluripotent stem cells that can proliferate in a pluripotent state but have limited ability to differentiate. We termed these "lineage-restricted pluripotent stem cells" (LR-PSCs). Specifically, we focused on the AP axis of the developing neural tube as midbrain dopaminergic precursors require incremental expression, and investigated transcription factors that regulate cell fates in the hindbrain and spinal cord. The anterior hindbrain has been shown to require Gbx2, and the spinal cord is dependent on Cdx family members. By deleting those genes, we increased the proportion of cells that take on a midbrain identity and prevented specification to a spinal cord fate. The results show that the approach can reliably generate functional mesDA neurons without the strict requirement for specific concentrations of exogenous factors. Figures 7-10 show how lineage-restricted stem cells can be generated by deleting specific genes from cells.

[0011] In summary, the present invention relates to pluripotent stem cells that have a restricted ability to differentiate into cell lineages different from dopaminergic neural progenitor cells and their derivatives by specific gene knockouts. In particular, the present invention relates to dopaminergic neural progenitor cells or cell derivatives thereof obtained from these lineage-restricted pluripotent stem cells, and uses thereof.

[0012] Therefore, an object of the present invention is to provide stem cells whose differentiation potential is restricted.

[0013] In particular, it is an object of the present invention to provide midbrain dopaminergic (mesDA) neuronal cells with medical uses.

[0014] Thus, in its broadest aspect, the invention relates to "lineage-restricted" pluripotent stem cells as well as dopaminergic neural progenitor cells or cellular derivatives thereof obtained from lineage-restricted pluripotent stem cells and uses thereof. In one embodiment, lineage restriction is obtained by inactivating one or more master regulator genes in the pluripotent stem cells. Examples of master regulator genes are provided in the various aspects and embodiments of the invention.

[0015] Furthermore, inactivation of one or more master regulator genes results in the pluripotent stem cells no longer being able to differentiate (or at least being inhibited from differentiating) into many cell types (other than dopaminergic neural progenitor cells or their cellular derivatives) compared to similar cell types in which one or more master regulator genes have not been inactivated.

[0016] In essence, cell lines in which one or more "master regulator" genes have been inactivated become "lineage restricted."

[0017] Thus, one aspect of the present invention relates to a pluripotent stem cell characterized in that one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A (POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 are inactivated.

[0018] Another aspect of the present invention relates to the use of the pluripotent stem cells according to the present invention for lineage-restricted cell differentiation of pluripotent stem cells.

[0019] Yet another aspect relates to the use of the pluripotent stem cells according to the invention for the generation of dopaminergic neural progenitor cells or cellular derivatives thereof.

[0020] Yet another aspect of the present invention is to provide a dopaminergic neural progenitor cell or a cellular derivative thereof differentiated from the pluripotent stem cell according to the present invention.

[0021] In yet another aspect, the present invention relates to a dopaminergic neural progenitor cell or a cell derivative thereof, characterized in that one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A (POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 are inactivated.

[0022] Yet another aspect of the present invention is to provide a dopaminergic neural progenitor cell or a cell derivative thereof according to the present invention for use as a medicament, such as in stem cell therapy.

[0023] In another aspect, the present invention relates to a dopaminergic neural precursor cell or a cell derivative thereof according to the present invention for use in the treatment, prevention or alleviation of a neurodegenerative disease.

[0024] In one aspect, the present invention relates to the use of the dopaminergic neural progenitor cells according to the invention or cell derivatives thereof in drug screening assays.

[0025] A further aspect of the present invention relates to a method for providing pluripotent stem cells according to the present invention, the method comprising inactivating at least one gene selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A (POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 in the pluripotent stem cells.

[0026] Yet a further aspect of the present invention is a method for providing dopaminergic neural progenitor cells or cell derivatives thereof, comprising the steps of: - providing a pluripotent stem cell according to the invention; and - Differentiating the provided pluripotent stem cells into dopaminergic neural progenitor cells or cellular derivatives thereof. The present invention relates to a method comprising the steps of:

[0027] The advantages of the cells according to the invention are at least the following: - Limited differentiation increases the amount of correct (neuronal) cells (see for example Examples 2-5). - Limited differentiation limits the risk of dangerous unwanted differentiation after transplantation in patients. - Limited differentiation can result in the correct neuronal cell type if suboptimal concentrations of growth factors or small molecules are used or are absent (see e.g. Examples 3-4). - Limited differentiation allows the generation of electrophysiologically mature neurons (see, for example, Example 8). - Limited differentiation allows for a robust population of mesDA neurons in an in vivo rat model of Parkinson's disease, resulting in rapid motor recovery (see, e.g., Example 9). [Brief description of the drawings]

[0028] [Figure 1A]Figure 1 Differentiation into caudal neural precursors at day 4. A) Schematic of CNP differentiation protocol at day 4. B) QPCR analysis of OTX2 in H9, GBX2 and 4xKO conditions at CNP at day 4. C) QPCR analysis of CDX2 in H9, GBX2 and 4xKO conditions. D) Heatmap of pluripotency and neural genes representing anterior, midbrain, hindbrain and spinal cord regions of H9 GBX2 and 4xKO lines at CNP at day 4. E) Schematic of generation of 4xKO cell lines including single cell clonal selection of CRISPR modified hESCs. F) Generation of 8xKO knockout cell lines from 4xKO cell lines. 4XKO cell lines were transduced with lentivirus containing guide strands targeting GBX1, HOXA1, HOXA2 and HOXB1. [Figure 1B] Figure 1 continued [Figure 1C] Figure 1 continued [Figure 1D] Figure 1 continued [Figure 1E] Figure 1 continued [Figure 1F] Figure 1 continued [Figure 2A] Figure 2. Differentiation into caudal neural precursors on day 11. A-P) RNA expression analysis of midbrain genes OTX2, PAX2, PAX5, PAX8, and EN1; hindbrain genes IRX3, HOXA2, HOXB2, HOXB1, KROX20, MAFB, HOXA3, HOXA4; and spinal cord genes HOXC6, HOXB8, and HOXC10. Q) Schematic diagram of CNP differentiation protocol on day 11. [Figure 2B] Figure 2 continued [Figure 2C] Figure 2 continued [Figure 2D] Figure 2 continued [Figure 2E] Figure 2 continued [Figure 2F] Figure 2 continued [Figure 2G] Figure 2 continued [Figure 2H] Figure 2 continued [Figure 2I] Figure 2 continued [Figure 2J] Figure 2 continued [Figure 2K] Figure 2 continued [Figure 2L]Figure 2 continued [Figure 2M] Figure 2 continued [Figure 2N] Figure 2 continued [Figure 2O] Figure 2 continued [Figure 2P] Figure 2 continued [Figure 2Q] Figure 2 continued [Figure 3A] Figure 3. Differentiation into ventral midbrain precursors. A) Schematic of the caudal midbrain differentiation protocol. B-G) Expression of OTX2, LMX1A, FOXA2, EN1, CNPY1 and HOXA2 in day 16 H9 and 4xKO cells across a range of GSK3i (CHIR99021) concentrations. [Figure 3B] Figure 3 continued [Figure 3C] Figure 3 continued [Figure 3D] Figure 3 continued [Figure 3E] Figure 3 continued [Figure 3F] Figure 3 continued [Figure 3G] Figure 3 continued [Figure 4] Figure 4. Single cell sequencing of 1μM neural precursors. Examination of genes expressed along the AP axis of H9 and 4xKO cells at day 16 of midbrain differentiation using 1μM GSK3i (CHIR99021). [Figure 5A] Figure 5. Generation of 8xKO cell lines. A) Day 4 CNP differentiation protocol. B) OTX2 expression in H9, 4xKO, 8xKO day 4 CNP differentiated under 3 μM GSK3i. C) Percentage of cells expressing OTX2 in day 4 CNP differentiated under 3 μM GSK3i. D-E) Heatmaps showing log2 centered expression of selected genes across H9 hESC, H9 day 4, 4xKO day 4 and 8xKO day 4. [Figure 5B] Figure 5 continued [Figure 5C] Figure 5 continued [Figure 5D] Figure 5 continued [Figure 5E] Figure 5 continued [Figure 6A]Figure 6. A: Single cell analysis of 4xKO cell line compared to control (H9) cell line at day 16 of dopaminergic neuronal differentiation protocol. Percentage of cells expressing genes indicative of cell types along the dorsal-ventral axis are shown. FOXA2 is expressed in floor plate cells. The lateral floor plate and substratum are indicated by expression of either NKX6.1, NKX6.2, NKX2.2, PHOX2A, and PHOX2B. B: Single cell analysis of 4xKO cell line and control (H9) at day 28 of dopaminergic neuronal differentiation protocol. Single cell analysis of combined 4X and H9 cells at day 28 of dopaminergic differentiation revealed 15 cell clusters. Expression of anterior-ventral marker NKX2.1 and medial marker of dorsal-ventral axis PAX6 were examined. [Figure 6B] Figure 6 continued [Figure 7] Figure 7. Schematic overview showing differentiation of hESCs into dopaminergic neurons. Examples of cell types and extrinsic factors such as growth factors are shown. [Figure 8] Figure 8. Schematic overview showing differentiation of hESCs into dopaminergic neurons. Compared to Figure 7, it also includes examples of transcription factors controlling cell fate. [Figure 9] Figure 9. Schematic overview showing differentiation of hESCs into dopaminergic neurons. Compare with Figure 8 to illustrate how deleting specific transcription factors blocks certain cell fates. [Figure 10] FIG 10. Schematic overview showing differentiation of hESCs into dopaminergic neurons. Compared to FIG 9, it illustrates how an even more restricted differentiation potential is achieved by deleting additional specific transcription factors. [Figure 11A] Figure 11. Single cell analysis at 62 DIV. A) Heatmap of expression of selected genes showing cluster identity. B) Violin plot of LMX1A, TH, FOXA2 and EN1 in the cluster at 62 DIV. C) Violin plot of PDLIM1 and SEMA3C in the cluster at 62 Div. D) Violin plot of NR4A2, EN1, KCNJ6, CALB1 and FOXA2 in the TH subcluster at 62 DIV. [Figure 11B] Figure 11 continued [Figure 11C] Figure 11 continued [Figure 11D] Figure 11 continued [Figure 12A] Figure 12. Generation of functional ventral midbrain DA neurons in vitro. A) Schematic of long-term (62 DIV) neuronal differentiation protocol. B) Representative response (upper trace) to depolarizing current injection (lower trace) showing repetitive action potential firing. C) Example of spontaneous firing showing burst-like events at a resting membrane potential of -45 mV. Overshooting spikes occurred in groups interspersed with periods of subthreshold membrane oscillations. D) Frequency distribution of spontaneous cell firing showing firing frequencies ranging from 1 Hz to 5 Hz (n = 16 cells). E) Dopamine content of 4X and H9 cells at 79 DIV measured by HPLC (normatilized to protein concentration). Data are shown as mean ± SD; n = 3. Groups were compared using unpaired t-test. **P < 0.01. [Figure 12B] Figure 12 continued [Figure 12C] Figure 12 continued [Figure 12D] Figure 12 continued [Figure 12E] Figure 12 continued [Figure 13A]Figure 13. In vivo analysis of cells transplanted into Parkinson's disease model rats. A) Overview of the in vivo study. Unilateral 6-OHDA-induced MFB lesions were made (-4 weeks) and confirmed 3 weeks later by cylinder test and amphetamine-induced rotation test. Animals were subdivided into three groups with similar mean scores in the rotation test. Four weeks after lesioning (0 weeks), two of these subgroups were transplanted with 250,000 cells (H9 or 4X cells), while the third group (6-OHDA-lesioned group) was not transplanted. Rotation tests were performed again at 8 and 18 weeks after transplantation, and cylinder test was performed again at 18 weeks. Animals were killed 19 weeks after transplantation (23 weeks after lesioning) and histological analysis was performed. B-C) ​​Amphetamine-induced rotation (B) and cylinder test (C) 3 weeks after lesioning in 6-OHDA-lesioned rats. The behavior of the 6-OHDA, H9 and 4X groups was comparable before transplantation. B-C) ​​N=8 for 6-OHDA, N=9 for H9 and N=10 for 4X. D) Amphetamine-induced rotational asymmetry. Two-way repeated measures ANOVA followed by Sidak's multiple comparison test; time: F(1.689, 35.46)=19.50, P<0.0001; treatment: F(2, 21)=15.23, P<0.0001. **P<0.01 and ****P<0.0001 vs. 4X cell transplant group at the same time point. §§P<0.01 and §§§§P<0.0001 vs. same group at -1 week. E) Use of each forelimb (contralateral or ipsilateral) and both forelimbs in the cylinder test was analyzed by two-way repeated measures ANOVA with time and group as variables followed by Sidak's multiple comparison test. Time × group: both: F(2, 22) = 5.785, P = 0.009; ipsilateral: F(2, 22) = 8.800, P = 0.001; contralateral: F(2, 22) = 4.642, P = 0.021. *P < 0.05 and **P < 0.01 vs. same groups at week -1. $P < 0.05 and $$P < 0.01 vs. 6-OHDA-lesioned group at the same time points. £P < 0.05 and ££P < 0.01 vs. H9 cell transplanted group at the same time points. Data in D) and E) are shown as mean ± SEM. n = 7 rats for 6-OHDA-lesioned group, n = 9 rats for 4X cell transplanted group, and n = 8 rats for H9 cell transplanted group). F) Representative photographs of coronal sections of all three groups immunostained for TH.Higher magnification images of areas within the frames are shown on the right. Scale bar, 50 μm for all three photographs in a column. G) Estimated number of TH-positive cells within the graft. H) Yield of TH-positive neurons per 100,000 grafted cells. I) Volume of TH graft. J) Quantitative analysis of immunofluorescence data showing percentage of GIRK2 / TH and CALB1 / TH double positive cells within 4X cell grafts. Data are presented as mean percentage ± SD (n=9 rats). [Figure 13B] Figure 13 continued [Figure 13C] Figure 13 continued [Figure 13D] Figure 13 continued [Figure 13E] Figure 13 continued [Figure 13F] Figure 13 continued [Figure 13G] Figure 13 continued [Figure 13H] Figure 13 continued [Figure 13I] Figure 13 continued [Figure 13J] Figure 13 continued

