WO2018080925A1 - Methods of differentiation to neuronal cells and kits therefor - Google Patents

Abstract

Embodiments herein provide methods of differentiating neural stem cells to neuronal cells while concomitantly retarding neural stem cell proliferation. Resultant cultures demonstrate reduced clumping of cells, increased purity of neuronal cells and accelerated electrophysiology as compared to control methods.

Description

M ETHODS OF DI FFERENTIATION TO NEURONAL CELLSAND KITS

THEREFOR

FI ELD

[0001] Present embodiments relate to retarding proliferation of multipotent neural stem cells and concomitantly differentiating to neuronal cells, as well as differentiating primary cortical neurons and concomitantly retarding glial cell proliferation, methods, kits and uses therefor.

I NTRODUCTION

[0002] Neuronal cells differentiated from human pluripotent stem cell (hPSC)-derived neural stem cells (NSCs) are used to model the physiology of neurons and neurological diseases in vitro. When hPSCs are induced in vitro into neural stem cells (NSCs), and the NSCs then differentiated in vitro into neuronal cells, undifferentiated NSC's readily takeover the cul ture because they prol i f erate faster than the maturi ng neuronal cell s. U nchecked proliferation of undifferentiated NSCs results in increased cell density and resultant cell cl umpi ng. I mpure neuronal cul tures and cell cl umps i ncrease the di f f i cul ty of I ong-term mai ntenance of cultured neuronal cellsaswell as downstream analyses such as cell counting, cell imaging, and cell assays, for example.

[0003] Primary neuronal cells differentiated from neural progenitors isolated from rodent embryonic brain provide a classical cell model to study functions of neuronal cells/^'n vitro because isolated neural progenitors from rodent embryonic brain contain both neuronal and glial progenitors. In the differentiation of primary neuronal cells, the number of glial cells increases during the period of prolonged differentiation because of the proliferative property of glial progenitors. For researchers to use pure neuronal cells for thei r studies, contaminating glial cells present a problem that has been addressed in the past by culturing primary neuronal cultures for several days with anti-mitoticssuch asfluorodeoxyuridine (FDU) and/or cytosinearabinofuranoside (Ara-C) that kills all proliferating cells by disturbing DNA synthesis. However, these mitotics are also toxic to neuronal cells.

[0004] Embodi merits herei n address these probl ems and provi de sol uti ons that have unexpected benefits. SUM MARY

[0005] I n one aspect, embodi ments herei n address the probl em of unchecked proliferation of neural stem cellsduring differentiation to neuronal cellsand provide methods and kits that solve the problem with the unexpected benefits of reduced clumping during differentiation, and enhanced electrophysiology of resultant neuronal cells. In one aspect, cul tures resul ti ng from the methods herei n, as compared to cul tures not treated as descri bed herei n, have at I east one of the f ol I owi ng attri butes: greater than 50% of the cell s are neuronal cell s, greater than 50% of the cell s are M A P2 posi ti ve cell s, greater than 50% of the cells are HuC/D positive cells, cell s have average or better than average neurite length, cells have equivalent or better electrical activity, cells have accelerated excitability in response to a sti mul us, and greater than 50% of the cell s test negati ve for the SOX 1 marker at Day 14 of differentiation.

[0006] I n another aspect, embodi ments herei n address the probl em of unchecked proliferation of glial progenitors during differentiation to neuronal cellsand provide methods and kitsfor virtually eliminating glial progenitors.

[0007] I n some embodi ments, a method for accel erati ng di f f erenti ati on of at I east one neural stem cell to at least one neuronal cell and concomitantly retarding neural stem cell proliferation is provided, the method comprising, culturing the at least one neural stem cell in a different! ati on medium for a time and under conditions to form the at least one neuronal cell , wherei n the different! ati on medi urn compri ses a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, wherein excitability of the at I east one neuronal cell is accelerated as compared to culturing the at I east one neural stem cell in the different! ati on medium lacking the at least one gamma secretase inhibitor. In an embodiment, excitability of theat least one neuronal cell when differentiated in the presence of a gamma secretase inhibitor isaccelerated by greater than 100% compared to a basel i ne val ue at Day 7. I n that embodi ment, the control culture different! ated without presence of the gamma secretase i nhi bitor was measured at a 37% i ncrease i n exci tabi I i ty compared to a basel i ne val ue on Day 7.

[0008] A method of reducing cell clumping during differentiation of neural stem cells to neuronal cells isan aspect of embodi ments herein, the method comprising, culturing the neural stem cells in a differentiation medium for atimeand under conditions to form neuronal cells, wherein the differentiation medium comprises a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, wherein cell clumping is reduced at least 50% when compared to culturing the neural stem cells in the differentiation medium lacking the at least one gamma secretase inhibitor. In one embodiment, cell clumping is reduced at least 75%, or in another embodiment, reduced at least 90%, when compared to culturing i n the absence of the at least one gamma secretase inhibitor at Day 14 of differentiation.

