# Patent

## Title
Generation of functional basal forebrain cholinergic neurons from stem cells

## Abstract

The present invention provides method, compositions, and systems for generating basal forebrain cholinergic neurons (BFCNs) using FGF8, SHH, LXH8, GBX1, or vectors encoding these ligands, as well as using such BFCNs to treat neurological disorders such as Alzheimer's disease.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application No. 61/362,555, filed Jul. 8, 2010, which is incorporated by reference in its entirety.


STATEMENT REGARDING GOVERNMENT FUNDING
This invention was made with government support under grant number 5 P50 NS054287 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention provides method, compositions, and systems for generating basal forebrain cholinergic neurons (BFCNs) using FGF8, SHH, LHX8, GBX1, or vectors encoding these ligands, as well as using such BFCNs to treat neurological disorders such as Alzheimer's disease.
BACKGROUND
The basal forebrain cholinergic system is the predominant source of cortical cholinergic input and is comprised of cholinergic projection neurons travelling from the basal telencephalon through the neocortex to the hippocampus, olfactory system, cortical mantle, and amygdala. Alzheimer's disease-related tauopathies arise earliest in cholinergic neurons of the basal forebrain and loss of these neurons parallels cognitive decline (1-4). Human lesion (5-7) and MRI (8) studies have demonstrated the role of BFCN in memory function, while studies in monkeys, rats and mice using a specific BFCN immunotoxin have further demonstrated the role of these cells in memory function (9-12), hippocampal neurogenesis, and the survival of new neurons (13-15). Additionally, numerous animal models have demonstrated the necessity of BFCN for functional plasticity of the motor cortex after training (16) or damage (17), and also throughout the visual (18) and auditory (19) cortices, data which can be partially replicated with anticholinergic drugs in humans (20). Being of such import in Alzheimer's disease, memory formation, and cortical plasticity, the derivation of BFCN from human stem cells could be of great potential therapeutic benefit as it might allow the repopulation of BFCN after loss or damage.
SUMMARY OF THE INVENTION
The present invention provides method, compositions, and systems for generating basal forebrain cholinergic neurons (BFCNs) using FGF8, SHH, LHX8, GBX1, or vectors encode these ligands, as well as using such BFCNs to treat neurological disorders such as Alzheimer's disease or Parkinson's disease.
In some embodiments, the present invention provides methods of generating basal forebrain cholinergic neurons (BFCNs) comprising: treating neural progenitor cells with at least one ligand, or an expression vector that expresses the ligand, wherein the ligand is selected from the group consisting of: FGF8, a biologically active fragment or variant of FGF8; a FGF8 small molecule mimetic; sonic hedgehog (SHH), or a biologically active fragment or variant of SHH, or a SHH mimetic; LHX8 or a biologically active fragment or variant thereof (or a LHX8 mimetic), GBX1 or a biologically active fragment or variant thereof (or GBX1 mimetic); wherein the treating generates BFCNs. In certain embodiments, the neural progenitor cells are contacted with some or all of the above recited ligands. For example, in certain embodiments, the neural progenitor cells are contacted with both LHX8 and GBX1. In other embodiments, the neural progenitor cells are contacted with SHH and/or FGF8 and both LHX8 and GBX1, or vectors encoding some or all of the recited ligands.
In particular embodiments, the methods further comprise generating the neural progenitor cells by treating embryonic stem cells with retinoic acid. In certain embodiments, the ligand is a human ligand (e.g., human SHH or FGF8). In other embodiments, neural progenitor cells are human neural progenitor cells. In other embodiments, the methods further comprise treating the neutral progenitor cells with BMP9. In additional embodiments, the methods further comprise transfecting the neural progenitors with vectors that cause overexpression of Lhx8 (e.g., human accession number NM—001001933, or mouse accession number NM—130869), or biologically active fragment or variant thereof, and Gbx1 (e.g., human accession number NM—001098834) or a biologically active fragment or variant thereof.
In certain embodiments, the present invention provides methods of generating basal forebrain cholinergic neurons (BFCNs) comprising: transfecting neural progenitor cells with vectors that cause overexpression of Lhx8 and Gbx1, wherein the transfecting generates BFCNs. In some embodiments, the methods further comprise generating the neural progenitor cells by treating embryonic stem cells with retinoic acid. In further embodiments, the neural progenitor cells are human neural progenitor cells. In other embodiments, the methods further comprise treating the neural progenitor cells with at least one ligand selected from the group consisting of: FGF8, a biologically active fragment or variant of FGF8; a FGF8 small molecule mimetic; sonic hedgehog (SHH), or a biologically active fragment or variant of SHH, or a SHH small molecule mimetic.
In particular embodiments, the present invention provides methods of treating a patient with a neurological disorder comprising: implanting an isolated population of BFCN's into the brain area of a patient with a neurological disorder. In certain embodiments, the BFCN's are generated according to the methods described herein. In further embodiments, the neurological disorder is Alzheimer's disease or related disease. In certain embodiments, the neurological disorder is Parkinson's disease.
In some embodiments, the present invention provides system and kits comprising: a) neural progenitor cells, and b) at least one ligand selected from the group consisting of: FGF8, a biologically active fragment or variant of FGF8; a mimetic of FGF8 (e.g., a small molecule mimetic of FGF8), sonic hedgehog (SHH), or a biologically active fragment or variant of SHH, or a mimetic of SHH (e.g., small molecule mimetic).
In certain embodiments, the present invention provides system and kits comprising: a) neural progenitor cells, and b) at least one expression vector configured for expressing a ligand selected from the group consisting of: FGF8, a biologically active fragment or variant of FGF8; a mimetic of FGF8 (e.g., a small molecule mimetic of FGF8), sonic hedgehog (SI-1H), or a biologically active fragment or variant of SHH, or a mimetic of SHH (e.g., small molecule mimetic).
In some embodiments, the present invention provides systems comprising: a) neural progenitor cells, and b) at least one ligand, or at least one expression vector encoding the ligand, wherein the ligand is selected from the group consisting of: FGF8, a biologically active fragment or variant of FGF8; sonic hedgehog (SHH), or a biologically active fragment or variant of SHH; LHX8, or a biologically active fragment or variant of LHX8; and GBX1, or a biologically active fragment or variant of GBX1.