[0029] The present invention will now be described in more detail. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition Before describing the invention in further detail, the following terms and conventions will first be clarified.

[0031] Dopaminergic neural progenitor cells In the context of the present invention, "dopaminergic neural progenitor cells" should be understood as cells that can become dopaminergic neural cells, including midbrain precursor dopaminergic neural cells, caudal midbrain precursors, midbrain floor plate precursors, and the like.

[0032] The dopaminergic nerve cells may be a cell population containing other types of cells. The cell population is preferably a cell population that does not contain serotonin nerve cells. The dopaminergic nerve cells are preferably a cell population that contains FOXA2, LMX1A, EN1 and TH positive cells.

[0033] In one embodiment, the dopaminergic neural progenitor cells are a cell population preferably comprising FOXA2, LMX1A, OTX2, EN1, SPRY1, WNT1, CNPY1, PAX8, ETV5, PAX5, SP5, and / or TLE4 positive cells.

[0034] Derivatives of dopaminergic neural precursor cells In the context of the present invention, "derivatives of dopaminergic neural precursor cells" should be understood as cells that "dopaminergic neural precursor cells" can become, such as dopaminergic neural cells, midbrain dopaminergic neural cells, etc.

[0035] Midbrain dopaminergic neurons In the context of the present invention, the term "midbrain dopaminergic neurons" refers to cells derived from the midbrain region of the developing brain. Thus, midbrain dopaminergic neurons are a subset of dopaminergic neurons.

[0036] Genealogy Limited In the context of the present invention, the term "lineage restricted" refers to cells that can no longer differentiate (or are at least inhibited from differentiating) into many cell types compared to similar cell types that are not "lineage restricted", such as pluripotent stem cells.

[0037] In the context of the present invention, "lineage restriction" is achieved by inactivating one or more genes in the cell line (such as by gene knockout), as defined in the claims.

[0038] stem cells Stem cells are undifferentiated cells from which specialized cells subsequently arise.

[0039] The pluripotent stem cells that can be used in the present invention are stem cells that have the pluripotency to differentiate into any cell present in a living body, and also have the ability to proliferate. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer ("ntES cells"), embryonic germ cells ("GS cells"), germinal cells ("EG cells"), induced pluripotent stem (iPS) cells, and pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells). The pluripotent stem cells are preferably ES cells, ntES cells, or iPS cells.

[0040] The pluripotent stem cells used in the present invention are generated with the proviso that the process does not involve human cloning or industrial or commercial use of human embryos.

[0041] Embryonic stem cells have the capacity to differentiate into all three germ layers with extensive self-renewal and pluripotency. They are potentially useful for therapeutic purposes and provide an unlimited source of cells for tissue replacement therapy, drug screening, functional genomics and proteomics.

[0042] Induced pluripotent stem cells (also called iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from somatic cells.

[0043] It should be noted that in alternative aspects of the invention, pluripotent stem cells are also considered to include astrocytes or reprogrammed / reprogrammable astrocytes, as it is well known that astrocytes can be reprogrammed to differentiate into other cell types, such as neurons.

[0044] Master Regulator In genetics, a "master regulator" is a gene that is at the apex of a genetic regulatory hierarchy, particularly in regulatory pathways related to cell fate and differentiation. Master regulator genes may also be called "cell identity determinants" or "cell type determinants." Thus, genes that may be inactivated according to the present invention can be considered to include master regulator genes.

[0045] Neurodegenerative diseases Degenerative neurological disorders affect many activities of the body, including balance, movement, speech, breathing, and heart function. Many of these disorders are genetic. Sometimes the cause is a medical condition such as alcoholism, tumors, or stroke. Other causes may include toxins, chemicals, and viruses. Sometimes the cause is unknown.

[0046] In the context of the present invention, the term "neurodegenerative disease" includes Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and spinal muscular atrophy.

[0047] Neurodegeneration is the progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases develop as a result of the neurodegenerative process, including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, and prion diseases.

[0048] Parkinson's disease (PD) is a neurodegenerative disorder caused by a loss of midbrain dopaminergic (DA) neurons. Parkinson's disease is caused by a relatively localized degeneration of a specific type of midbrain dopamine (mesDA) neuron, making it a particularly attractive target for stem cell-based therapies. Proof of concept for cell replacement therapy for Parkinson's disease has been obtained in numerous clinical trials. Parkinson's disease (PD) is a neurodegenerative disorder caused by a loss of midbrain dopaminergic (DA) neurons.

[0049] In a preferred embodiment, the dopaminergic neural progenitor cells generated according to the invention are for use in the treatment or alleviation of Parkinson's disease.

[0050] Lineage-restricted pluripotent stem cells As outlined above, part of the present invention relates to pluripotent stem cells(s) whose ability to differentiate into different cell lineages from dopaminergic neural progenitor cells and their derivatives is restricted by knockout of certain genes.Specifically, the present invention relates to dopaminergic neural progenitor cells or their cell derivatives obtained from these lineage-restricted pluripotent stem cells and their uses.Thus, one aspect of the present invention relates to pluripotent stem cells characterized in that one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A(POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 are inactivated.

[0051] In a preferred embodiment, one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2 and HOXB1 are inactivated. As outlined in the Examples section, cell lines with one, four or eight genes knocked out have been generated. These cell types are inhibited from differentiating into lineages other than dopaminergic neural progenitor cells and their derivatives.

[0052] In one embodiment, both alleles of one or more of the listed genes are inactivated, which is likely to be most efficient if both alleles are inactivated.

[0053] In a further embodiment at least two genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1 and HOXB2 are inactivated, such as at least three genes, preferably such as at least four genes, such as at least five genes, such as at least six genes, such as at least seven genes, or more preferably such as at least eight genes are inactivated.

[0054] In another preferred embodiment, at least GBX2 and CDX2 are inactivated. By inactivating these genes, the differentiation of the caudal neuroepithelium is prevented. The data of GBX2 are shown, for example, in FIG. 1 and FIG. 2. Furthermore, CDX2 is expressed in most of the spinal cord and is considered to be the most important of the three CDX genes.

[0055] In a further embodiment, at least one of CDX1, CDX2 and CDX4 is inactivated, for example at least two of CDX1, CDX2 and CDX4 are inactivated, for example CDX1 and CDX2 are inactivated, for example CDX2 and CDX4 are inactivated, or CDX1 and CDX4 are inactivated, or preferably, for example at least CDX1, CDX2 and CDX4 are inactivated. By inactivating one or more of these genes, differentiation of caudal neural progenitor cells is prevented.

[0056] In yet another embodiment, at least one of HOXA1, HOXA2, HOXB1 and HOXB2 are inactivated, e.g., at least two of HOXA1, HOXA2 and HOXB1 are inactivated, e.g., HOXA1 and HOXA2 are inactivated, e.g., HOXA2 and HOXB1 are inactivated, or e.g., HOXA1 and HOXB1 are inactivated, or preferably, e.g., at least HOXA1, HOXA2 and HOXB1 are inactivated. By inactivating one or more of these genes, differentiation of hindbrain cells is prevented.

[0057] In yet another embodiment, at least GBX2, CDX1, CDX2, and CDX4 are inactivated. Examples 2-4 provide data on such cell lines (4xKO cell lines).

[0058] In another embodiment, at least GBX2, CDX1, CDX2, CDX4, GBX1, HOXA1, HOXA2 and HOXB1 are inactivated. Example 5 provides data on such cell lines (8xKO cell line).

[0059] In a further embodiment, at least the following genes are inactivated: - GBX2, CDX1, CDX2 and CDX4; or - GBX2, HOXA1, HOXA2 and HOXB1; or - GBX2, HOXA1, HOXA2, HOXB1 and HOXB2; or - GBX2, CDX2, HOXA1, HOXA2, HOXB1 and HOXB2.

[0060] In yet another embodiment, one or more of the following genes are inactivated: - PAX6, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A(POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1.

[0061] The rationale for inactivating these genes is explained in more detail in Example 6. In summary, the genes listed represent cell types along the dorsal-ventral axis.

[0062] Different genes are inactivated by different methods.Thus, in one embodiment, at least one gene is inactivated by gene knockout or introduction of premature stop codon, for example by CRISPR, or by gene silencing by blocking transcription or translation of gene, for example by siRNA, CRISPR inhibition, or by introduction of dominant negative version of gene, preferably using CRISPR.In the example section, inactivation is carried out by CRISPR, which introduces indel mutation into gene(s), resulting in either frameshift mutation or deletion of part of sequence, resulting in loss of one or more amino acids.