[0009] A method of di f f erenti ati on of pri mary cellsto Hu C & Hu D posi ti ve neuronal cells and concomitantly retarding differentiation of the pri mary cells to GFAP positive astrocytes is a further embodiment herein, the method comprising, culturing the pri mary cell s i n a diff erenti ati on medi um for a ti me and under condi ti ons to form H u C & H u D positive neuronal cells, wherein the differentiation medium comprises at least one serum- free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, wherein differentiation of the primary cells to GFAP positive astrocytes is reduced when compared to culturing the primary cells in the differentiation medium lacking the at least one gamma secretase inhibitor. In an embodiment, the reduction in GFAP positive astrocytes is at least 50% as compared to culturing in the different! ati on medi um I acki ng the at I east one gamma secretase i nhi bi tor.

[00010] I n some aspects of the above descri bed methods, the serum-free suppl ement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof. In another embodiment, the serum-free suppl ement comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027,

LY411575, a salt thereof, and a combination thereof. In another aspect of the above described methods, the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, a sal t thereof, and a combi nati on thereof. I n another aspect of the above descri bed methods, the serum-free supplement of the differentiation medium comprises the gamma secretase inhibitor YO-01027, or a salt thereof. In another aspect of the above descri bed methods, the serum-free supplement of the differentiation medium comprises the gamma secretase inhibitor Compound E, or a salt thereof. [00011] I n another aspect, the gamma secretase i nhi bitor is present i n the

differentiation medium at a concentration of 0.1 micromolar to 80 micromolar, 0.1 micromolar to 40 micromolar, a concentration of 0.1 micromolar to 20 micromolar, a concentration of 0.2 micromolar to 10 micromolar, a concentration of 0.2 micromolar to 2.0 micromolar, a concentration of 0.2 to 1.0 micromolar, or a concentration of 0.2 to 0.5 micromolar.

[00012] The at I east one neural stem cell of the above descri bed method i s deri ved from an induced human pi uri potent stem cell, or alternatively, the at least one neural stem cell is derived from a human embryonic stem cell.

[00013] In some embodiments, the at least one neural stem cell isa SOXI positive neural stem cell, a SOX2 positive neural stem cell, and/or a NESTIN positive neural stem cell, and the neuronal cell isa MAP2 positi e neuronal cell, a DCX positive neuronal cell and/or a H U C& D positive neuronal cell .

[00014] In someaspectsof method embodiments, theat least one neuronal cell is maintained in culture for at least a period of five weeks.

[00015] A kit for accelerating differentiation of at least one neural stem cell to at least one neuronal cell and concomitantly retarding neural stem cell proliferation isafurther embodiment herein, the kit comprising, at least one serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, and opti onal I y , reagents and i nstructi ons pertai ni ng to use of the ki t. In some embodi ments, the serum-free suppl ement of the ki t compri ses a gamma secretase i nhi bi tor sel ected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combi nation thereof. I n some aspects, the serum-free supplement of the kit compri ses a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, and LY411575, a salt thereof, and a combi nation thereof. In an embodiment, the serum-free supplement has a 100X concentration such that, e.g., 5 ml_ of the 100X concentration is added to the serum-free neural stem cell culture medium to form the differentiation medium.

[00016] A method of treati ng a subj ect havi ng a neurodegenerati ve condi ti on i s an aspect herein, the method comprising administering to the subject a pharmaceutical composi ti on compri si ng neuronal cell s made by any one of the methods descri bed above. I n one aspect, the neuronal cell s are deri ved from a neural stem cell autol ogous for the subj ect and differentiation is ex vivo prior to administration, e.g., by transplantation. I n another aspect, the neuronal cells are deri ved from a neural stem cell allogeneic for the subj ect and differentiation is ex vivo prior to administration, e.g., by transplantation. The

neurodegenerative condition may be, e.g., Parkinson's disease, Huntington's disease, stroke effects, or dementia such as Alzheimer's conditions.

DRAWI NGS

[00017] The ski I led artisan will understand that the drawings, described below, are for illustration purposesonly. The drawings are not intended to limit the scope of the present teachi ngs i n any way.

[00018] FIG. 1A - FIG. 1B provide phase-contrast imagesof cellswithout (FIG. 1A) and with (FIG. 1B) the presence of Compound E at Day 5 of neuronal differentiation. Note the neurite outgrowth of cells differentiated i n the presence of Compound E.

[00019] Fl G. 1 C - Fl G. 1 H provi de i mmunof I uorescent i mages of cell s at Day 7 of differentiation without Compound E (left column) and with Compound E (right column). DAPI isa nuclear stain, NESTIN isastain for neural stem cellsand DCX isa stain for neuronal cells.