DESCRIPTION OF THE FIGURES
FIG. 1 shows generation of BFCN through BMP9 treatment. a,b: Confocal microscopy demonstrates that cells generated through BMP9 treatment of FGF8/SSH pretreated neural progenitors express ChAT, p75, and MAP2 and have a projection neuron morphology, while control neurons are only MAP2 positive. c,d: Confocal microscopy of equivalent cells stained only for ChAT and MAP2 show the same BMP9 response and long ChAT+, MAP2-axons. All scale bars=20 uM. e: qRT-PCR analysis shows 12-40 fold increases of RNA levels for markers characteristic of the BFCN. Bars are standard error, N=4. All increases were significant by ANOVA (p values: *<0.0001, **=0.0014, ***<0.0001,****<0.0001). Data are from four replicate experiments; error bars show s.e.m. fig: BMP9-mediated ChAT immunopositivity is associated with expression of the vesicular acetylcholine transporter (VChaT). Lower magnification analysis of fields of neurons stained for VChAT. Scale bars=100 uM. Data in a-d,f-g are from five replicate experiments, data in e is from 4 replicate experiments; error bars show s.e.m.
FIG. 2 shows generation of BFCN through transcription factor overexpression. a: Confocal analysis of FACS-purified neurons shows strong expression of ChAT, p75, and MAP2. b: Confocal microscopy shows FACS-purified neurons immunostained only for ChAT and MAP2. c: FACS-purified neurons shown at higher power contain large numbers of vesicles positive for VChAT. All scale bars=20 uM. d: qRT-PCR analysis shows large increases in ChAT and p75 RNA transcript levels. Bars show s.e.m., N=4. Both increases were shown to be significant by ANOVA (p values: *=0.0002, **=0.0031).
FIG. 3 shows LhxB siRNA blocks BMP9 effects on BFCN differentiation. a: LhxB siRNA nucleofection blocks the BMP9 mediated increase in LhxB levels, causing a reduction in LhxB transcript to levels below basal expression when compared to scrambled siRNA nucleofection after BMP9 treatment of dissociated and plated neural progenitors. b: qRT-PCR analysis indicates that LhxB siRNA inhibits the BMP9-mediated BFCN differentiation of human neural progenitors, with only a 2-fold but still significant increase in levels of ChAT mRNA (p*=0.0225) after the siRNA treatment. c: Neurons generated from LhxB siRNA-expressing neural progenitors fail to become ChAT immunopositive. Scale bar=20 uM. Data in b-c are from three replicate experiments; error bars show s.e.m.
FIG. 4 shows quantification of neuronal differentiation into BFCN. a: A high percentage of neurons from the FACS-purified (94.00±1.53%) or BMP9-treated (85.59±1.31%) populations are ChAT immunopositive, while control (0.89±0.24%) and LhxB siRNA-treated (1.26±1.33%) populations fail to express ChAT. All populations are significantly different by Mann-Whitney U Test (p values: *=0.035, **<0.001, ***=0.001,****=0.006) except BMP9 vs Nucleofected (#, p=0.066). N=4,700 control, 2,565 siRNA, 2,582 BMP9, or 1,718 nucleofected cells from 4 (control, BMP9 and nucleofected) or 3 (siRNA) replicate cultures. Error bars show s.e.m. b: Representative ChAT immunohistochemistry demonstrates the clear distinction of ChAT immunopositivity between positive and negative cells. Scale bar=20 uM.
FIG. 5 shows immunohistochemical evidence for, and characterization of, functional synaptic transmission after engraftment of FACS-purified neurons into murine ex vivo hippocampal slice cultures. a: FACS-purified neuronal populations stably engraft in mouse hippocampal ex vivo slice cultures and project long networks of axons. All green fluorescence in FIG. 5 is the eGFP expression from the adenovirally-labeled FACS-purified LhxB/Gbx1 transiently overexpressing neurons. Scale bar=50 uM. b: Murine presynaptic terminals (synapsin1, red channel) line the axons of the engrafted cells, giving immunohistochemical verification of the electrophysiologically detected synaptic inputs to these cells. Scale bar=5 uM. c: Transcription factor-generated basal forebrain cholinergic neurons contain presynaptic terminals (synapsin1, red channel) within their axons, indicating that they are generating synapses with other neurons.
FIG. 6 show electrophysiological recordings confirm functional cholinergic neurotransmission in LhxB/Gbx1-generated basal forebrain cholinergic neurons a: Confocal microscope image of eGFP expressing neurons engrafted into mouse dentate gyrus (DG) slice cultures. b: Spontaneous action potentials in the presence of 4-AP (1OOI1M), recorded from an eGFP expressing neuron. The cell was held at −60 mVin current clamp mode, (scale bars, 200 ms, 2 pA, n=3). Lower trace illustrates action potentials from the same cell displayed on an extended scale (100 ms). c: Representative 5, traces of spontaneous GABAergic post-synaptic currents (PSCs) detected in eGFP-expressing cells under voltage clamp conditions. Using high KCl in the pipette, at −70 mV eGFP-positive cells displayed numerous PSCs. The frequency and amplitude of PSCs was significantly reduced after application of bicuculline (BIC, 100  ̃M, *p<0.01, 5 out of cells) and CNQX (10  ̃M). PSCs reappeared after 10 minutes washing (lower trace). d: In eGFP-expressing cells the frequency (**p<0.01) and amplitude (*p<0.05) of PSCs were significantly blocked by BIC (100 ̃M) plus CNQX (10 J1.M,n=5) but not by methyllycaconitine (MLA, 10 nM) or dihydro-1S-erythroidine (DH ̃E, 1J1.M, Not Shown). e: (i) PSCs recorded from a murine neuron in close proximity to an eGFP-expressing cell. PSCs were recorded under whole cell voltage clamp (−70 m V) conditions. (ii) These PSCs were partially blocked by BIC (100  ̃M) and CNQX (10 ̃M). (iii,iv) The frequency of PSCs was further blocked by the nicotinic antagonists MLA (10 nM) and DH ̃E (1  ̃M). (v) PSCs reappeared following washout of these drugs. f: In non-eGFP expressing cells juxtaposed to eGFP expressing cells MLA and DHβE produced a significant decrease in PSC frequency, (n=7). Error bars in d and f show s.e.m.
FIG. 7 shows HB9 Immunohistochemistry. a,b: At maximum 594 nM laser intensity on a confocal microscope, presumptive BFCN generated through BMP9 treatment are not HB9 (HLXB9) immunopositive, indicating that they are not cholinergic motor neurons. Scale bar=20 uM.
FIG. 8 shows BMP9 activity changes hNSC-derived neurons from a glutamatergic to cholinergic phenotype. Control neurons express high levels of VGlut1 (a) but low levels of VChAT (c,e), while 72 hours of 10 ng ml-1 BMP9 treatment after SHH/FGF8 pretreatment results in an opposite phenotype, with low levels of Vglut1 (b) and high expression of VChAT (d,f). These data suggest that the control neurons generated in these experiments default to a glutamatergic phenotype, and BMP9 is able to alter this default lineage commitment towards a cholinergic phenotype. Scale bars: a,f=20 ̃M, b-e=100 uM
FIG. 9 shows culture conditions utilized only support neuronal growth. Combinatorial staining for MBP and MAP2 (a), GFAP and MAP2 (b), or GFAP and MBP (c) show that the cultures generated through the BMP9 treatment paradigm contain only neurons after 16 days in these media conditions, and are free of oligodendroglial (MBP) or astroglial (GFAP) cells. Scale bar=100 uM.