[0063] Various pluripotent stem cells can be used in the present invention. Thus, in one embodiment, the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells, preferably of mammalian origin, even more preferably of human origin.

[0064] Pluripotent stem cells can be characterized by the expression patterns of various genes. Thus, in one embodiment, the pluripotent stem cells according to the present invention are characterized by the expression patterns of NANOG + , POU5F1 + (OCT4 + ) and / or SOX2 + , preferably NANOG + , POU5F1 + (OCT4 + ) and SOX2 + Those skilled in the art may identify other expression patterns known in the art.

[0065] As also outlined above, it should be noted that in alternative aspects of the invention, pluripotent stem cells are also considered to include astrocytes or programmed / programmable astrocytes. It is well known that astrocytes can be programmed to differentiate into other cell types, such as neurons. For example, it has been shown that astrocytes can be converted into midbrain dopaminergic neurons by overexpression of specific genes (Rivetti et al., 2017). Corti et al. (Corti et al. 2012) also demonstrated the dedifferentiation of human cortical astrocytes into a neural stem / progenitor phenotype to obtain precursor and mature cells with a neural fate.

[0066] Therefore, the restoration of pluripotency from human astrocytes may have potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders, and this possibility can be further explored in combination with the present invention.

[0067] Therefore, methods to enhance dopaminergic neuronal generation by knocking out genes and limiting their capacity may also be applicable to converting (reprogrammed / reprogrammable) astrocytes into midbrain dopaminergic neurons.

[0068] It should be understood that this aspect may also be combined with other aspects of the invention.

[0069] Use of pluripotent stem cells The pluripotent stem cells according to the present invention can be used for various purposes. Thus, one aspect of the present invention relates to the use of the pluripotent stem cells according to the present invention for lineage-restricted cell differentiation of pluripotent stem cells.

[0070] Another aspect relates to the use of the pluripotent stem cells according to the invention for the generation of dopaminergic neural progenitor cells or cellular derivatives thereof.

[0071] In one embodiment, the dopaminergic neural progenitor cells or cell derivatives thereof are selected from the group consisting of dopaminergic neural cells, such as midbrain dopaminergic neural cells, midbrain dopaminergic progenitor cells, caudal midbrain progenitor cells, and midbrain floor plate progenitor cells.

[0072] In another embodiment, said cellular derivative is a dopaminergic neuronal cell, such as a midbrain dopaminergic neuronal cell.

[0073] Dopaminergic neural precursor cells or their cell derivatives In particular, the present invention relates to dopaminergic neural progenitor cells or cellular derivatives thereof obtained from the lineage-restricted pluripotent stem cells according to the invention. Accordingly, one aspect further relates to dopaminergic neural progenitor cells or cellular derivatives thereof differentiated from the pluripotent stem cells according to the invention. Again, as outlined in the Examples section, the lineage-restricted stem cells of the present invention can be efficiently differentiated into dopaminergic neural progenitor cells or cellular derivatives thereof.

[0074] Thus, in yet another aspect, the present invention relates to a dopaminergic neural progenitor cell or a cell derivative thereof, characterized in that one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A (POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 are inactivated.

[0075] In one embodiment, the dopaminergic neural progenitor cells or cell derivatives thereof have one or more genes inactivated selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2 and HOXB1.

[0076] In another embodiment, the dopaminergic neural progenitor cells or cell derivatives thereof are selected from the group consisting of dopaminergic neural cells, such as midbrain dopaminergic neural cells, midbrain dopaminergic progenitor cells, midbrain floor plate progenitor cells, and caudal midbrain progenitor cells.

[0077] In a related embodiment, the cellular derivative is a dopaminergic neuronal cell, such as a midbrain dopaminergic neuronal cell.

[0078] It should be understood that the list of inactivated genes given for the embodiment relating to pluripotent stem cells also applies to this embodiment.

[0079] Dopaminergic neural progenitor cells or their cell derivatives can be characterized by distinctive expression patterns. In one embodiment, neural cells (dopaminergic neural progenitor cells) express FOXA2, LMX1A, EN1 and / or TH.

[0080] In related embodiments, the dopaminergic neural progenitor cells express (high levels of) FOXA2, LMX1A, OTX2, EN1, SPRY1, WNT1, CNPY1, PAX8, ETV5, PAX5, SP5, and / or TLE4.

[0081] It may be advantageous if the generated cells can be stored prior to use, thus in one embodiment the neural cells are cryopreserved.

[0082] Medical Use The neural cells generated according to the invention may have various medical applications. Thus, one aspect of the invention relates to dopaminergic neural progenitor cells according to the invention or cell derivatives thereof for use as a medicament, such as in stem cell therapy.

[0083] In another aspect, the present invention relates to a dopaminergic neural precursor cell or a cell derivative thereof according to the present invention for use in the treatment, prevention or alleviation of a neurodegenerative disease.

[0084] In one embodiment, the dopaminergic neural progenitor cells or cell derivatives thereof are transplanted into the brain of a subject, preferably a mammalian subject, more preferably a human subject.

[0085] In another embodiment, the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body disease, preferably Parkinson's disease. In a preferred embodiment, the neurodegenerative disease is Parkinson's disease.

[0086] The dopaminergic neural progenitor cells or cell derivatives thereof can be used in various forms of grafts. Thus, in one embodiment, the use is as an autograft, a xenograft or an allograft.

[0087] In vitro use The dopaminergic neural progenitor cells or their cell derivatives according to the present invention can also be used in vitro. Thus, one aspect of the present invention relates to the use of the dopaminergic neural progenitor cells or their cell derivatives according to the present invention in drug screening assays, such as drugs that prevent oxidative stress, drugs that prevent mitochondrial damage, drugs that promote the degradation of misfolded proteins, drugs that prevent the secretion of misfolded proteins, and drugs that protect the nerves.

[0088] Methods for Providing Lineage-Restricted Pluripotent Stem Cells - Patent application Furthermore, one aspect of the present invention relates to a method for providing a pluripotent stem cell according to the present invention, the method comprising inactivating at least one gene selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2, HOXB1, HOXB2, HOXA3, NKX6.1, NKX6.2, NKX2.1, NKX2.2, PAX6, BRN3A (POU4F1), PHOX2A, PHOX2B, PITX2, DBX1, and SIM1 in the pluripotent stem cell.

[0089] In one embodiment, one or more genes selected from the group consisting of GBX2, CDX2, CDX1, CDX4, GBX1, HOXA1, HOXA2 and HOXB1 are inactivated in the pluripotent stem cells.

[0090] Again, it should be understood that the list of inactivated genes given in the embodiment relating to pluripotent stem cells also applies to this embodiment.

[0091] In a further embodiment, the provided pluripotent stem cell(s) comprising at least one inactivated gene is limited in its ability to differentiate into cell lineages distinct from dopaminergic neural progenitor cells or cell derivatives thereof.

[0092] In a related embodiment, the provided pluripotent stem cells comprising at least one inactivated gene are limited in their ability to differentiate into a cell lineage selected from the group consisting of caudal neural progenitor cells, spinal cord progenitor cells, hindbrain progenitor cells, hindbrain progenitor cells, and myelencephalon progenitor cells.

[0093] In one embodiment, at least one gene is inactivated by gene knockout or introduction of a premature stop codon, e.g. by CRISPR, or by disrupting transcription or translation of the gene, e.g. by gene silencing, by siRNA, or by CRISPR inhibition, or by expression of a dominant negative version of the gene, preferably using CRISPR.

[0094] A further aspect of the present invention relates to pluripotent stem cells obtained / obtainable by the method according to the present invention.

[0095] Methods for Providing Dopaminergic Neural Progenitor Cells Yet a further aspect of the present invention is a method for providing dopaminergic neural progenitor cells or cell derivatives thereof, comprising the steps of: - providing a pluripotent stem cell according to the invention; and - Differentiating the provided pluripotent stem cells into dopaminergic neural progenitor cells or cellular derivatives thereof. The present invention relates to a method comprising the steps of:

[0096] The advantage of the method according to the invention is that the amounts of various components in the cell differentiation medium may be less limiting. For example, the concentration of CHIR99021 is less limiting, as shown in Example 3. Therefore, the desired cell type can be generated even if suboptimal concentrations of growth factors or small molecules are used.

[0097] In a further embodiment, the provided dopaminergic neural progenitor cells or cell derivatives thereof are cryopreserved.

[0098] In another embodiment, the dopaminergic neural progenitor cells or cell derivatives thereof are selected from the group consisting of dopaminergic neural cells, such as midbrain dopaminergic neural cells, midbrain dopaminergic progenitor cells, midbrain floor plate progenitor cells, and caudal midbrain progenitor cells.

[0099] In a further embodiment, the cellular derivative is a dopaminergic neuronal cell, such as a midbrain dopaminergic neuronal cell.

[0100] In yet another embodiment, the methods of the invention are carried out in vitro and / or ex vivo.

[0101] Other aspects of the invention A further aspect of the invention relates to a method for treating or alleviating a subject suffering from a neurodegenerative disease, comprising transplanting dopaminergic neural precursor cells according to the invention or cellular derivatives thereof into the brain of the subject.

[0102] It should be noted that embodiments and features described in the context of one aspect of the invention also apply to other aspects of the invention.

[0103] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0104] The invention will now be described in further detail in the following non-limiting examples. EXAMPLES

[0105] Example 1 – Materials and Methods Human pluripotent stem cell culture Human embryonic stem cells (hESCs; H9 cell line, WiCell) were maintained on irradiated human fibroblasts in KSR medium consisting of DMEM / nutrient mix F-12 supplemented with 1% non-essential amino acids (NEAA), 2 mM glutamine, 0.1 mM β-mercaptoethanol, 0.5% pen / strep, and 20% knockout serum replacement. KSR medium was supplemented with FGF2 (15 ng / ml; Peprotech) and activin A (15 ng / ml; R&D Systems). Every 7 days, cells were manually passaged and fragments were transferred to freshly prepared gelatin-coated dishes containing irradiated fibroblasts.

[0106] Differentiation into caudal neural precursors on days 4 and 11 hESCs were differentiated into caudal neural precursors as previously described (Denham et al., 2015). Briefly, hESC fragments were dissected from colonies growing on feeders (CCD-1079 Sk, ATCC) and plated on vitronectin-coated plates in N2B27 medium containing 1:1 Neurobasal Medium (NBM) and DMEM / F-12 supplemented with 1% N2 supplement, 1% B27 supplement-vitamin A, 1% insulin / transferrin / selenium-A (ITS-A), 0.3% glucose, 1% Glutamax supplement, and 0.5% penicillin / streptomycin (all from Life Technologies). The medium was supplemented with SB431542 (SB; 10 μM, Tocris Bioscience) and LDN193189 (LDN; 100 nM, Stemgent) and CHIR99021 (CHIR; 3 μM, Stemgent) for 4 days. For day 11 caudal neural precursors, cells were cultured as above until day 4, and then colonies were cut into 0.5 mm pieces and cultured in suspension in low-attachment 96-well plates (Corning, Corning, NY, USA, www.corning.com) in N2B27 medium supplemented with FGF2 (20 ng / ml; Peprotech) and 400 nM SAG (Millipore).