[00020] FIG. 11 - FIG. 1N provi de i mmunof I uorescent i mages of cellsat Day 14 of differentiation without Compound E (left column) and with Compound E (right column). DAPI isa nuclear stain, NESTIN isastain for neural stem cellsand DCX isa stain for neuronal eel la

[00021] Fl G. 2A - Fl G. 2F provide images at Day 14 of neuronal differentiation of H9 human ESC-derived NSCs without (first column) or with (second column) the presence of Compound E during differentiation.

[00022] Fl G. 2A provi des a phase-contrast i mage of cd I s without the presence of Compound E during neuronal differentiation that shows cell clump formation. Compare this i mage wi th the phase-contrast i mage of cell s different! ated i n the presence of Compound E i n FIG. 2B. Evenly distributed cells are evident without formation of cell clumps. FIG. 2C provides an immunofluorescent image of cells differentiated without Compound E and stained with antibodies against the neuronal marker MA P2. Compare this image with the image of FIG. 2D that shows cells differentiated in the presence of Compound E stained with antibodies against the neuronal marker MAF2. FIG. 2E provides an immunofluorescent image of cells differentiated without Compound E and stained with antibodies against the neural stem cell marker SOX1. Compare this image with the image of FIG. 2F that shows cells differentiated in the presence of Compound E stained with antibodies against the neural stem cell marker SOX1.

[00023] Fl G. 3 provi des a quanti tati ve measure of the degree of cell cl umpi ng after two weeks of culture i n the differenti ation medi um without and with the presence of

Compound E. A 90% reduction in clumping is found when Compound E is present in the differentiation medium.

[00024] FIG. 4A and FIG. 4B provide images that assess proliferation of cellsat six days of differentiation without (FIG. 4A) and with (FIG. 4B) Compound E.

[00025] Fl G. 4C and Fl G. 4D provi de i mages that assess apoptoti c cell death at six days of differentiation without (FIG. 4C) and with (FIG. 4D) Compound E.

[00026] FIG. 5A and FIG. 5B provide quantitative analyses of thedataof FIG. 4A - FIG. 4D. FIG. 5A demonstrates that proliferation of neural stem cells is substantially arrested i n the presence of Compound E and Fl G. 5B shows that no signif i cant amount of cell death has occurred i n either the absence or the presence of Compound E.

[00027] Fl G. 6 provides data to compare the effect of a number of gamma secretase inhibitors on cell clumping during differentiation in the absence and the presence of the inhi bitor. Various concentrations of the i nhibitors were studied rangi ng from 0.01 mi cromol ar to 160 mi cromol ar. See Exampl e 4 for the concentrati ons of i nhi bi tors used to generate the data of this figure. The double asterisks show the statistical analyses as carried out by the All F¾i rs Tukey-Kramer analyses with a p val ue less than 0.01. [00028] Fl G. 7A and Fl G. 7B show that differentiated neuronal cells detached from the culture plate at three weeks without the presence of Compound E (FIG. 7A) while neuronal cells differentiated in the presence of Compound E can be maintained to at least five weeks (FIG. 7B).

[00029] Fl G. 8A - Fl G. 8D show i mmunof I uorescent i mages for the astrocyte marker GFAP (FIG. 8A and FIG. 8B) and for the neuronal marker HuC&HuD (FIG. 8C and FIG. 8D) for primary rat cortical neuronal cells differentiated without and with presence of Compound E in the differentiation medium. Presence of Compound E in the differentiation medium substantially eliminated the astrocytes.

[00030] FIG. 9A and FIG. 9B provide measurements of cell proliferation of control NSC cultures (n=8) compared to cultures differentiated i n the presence of Compound E to neuronal cells (n=7) on Day 4 (FIG. 9A) and on Day 7 (FIG. 9B) using the CY QUANT™ Di rect Cel I Prol if eration Assay as descri bed i n Example 7.

[00031] FIG. 9C provides a plot of signal vstimefor measuring calcium flux using the Fluo-4 Calcium Imaging Kit. Representative traces of fluorescent response to the addition of the 30 mM KCI sti mul us to control NSC cultures and to cultures different! ated i n the presence of Compound E to neuronal cells are provided. The signal is measured at 1 hZ and plotted in a running average of multiple wells as fold increase, designated as (signal max - signal min)/signal min.

[00032] Fl G. 10A and Fl G. 10B provi de tabul ar data showi ng the averaged peak calcium responses (n=4-8 each) to NSCs cultured without (dotted bars) and to cultures different! ated i n the presence of Compound E to neuronal cell s (I i ned bars) across a range of potassium sti mul us strengths at Day 4 (FIG. 10A) and at Day 7 (FIG. 10B).

DESCRI PTI ON OF VARI OUS EM BODI M ENTS

[00033] 11 i s to be understood that both the f oregoi ng general descri pti on and the following detailed description are exemplary and explanatory only and are not intended to I i mi t the scope of the current teachi ngs. I n thi s appl i cati on, the use of the si ngul ar i ncl udes the plural unlessspecifically stated otherwise. The use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to De limiting. Use of "or" means"and/or" unless stated otherwise. The term "and/or" means that the terms before and after can be taken together or separately. For i 11 ustration purposes, but not as a I i mitation, "X and/or Y" can mean "X" or "Y" or "X and Y." Asused herein and unless otherwise indicated, the terms "a" and "an" are taken to mean "one," "at least one" or "one or more."