FIG. 10 shows FORSE1 immunohistochemistry demonstrates the generation of forebrain progenitor cells. FORSE1 is an antibody specific to forebrain progenitor cells which binds to the Lex phosphacan, a brain-specific chondroitin sulfate proteoglycan. a: Neurospheres generated using the culture system are almost uniformly nestin positive (red immunofluorescence), but the control retinoic acid-derived cells do not express FORSE1 (green immunofluorescence), which correlates with their inability to be driven towards a BFCN lineage. b: Cells pretreated for 72 hours in 200 ng ml−1 SHH alone show a small increase in FORSE1 staining, and some ability to be made into BFCN (Not Shown). c: Neurospheres pretreated for 72 hours in 100 ng ml-i FGF8 and 200 ng ml−1 SHH show a marked increase in both FORSE1 expression and the ability to become BFCN (FIGS. 1,2,4). Scale bars=20 uM.
FIG. 11 shows the sequence of full length Gbx1 determined through RACE experiments. a: Nucleotide sequence of the novel human Gbx1 sequence (SEQ ID NO:1). b: The human (upper sequence; SEQ ID NO:2) and murine (lower sequence; SEQ ID NO:3) Gbx1 proteins have a high level of homology.
FIG. 12 shows the transcription factor induction following BMP9 treatment. a: Representative qRT-PCR curves showing the distinct upregulation of LhxB expression between 3 and 12 hours after 10 ng ml−1 BMP9 treatment of human neurospheres. b: Time course of transcription factor response to BMP9 treatment. LhxB levels increased to 72× baseline at 6 hours, while Gbx1 levels gradually increased to 4.62× baseline over 48 hours. Related factors, such as Lhx6 and Isl1 were unchanged following BMP9 treatment.
FIG. 13 shows representative FACS data. After gating to remove debris and doublets, neurons were FACS-purified based on GFP expression; only cells with expression levels markedly above the negative control were kept while minimally-expressing cells were discarded.
FIG. 14 shows orthogonal views of z-stacked confocal images. a,b,c: Orthogonal views of z-stacked confocal images indicate ChAT immunopositivity (green channel) is entirely encapsulated within MAP2 immunopositivity (red channel), demonstrating that ChAT immunopositivity is localized within the neurons and is neither background staining within the cells nor on their surface (side/top bar beside each image). Scale bar=20 uM. d: An orthogonal view of z-stacked confocal images confirms the generation of presynaptic terminals in nucleofected neurons expressing LhxB and Gbx1 after engrafting into murine hippocampal slice cultures. An enlarged and flattened rendering (i) of a synapse (ii), showing the total inclusion of synapsin1 within the axon. Inclusion is confirmed with individual x-axis (iii) and y-axis (iv) renderings of the synapse showing complete overlap of the punctate synapsin1 staining (red channel) within the eGFP-expressing engrafted axon. Scale bar=5 uM.
FIG. 15 shows a schematic of the timeline of media transitions.
FIG. 16 shows representative images of cells during differentiation. a: Typical untreated hESC colony morphology on matrigel. Colonies with a rim of differentiating cells, internal separation between the hESC, or non-circular colony shape were discarded. b: 10 uM retinoic acid treatment alters the morphology of the hESC, generating cells with both larger nuclei and expanded cytoplasm. c: Neurospheres after 4 days in neurosphere media. d: Neurons 5 days after FACS-purification. All scale bars 200 uM.
FIG. 17 shows expression vector used for transcription factor overexpression. a: A vector based on the pBudCe4.1 backbone (Invitrogen) was generated to express LhxB from the CMV promoter and Gbx1 from the EF-1a promoter. A constitutively active CAGGFP cassette was added into the vector backbone to allow FACS-purification, as either a GFP-fusion construct or IRES sequence could have altered gene function or expression levels. b: 24 hours after nucleofection, expression levels of LhxB and Gbx1 increase by 1,002.93× and 948.83× when compared with nucleofection of the GFP-only empty vector control, indicating that all three promoters cause functional transcription in the hNSC used for these experiments.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides method, compositions, and systems for generating basal forebrain cholinergic neurons (BFCNs) using FGF8, SHH, LHX8, GBX1, or vectors encoding these ligands, as well as using such BFCNs to treat neurological disorders such as Alzheimer's disease.
An early substantial loss of basal forebrain cholinergic neurons (BFCN) is a constant feature of Alzheimer's disease and is associated with deficits in spatial learning and memory. The ability to selectively control the differentiation of human embryonic stem cells (hESC) into BFCN would be a significant step towards a cell replacement therapy. Work conducted during development of embodiments of the present invention demonstrated a method for the derivation of a predominantly pure population of BFCN from hESC cells using diffusible ligands present in the forebrain at developmentally relevant time periods. Overexpression of two relevant human transcription factors (Lhx8 and Gbx1) in hESC-derived neural progenitors also generates BFCN. These neurons express only those markers characteristic of BFCN, generate action potentials, and form functional cholinergic synapses in ex vivo murine hippocampal slice cultures. siRNA-mediated knockdown of the transcription factors blocks BFCN generation by the diffusible ligands, clearly demonstrating the factors both necessary and sufficient for the controlled derivation of this neuronal population.
Although 14 distinct regional subpopulations of BFCN exist in murine cortex, the group arising from the median ganglionic eminence (MGE) is the largest and best characterized (21, 22); derivation of a human population of such cells was thus the focus of the Example below. Several proteins are characteristic of BFCN in vivo and in vitro. Choline acetyltransferase (ChAT), which catalyzes the formation of acetylcholine, is expressed by cholinergic neurons of both the basal forebrain and the motor system. In the cortex, TrkA, the high-affinity nerve growth factor receptor, is expressed by BFCN from development through adulthood (23), and is necessary for NGF-mediated survival of these neurons (24). The low-affinity neurotrophin receptor (p75NTR, p75), is also expressed by more than 95% of ChAT-positive cells in the basal forebrain (25). BFCN should also express acetylcholinesterase (AChE), but not NADPH diaphorase, somatostatin, or HB-9, which are specific to cortical, amygdalar, and motor cholinergic neuronal subpopulations.
Although BFCN are neurotrophin responsive, these neurons are still present in murine knockouts of TrkA (26), or p75 (27, 28), demonstrating that neither these receptors nor NGF (29) are required for BFCN lineage commitment. However bone morphogenetic protein-9 (BMP9, Growth and Differentiation Factor 2 (GDF2)), a member of the transforming growth factor-β (TGF-β) superfamily of signaling factors (30), is transiently expressed in vivo in the septum during the period of BFCN development, and treatment of mouse septal cultures with BMP9 increases expression of cholinergic markers. Injection of BMP9 into E14 and E16 mouse ventricles increases levels of acetylcholine detectable in the forebrain (31), and BMP9 induces the transcriptome of BFCN in cultured murine septal progenitors (32). The effects of BMP9 on cholinergic neurogenesis are both spatially and temporally limited, having minimal effects outside of the E14-16 septum (31).