[0107] Differentiation into midbrain dopaminergic neurons Generation of mesDA neurons was achieved by performing a previously reported protocol with minor modifications (Nolbrant et al., 2017). Briefly, from day 0 to day 9, cells were grown in N2B27 medium supplemented with 10 μM SB431542, CHIR99021 (0.5–1 μM), 0.1 μM LDN-193189 (Stemgent), and 400 nM SAG (Millipore). On day 4, colonies were cut into fragments and cultured in suspension. From days 9–11, the medium supplement was changed to FGF8 (100 ng / ml; R&D Systems). From day 11, the medium was supplemented with FGF8 (100 ng / ml), LM22A4 (2 μM), and ascorbic acid (200 μM; Sigma). From day 16 onwards, cells were detached with Accutase and cultured on culture plates coated with polyornithine, fibronectin and laminin (all from Sigma). Neural differentiation medium consisted of B27 1%, pen / strep 25U / mL and glutamax 0.5%. NDM was supplemented with 200μM ascorbic acid, LM22A4 (2μM), 1μM DAPT (Tocris bioscience), GDNF (10ng / ml) and dcAMP (500μM). Medium was changed every other day until the end of the experiment. Alternatively, cells were kept in suspension culture to generate organoids on day 16.

[0108] Production of lentivirus Third generation lentiviruses were generated as previously reported (Gill and Denham, 2020). Briefly, five lentiviral plasmids were used to generate lentiviruses: pLV-4gRNA-GBX2-RFP, pLV-hUbC-GBX2-CDX124-Cas9-T2A-GFP, pLV-HOXA1+2-HOXB1-hUbC-dsRED, pLV-Puro-U6-GBX1-G3-U6-GBX1-G1 and lentiCas9-Blast (Addgene#52962).

[0109] Generation of knockout cell lines To generate gene knockout cell lines, a multiplex CRISPR / Cas9-based single lentiviral vector system was used (Kabadi et al., 2014). The construction of lentiviral plasmids containing guide sequences required the use of Golden Gate cloning methods. Specifically, the following donor plasmids were used: ph7SK-gRNA, phU6-gRNA, pmU6-gRNA, phH1-gRNA and destination vectors pLV hUbC-Cas9-T2A-GFP, pLV GG hUbC-dsRED (Addgene; 53189, 53188, 53187, 53186, 53190, 84034). The donor plasmids were digested with BbsI and a pair of single-stranded oligos containing the genomic target sequence of the gene were annealed together and cloned into the plasmid. A set of four Golden Gate compatible donor vectors was then selected and used to generate the final destination vectors. Briefly, all selected donor and destination vectors were digested with BsmBI, the fragments were ligated with T4 ligase, transformed, and colonies were selected with ampicillin. The final destination vectors generated were pLV-GBX2-CDX1+2+4-hUbC-Cas9-T2A-GFP and pLV-HOXA1+2-HOXB1-hUbC-dsRED. For GBX1, a lentiviral dual guide puromycin vector, pLV-Puro-U6-GBX1-G3-U6-GBX1-G1, was designed and purchased from Vectorbuilder (vector ID: VB190322-1078gcv), with the guide sequences in Table 1.

[0110] Briefly, H9 cells, a GBX2 knockout cell line, were transduced with LV-4gRNA-GBX2-RFP and lentiCas9-Blast, and after 3 days, transduced cells were selected using 10 μg / ml blasticidin for 6 days. Single RFP-positive cells were then isolated in 96-well plates using FACS with the 561 nm laser of a FACSAriaIII (BD Biosciences, San Jose, CA). Indels at the corresponding target sites in clones were analyzed by genomic PCR. To generate 4X knockout cell lines, H9 cells were infected with LV-hUbC-GBX2-CDX124-Cas9-T2A-GFP, and after 7 days, single GFP-positive cells were sorted by FACS. 8X knockout cell lines were generated in a similar manner. Allele-specific mutations were confirmed using whole exome analysis. Whole exome analysis and mapping were performed by BGI (BGI, Copenhagen). Allele-specific mutations were revealed using Integrated Genome Browser V2.10.0. To reveal large deletions that could not be mapped by the alignment tool, individual sequencing reads were extracted from the FastQ files using Grep and analyzed manually.

[0111] Based on the above, one skilled in the art would be able to design knockout cell lines for other relevant genes listed but not tested here.

[0112] [Table 1]

[0113] QPCR and Nanostring QPCR and NanoString experiments were performed as commonly known to those skilled in the art. Briefly, RNA was extracted using Qiagen RNeasy mini kit and treated with DNase I according to standard protocols. cDNA was generated from 500 ng of total RNA using Superscript III and random primers according to the manufacturer's instructions. For QPCR, TaqMan Universal Master Mix II without UNG and TaqMan probes were used. NanoString experiments were performed using a NanoString nCounter SPRINT (NanoString Technologies) according to the manufacturer's instructions. Briefly, 200 ng of total RNA was used. Reporter probes were hybridized at 65°C for 20 hours. A custom-designed NanoString CodeSet consisting of a panel of capture and reporter probes designed to target 100 nucleotides of the gene of interest and a panel of housekeeping genes was used. RNA expression data were normalized to the expression of housekeeping genes.

[0114] Immunofluorescence Immunofluorescence was performed as commonly known to those skilled in the art. Briefly, cells cultured on glass coverslips or suspended in culture plates as spheroids were collected. Samples were washed twice with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 15 min (glass coverslips) or 2 h (spheroids) at 4°C, and washed three times with PBS for 10 min each. Spheroids were transferred to 20% sucrose in PBS, incubated overnight at 4°C, and embedded in OCT (Tissue-Tek). Sections were cut at a thickness of 10 μm using a cryostat (Crostar NX70) at -20°C. Coverslips and sections were incubated in 0.25% Triton-X in PBS (PBST) for 10 min and blocked with 5% donkey serum (Almeco) in PBST for 1 h at room temperature. The following primary antibodies were applied overnight at 4°C: goat anti-OTX2 (1:500, R&D Systems, Cat. No. AF1979), mouse anti-CDX2 (1:200, BioGenex, Cat. No. MU392-UC), mouse anti-Engrailed 1 (EN1, 1:40, DSHB, Cat. No. 4G11-s), rabbit anti-EN1 (1:50, Merck, Cat. No. HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, Cat. No. 8186), goat anti-FOXA2 (1:200, R&D Systems, Cat. No. AF2400), rabbit anti-LMX1A (1:5000, Millipore, Cat. No. AB10533), mouse anti-TH (1:2000, Millipore, Cat. No. MAB318), rabbit anti-TH (1:1000, Pel Freez, catalog no. P40101-150), rabbit anti-GIRK2 (1:500, Almon, catalog no. APC-006), mouse anti-CALB1 (1:5000, SWANT, catalog no. 300), rabbit anti-collagen 3A1 (1:1000, NovusBio, catalog no. NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, catalog no. AF6220), and mouse anti-HNA (1:200, Abcam, catalog no. ab191181).Cells were washed three times with PBST for 10 min each, and then the corresponding secondary antibodies (1:200, Jackson ImmunoResearch Laboratories or 1:1000, Invitrogen) were applied for 1 h at room temperature. After removing the secondary antibodies, cells were washed three times with PBST in the dark for 10 min each. Nuclei were counterstained with DAPI (1 μg / ml, Sigma) and rinsed three times with PBS for 5 min each. Slides or coverslips were mounted with PVA-DABCO. Images were taken with a confocal microscope (Zeiss LSM780) and Zen software.

[0115] Flow cytometry analysis Flow cytometry analysis was performed as commonly known to those skilled in the art. Briefly, cells were washed twice with PBS- and dissociated with Accutase to obtain single cells. Centrifuged at 300×g for 4 min and resuspended in 4% PFA for 10 min at room temperature. Cells were then washed with PBS-, centrifuged, resuspended in PBST, centrifuged again, and blocked with 5% donkey serum for 30 min at room temperature. Primary antibody in blocking solution was added to cells and incubated for 2 h at room temperature. Washed once with PBST, resuspended in secondary antibody in blocking solution, and incubated in the dark for 30 min at room temperature. Cells were washed overnight with PBST at 4° C. and resuspended in PBS for flow cytometry using a NovoCyte Quanteon analyzer (Acea Biosciences Inc., Santa Clara, CA). Data was analyzed using FlowJo software (v.10, Ashland, OR).

[0116] Quantification of immunofluorescence images Quantification of immunofluorescence images was performed as commonly known to those skilled in the art. Briefly, the percentage of OTX2 / DAPI double positive, GIRK2 / TH double positive and CALB1 / TH double positive cells either in culture or in the explants was quantified using ImageJ software (1.53) by semi-automated object-based colocalization analysis (Lunde et al., 2020). Multichannel immunofluorescence images were processed into multichannel binary and grayscale output images using the Colocalization Image Creator Plugin. Binary output images were generated by processing the input channels of ImageJ filters applying automatic local intensity threshold, radius outlier removal, watershed segmentation, eroding, hole filling, Gaussian blur and maximum algorithms. Furthermore, binarized objects with inappropriately small size were removed from the output images by a defined minimum area size. To improve the visualization of colocalization signals, object overlap was restricted to the nuclei of the cells. The accuracy of binary object segmentation was visually verified via the grayscale output image. After verification, binary objects representing individually labeled or co-labeled cells were automatically quantified using the Colocalization Object Counter plugin. All immunofluorescence images were analyzed with the saved binary object segmentation settings. A minimum of four randomly selected fields taken at 20x and 63x magnification were used for quantification of OTX2-positive cells in culture. Quantification of GIRK2 / TH and CALB1 / TH double positive cells in the grafts was performed blindly by analyzing four non-overlapping images taken at 20x magnification from two graft sections per animal.

[0117] RNA sequencing and data analysis RNA sequencing and data analysis were performed as commonly known to those skilled in the art. Briefly, library construction, sequencing, and initial data filtering including adapter removal were performed by the BGI Europe Genome Center. Total RNA was subjected to oligo-dT-based mRNA enrichment. Sequencing of 100 bp paired-end reads was performed on the DNBseq platform. More than 20 million clean reads were obtained per sample. Alignment was performed to human genome build hg38 (ensemble release 92) using the HISAT2 aligner (v2.1.0). Transcript quantification was performed using FeatureCount (v1.6.4), and read counts were normalized by effective gene length and sequencing depth to obtain Transcripts per kilobase million (TPM). Differentially expressed genes were identified from the count tables using edgeR (v3.32). Heatmaps were constructed by centering and unit variance scaling of the TPM values ​​and then subjected to principal component analysis (PCA) with Clustvis using SVD with imputation (Metsalu et al., 2015).