[00034] Whenever a range of val ues i s provi ded herei n, the range i s meant to i ncl ude the starti ng val ue and the endi ng val ue and a val ue or val ue range there between unl ess otherwise specifically stated. For example, "from 0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3 - 0.4, 0.2 - 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 - 0.39; and the like.

[00035] The secti on headi ngs used herei n are for organi zati onal purposes onl y and are not to be construed as limiting the subject matter described in any way. All literature and si mi I ar materi al s ci ted i n thi s appl i cati on i ncl udi ng, but not I i mi ted to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. I n the event that one or more of the i ncorporated I i terature and si mi I ar materials defines or uses a term in such away that it contradicts that term's definition in this appl i cati on, thi s appl i cati on control s. Whi I e the present teachi ngs are descri bed i n conj uncti on with vari ous embodiments, it is not i ntended that the present teachi ngs be I i mi ted to such embodimenta On the contrary, the present teachi ngs encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[00036] The term "or combi nati ons thereof" as used herei n refers to al I permutati ons and combi nati ons of the I i sted i terns precedi ng the term . For exampl e, " A , B , C, or combi nati ons thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CA B . Conti nui ng wi th thi s exampl e, expressl y i ncl uded are combi nati ons that contai n repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC,

CBBAAA, CABABB, and so forth. The ski I led artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. [00037] Certai n embodi merits herei n rel ate to i mprovi ng the process of di f f erenti ati on of neural stem cellsto differentiated cell shaving desired cell markers. The methods presented herein improve the yield of differentiated cells, improve the ratio of differentiated cellsto undifferentiated cells, improve the purity of the differentiated cell population, and reduce clumping of cells, thereby facilitating quantitation and imaging of the differentiated cells. The methods are particularly applicableto differentiation of neural stem cellsto neuronal cells

[00038] Further embodi ments herei n rel ate to methods for removal of contami nati ng cell types in primary cell cultures, e.g., removal of astrocytes from primary neuronal cell cultures.

[00039] I n embodi ments herei n, the term , " neural stem cell s" means SOX 1 posi ti ve neural stem cells (NSCs). NSCs can be obtained in a number of ways, e.g., from human i PSCs usi ng the Gl BCO™ -PSC Neural I nduction M edi um as provided by the manufacturer (Cat. No. A1647801, Thermo Fisher Scientific, Waltham MA), or using the STEMDI FF^M Neural Induction Medium as provided by the manufacturer (Cat. no. 05835, StemCell Technologies, Cambridge, MA), for example. The neural stem cells of embodi ments herein are characterized by using cell -type specific antibody markers commonly used for immunocytochemical (ICC) analysissuch as those from Thermo Fisher Scientific, for example, use of Antibody Cat. No. PA5-23351 or PA5-23370 having binding specificity and affinity for antigen SOX1, use of Antibody Cat. No. MA1-014, MA1-014D488, MA1- 014D550, MA1-014D650, MA1-014HRP, or PA1-094 having binding specificity and affinity for antigen SOX2, or use of Antibody Cat. No. MA1-110 having binding specificity and affinity for antigen NESTI N, for example.

[00040] Embodiments of the method provided herein providefor differentiation of SOX1 positive NSCs to neuronal cells. As used herein, the term "neuronal cells" means cells characterized by usi ng cell -type specific anti body markers used for I CC analysis such as those from Thermo Fisher Scientific, for example, use of Antibody Cat. No. MA5-12823, 13- 1500, or MA5-12826 having binding specificity and affinity for antigen MAR2, Antibody Cat. No. A21271 or A21272 having binding specificity and affinity for antigen HuC/D or Antibody Cat. No. 48-1200 having binding specificity and affinity for antigen DCX, for example. In some embodiments herein, the neuronal cell ispositivefor MAP2 and/or DCX markers.

[00041] The phrase " retardi ng neural stem cell prol iferati on" , as used herei n, means arresting, slowing, hindering, or impeding the increase in number of neural stem cells without causing significant cell death. Cell proliferation can be measured by incorporation of the nucleoside thymidine analog EdU which is incorporated into DNA during active DNA synthesiswith the result that proliferating cells become labeled. Such measurements are compared from cellscultured with and without presence of the gamma secretase inhibitor as shown in Example 3 herein. The retardation of neural stem cell proliferation, in some embodiments, is at least 10% to at least 20% reduction, at least 20% to at least 40% reduction, at least 20% to at least 40% reduction, or in some embodiments, at least 40% to at least 60% reduction of neural stem cell number in the presence of a gamma secretase inhi bitor as compared to the absence of a gamma secretase inhibitor at six days of differentiation. The term "concomitantly," as used herein, means that the retardation of prol i f erati on of some neural stem cell s occurs i n the same cul ture i n whi eh some cell s are differentiating to neuronal cell a