Several transcription factors have been implicated in the differentiation of MGE-derived BFCN. Lhx8, a LIM-family homeodomain transcription factor, is expressed in the developing MGE (33); although sequentially related to Lhx6 (34), and with overlapping domains of expression, Lhx8-expressing cells become cholinergic neurons while Lhx6-expressing cells become GABAergic interneurons (35, 36). Lhx8-positive cells which become BFCN also express Gbx1 (35). Different murine Lhx8 knockout lines (37-39), have slightly different phenotypes, but all have distinct and specific reductions in BFCN with minimal effects on other neuronal subtypes. Transgenic cells expressing dysfunctional Lhx8 neither multiplied, died, nor differentiated, and continued to induce Gbx1 mRNA, demonstrating both Lhx8's involvement in differentiation of committed progenitors into a cholinergic phenotype without altering proliferation or survival, and Gbx1's independent induction and insufficiency for BFCN formation (38, 40). Nkx2.1, necessary for encoding the regional identity of the M GE, is expressed during development in all cells derived from this region (41). Nkx2.1 knockout animals lack BFCN (42) and all other populations generated in or migrating through the MGE.
EXAMPLES
Example 1
Controlled Generation of Functional Basal Forebrain Cholinergic Neurons from Human Embryonic Stem Cells
This example describes the generation of functional basal forebrain cholinergic neurons from human embryonic stem cells.
Results
Generation of Basal Forebrain Cholinergic Neurons Using Diffusible Ligands
Methods were initially developed to differentiate hESCs into BFCN through treatment with diffusible ligands known to be expressed in the developing murine MGE. hESC-derived neural progenitors (hNSCs) were first generated using retinoic acid and published protocols (43). As FGF8 and sonic hedgehog (SHH) are necessary for patterning the developing neural tube and specification of the primordial forebrain (44), and together induce the transcriptome of the MGE while inhibiting LGE-specific factors (45), pretreatment with SHH and FGF8 was used to differentiate hNSCs towards a forebrain progenitor fate. FGF8/SHH preprogrammed hNSCs were then dissociated and treated with BMP9. qRT-PCR analysis of these neurons at D16 (FIG. 1e) shows a large, significant increase in the expression of markers for the BFCN lineage, including ChAT, p75, TrkA, and AChE, while markers for other populations of cholinergic neurons, such as somatostatin and nitric oxide synthase, are expressed at levels below control neuronal cultures. TuJl levels remain unchanged, indicating that BMP9/FGF8/SHH treatment alters lineage selection within progenitors already committed to a neural fate. Immunostaining at D19 for ChAT or p75 shows a vast increase in numbers of Map2+/marker+ cells, (immunocytochemistry FIG. 1a; counts FIG. 4a) with 85.59±1.31% of cells becoming ChATpositive neurons with a projection neuron morphology, while 9.24±1.19% become ChATnegative cells with an interneuron morphology (N=5 replicate cultures; 2,582 cells). There is no HB9 (HLXB9) immunopositivity, indicating that these cholinergic neurons are not motor neurons (FIG. 7a,b). Control neurospheres, derived in parallel but neither pretreated with FGF8/SHH nor treated with BMP9, yielded 0.89±0.24% cholinergic neurons (N=4 cultures; 4,700 cells) (FIG. 1b,c), and expressed markers consistent with glutamatergic neurons (FIG. 8). Neither control nor BMP9 treated cultures contained cells immunopositive for markers of an astroglial (GFAP) or oligodendroglial (MBP) lineage (FIG. 9a-c) at D16. BMP9-derived neuronal axon growth and RNA expression were NGF-responsive, indicating their expression of TrkA. Direct treatment of hESC (instead of the FGF8/SHH restricted hNSC) with BMP9 did not generate neurons; without SHH/FGF8 pretreatment neurospheres generate progenitors which fail to become BFCN after BMP9 treatment. Pretreating with SHH alone before BMP9 treatment yielded neurons with BFCN-like RNA expression patterns, but with much lower neuronal survival than when FGF8 was added. These data indicate that a temporally precise regime of neural restriction, followed by pretreatment with forebrain-specifying factors, followed by exposure to a factor expressed in the MGE during BFCN generation is able to generate a population of neurons significantly enhanced for markers of the BFCN lineage. Although this diffusible ligand treatment greatly increases the percentage of BFCN generated from hESC cells (85%), derivation of a pure population of any single cell type using extracellular signaling molecules may potentially not be possible due to heterogeneity of both hESC and hESC-derived neural progenitors. Although staining for FORSE1, an immunohistochemical marker of forebrain progenitors (46), increases greatly after FGF8/SHH pretreatment (FIG. 10), expression is not uniform, and progeny from these neurospheres vary depending on the hESC passage number and the duration in culture between neurosphere generation and dissociation.
Generation of Basal Forebrain Cholinergic Neurons Through Controlled Gene Expression
Because hNSC had variable, highly timing-dependant responses to BMP9 treatment (only 85% BFCN) it was wondered if an increased purity of BFCN could be generated through controlled expression of specific human genes, potentially bypassing differences in hNSC expression of receptors or downstream signaling components. Based on murine embryology, the transcription factors Lhx8 and Gbx1 were chosen for further study. Since no complete human Gbx1 sequence has been published, the full-length human sequence, with an ORF encoding 439 amino acids (FIG. 11), was derived through RACE experiments using a human 13-week fetal brain mRNA library. Involvement of these genes as downstream effectors of the BMP9 signaling cascade was confirmed through treating either control or FGF8/SHH-pretreated neurospheres with BMP9 and analyzing the time courses of individual transcription factor responses (FIG. 12). Lhx8 transcripts increased 72-fold at 6 hours following BMP9 treatment and remained elevated for 12 hours before decreasing. Gbx1 had a delayed 4.62-fold increase at 48 hrs after treatment, while factors with high sequence homology (Lhx-6) or related to other neuronal populations (Is)etl), were unaffected by BMP9 treatment. Control (non-FGF8/SHH pretreated) neurospheres had negligible changes in transcription factor expression following BMP9 treatment, mirroring their inability to generate cholinergic neurons. Although not detected in intact neurospheres, Nkx2.1 RNA expression was transiently detected 12 hours after neurosphere dissociation, indicating that, as expected, these neurons are derived from an Nkx2.1-expressing progenitor lineage.
Since Lhx8 and Gbx1 are specifically upregulated by BMP9 treatment of hNSCs, a technique was developed for nucleofection-based overexpression of these factors with a constitutively-active eGFP (enhanced-Green Fluorescent Protein) in FGF8/SHH-pretreated hNSCs, allowing for FACS-based purification of overexpressing cells. Expression of each factor individually in neural progenitors resulted in neurons with reproducible morphologies and RNA expression patterns, but without the BFCN phenotype. FGF8/SHH-treated forebrain progenitors were nucleofected and grown without BMP9. For RNA experiments, populations of cells were used without FACS-purification. Although only  ̃40% of cells were successfully nucleofected in each group (FIG. 13), this was sufficient to significantly increase expression of markers for the BFCN lineage (FIG. 2); these data were confirmed with FACS-purified neurons. The nucleofected cells were FACS purified 48 hours after nucleofection and then cultured for 19 days. Immunostaining for ChAT showed that 94.00±1.53% of cells became cholinergic neurons with long projecting axons, while the remainder became neurons without ChAT expression or with an interneuron morphology (N=3 cultures, 1,718 cells). Confocal analysis confirmed that the ChAT immunopositivity was entirely contained within the neuronal cytoplasm (FIG. 8a-c, FIG. 10b). Dual immunostaining for ChAT/p75 demonstrated that these cells are positive for both markers, whereas control neurons are 18.67±0.88% p75 positive. These RNA and immunohistochemical data show that the FACS-purified transiently Lhx8/Gbx1-overexpressing cells from SHH/FGF8 pretreated neural progenitors, when grown for 19 days in culture, are a highly purified population of human neurons expressing only those markers characteristic of the BFCN.