[0118] Single-cell RNA-seq and data analysis Single cell RNA-seq and data analysis were performed as commonly known to those skilled in the art. Briefly, on days 16, 28 and 62, cultured cells were dissociated into single cells using Accutase. Neurospheres (n=10 biological replicates per cell line) were pooled together on day 16, 4 biological replicates per cell line were pooled together on day 28, and 4 biological replicates per cell line were pooled together on day 62. To construct the libraries, 10x Genomics Chromium Next GEM Single Cell 3' kit v3.1 was used according to standard protocols. Each of the six groups (H9 cells on day 16, 4X cells on day 16, H9 cells on day 28, 4X cells on day 28, H9 cells on day 62, and 4X cells on day 62) was run in a separate lane on a Chromium controller, with a total of 8000 cells loaded per lane. Next generation sequencing was performed on an Illumina NovaSeq instrument. Cell Ranger Single-Cell Software Suite (v3.1.0) was used for demultiplexing, barcoding and single-cell 3′ gene counting. Reads were aligned to the human GRCh38 reference genome. Further analysis, including quality filtering, dimensionality reduction and application of standard unsupervised clustering algorithms, was performed using the Seurat R package (v3.2.1). To exclude outlier cells, the number of genes expressed in each cell was plotted per sample to select the optimal minimum number of allowed genes per cell. The minimum number of genes per cell was set at 3000 for H9 cells at day 16, 2000 for 4X cells at day 16, 3000 for H9 cells at day 62 and 3000 for 4X cells at day 62. We also removed cells with a high percentage of reads mapping to mitochondrial genes. For day 16 and day 28 samples, we removed all cells with more than 10% mitochondrial RNA, and for day 62, we set a limit of 15%. Cell doublets were removed from the single-cell transcriptome data using the R package DoubletFinder (v.2.0.3), with the expected proportion of doublet cells set at 7.5%.Single-cell data were normalized by dividing the gene counts of each cell by the total number of cells in that cell, multiplying by a scaling factor of 10,000, and natural log-transforming. Dimensionality reduction was performed using the UMAP method. Clustering was performed by Seurat's graph-based clustering method using the FindClusters function with the resolution set to 0.6. Plots of the various single cells were created using Seurat in R.

[0119] Electrophysiology Electrophysiological recordings of 4X cells were performed at 80–84 DIV. 4X cells co-cultured with astrocytes on 13 mmφ coverslips were transferred to the recording chamber by stepwise transition from culture medium to artificial cerebrospinal fluid (aCSF) and then adding 5 drops (200 μL each) of aCSF to the culture medium over 20 s. After transfer to the recording chamber, the coverslips were continuously perfused at room temperature with aCSF containing 119 parts NaCl, 2.5 parts KCl, 26 parts NaHCO3, 1 part NaH2PO4, 1 part D-glucose, 2 parts CaCl2, and 2 parts MgCl2 (adjusted to pH 7.4).

[0120] The recording chamber was mounted on an upright microscope (Scientifica) linked to a digital camera (QImaging Exi Aqua). A 63x water-immersion objective (Olympus, LumiPlan) was used to visualize cells 4X. Cells selected for electrophysiological recordings exhibited a neuronal-like morphology with finely branched neurites. Clusters of aggregated cells were avoided. Acquisitions were performed in current clamp mode in whole-cell configuration using Clampex 10.6 software connected to a Multiclamp 700B amplifier via a Digidata 1550A digitizer (Molecular Devices). Data were low-pass filtered at 200 Hz, digitized at 10 kHz, and corrected for whole-cell capacitance. Patch pipettes (resistance of 5–10 MOhm) were filled with an internal solution containing (in mM) 153 potassium gluconate, 10 HEPES, 4.5 NaCl, 9 KCl, 0.6 EGTA, 2 MgATP, and 0.3 NaGTP. The pH and osmolality of the internal solution were close to physiological conditions (pH 7.4, osmolality 297 mOsm). The access resistance of the cells in the sample was ~30 MOhm. Of the 30 neuronal recordings obtained, 16 were retained for analysis. The remaining recordings were either from neurons that did not respond to the depolarizing step (putative astrocytes), were unstable, or did not show spontaneous activity, and therefore those recordings were excluded from analysis.

[0121] Spontaneous excitatory postsynaptic potentials (sEPSPs) were recorded in current-clamp gap-free mode (clamped at -45 mV). Current-clamp recordings of evoked action potentials (-60 mV) were performed by applying repeated current pulses (800 ms) of increasing amplitude (20 pA).

[0122] Data analysis was performed using Clampfit 10.6 software (Molecular Devices). To visualize the distribution of firing frequency, the number of spontaneous spikes per second in current clamp mode was counted over 1 min using the threshold tool of Clampfit software and sorted into bins with width equal to 1 (corresponding to 1 Hz). Data were visualized using the frequency distribution mode of GraphPad Prism V9 software.

[0123] HPLC analysis of dopamine content At 80 DIV, one to two organoids per sample were collected and homogenized in 100 μl of 0.2 M HClO4. Samples were then centrifuged, and the supernatants were collected and spun through a 0.2 μm spin filter (Costar Spin-X, Merck) at 14000×g for 1 min at 4°C and loaded onto an HPLC system (Thermo Scientific Ultimate3000). The mobile phase was 12.5% ​​acetonitrile buffer (pH 3.0, 86 mM sodium dihydrogen phosphate, 0.01% triethylamine, 2.08 mM 1-octanesulfonic acid sodium salt, and 0.02 mM EDTA). The flow rate of the mobile phase was adjusted to 1.5 ml / min. Dopamine levels were calculated using a standard curve generated using an external DA standard (coefficient of determination of the standard curve was 0.99946). Dopamine content was then normalized to protein concentration and expressed in nmol / g.

[0124] Preparation of cells for in vivo transplantation For each batch of single cells, 10 neurospheres were collected at 16 DIV from each cell line (H9 and 4X) and washed twice with PBS. Then, 500 μl of Accutase (supplemented with 100 μg / ml DNase) was added and the cells were incubated at 37°C for 10 min. The neurospheres were pipetted first with a 1 ml pipette and then with a 200 μl pipette to obtain a solution of single cells. 500 microliters of washing medium (DMEM / F12 supplemented with 1% human serum albumin) was added and the cells were spun down at 400×g for 5 min at room temperature. The cell pellet was resuspended in HBSS (supplemented with 100 μg / ml DNase) at a concentration of 100,000 cells / μl and kept on ice. The cell suspension was kept on ice for up to 3 h, after which a new batch of cells was prepared.

[0125] In vivo transplantation Adult (9 weeks old) males (225-300 g) (n=30) NIH (NTac: NIH-Foxn1) were purchased from Taconic Biosciences A / S. rnu Nude rats were group-housed in ventilated cages in a clean room under a 12-h light / dark cycle and had free access to sterile food and water. In addition to standard rat food, they were given peanuts to increase caloric intake.

[0126] Rats were anesthetized with isoflurane (5% for induction, 2–3% for maintenance), 1.2 L / min O2, and 0.6 L / min air, placed in a stereotaxic frame (Stoelting), and unilaterally injected with 6-OHDA (Sigma-Aldrich A / S) (2 μl of 7 μg / μl free base in saline containing 0.02% ascorbic acid) (Tentilier et al., 2016) into the right MFB (anteroposterior (AP), -4.4; mediolateral (ML), -1.1; dorsoventral (DV), -7.6; tooth bar, 3.3) using a Hamilton syringe fitted with a glass cannula. After injection, the cannula was left in place for 5 min before being slowly withdrawn. The incision was sutured, and the animals were injected with buprenorphine (0.36 mg / kg) as an analgesic. Once the animals were fully awake they were placed back into their cages containing wet food and 0.009 mg / ml Temgesic in the water.

[0127] Lesion formation efficiency was evaluated using the amphetamine-induced rotation test 3 weeks after surgery, and animals that showed >5 rotations / min were used for further experiments. The selected rats were divided into three groups with similar average amphetamine-induced rotations: 6-OHDA lesion (without implantation) group (n=8), H9 cell implantation group (n=9), and 4X cell implantation group (n=9) (Figure 13B-C). Four weeks after lesion formation, animals in the H9 cell implantation group and the 4X cell implantation group were stereotactically injected with 250,000 cells of each cell type in a volume of 2.5 μl into the striatum (AP, +0.5; ML, -3; DV, -4.6 / 4.8) using a protocol similar to the one described above. All these three groups were sacrificed at 22 weeks after lesion formation (i.e., 18 weeks after implantation). Two transplanted rats did not complete the study and were euthanized due to health problems: one in the 4X cell transplant group due to a broken tail (8 weeks after transplantation) and one in the H9 cell transplant group due to hind leg paralysis (17 weeks after transplantation).

[0128] Amphetamine-induced rotation test One week before implantation and at weeks 8 and 18 after implantation, the amphetamine-induced rotation test was performed to assess the effects of the lesion as previously reported (Bjorklund et al., 2019). Animals were injected intraperitoneally (ip) with 5 mg / kg D-amphetamine and connected to a rotameter (LE902, PanLab, Harvard Apparatus) linked to a LE3806 multicounter (PanLab, Harvard Apparatus). The number of body rotations was recorded over a 90 min period.

[0129] Data are expressed as the final number of whole-body rotations per minute, with ipsilateral rotations having positive values ​​and contralateral rotations having negative values. Animals showing >5 rotations / min were considered to have successfully been lesioned. One rat had technical problems with one of the rotation tests and was excluded from that behavioral testing.

[0130] Cylinder Test Three weeks after lesioning (one week before grafting) and 18 weeks after grafting, limb use asymmetry was assessed using the cylinder test: animals were placed in a transparent Plexiglas cylinder (30 cm high, 20 cm diameter) with two mirrors placed behind the cylinder to allow full visibility of the cylinder surface.

[0131] Locomotor activity was video recorded for a total of 5 min. Data analysis was performed by an investigator blinded to the groups using VCL Media Player software in slow motion as previously reported (Schallert et al., 2000). Because the majority of the animals' exploratory motor activity was confined to the first 2 min, with little movement after this time point, the first 2 min of activity was analyzed, and activity after this time point was only analyzed if the animals showed less than 10 movements (wall contacts and rearing).

[0132] The following behaviors were scored to determine the degree of asymmetry in forelimb use (Schallert et al., 2009): a) use of the left or right forelimb alone to contact the wall during full rearing or to land on the floor after rearing, and b) use of both the left and right forelimbs simultaneously to contact the wall of the cylinder during full rearing, to move laterally along the wall (wall stepping), and to land on the floor after rearing. Data are expressed as the percentage of time that each forelimb (left or right) or both forelimbs were used relative to total movements (wall and floor).

[0133] Immunohistochemical analysis of brain sections Rats were killed by an overdose of pentobarbital (50 mg / kg i.p.) 23 weeks after 6-OHDA-induced lesioning. During respiratory arrest, rats were perfused via the ascending aorta with ice-cold saline followed by cold 4% PFA (in 0.1 M NaPB, pH 7.4). Brains were extracted, postfixed in PFA for 2 h, and transferred to 25% sucrose solution (in 0.02 M NaPB) overnight. Brains were cut into 35 μm-thick coronal sections on a freezing microtome (Microm HM 450, Brock and Michelsen), divided into serial coronal sections (series of 8 for striatum and substantia nigra), and stored at -20°C.

[0134] Immunohistochemical staining was performed on free-floating brain sections using the following primary antibodies: mouse anti-rat TH (1:4000, MAB318, Merck Millipore), rabbit anti-Girk2 (1:500, APC-006, Almon), rabbit anti-TH (1:1000, PelFreeze), mouse IgG1 anti-CALB1 (1:5000, 28k, SWANT), mouse IgG1 anti-HNA (1:200, 151181, Abcam), goat anti-FOXA2 (1:200, AF2400), sheep anti-hCOL1A1 (1:200, R&D Systems), rabbit anti-hCOL3A1 (1:1000), rabbit anti-EN1 (1:50), and rabbit anti-LMX1A (1:5000).