[00042] I n some embodi ments as shown by Exampl e 7, the retardati on i s at I east a 2- fold reduction of neural stem cell number when differentiation occurs in the presence of a gamma secretase i nhi bitor as compared to when differentiation occurs i n the absence of a gamma secretase i nhi bitor at four days of differentiation. I n some embodi ments, the retardation isat least a5-fold reduction of neural stem cell number when differentiation occurs i n the presence of a gamma secretase i nhi bitor as compared to differentiation i n the absence of a gamma secretase i nhi bi tor at seven days of di f f erenti ati on.

[00043] Cel I death can be measured by stai ni ng cell s wi th anti bodi es agai nst the apoptotic cell death marker caspase 9 as shown by Example 3 herein. The term "without causing significant" cell death means that differentiation in the presence of the gamma secretase i nhi bi tor i n an N SC cell cul ture does not cause an i ncrease i n cell death of more than 2- to 4-fold, of more than 4- to 6-fold, of more than 6- to 8-fold or more than 8- to 10- f ol d as compared to diff erenti ati on of the NSC culture i n the absence of gamma secretase inhi bitor. [00044] The term "accelerating differentiation" of a neural stem cell, as used herein, means that a greater calcium signal is obtained in response to a stimulus when differentiation of neural stem cell s to neuronal cell s i s carri ed out i n the presence of the gamma secret ase inhi bitor as compared to when differentiation occurs in the absence of the gamma secret ase inhibitor as measured, for example, by using components of the Fluo-4 Calcium Imaging Kit (Cat. No. F10489, Molecular Probes, Eugene OR). Calcium signals elicited by potassium depol ari zati on are measured and the measure provi des an esti mate of the rel ati ve number of voltage gated calcium ion channels present on cells in the culture, which isa "proxy" for neuronal signaling and developmental maturity. Mature, excitablecellsexpress large numbers of voltage gated channelsthat can be opened by depolarizing the cellular membrane with extracellular potassium addition, while immature or non-excitable cells express few or no voltage gated calcium ion channels avail able for opening with a potassium depolarization stimulus.

[00045] This greater calcium signal is observed at I east as early as Day 4 of differentiation. Example7 herein provi des data showing that the control culture provided an i ncrease i n cal ci urn si gnal of 23%, 37%, 72% and 112.4% for Day 4, 7, 14, and 21 , respectively, whilethetest culture differentiated in the presence of Compound E provided an increasein calcium signal of 57%, 128%, 116.9% and 286.6% for Day 4, 7, 14, and 21, respectively. These data demonstrate that cells differentiated in the presence of Compound E have greater excitability i n response to a sti mul us as compared to cells i n the same medi urn without Compound E, indicating that the maturity of the neuronal cells is accelerated.

[00046] An accelerated differentiation signal is seen by comparing the signal at Day 4 of 23% in control cultures with the signal of 57% at Day 4 in test cultures, i .a, a culture having a gamma secretase inhibitor present during differentiation. Similarly, an accelerated differentiation signal is seen by comparing the signal at Day 7 of 37% in control cultures with the signal of 128% at Day 7 in test cultures, i.e., a culture having a gamma secretase inhi bitor present during differentiation. An accelerated differentiation signal is also seen by comparing the signal at Day 14 of 72% in control cultures with the signal of 116.9% at Day 14 i n test cul tures, i .e. , a cul ture havi ng a gamma secretase i nhi bi tor present duri ng differentiation. An accelerated differentiation signal isalso seen by comparing the signal at Day 21 of 112.4% in control cultures with the signal of 286.6% at Day 21 in test cultures, i.e., a culture having a gamma secretase i nhi bitor present duri ng differentiation. [00047] I n one aspect, the diff erenti ati on medi um of embodi ments herei n compri ses at least one 9erum-f ree neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor. The differentiation medium, in some embodiments, lacks presence of at least one of leukemia inhibitory factor (LI F), an inhibitor of glycogen synthase kinase3 (GSK3), and an inhibitor of transforming growth factor bets (TGF-beta). I n some embodi ments, the di ff erenti ati on medi um I acks presence of f i brobl ast growth factor/epidermal growth factor (FGF2/EGF). Every 2-3 days, half spent medium was removed from each welI of the culture pi ates and the same vol ume of fresh medi um was added into each well.

[00048] An exempl ary serum-free neural stem cell culture medi um compri ses, for example, NEUROBASAL™ Medium (Brewer et a/., J. Neuroscience Res., 35:567-576, 1993; Cat. No. 21103, Thermo Fisher Scientific, Waltham MA), 2% B27 Supplement (Cat. No. 17504, Thermo Fisher Scientific, Waltham MA), 1% GLUTAMAX™ Supplement (Cat. No. 35050, Thermo Fisher Scientific, Waltham MA) and 200 micromolar ascorbic acid (Cat. No. A8960, Sigma-Aldrich, St. Louis MO).