BMP9 and Lhx8/Gbx1 Overexpression Function Through One Pathway
Although these experiments demonstrate that the transcription factors Lhx8 and Gbx1 respond to BMP9 signaling after FGF8/SHH pretreatment and are able to drive progenitors towards a BFCN phenotype, it remained to be shown that the transcription factors were the downstream mediators of BMP9 signaling along a common pathway. To determine this, an siRNA-mediated knockdown of Lhx8 was performed on FGF8/SHH pretreated neural progenitors. Simultaneously with the BMP9 treatment paradigm. Co-nucleofection of the Lhx8 siRNA with a fluorescently-labeled scrambled siRNA indicated that over 900/0 of surviving cells were successfully treated with the siRNA. The siRNA blocked the BMP9 mediated upregulation of Lhx8 levels, reducing Lhx8 levels below basal expression (FIG. 3a). qRT-PCR analysis at D16 after Lhx8 knockdown demonstrates greatly reduced BFCN marker expression compared to equivalent BMP9 treated cells without siRNA (FIG. 3b), and immunohistochemistry showed an equivalent reduction to 1.26±1.33% of MAP2-positive neurons expressing ChAT (immunocytochemistry FIG. 3c, counts FIG. 4a) (N=3 cultures, 2,565 cells). The ability of Lhx8 knockdown to block the effects of BMP9 treatment indicates that it is a necessary downstream effector of the BMP9-mediated signaling cascade in FGFS/SHH pretreated neural progenitors, and demonstrates that the BMP9 treatment and the Lhx8/Gbx1 overexpression studies function through the same pathway. That the Lhx8-specific knockdown blocks the effects of BMP9 further supports the conclusion that the ChAT+, p75+, Map2+ cells generated by both the small molecule and transcription factor treatments are BFCN since this is the only neuronal population specifically lost after null mutation of Lhx8 (37-39).
ChAT Staining Correlates with Enzymatically Functional ChAT
The functionality of the ChAT detected by immunohistochemistry was confirmed through direct detection of acetylcholine. The cytoplasm of neurons from both derivation methods had markedly increased ACh levels: 5.11±0.66 ngACh/ugProtein for BMP9 treatment and 6.94±3.06 ngACh/ugProtein from nucleofections vs. 0.83±0.02 ngACh/ugProtein for control neurons (N=3 replicate cultures/condition); as expected, Lhx8 siRNA expression reduced acetylcholine levels towards basal levels—1.79±0.16 ngACh/ugProtein. These data confirm that neuronal ChAT immunopositivity correlates to functional ChAT enzymatic activity.
Neurons Engraft in Mouse Ex Vivo Slice Cultures
Although neurons generated through Lhx8/Gbx1 nucleofection almost uniformly express markers characteristic of BFCN and produce acetylcholine, it remained to be determined whether these cells are functional ex vitro. To address this, a FACS-purified population was virally labeled with eGFP and allowed to engraft in entorhinal-hippocampal murine ex vivo cortical slices for 7-19 d. The cells migrated and extended long axonal projections deep into the cortical slices (FIG. 5a). These neurons continued to express markers of the BFCN lineage and formed synaptic structures with mouse cortical neurons. Immunohistochemistry for synapsin1, a marker specific for the presynaptic terminal, showed a large number of presynaptic regions directly abutting neurite outgrowths from the engrafted cells (FIG. 5b), indicating, as confirmed electrophysiologically below, that murine neurons form functional synapses with these neurons. Confocal analysis also confirmed the presence of presynaptic regions within the engrafted cells (FIG. 5c; orthogonal view confirming synapsin1 inclusion FIG. 14d), suggesting that the BFCN are initiating synaptic transmission with murine cells. To better characterize these synapses, live cultures were treated with fluorescently-conjugated α-bungarotoxin, a highly selective and permanent α7 nicotinic cholinergic receptor antagonist. Axons from the engrafted BFCN terminated on α-bungarotoxin-positive processes (FIG. 5d), confirming that these neurons are capable of generating cholinergic synapses.
Electrophysiological Function after Engraftment in Murine Slice Cultures
To demonstrate that the neurons that were generated are functional, electrophysiological recordings were performed from FACS-purified eGFP-expressing cells engrafted into hippocampal slice cultures (FIG. 6a). Recording from engrafted cells under current clamp conditions in the presence of 4-Aminopyridine (4-AP), numerous spontaneous action potentials were observed (FIG. 6b), consistent with their neuronal phenotype. Furthermore, engrafted eGFP-positive cells were observed that exhibited numerous postsynaptic currents (PSCs) under voltage clamp conditions. These currents were blocked by a combination of the GABA-A receptor antagonist bicuculline and the glutamate receptor blocker CNQX (FIG. 6c,d). No PSCs recorded from engrafted neurons were sensitive to nicotinic blocking drugs including methyllycaconitine (MLA), a blocker of α7 nicotinic receptors, or dihydro-β-erythroidine (DHβE), a blocker of α4β2 nicotinic receptors. By contrast, murine neurons in close proximity to these eGFP-expressing neurons were recorded, it was always observed that a significant percentage of PSCs were blocked by MLA or DHβE, indicating the presence of nicotinic cholinergic synapses on these cells (FIG. 6e,f), while recordings from murine neurons distal to eGFP-expressing cells never showed cholinergic PSCs (Not Shown, n=5). Thus neurons generated through Lhx8/Gbx1-overexpression are electrically excitable, being capable of generating action potentials; they receive synaptic inputs, all of which are mediated by glutamate or GABA, and they are capable of quantal release of acetylcholine at nicotinic synapses formed with other neurons: these properties establish these cells as bona fide cholinergic neurons.
Discussion
These experiments demonstrate, for example, the controlled generation of functional human BFCN from pluripotent stem cells. Treatment of hESC-derived neural progenitor cells with human orthologs of ligands known to be present in the MGE during murine embryogenesis generated a relatively pure (85%) population of these neurons. Higher yields and purity of these cells (94%) could be achieved through transient over-expression of the transcription factors Lhx8 and Gbx1 in hNSC. These neurons express all relevant markers of the BFCN lineage at both the RNA and protein levels, produce acetylcholine in vitro and do not express markers of other cholinergic lineages. Further, they generate electrophysiologically functional cholinergic synapses and spontaneous and induced action potentials ex vivo when engrafted into murine hippocampal slice cultures. Because acetylcholine produced by BFCN functions as a hippocampal neuromodulator (47, 48) it may not be necessary for transplanted neurons to replace the exact synapses lost through Alzheimer's disease related cell death in order to enhance memory function if it is only necessary to replace hippocampal cholinergic drive. These functional human cholinergic neurons thus could be use as a replacement for cells lost in Alzheimer's disease. The ability to derive these cells with high efficiency will also allow study of factors regulating the survival and function of this critical population of human neurons.