[0135] Immunohistochemistry was performed as previously reported (Tentilier et al., 2016) using avidin-biotin-peroxidase complex (ABS Elite, Vector Laboratories) and 3,3-diaminobenzidine (DAB) as chromogen to visualize signals. Sections were mounted on chrome-alum gelatin-coated slides, dehydrated, and covered with a coverslip. Slides were analyzed using an Olympus VS120 Slide Scanner (upright widefield fluorescence) with a 20x objective.

[0136] For immunofluorescence, free-floating sections were blocked with 5% normal donkey serum in 0.25% Triton X-100 in KBPS and then incubated overnight at room temperature with the selected primary antibody in 2.5% donkey serum and 0.25% Triton X-100 in KBPS. Sections were washed with KPBS, preblocked for 10 min with 1% donkey serum and 0.25% Triton X-100 in KPBS, and incubated for 2 h with the following species-specific fluorochrome-conjugated secondary antibodies made in donkey: Alexa Fluor488-conjugated anti-mouse IgG (1:200, Jackson ImmunoResearch), Alexa Fluor568-conjugated anti-goat IgG (1:1000, A11057, Invitrogen), Alexa Fluor647-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch), Alexa Fluor568-conjugated anti-rabbit IgG (1:1000, A10042, Invitrogen), Alexa Fluor647-conjugated anti-mouse IgG (1:1000, A-31571, Invitrogen), and Alexa Fluor568-conjugated anti-mouse IgG1 (1:1000, A10037, Invitrogen). Fluor568-conjugated anti-sheep IgG (1:1000, Invitrogen). DAPI (1:2000, Sigma-Aldrich A / S) was used for nuclear staining. Sections were mounted on chrome-alum gelatin-coated slides using Dako Fluorescent mounting medium.

[0137] Microscopic analysis (number and yield of TH positive cells and graft volume) Coronal sections (1:8) from each animal were immunostained for TH to analyze DA neurons in the grafts. Images of slides were acquired using an Olympus VS120 Slide Scanner (upright widefield fluorescence) with a 20x objective. All sections in which the graft was visible were selected: 3–5 sections per H9 cell-transplanted animal and 4–8 sections per 4X cell-transplanted animal. Regions in which the number of TH-positive cells was quantified included the striatum and globus pallidus, but not TH-positive cells in the cortex and corpus callosum. Images were analyzed by identifying cells within regions of interest (ROIs) using QuPath software (Bankhead et al, 2017). Settings were varied for each section depending on the stain, and the following settings were used: detection image = optical density sum, required pixel size = 0.5 μm, background radius = 15-30 μm, threshold = 0.15-0.3, median filter radius = 0-3 μm, sigma = 0.7-2 μm, minimum area = 85-130 μm 2 , maximum area=500~1200μm 2 , maximum background intensity = 2, cell expansion = 2 μm. Cells were classified by shape, including the shape of the nucleus, and the boundaries were smoothed.

[0138] To estimate the number of cells in the entire graft, the total number of TH-positive cells per animal was determined by QuPath software, multiplied by 8, and corrected for double counting of cells across two or more sections using the Abercrombie method (Abercrombie, 1946). The Abercrombie factor for each group was calculated by dividing the mean thickness per section by (mean thickness + mean TH-positive cell size). These numbers were calculated from three different animals per group, sampling three sections and 18 cells per animal. The total number of cells in the graft was calculated as Abercrombie factor × total number of TH-positive cells × 8. The number of viable cells (yield) per 100,000 grafted cells was estimated. The volume of each graft was calculated as V = A1T1 + A2T1 + … + A nIt was estimated as T1, where V is the estimated volume, T1 is the sampling interval of 1 / 8 series (8 × 35 μm), and A(n) is the area of ​​the TH-positive region in slice (n) (Piao et al., 2021).

[0139] statistical analysis All statistical analyses were performed using GraphPad Prism v9.1.1.225. One-way or two-way ANOVA was performed, and Sidak's test was used for post-hoc analysis when appropriate. When comparing transplanted groups only, unpaired two-tailed t-tests were used. Data are presented as mean ± standard error of the mean (SEM) or ± standard deviation (SD) (as indicated). P < 0.05 was considered significant.

[0140] Example 2 – Deleting GBX2 and CDX1 / 2 / 4 results in increased midbrain identity Research Objective To clarify whether deleting GBX2 and CDX1 / 2 / 4 results in increased midbrain identity.

[0141] Materials and Methods See Example 1.

[0142] result To enhance differentiation of hESCs into dopaminergic neurons, we focused on knocking out transcription factors that control cell fate along the AP axis. We first investigated whether knocking out GBX2- / - in hESCs (H9 cell line) would result in an increase in midbrain cell types when differentiated under conditions known to give rise to hindbrain and spinal cord cell types. GBX2 - / - We generated human embryonic stem cell lines that, in their undifferentiated state, were transcriptionally indistinguishable from control hESCs and were capable of differentiating into neural precursors.

[0143] GBX2 - / - Using our previously reported protocol for generating caudal neural progenitors (CNPs; (Denham et al., 2015)), GBX2 - / - Cells were differentiated into CNPs for 4 days and compared to hESC-derived CNPs (Figure 1A). Immunostaining of H9 CNPs at day 4 showed no expression of OTX2 and positive staining for CDX2. GBX2- / - CNPs at day 4 showed fewer OTX2-positive cells and positive staining for CDX2. 4xKO CNPs had a small number of cells positive for OTX2 and no cells positive for CDX2 (photos of cells not shown). Indeed, when differentiated in the presence of a GSK3B inhibitor (GSK3i; CHIR99021) at a concentration (3 μM) known to give rise to the hindbrain and spinal cord, GBX2 - / - The strain showed a slight but significant increase in the forebrain / midbrain marker OTX2 (Fig. 1B) and a significant decrease in CDX2 transcripts (Fig. 1C) compared to H9. Despite the increase in OTX2, the GBX2 - / - CNPs still expressed HOX genes associated with hindbrain and spinal cord cell types (Fig. 1D), and FACS analysis revealed only a slight increase in the number of OTX2-positive cells and a slight decrease in the percentage of CDX2-positive cells (data not shown).

[0144] GBX2 - / - CDX1,2,4 - / - GBX2 - / - Based on the results from the line, we attempted to further define the cell potential along the AP axis by knocking out the CDX gene family. Cdx2 is an upstream regulator of posterior Hox genes and is a key determinant of the spinal cord (Skromne et al., 2007). To ensure there was no expression or compensation from other CDX family members, homozygous knockouts of all three CDX family members CDX1 / 2 / 4 were generated by targeting their DNA-binding domains using CRISPR (Figure 1E). The resulting hESC line, GBX2 - / - CDX1,2,4 - / -(hereafter referred to as 4xKO) were differentiated for 4 days using the same CNP protocol (Figure 1A). As expected, neither CDX2 transcripts nor CDX2-positive cells could be detected in 4xKO CNP (Figure 1C). Remarkably, 4xKO neural precursors at day 4 were significantly more abundant than H9 and GBX2. - / - showed a significant increase in OTX2 transcripts compared with the derived CNP (Fig. 1B).

[0145] To further understand the effects on gene expression, three cell lines (H9, GBX2 - / - We performed RNA sequencing of all CNPs (4xKO and 4xKO). Examination of the HOX gene profile revealed that 4xKO CNPs had a significant restriction in the expression of caudal HOX genes, with no expression of HOXA4 or HOX genes posterior to HOXA13 (Figure 1D). These results indicate that the 4xKO cell lines were unable to generate precursor cell types associated with and caudal to rhombomere R4.

[0146] Next, we wanted to know the extent to which there were changes in anterior genes. First, we examined the expression of forebrain genes in 4xKO CNPs and found that there were no significant changes in the expression of SIX3, DLX2, and FOXG1. However, the transcripts of the forebrain / midbrain gene OTX2 were significantly higher than those of H9 and GBX2. - / - The midbrain genes PAX2, PAX5, and EN1 / 2 were also significantly increased compared to H9 and GBX2. - / - The increase was significantly greater than that in GBX2 (Fig. 1C). - / - In 4xKO cells, there was a decrease in anterior rhombomere genes, such as EGR2 and MAFB, whereas in 4xKO, these genes were increased in agreement with the decrease in posterior HOX genes. Overall, 4xKO cell lines showed a posterior limit equivalent to rhombomere R4 and significantly higher expression of midbrain and anterior rhombomere genes (Figure 1C).

[0147] To further explore the capabilities of the 4xKO line, 4xKO cells and GBX2 - / -The lines were differentiated under ventralized conditions to CNP on day 11 (Figure 2Q). As at day 4, the 4xKO lines were differentiated to CNP on day 11 using either H9 or GBX2 - / - In 4xKO, the transcripts of OTX2 were higher than those of H9 and GBX2 (Fig. 2A). - / - We found that the midbrain genes PAX2, PAX5, and PAX8 were all upregulated in 4xKO compared to H9 and GBX2 (Fig. 2B, C, E). EN1, which spans the caudal midbrain and rhombomere R1 during development, was also significantly upregulated in 4xKO compared to H9 and GBX2 (p<0.01 and p<0.05, respectively; Fig. 2D). In the case of the hindbrain gene KROX20, which is expressed in rhombomeres R3 and R5, it was significantly upregulated (p<0.01; Fig. 2J), while MAFB, a marker of rhombomeres R5 and R6, did not change significantly (Fig. 2K). IRX3 was still present, but was downregulated in H9 and GBX2. - / - There were significantly fewer transcripts in 4xKO compared to H9 (Fig. 2F). These results showed that the HOX profile was also restricted to the posterior limit in 4xKO to HOXA3, but HOXA3 transcripts were low and the more posterior HOX genes were not detected (Fig. 2L,M,N,O,P). Interestingly, the anterior HOX gene HOXA2 (Fig. 2G) maintained its expression in 4xKO day 11 CNP, while HOXB2 (Fig. 2H) and HOXB1 (Fig. 2I) were significantly upregulated compared to H9. These results indicate that there was an anterior shift in the population, where cells preferentially acquired a midbrain or anterior rhombencephalic identity.

[0148] Immunofluorescence staining confirmed the population shift toward midbrain identity. OTX2 in 4xKO cells at day 11 + / EN1 + The presence of positive caudal midbrain precursors was seen but was not detected in H9 control cells (data not shown).

[0149] These results indicate that under differentiation conditions known to generate CNP, the 4xKO line fails to generate spinal fates and has a limited HOX profile up to rhombomere R4. Furthermore, the cell type distribution was shown to be anteriorly shifted by the presence of OTX2+ / EN1+ cells, which were not detected in the control cell line under CNP differentiation conditions.

[0150] conclusion The data obtained showed that knocking out the gene alone (GBX2 - / - To generate cell lines, we either knocked out the four genes GBX2, CDX1, CDX2, and CDX4 (GBX2 - / - CDX1, 2, 4 - / - It is shown that the method (generating cell lines) has produced cell lines with limited differentiation potential that can more efficiently generate midbrain dopaminergic neuronal precursors or midbrain dopaminergic neurons.

[0151] Example 3 – Lineage-restricted PSCs efficiently generate caudal midbrain precursors Research Objective To determine whether the 4xKO line can generate mesDA neurons more efficiently when differentiated using a dopaminergic neuron protocol.

[0152] Materials and Methods See again Examples 1 and 2.