[00049] A further exemplary serum-free neural stem cell culture media comprises DMEM/F12, 1xN2, 1xB27 Supplement (Cat. No. 17504, Thermo Fisher Scientific, Waltham MA), 300 ng/mL cAMP(Sigma-Aldrich) and 0.2 mM Vitamin C (Sgma-A I drich) (Li et al. PNAS 108:20, 8299-8304).

[00050] Another exemplary serum-free neural stem cell culture media isthe

STEM PRO™ NSC SFM (Cat. No. A1050901, GIBCO, Grand Island, NY) that contains STEM PRO™ Neural Supplement.

[00051] The serum-free supplement of the differentiation medium comprises at least one gamma secretase inhibitor. Known gamma secretase inhibi tors (GSIs) include, e.g., GSI I, Z-Leu-Leu-Norleucine-CHO; GSI I I; GSI III , N-benzyloxycarbonyl-Leu-leucinal; GSI IV, N-(2-naphthoyl)-Val-phenylalaninal; GSI V, N-benzyloxycarbonyl-Leu-phenylalaninal; GSI

VI, 1-(S)-endo-N-(1,3,3)-trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenyl sulfonamide; GSI

VI I, menthyloxycarbonyl-LL-CHO; GSI IX, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S- phenylglyci net-butyl ester (DAFT); GSI X, { 1S-Benzyl-4R-[1-(1S-carbamoyl-2- phenethyl carbamoyl )-1S-3-methyl butyl carb- amoyl]-2R-hydroxy-5-phenylpentyl} carbamic acid tert-butyl ester; GSI XI , 7-aminc>-^chlorc>-3-methoxyisocoumarin; GSI XII , Z-lle-Leu- CHO; GSI XII I, Z-Tyr-lle-Leu-CHO; GSI XIV, Z-Cys(t-Bu)-lle-Leu-CHO; GSI XVI, N-[N- 3,5-difluorophenacetyl]-L-alanyl-S-phenylglycine methyl ester; GSI XVII ; GSI XIX, (2S,3R)-3-(3,4-Difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo- -5-phenyl-2,3- dihydro-1H-benzo[e][1,4]diazepin-3-yl)-butyramide; GSI XX, (S,S)-2-[2-(3,5- dif I uorophenyl )-acetyl ami no] -N-(1-methyl -2-oxo-5-phenyl -2- ,3-di hydro- 1 H- benzo[e] [ 1 ,4]diazepi n-3-yl )-propionami de (Di benzazepi ne (DBZ) (YO-01027,

SelleckChem); and the hydroxylated form: N-2((2S)-2-(3,5-dif I uorophenyl )-2- hydroxyethanoyl )-N 1 -((7S)-5-methyl -6-oxo-6,7-di hydro-5H-di benzo[b,d] azepi n-7-yl )-l - alaninamide (LY411575, Sigma-Aldrich, STEMGENT™); GSI XXI, (S,S)-2-[2-(3,5- dif I uorophenyl )-acetyl ami no] -N-(1-methyl -2-oxo-5-phenyl -2- ,3-di hydro- 1 H- benzo[ e][1, 4] di azepi n-3-yl)-propionamide (Compound E, EMD M i 11 i pore, Enzo Life Sciences); gamma40 secretase inhibitor I, gamma40 secretase inhibitor II and RO4929097.

[00052] I n one aspect, the gamma secretase i nhi bitor i s YO-01027. I n another aspect, the gamma secretase i nhi bi tor i s Compound E. In one aspect, the gamma secretase i nhi bi tor is LY411575. I n an aspect, the gamma secretase inhibitor is MK-0752. I n another aspect, the gamma secretase inhibitor isLY450139 (Semagacestat). In another aspect, the gamma secretase i nhi bi tor i s RO4929097. Gamma secretase i nhi bi tors are commerci al I y avai I abl e from, e.g., EM D Millipore (Billerica MA), APExBIO (Houston TX), or from SelleckChem, for example

[00053] I n some embodi ments, the gamma secretase i nhi bitor i s other than that of group IX (GSI IX), i.e., other than that of DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S- phenylglyci net-butyl ester.

[00054] I n some embodi ments, the amount of gamma secretase i nhi bitor i n the suppl ement i s such that di I uti on thereof i nto the serum-free neural cell cul ture medi urn to generate the differentiation medium provides an amount of inhibitor effective to accelerate differentiation of a neural stem cell to a neuronal cell while retarding neural stem cell prol iferation. I n some embodi ments, the gamma secretase i nhi bitor is present i n the differentiation medium at a concentration of 0.1 micromolar to 80 micromolar, 0.1 micromolar to 40 micromolar, a concentration of 0.1 micromolar to 20 micromolar, a concentration of 0.2 micromolar to 10 micromolar, a concentration of 0.2 micromolar to 2.0 micromolar, a concentration of 0.2 to 1.0 micromolar, or a concentration of 0.2 to 0.5 micromolar. In some embodiments, the final concentration of the inhibitor in the differentiation medium is 0.2 micromolar to I ess than 0.5 micromolar.