Although nucleofection experiments reliably generate a very high proportion of BFCN, the potential exists to increase the efficacy of these protocols. Altering the periods of growth in different media, especially during exposure to FGF8/SHH, has distinct consequences on the success of BMP9 treatment. Similar to the narrow spatial and temporal window of cortical BMP9 response during embryogenesis, almost no BFCN were generated either 24 hours before or after the times used here or with other departures from the protocol; further analysis of the timing or hNSC derivation might potentially increase the purity or survival of BFCN. Complete dissociation of neurospheres is important as undissociated fragments generate bFGF-responsive non-neuronal cells able to overgrow the cultures; similarly, cell cell signaling in progenitors plated at too high a density blocks their proper differentiation into BFCN. The BMP9-derived BFCN population contains some cells not committed to neuronal lineages but all cells generated from the FACS-purified nucleofections differentiated into neurons, suggesting that this population is more suitable for transplantation and might pose negligible transplantation risk; if necessary, established protocols for FACS-purification based on p75 expression (49) could remove the 6% nonBFCN neurons. These protocols were effective with the H1 hESC line in addition to the H7 hESC line used here. These studies used hESC between passage 29 and 35; BMP9 treatment had no effect on BFCN differentiation in cells above p40, and gene overexpression became progressively less efficient with increased passaging.
It remains unclear if Lhx8 and Gbx1 activity are the final mediators of signaling towards the BFCN lineage or if they are upstream of other genes along a common pathway. The differing responses of transcripts for these factors after BMP9 treatment suggests independent regulation, as does the prevalence of Gbx1 expression in presumptive BFCN in Lhx8 null mutants (38). Individual overexpression of Lhx8 or Gbx1 caused a small upregulation of the other factor but each factor individually was unable to generate cholinergic neurons. This example demonstrate that Lhx8 and Gbx1 are both necessary and sufficient to drive the differentiation of human neural progenitors into functional basal forebrain cholinergic neurons.
Methods
Generation and Programming of Neural Progenitors with BMP9 Treatment.
Using retinoic acid, neural progenitors were generated from hESC using modifications of an existing protocol (43). Cells from the H7 hESC line were grown in adherent culture on matrigel in hESC media conditioned, for 24 hours on a feeder layer of 56,000 gamma-irradiated mouse embryonic fibroblasts Jcm2 and supplemented to 4 ng m−1 bFGF. Prior to splitting, all colonies were assayed for morphology and all imperfect colonies were manually removed (FIG. 16). Two days after splitting, cells were treated with 10 μM RA in conditioned media for 7 days. Fresh RA aliquots were used every week, and RA, stock was made at 6 mM in 100% EtOH. RA-treated hESC were dissociated for 4 minutes with 5 mls accutase/10 cm dish at 370 followed by enzyme inactivation with media and gentle scraping. After pelleting at 150×G for 7 minutes, the small clumps were grown in non-adherent conditions for 4 days in hESC media without either conditioning, RA, or bFGF to begin neurosphere formation. These nascent neurosphere cultures were expanded in non-adherent dishes in neurosphere media for four days with 1⁄2 volume replaced after 48 hrs. Plates were kept on a slow rotary shaker to minimize neurosphere aggregation. Individual neurospheres were moved using a pipettor under a sterile microscope to the same media supplemented with 100 ng m−1 FGF8 and 200 ng m−1 SHH for 72 hrs with 1⁄2 volume replaced after 48 hrs. Neurospheres were dissociated in accutase at 37° for 10 minutes, with gentle agitation at 5 and 10 minutes, pelleted, then treated with trypsin inhibitor at 370 for a further 10 minutes. Cells were rinsed in HBSS and titrated gently through a p200 tip until all neurospheres had dissociated. Cells were plated on PDL-laminin in neuron media 1 for 5 days. For the first 24 hrs, media was supplemented with 100 ng m−1 SHH, 100 ng m−1 FGF8 and 10 ng ml-1 BMP9. For the next 48 hrs, media was supplemented with only B-MP9. Cells grew without additional mitogens for a subsequent 48 hrs when they were moved to neuron media 2, which has been shown optimal for the growth of murine BFCN (50), from D5 to D16-19. From D5 to D10, media was supplemented with 2.66 uM AraC to eliminate the growth of bFGF-responsive cells arising from fragments of undissociated neurospheres.
Generation of BFCN Through Nucleofection.
For nucleofections, 4 ug of DNA was used (a plasmid encoding both Lhx8 and Gbx1 expressed from separate promoters with a third promoter driving eGFP (FIG. 17) in hESC Nucleofection Solution-1 (Lonza) with program C-30 on an Amaxa nucleofector. Neurospheres were similarly pretreated for 72 hours in FGF8 and SHH, and were again plated into 100 ng ml−1 SHH and 100 ng ml−1 FGF8 for 24 hours following nucleofection, but in the absence of BMP9. Control neurons were nucleofected with the same vector without the Lhx8 and Gbx1 sequences. After 48 hours the neurons were detached with a combination of accutase and trypsin, then FACS-purified at low pressure using a Beckman Coulter MoFlo after gating to exclude debris and dead cells. Neurons were grown in the same media for the same periods as the BMP9 cells above, but without the AraC treatment as all of the FACS-purified cells differentiated following transcription factor expression.
siRNA Studies.
100 pg (5 ul of 20 nM stock) of Dharmacon ON-TARGETplus SMARTpool siRNA specific to human Lhx8 was used for each replicate siRNA nucleofection, while control replicates received an equal amount of scrambled non-specific ON-TARGETplus Non-Targeting Pool. To determine siRNA nucleofection efficiency, an equivalent amount of siGLO Red Transfection Indicator siRNA was used in separate experiments. To show the efficacy of the Lhx8 siRNA, dissociated neurospheres were nucleofected with either Lhx8 specific or non-targeting siRNA, plated for 6 hours, then treated with BMP9 for 6 hours before harvesting the cells for RNA.
Cell Analysis.
RNA was extracted with the RNAqueous-4PCR kit (Ambion), treated with TurboDNAse (Ambion) for 30 minutes, then reverse transcribed with Thermoscript for 90 minutes (Invitrogen). qRT-PCR was performed using an Eppendorf Realplex thermocycler and SybrGreen mastermix (Applied Biosystems). Before harvesting, all neurons were grown without NGF for 48 hrs and then again with NGF for the last 24 hrs; because ChAT is a 2.1 kb (kilo base pair) cDNA comprising 15 exons spliced from a 56 kb locus it is very stable and thus has low basal expression levels: this transient NGF withdrawal allows for accurate determination of gene expression levels. The sequences for all qRT-PCR primers are provided (Table 1).