[0153] result We compared H9 and 4xKO and differentiated them using one of the most recent mesDA protocols (Nolbrant et al., 2017) (Figure 3A). We started by adjusting the concentration of GSK3i from 0.5 μM to 1 μM to determine the optimal concentration for generating mesDA precursors using H9. H9 and 4xKO cells were differentiated until day 16 (cell diagrams not shown).

[0154] In our laboratory, the optimal concentration of H9 was found to be 0.6 μM, and when assessed at this concentration on day 16, the levels of OTX2 transcripts were greatest at 0.6 μM and decreased at higher concentrations of GSK3i (Figure 3B). Furthermore, transcript levels of HOXA2 were lowest at 0.5-0.6 μM GSK3i (Figure 3G). Concentrations of the caudal midbrain marker CNPY1 were highest at 0.5-0.6 μM and decreased significantly when reaching 1 uM (Figure 3F). EN1 was also significantly higher at 0.5-0.6 μM and decreased significantly from 0.7 μM and 1 μM (Figure 3E). These results were consistent with previous reports (Kirkeby et al., 2012) that showed that concentrations below 1 uM of GSK3i were necessary for midbrain specialization, and that concentrations approaching 1 μM or higher resulted in a dramatic shift toward hindbrain identity.

[0155] Comparing 4xKO cells at the same GSK3i concentrations, striking differences were observed relative to H9. The 4xKO line had significantly more transcripts for OTX2, EN1, and LMX1A across all concentrations, including 0.5 μM up to 1 μM, compared to H9 (Figures 3B, 3E, and 3C). The caudal midbrain marker CNPY1 was significantly higher across the range of 0.65 μM up to 1 μM, compared to H9 (Figure 3F). These results indicated that the limited capacity of the 4xKO line resulted in the generation of midbrain precursors across a broader range of GSK3i concentrations. Furthermore, the 4xKO line was also able to generate significantly more midbrain transcripts than H9 could generate at the optimal concentration (cell diagrams not shown).

[0156] To further explore why 4xKO is more efficient than H9 even under optimal conditions, we investigated the range of cell types along the AP axis that control the cell types generated even under the most optimal conditions. We found that control cells still expressed transcripts of HOXA2 corresponding to the hindbrain (Figure 3G), and even with the optimized protocol, the H9 line generated a large distribution of cell types along the AP axis, including hindbrain cell types. These results were consistent with previous reports showing expression of hindbrain cell types in cell lines even at optimal concentrations (Kirkeby et al., 2012). However, 4xKO cells expressed significantly lower concentrations of HOXA2 compared to H9. Furthermore, HOXA2 levels in H9 were significantly increased when higher concentrations of GSK3i were used, whereas in the 4xKO line, HOXA2 levels remained unchanged throughout all concentrations (Figure 3G).

[0157] Immunofluorescence staining confirmed the above results, showing that EN1-positive cells were abundant at GSK3i concentrations of 0.6 μM to 1 μM in the 4xKO line (data not shown), but in the H9 line, EN1-positive cells were limited to a concentration of 0.6 μM, indicating a broader distribution of cells along the AP axis in H9 cells.

[0158] conclusion These results indicate that 4xKO fails to give rise to spinal cell types and the majority of cells are restricted to midbrain and anterior hindbrain identity, resulting in a higher proportion of mesDA precursors, even at suboptimal concentrations of GSK3i.

[0159] Example 4 – Lineage-restricted PSCs differentiated in hindbrain conditions generate midbrain precursors and mesDA neurons Research Objective To determine the cell types produced in the 4xKO line under conditions that normally give rise to predominantly hindbrain cell types.

[0160] Materials and Methods Please refer again to Examples 1-3.

[0161] result H9 and 4xKO cells at a concentration of 1 μM of the GSK3i inhibitor were compared by single-cell analysis at day 16. As expected, both conditions revealed a significant proportion of cells ventral to the floor plate (H9 48%, 4xKO 73%). However, a significantly greater difference was observed in the distribution of cells along the AP axis between the two cell lines (p<0.00001; Figure 4). In 4xKO cells, there were significantly more cells expressing the anterior gene OTX2 (H9 1%; 4xKO 43%). Furthermore, in the 4xKO condition, caudal midbrain cells were highly enriched (OTX2+ / En1+; H9 0%; 4xKO 35%). Cells corresponding to rhombomere R1 (Otx2- / En1+) were also enriched in 4xKO cells (H9 3%; 4xKO 39%). The rhombencephalic and more caudal cell types indicated by expression of HOX genes were significantly higher in H9 compared to 4xKO cells (H9 85%; 4xKO 2%), indicating that the majority of cells in H9 correspond to a rhombencephalic identity, whereas the distribution of 4xKO cells corresponds to the caudal midbrain and anterior rhombencephalon (Figure 4 ).

[0162] Next, we wanted to know whether the 4xKO cell line could generate mesDA neurons under these caudalization conditions. Single cell analysis showed that at 1 μM GSK3i, the 4xKO line was able to generate approximately 11.6% dopaminergic neurons compared to 2.8% in the control. Immunofluorescence analysis of H9 and 4xKO day 100 organoids also showed that the majority of dopaminergic neurons also expressed the ventral marker FOXA2, indicating successful generation of mesDA neurons. 4xKO organoids contained dopaminergic neurons TH and FOXA2 positive. No FOXA2 and TH double positive cells could be detected in H9 organoids (data not shown). Day 100 midbrain organoids differentiated from 4xKO and H9 using 1 μM GSK3i, 4xKO organoids contained dopaminergic neurons TH and FOXA2 positive. In H9 organoids, no FOXA2 and TH double-positive cells could be detected.

[0163] conclusion These data suggest that GBX2 - / - CDX1, 2, 4 - / - It is further supported that starting from a cell line it is possible to generate mesDA neurons more efficiently compared to a control cell line.

[0164] Example 5 – Knockout of anterior HOX genes further restricts PSC potential and increases midbrain cell types – 8xKO Research Objective To determine whether knockout of anterior HOX genes would further restrict PSC potential and increase midbrain cell types.

[0165] Materials and Methods Please refer again to Examples 1-4.

[0166] result Based on single-cell data, differentiation of 4xKO cells into mesDA progenitors gave rise to only 1% HOX gene-expressing cells. However, under highly caudalized conditions the cell line was still able to express hindbrain HOX genes (Figure 1C). The CDX gene family does not control anterior HOX genes in rhombomeres R2-R4. Therefore, we selected HOXA1 / 2, HOXB1 / 2, and GBX1 as additional genes to knock out. We successfully targeted HOXA1 / 2, HOXB1, and GBX1 (Figure 1F). Nevertheless, we continued with the new 4xKO and HOX combined knockout cell lines (hereafter referred to as 8xKO) and differentiated them into CNP at day 4 (Figure 5A). In 8xKO cells at day 4, the transcript level of OTX2 was significantly increased, as evidenced by the large increase in OTX2-positive cells (30%) compared to 2% in 4xKO cells (Figure 5B-C). Furthermore, analysis by RNA sequencing showed that expression of midbrain genes was significantly increased compared to the 4xKO strain (Figure 5D-E).

[0167] conclusion HOXA1 / 2, HOXB1 and GBX1 were further knocked out, thereby - / - CDX1, 2, 4 - / - HOXA1, 2 - / - HOXB1 - / - and GBX1 - / - Generating a cell line that is .gtoreq.4xKO results in a significant increase in expression of midbrain genes compared to the 4xKO line.

[0168] Example 6 – Genes representing cell types along the dorsal-ventral axis Research Objective To assess the effects of expression of genes representing cell types along the dorsal-ventral axis.

[0169] Materials and Methods See the previous examples.

[0170] result Analysis of the 4xKO cell lines (knockout of GBX2, CDX1, CDX2, and CDX4) showed that cell specification along the anterior-posterior axis was prevented, resulting in cells that generate more midbrain cell types. When expression of genes representing cell types along the dorsal-ventral axis was analyzed, control and 4xKO cell lines showed no significant changes in the proportion of cells generated (Figure 3D). Notably, we found that both cell lines were able to generate basal plate cells, as indicated by expression of NKX gene family members and PHOX2A / B (Figure 6A).

[0171] Single-cell analysis on day 28 of dopaminergic differentiation revealed 15 cell clusters in 4X and H9 cells combined. The anterior-ventral marker NKX2.1 was predominantly expressed in clusters 11 and 6, whereas the dorsal-ventral medial marker PAX6 was expressed in cluster 12 (Fig. 6B).

[0172] conclusion Midbrain dopaminergic neurons arise from the floor plate, the most ventral population of the neural tube, and NKX family members are important for the specification of the lateral (substrate) population of the neural tube.

[0173] Based on the data presented, we conclude that the same knockout approach can be extended to NKX family members and other substrate markers to silence their cell types and enhance their specification to midbrain dopaminergic neural precursors.

[0174] Example 7 – LR-PSCs efficiently generate mesDA neurons under conditions favoring hindbrain identity Research Objective To investigate the extent to which midbrain floor plate precursors can generate mesDA neurons.

[0175] Materials and Methods See the previous examples.

[0176] result The 1 μM GSK3i protocol was extended to 62 DIV. At 62 DIV, the two cell lines occupied almost entirely separate clusters (chi-squared, P<0.0001) (data not shown). 4X cells were largely separated into two major cell types: the hindbrain r1 floor plate cluster (clusters 4, 5, and 6) expressing FOXA2, SHH, NETRIN1, SPON1, and EN1, and the neuronal cluster (clusters 0 and 8; Figure S11A). This neuronal population contained mesDA neurons identified by expression of TH, FOXA2, LMX1A, and EN1 (Figure S11A-B). Clusters 0 and 8 were almost entirely composed of 4X cells (4X: 82% and 77%, H9: 18% and 23%) (data not shown). Closer examination of the differences between clusters 0 and 8 identified a subset of cells within cluster 8 that expressed NKX2.1, a marker for hypothalamic neurons.

[0177] In contrast to 4X cells, H9 cells formed one major connected cluster (clusters 1, 2, 3, and 7) and two small isolated clusters (clusters 9 and 10) (data not shown). All six clusters were predominantly populated by cells expressing markers indicative of vascular leptomeningeal cysts (VLMCs), namely COL3A1, IFITM2, and S100A11 (Fig. 11A). Interestingly, EN1 was almost absent in the VLMC clusters (data not shown). Cluster 7 also contained a population of cells (41%) expressing STMN2, SEMA3C, and PDLIM1 (Fig. 11A and 11C), corresponding to a subtype of peripheral sensory neurons according to the single-cell brain atlas.

[0178] We next sought to investigate the subtypes of mesDA neurons by subclustering TH-positive neurons (data not shown). To distinguish between substantia nigra and ventral tegmental area (VTA) DA neurons, we assessed the expression of GIRK2 (also known as KCNJ6) and calbindin D (CALB1). GIRK2 was highly expressed in subclusters 1 and 3, accounting for 38% of the TH population, and only a small proportion of TH neurons expressed CALB1 (13.6%; Fig. S1D).

[0179] To further support the single-cell sequencing results, histological analysis was performed. The same growth factor paradigm was used, but the differentiation protocol was adapted to generate organoids, providing an optimal environment for neuronal survival (Figure 12A). At 83 DIV, a large population of FOXA2 / TH double-positive DA neurons was observed within organoids made from 4X cells (data not shown). In contrast, TH-positive cells were occasionally scattered throughout organoids made with H9 cells. However, few TH-positive cells co-expressing FOXA2 were detected (data not shown). These results were consistent with histological analysis of 2D cultures at 62 DIV (data not shown). Further examination of TH-positive neurons showed that, consistent with the single-cell data, the most abundant population of 4X cell-derived TH neurons co-expressed GIRK2 (data not shown) and that a small population of CALB1 / TH double-positive neurons was present (data not shown).