[00055] I n certai n embodi ments, when the gamma secretase i nhi bi tor i s YO-01027, Compound E, or LY411575, the concentration of the inhibitor in the differentiation medium is 0.01 micromolar to 20 micromolar, 0.1 micromolar to 20 micromolar, 0.2 micromolar to 10 micromolar, 0.2 micromolar to 5 micromolar, 0.2 micromolar to 2.0 micromolar, 0.2 to 1.0 micromolar, or a concentration of 0.2 to 0.5 micromolar. In some embodi ments, the final concentration of the inhibitor in the differentiation medium is 0.2 micromolar to less than 0.5 micromolar.

[00056] I n another aspect, when the gamma secretase i nhi bi tor i s RO4929097, LY450139 (Semagacestat) or MK-0752, the concentration in the differentiation medium is 0.8 micromolar to 80 micromolar, 1.0 micromolar to 60 micromolar, 5 micromolar to 60 micromolar, 10 micromolar to 50 micromolar, or 20 micromolar to 50 micromolar.

[00057] Serum-free suppl ement i ngredi ents may i ncl ude i n addi ti on to the gamma secretase inhibitor, optionally, one or more of progesterone, sodium selenite, recombinant human insulin, putrescine, and human transferrin holoenzyme. In some embodi ments, the serum-free supplement may include, in addition to the gamma secretase inhibitor, the N-2 supplement (Thermo Fisher Scientific, Waltham MA).

[00058] Cellsare plated generally on a matrix such as laminin, collagen IV, fibronectin, vitronectin, polylysine, polyornitine, or a combination thereof such as, for example, basement membrane matrices avail able such as GEL TREX™ (Thermo Fisher Scientific, Waltham MA) or MATRIGEL™ (Fisher Scientific, Waltham MA). In one embodi ment, the matri x i ncl udes I ami ni n.

[00059] As used herei n, the term " sal t thereof" wi th reference to a gamma secretase inhibitor refers to an acid or base addition salt that retains the biological efficacy and properti es of the i nhi bi tor and i s made usi ng aci ds or bases as appropri ate. Exempl ary aci d addition salts include, e.g., HCi, HBr, HI, H2SO4, nitric acid, phosphoric acid and sulfamic acid, as well as those made using acids such as citric acid, fumaric acid, lactic acid, malic acid, met hanesulfonic acid, oxalic acid, salicylic acid, succinic acid, p-toluenesulfonicacid, and the like. Exemplary base addition salts include, e.g., those made using hydroxides of ammonia, potassium, or sodium, such as tetramethy I ammonium hydroxide.

[00060] In a method of treating a subject having a neurodegenerative condition, the administration of a pharmaceutical composition containing neuronal cellsmadeby methods as descri bed herei n may be by i mpl antati on, i nj ecti on, or transpl antati on, for exampl e. I n one aspect, the neuronal cells are differentiated from neural stem cells autologous for the subject, that i s, the neural stem cell s are autol ogous by way of i nducti on of stem cell s obtai ned from the subject to neural stem cells. The differentiation is ex vivo prior to administration. In another aspect, the neuronal cells are derived from a neural stem cell induced from stem cells al I ogenei c for the subj ect and di f f erenti ati on i s ex vi vo pri or to admi ni strati on. The neurodegenerative condition may be, e.g., Parkinson's disease, Huntington's disease, stroke effects, or dementia such as Alzheimer's conditions, and administration may be, e.g., to the central nervous system.

[00061] A "pharmaceutical composition" of a gamma secretase inhibitor is substantially non-toxic to the subject to which the composition is administered. A

"therapeutically effective amount" is an amount of gamma secretase inhibitor, or the amount of a composition or pharmaceutical composition containing the gamma secretase inhibitor that is effective for producing a desired therapeutic effect upon administration to a patient.