TABLE 1






Forward
Reverse
SEQ ID NOs:







Acetylcholinesterase
GGAACCGCTTCCTCCCCAAATTG
TGCTGTAGTGGTCGAACTGGTTCTTC
SEQs 4 and 5


 


β-III Tubulin
ATCAGCGTCTACTACAACGAGGCC
CAAAGATGAAATTGTCAGGCCTGAAGAGATGT
SEQs 6 and 7


 


CNPase
AAGATGGACTTGGTCACCTACTTTGGAAAG
CGTCTTGGGTGTCACAAAGAGGG
SEQs 8 and 9


 


ChAT 5′
TGCCGCCTACTGAGAGCAA
GTGGCAGGAGTCAAGGTTGGT
SEQs 10 and 11


 


ChAT 3′
CATGAAGCAATACTATGGGCTCTTCTCCTC
GACGGCGGAAATTAATGACAACATCCAAG
SEQs 12 and 13


 


GAD1
CCAGAAAACTGGGGCTCAAGATCTG
GCAAACAGATTAGAGAAGTCAGTCTCTGTGC
SEQs 14 and 15


 


GAPDH
GAGCACAAGAGGAAGAGAGAGACCC
GTTGAGCACAGGGTACTTTATTGATGGTACATG
SEQs 16 and 17


 


Gbx1
GCTGGAAGCTGATGAGCTGCT
CTTCTCCTCATCTGAGCTGTACACCTTC
SEQs 18 and 19


 


Gbx1 Endogenous
GAAACCCCAAGATTGTTGTCCCCATAC
CAGATCCCTCGCCTTCCTAAGTTCTTG
SEQs 20 and 21


 


GFAP
CTGGATCTGGAGAGGAAGATTGAGTCG
CTCATACTGCGTGCGGATCTCTTTCA
SEQs 22 and 23


 


Islet1
TGAAATGTGCGGAGTGTAATCAGTATTTGGAC
CACACAGCGGAAACACTCGATGTG
SEQs 24 and 25


 


Lhx8
GTTTCAGAATTGTAGAGCACGCCACAAG
CTATGCAGCGCAGTTAACATCGTTCC
SEQs 26 and 27


 


Lhx8 Endogenous
GTTACCCCATTCAATGACACAACTGCC
CAGCAAAGTGATGTTGGAAATGCTTTAGGTG
SEQs 28 and 29


 


Lhx6
CACGGCTACATCGAGAGTCAGGTAC
CAATCTGGCTCCATTTACCTTCTCAC
SEQs 30 and 31


 


Nkx2.1
CTACTGCAACGGCAACCTGGG
CCATGAAGCGGGAGATGGCG
SEQs 32 and 33


 


NOS
CAGGCTGTGACTGATGACCACATC
AGGTCATGTTTGGAGATGACCCTTGAG
SEQs 34 and 35


 


P75NTR
GGAGAAAAACTCCACAGCGACAGTG
AGAGCCGTTGAGAAGCTTCTCCAC
SEQs 36 and 37


 


Somatostatin
CAGACTCCGTCAGITTCTGCAGAAG
CTTCAGGTTCCAGGGCATCATTCTC
SEQs 38 and 39


 


TrkA
GAGGTCTCTGTTCAGGTCAACGTCT
CTCAGTGAAGATGAAGCTGGTCTCATTGA
SEQs 40 and 41


 


Tyrosine Hydroxylase
AGTGTCATCACCTGGTCACCAAGTTC
CTTCAGCGTGGTGTAGACCTCCTT
SEQs 42 and 43


 


VGlut1
GCTACATTGTCACTCAGATTCCAGGAGG
ATCCTCACGAAGATGACACAGCCATAG
SEQs 44 and 45





qRT-PCR primer sequences. All primers have a melting temperature of 60° ± 0.3°. All primer sets (except GAPDH) span an intron.