[0180] Single-cell sequencing data showed that the majority of H9 cells were VLMCs (clusters 1, 2, 3, 7, 9, and 10; 93% of H9 cells). To confirm this result, we examined the expression of VLMC markers in organoids at 83 DIV and identified a large population of COL3A1 / COL1A1 double-positive cells with non-neuronal morphology among H9 cells (data not shown). No cells positive for COL3A1 or COL1A1 were found among 4X cells (data not shown).

[0181] conclusion mesDA neurons can be generated from 4x cells under caudalized conditions.

[0182] Example 8 – Genes representing cell types along the dorsal-ventral axis LR-PSC-derived DA neurons exhibit pacemaker activity Research Objective To investigate the electrophysiological properties of DA neurons.

[0183] Materials and Methods See the previous examples.

[0184] result At DIV80–DIV84, in vitro electrophysiological recordings were performed in whole-cell patch-clamp configuration (data not shown). We observed that the cells had become electrophysiologically mature neurons, as measured by their ability to produce repetitive action potentials upon somatic current injection (Figure 12B). Recordings in current-clamp mode revealed spontaneous pacemaker activity characteristic of DA neuronal identity, consisting of a mixture of single spikes and phasic bursts (Figure 12C). Membrane oscillations were attenuated at potentials below -50 mV (data not shown). Firing frequencies of samples ranged from 1 to 5 Hz (Figure 12D). Furthermore, HPLC analysis of cell extracts showed that the DA content of 4X cells was significantly higher than that of H9 cells (287.4 nmol / g of 4X cells vs. 65.1 nmol / g of H9 cells, P = 0.002; Figure 12E).

[0185] conclusion Based on the present data, it is concluded that dopaminergic neurons prepared from 4X cells can develop into electrophysiologically mature neurons.

[0186] Example 9 – Genes representing cell types along the dorsal-ventral axis In vivo analysis of 4X cells in a rat model of Parkinson's disease Research Objective Using current DA neuron differentiation protocols, DA neurons only make up a small percentage of the total cells in the graft after in vivo transplantation of mesDA precursors. We investigated how 4X LR-PSCs behave in vivo when transplanted into a rodent model of Parkinson's disease.

[0187] Materials and Methods See the previous examples.

[0188] result To confirm the results of the single cell analysis, the same midbrain differentiation protocol was used, but with a non-favorable caudalizing concentration of GSK3i (1 μM).

[0189] Four weeks after lesioning, a total of 250,000 4X or H9 cells were transplanted into the striatum of nude rats with 6-OHDA-induced medial forebrain bundle (MFB) lesions. A third group of lesioned rats that did not receive transplants served as lesion controls (6-OHDA, see Figure 13A for study design). Upon transplantation, all three groups of rats showed the same amphetamine-induced ipsilateral rotations / min (limit for inclusion: 5 rotations / min, Figure 13B), confirming a significant reduction in DA striatal innervation. All three groups showed forelimb asymmetry in the cylinder test, with rats mostly touching the wall or landing on the floor after rearing with the ipsilateral forelimb (6-OHDA total 52.4%; H9 70.4% and 4X 67.4%) and rarely with the contralateral forelimb (6-OHDA total 1.3%; H9 1.3% and 4X 0%), further supporting the induction of DA depletion by 6-OHDA (Figure 13C).

[0190] At 8 weeks after transplantation, rats receiving 4X cells showed complete correction of amphetamine-induced ipsilateral rotations (pre-transplant: 10.6 rotations / min vs. week 8: 0.35 rotations / min), suggesting that sufficient amounts of dopamine had been released into the striatum to normalize their behavior (Fig. S13D) or even overcompensate, as suggested by the number of contralateral rotations observed at 18 weeks after transplantation (-3.12 rotations / min). However, H9 cell-transplanted rats showed statistically the same number of ipsilateral rotations as the control 6-OHDA-lesioned group throughout the experiment (pre-transplant: 9.8; week 8: 9.7 and week 18: 9.2 rotations / min), and only showed a significant decrease in the number of rotations at 18 weeks compared to pre-transplant values ​​(pre-H9 transplant: 11.5; week 8: 12.6 and week 18: 5.6 rotations / min, Fig. S13D). Analysis of locomotor behavior in the cylinder test confirmed a significant improvement in 4X cell-implanted rats, as they used the contralateral forelimb alone (9.7% overall) or together with the ipsilateral forelimb (46.3% both) during the test at 18 weeks (Figure 13E). However, both H9 cell-implanted and 6-OHDA-lesioned rats used the ipsilateral forelimb most of the time (76.1% and 79.3% overall, respectively) as observed before transplantation, and used both forelimbs less than 30% of the time, but rarely used the lesioned contralateral forelimb when housed in the cylinder (Figure 13E). Thus, 4X cell transplantation significantly improved both drug-induced and locomotor behaviors after 6-OHDA-induced lesioning of the MFB.

[0191] Histological analysis of postmortem brains showed that rats transplanted with 4X cells had graft-derived TH-positive cells in the area of ​​injection, i.e., the striatum, as well as in the globus pallidus, corpus callosum, and areas of the cortex above the striatum (Figure 13F). However, H9-derived TH-positive cells remained mostly in the striatum, and in a few animals were also found in the globus pallidus. Quantification of graft-derived TH-positive cells (in the striatum and globus pallidus) showed that 4X cell-transplanted rats (average 23,520 TH-positive cells per graft) had significantly more TH-positive cells per graft than H9 cell-transplanted rats (1,898 TH-positive cells per graft), resulting in a higher yield (9,408 TH-positive cells per 100,000 transplanted 4X cells vs. 759 TH-positive cells per 100,000 transplanted H9 cells) (Figure 13G-H). The TH-positive 4X cell grafts spread across six to eight coronal AP striatal sections (in a series of 8), whereas the H9 cell grafts occupied four to five sections. Thus, the estimated graft volume was 10.11 mm in the 4X cell-grafted rats (20.46 mm 3 ) in rats with H9 cells transplanted (12.69 mm 3 ) was 61% greater than that in the 4X cell transplant group (Figure 13I). The increase in the number of TH-positive cells led to a significantly higher density of TH cells in the grafts in the 4X cell transplant group (1,090 ± 464 cells / mm 3 143±49 cells / mm in the H9 cell transplant group 3 ; P<0.0001), consistent with the rapid and robust behavioral recovery observed in the 4X cell transplant group.

[0192] Further examination of the grafts demonstrated that all TH-positive neurons found in 4X and H9 cell grafts co-expressed the human nuclear marker human nuclear antigen (HNA) (data not shown). In grafts from 4X cell-transplanted rats, TH-positive neurons co-expressed FOXA2, LMX1A, and EN1, indicating that they were mesDA neurons (data not shown). To distinguish A9 and A10 neurons, we calculated the percentage of TH-positive neurons expressing GIRK2 and CALB1, and found that 75.4% ± 4.99 of TH-positive neurons were GIRK2 positive (Figure 13J).

[0193] Interestingly, H9 cell-derived TH-positive neurons were also found to be positive for FOXA2, LMX1A, and EN1 (data not shown). This is in contrast to in vitro experiments in which H9 cell-derived TH-positive neurons expressed little FOXA2 (data not shown), suggesting that the in vivo environment is more permissive for the development and survival of TH-positive neurons than the in vitro environment. Since the in vitro data showed that H9 cells generated a large number of VLMCs, we examined the expression of vascular markers in both 4X and H9 cell transplanted rat grafts. A large population of COL3A1 / COL1A1 / HNA triple positive cells was found in H9 cell grafts, whereas 4X cell grafts showed few detectable COL1A1 positive cells co-expressing the marker HNA (data not shown).

[0194] conclusion Overall, the in vivo histological data demonstrated that 4X cells were capable of generating a robust population of mesDA neurons, consistent with the rapid locomotor recovery seen at 8 weeks.

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Claims

1. A pluripotent stem cell characterized by inactivation of GBX2.

2. The pluripotent stem cell according to claim 1, in which GBX2, CDX1, CDX2 and CDX4 are inactivated.

3. A pluripotent stem cell as described in claim 1 or 2, in which GBX2, CDX1, CDX2, CDX4, GBX1, HOXA1, HOXA2 and HOXB1 are inactivated.

4. The pluripotent stem cell according to any one of claims 1 to 3, wherein the gene is inactivated by gene knockout or introduction of a premature stop codon.

5. Use of the pluripotent stem cells described in any one of claims 1 to 4 for lineage-restricted cell differentiation of the pluripotent stem cells and / or for generating dopaminergic neural progenitor cells or their cell derivatives.

6. A dopaminergic neural progenitor cell or a cell derivative thereof differentiated from the pluripotent stem cell according to any one of claims 1 to 4.

7. A dopaminergic neural progenitor cell or a cell derivative thereof, characterized in that GBX2 is inactivated.

8. The dopaminergic neural progenitor cell or its cell derivative according to claim 7, wherein GBX2, CDX1, CDX2 and CDX4 are inactivated, or GBX2, CDX1, CDX2, CDX4, GBX1, HOXA1, HOXA2 and HOXB1 are inactivated.

9. The dopaminergic neural progenitor cell or cell derivative thereof according to any one of claims 6 to 8, which expresses FOXA2, LMX1A, OTX2, EN1, and / or TH, and / or expresses FOXA2, LMX1A, OTX2, EN1, SPRY1, WNT1, CNPY1, PAX8, ETV5, PAX5, SP5, and / or TLE4 positive cells.

10. A dopaminergic neural progenitor cell or a cell derivative thereof described in any of claims 6 to 9, wherein the dopaminergic neural progenitor cell or a cell derivative thereof is selected from the group consisting of dopaminergic neural cells, midbrain dopaminergic progenitor neural cells, midbrain floor plate progenitor cells, and caudal midbrain progenitor cells.

11. The dopaminergic neural progenitor cell or a cell derivative thereof according to any one of claims 6 to 10 for use as a pharmaceutical.

12. The dopaminergic neural progenitor cell or cell derivative thereof according to any one of claims 6 to 10 for use in stem cell therapy and / or for use in the treatment, prevention or alleviation of a neurodegenerative disease.

13. Use of a dopaminergic neural progenitor cell or a cell derivative thereof according to any one of claims 6 to 10 in an in vitro drug screening assay.

14. A method for providing a pluripotent stem cell according to any one of claims 1 to 4, comprising inactivating GBX2 in the pluripotent stem cell.

15. The method of claim 14, comprising inactivating GBX2, CDX1, CDX2 and CDX4, or inactivating GBX2, CDX1, CDX2, CDX4, GBX1, HOXA1, HOXA2 and HOXB1.

16. Pluripotent stem cells obtained / obtainable by the method according to claim 14 or 15.

17. A method for providing a dopaminergic neural progenitor cell or a cell derivative thereof, comprising providing a pluripotent stem cell according to any one of claims 1 to 4 or 15 to 16, and differentiating the provided pluripotent stem cell into a dopaminergic neural progenitor cell or a cell derivative thereof.

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