Cells for immunohistochemistry were rinsed in 4% (wt/vol) PFA (paraformaldehyde) then fixed in 4% PFA for 20 minutes followed by 30 minutes of permeabilization with 0.10% (vol/vol) Triton X-100 (Sigma). After blocking in 10% (vol/vol) goat serum for 30 minutes, primary antibodies (ChAT 1:666 Ayes Labs, Map2 1:500 Abcam, p75 1:666 Abcam, VChAT 1:250 SYSY, VGlut 1:3000 SYSY, HB9 1:1000 Abcam, GFAP 1:1000 Sigma, GFP 1:500 Abcam, MBP 1:666 Sternberger Monoclonals Inc., Synapsin 1:500 SYSY, FORSE1 1:75 Developmental Studies Hybridoma Bank, Nestin 1:500 Abcam) were added for 60 minutes at room temperature. Appropriate fluorescent secondary antibodies (Ayes Labs (FITC)-conjugated anti-Chicken IgY 1:500 for ChAT, Molecular Probes 1:1000 for all others) were added for 45 minutes. All photomicroscopy and counts of ChAT, p75, Map2, synapsin1, α-bungarotoxin, and HB9 were performed using a Zeiss LSM 510UV META laser scanning confocal microscope while VChat, VGlut, GFAP and MBP analysis used a Zeiss epifluorescence microscope. Acetylcholine levels in cultured neurons were determined with an AmplexRed detection kit (Invitrogen) and correlated with protein levels determined using the FluoroProfile Protein Quantification Kit (Sigma). For α-bungarotoxin labeling, live murine cortical cultures with engrafted BFCN were rinsed with HBSS, incubated with 3 mls 0.05 mg ml-1-1 AlexaFluor594-conjugated a-bungarotoxin (Invitrogen) for 30 minutes in final neuron media, rinsed in HBSS, rinsed in 0.1% Triton X-100 in PBS, then fixed in 4% PFA for 30 minutes.
Preparation of Murine Organotypic Hippocampal Slice Culture.
P5-P6 CD1 mice were chilled on ice and sacrificed by decapitation; brains were removed under aseptic conditions followed by the separation of the hippocampus and enthorinal cortex (HEC) from the two hemispheres as previously described (50). The HEC tissue blocks were cut using a McIlwain tissue chopper into 350 11 m-thick coronal slices. Slices were placed on semiporous membrane inserts (Millicell-CM, 0.4 11 m) and transferred to six-well culture plates with 1.2 ml of MEM supplemented with 25% (vol/vol) horse serum, 6.5 mg ml−1 D glucose, and 0.5 mM L-glutamine. After 3-4 d in culture, the medium was changed to final neuron media supplemented with 40/0 knockout serum replacement, and the media changed every 72 hrs.
Micrografting of BFCN Derived from HESC into Murine Slice Cultures.
Presumptive BFCN generated through Lhx8/Gbx1 nucleofection were FACS-purified at 48 hours and subsequently grown in culture for 5-7 days. To maintain fluorescent signal intensity beyond that from the transient nucleofection, purified cells were permanently labeled with an adenovirus encoding eGFP. Cultures were dissociated using accutase, counted, and loaded on the tip of a 0.5 μl Hamilton syringe mounted on a micromanipulator. After the slices had been transferred into the serum-supplemented final neuron media, suspensions of these FACS-purified cells (3-4,000 cells/0.2 ul) were seeded locally onto the murine hippocampal cultures in the area of the dentate gyrus (DG). After 2-3 d in culture, grafted cells were continuously monitored for GFP fluorescence using live confocal microscopy and processed for electrophysiological recording and eventual immunohistochemistry.
Electrophysiological Recordings of BFCN Derived from hESC into Murine Slice Cultures.
Organotypic slice cultures 2-19 days old were incubated for 30 min at 37° C. in oxygenated standard artificial CSF (ACSFl containing (in mM): 130 NaCl, 24 NaHC03, 3.5 KCt 1.25 NaH2P04, 1.5 CaCl2, 1 MgS04, and 10 glucose, saturated with 95% 02 and 5% C02 at pH 7.4 and then maintained at room temperature until being transferred to the recording chamber in oxygenated standard ACSF. The eGFP-positive engrafted cells were observed with the aid of a fluorescence microscope (BX-50WI; Olympus) and visualized with a chilled charge-coupled device video camera (Dage-MTI) with a 40× water-immersion differential interference contrast objective. Whole cell patch clamp recordings were performed either from eGFP-expressing cells or neighboring murine cells (not eGFPexpressing) located in the subgranular zone of the dentate gyrus. For whole cell voltage clamp recordings, patch electrodes with a resistance of 5-7 MΩ were pulled from borosilicate capillaries (World Precision Instruments; PG52165-glass). Patch pipettes were filled with a solution of (in mM) 150 KCt 10 HEPES, 4 Mg2ATP, 0.5 NaGTP, and 10 phosphocreatine. The pH was adjusted to 7.3 with KOH. For whole cell current clamp experiments the pipette solution was potassium gluconate, 100; EGTA, 10; MgCl2, 5; Hepes, 40; ATP, 3; GTP, 0.3; pH adjusted to 7.2 with KOH and osmolarity to 295 mOsm with sucrose. Whole-cell voltage clamped recordings were obtained from the fluorescence-labeled cells using an Axopatch 200B patch-clamp amplifier (Molecular Devices) and the data were captured with pClamp 9.0 software (Molecular Devices).
Data Analysis.
Data were filtered at 2 kHz and digitized at 10 kHz using a Digidata 1322A analog-to-digital board. Analysis was performed using the pClamp 9.0 (Molecular Devices), MiniAnalysis (Synaptosoft), or Sigmaplot. For the detection and measurement of PSCs, all PSCs were detected in 1-3 min recording segments under the appropriate experimental configuration (baseline control, drug application). Event frequency and amplitude were determined by MiniAnalysis software (Synaptosoft).
Drugs Used
Bicuculline methiodide, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dihydro-β-erythroidine HBr (DHbE), methyllycaconnitine (MLA), 4-aminopyridine (4-AP) (all Sigma) were applied by either focal or bath application. All experiments performed here were performed in accordance with animal experimentation protocols approved by the National Institutes of Health and institutional protocols.
Statistical Analysis.
The RNA data (FIGS. 1-3) were analyzed for statistical significance using the Analyses of Variance method CANOVA) with version 9.1 of SAS. For the counts of ChAT-positive neurons (FIG. 4), the ratios violated the assumptions of parametric hypothesis tests with respect to normal distributions and homogeneity of variance, consequently non-parametric alternative tests were carried out. The Kruskal-Wallis test was used to test the global null hypothesis that there was no significant difference between the median ratios with respect to the four groups. Mann-Whitney U tests were used to test the null hypotheses that there were no significant differences between the median ratios when pairs of treatments were compared. The decision rule was to reject the null hypothesis if the p value of each of the Kruskal-Wallis or the Mann-Whitney U tests was less than the prescribed level of significance of a=0.05. The hypothesis tests were performed using SPSS version 17.0. Paired and N on-paired Student's T-Tests were used to determine the significance of the changes in electrophyiological response following specific receptor antagonist treatment (FIG. 6d,f).
Supplemental Methods
Media Formulations (All Percents are (Vol/Vol))
hESC Media
50% DMEM-F12 (+L-Glutamine)
1% Non-essential amino acids
16.66% Knockout Serum Replacement (Invitrogen)
100 uM β-mercaptoethanol
Neurosphere Media
50% DMEM-F12 (+L-Glutamine)
1% amino acids
1% N2 supplement
100 ̃M β-mercaptoethanol
8 ug ml−1 Heparin
20 ng ml−1 bFGF
20 ng ml−1 EGF
Neuron Media 1
30% DMEM-F12 (+L-glutamine)
2% B-27 Supplement
100 ng ml−1 NGF
Neuron Media 2
Neurobasal Media
1% B-27 Supplement
0.5% Glutamax
0.5% Glutamine
20 ng ml−1 bFGF
100 ng ml−1 NGF
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All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

## Claims

1. A method of generating basal forebrain cholinergic neurons (BFCNs) in vitro comprising: treating isolated neural progenitor cells with: i) an effective amount of FGF8, or a biologically active fragment or variant thereof, and ii) an effective amount of Sonic Hedgehog (SHH) or a biologically active fragment or variant thereof, wherein said treating generates BFCNs.

2. The method of claim 1, further comprising generating said neural progenitor cells by treating embryonic stem cells with retinoic acid.

3. The method of claim 1, wherein said FGF8 and SHH are human.

4. The method of claim 1, wherein said neural progenitor cells are human neural progenitor cells.

5. The method of claim 1, further comprising treating said neural progenitor cells with BMP9.

6. The method of claim 1, further comprising transfecting said neural progenitor cells with vectors encoding Gbx1 and/or Lhx8.