[00062] Embodi ments herei n are further i 11 ustrated by the f ol I owi ng exampl es, whi ch are not to be construed as i mposi ng I i mi tati ons upon the scope of the appended cl ai ma

Example 1 - Retarding Proliferation Concomitantly with Differentiating iPSC-Der ived Neural Stem Cells to Neuronal Cells

[00063] Human induced pluripotent stem cells were derived from human fibroblasts using the CYTOTUNE™-i PS 2.0 Sendai Reprogramming Kit (Cat. No. A16517,

IN VITROGEN™ , a part of Thermo Fisher Scientific, Waltham MA) and were induced to neural stem cells (NSCs) using the xeno free version of the GIBCO™ PSC Neural Induction Medium (Cat. No. A1647801, Thermo Fisher Scientific, Waltham MA). [00064] To differentiate the NSCs into neuronal cells, cryo-preserved iPSC-derived NSCs were recovered and NSC passage three cells were plated on a 24 wel I pi ate (Cat. No. 087721, Thermo Fisher Scientific, Waltham MA) coated with human laminin (Cat. No. L6274, Sgma-Aldrich, St. Louis MO) at adensity of 1~5x10⁴ cells/cm². The cultures were maintained in neuronal differentiation medium containing NEUROBASAL™ Medium (Cat. No. 21103, Thermo Fisher Scientific, Waltham MA), 2% B27 Xeno Free Supplement (Cat. No. A1486701, Thermo Fisher Scientific, Waltham MA), 200 micromolar ascorbic acid (Cat. No. A8960, Sigma-Aldrich, St. Louis MO) with or without the gamma-secretase inhibitor Compound E (0.1 micromolar, Cat. No. 565790, EMD Millipore, Billerica MA). Every 2-3 days, medium was replenished with respective medium.

[00065] Phase images were monitored and differentiated cells were fixed at Day 7 and Day 15 with 4% paraformaldehyde and stained with antibodies against the neuronal marker DCX (polyclonal, Cat. No. 48-1200, Thermo Fisher Scientific, Waltham MA) and the neural stem cell marker NESTIN (Monoclonal, Cat. No. 611658, BD Biosciences, Franklin Lakes NJ) according to the manufacturers' protocol &

[00066] Differences between cultures with and without Compound E were observed as early as 5 days of differentiation as shown by compari ng the i mage of Fl G. 1 A of cells without Compound E with the image of FIG. 1B of cells differentiated in the presence of Compound E. Cellswithout Compound E lack marked neu rite outgrowth whereas cells with Compound E developed neuronal cells with extended neurite outgrowth.

[00067] FIG. 1C shows cells stained with DAPI, a nuclear stain, thereby showing all cells in the culture at Day 7 of differentiation without Compound E. FIG. 1E shows the same set of ceJIsstained with NESTIN, a markerfor neural stem cells, while FIG. 1G shows again the same set of cell s stai ned wi th DCX , a neuronal marker. Cel I s di f f erenti ated wi thout the presence of Compound E have a mixed phenotypeof neural stem cells and neuronal cells. Fl G. 1 D shows cell s stai ned with DA PI , the nucl ear stai n, thereby showi ng al I cell s i n the culture at Day 7 of differentiation i n the presence of Compound E. The phenotype was further examined with imaging the same set of cells using the neural stem cell marker NESTIN (FIG. 1 F) and the neuronal marker DCX (FIG. 1H) at Day 7 of differentiation. Cel Is different! ated in the presence of Compound E have had proliferation of NESTIN positive cellssubstanti ally retarded while most of the cells present have adopted a neuronal phenotype as shown by the stai n for D CX .

[00068] At Day 14, the effect of the presence of Compound E i n the differentiation medium is more apparent. Imagesof FIG. 11 (DAPI is a marker for nuclei), FIG. 1K

(NESTIN is a marker for neural stem cells) and FIG. 1M (DCX is a marker for neuronal cell s) show that cell s are prol if erati ng and dif f erenti ating in the absence of Compound E. I n contrast, fewer prol if erati ng progenitor cells are present i n the i mages of cultures havi ng Compound E present (FIG. 1 J, FIG. 1L and FIG. 1N). However, most of those cells appear to be neuronal cell s as i ndi cated by the stai n for neuronal marker DCX .

Example 2 - Retarding Proliferation Concomitantly with Differentiating hESC -Derived Neural Stem Cells to Neuronal Cells

[00069] H9 human embryonic stem cell (ESC)-derived neural stem cells (NSCs) were induced and expanded using the Gl BCO™ -PSC Neural I nduction Medi urn (Cat. No.

A1647801, Thermo Fisher Scientific, Waltham MA), a serum-free medium in which human PSCs are converted into NSCs in one week with high efficiency and without the laborious processes of embryonic body (EB) formation and mechanical NSC isolation.

[00070] To differentiate the NSCs into neuronal cells, cryo-preserved H9 ESC-derived NSCs were thawed and plated on poly-D-lysine 96-well plates(Cat. No. 08-774-255, Thermo Fisher Scientific, Waltham MA) coated with laminin (Cat. No. 23017, Thermo Fisher Scientific, Waltham MA) at a density of 5x1ο⁴ cells/cm². The cultures were maintained in the neuronal differentiation medium containing NEUROBASAL™ Medium (Cat. No. 21103, Thermo Fisher Scientific, Waltham MA), 2% B27 Supplement (Cat. No. 17504, Thermo Fisher Scientific, Waltham MA), 1% GLUTAMAX™ Supplement (Cat. No. 35050, Thermo Fisher Scientific, Waltham MA) and 200 micromolar ascorbic acid (Cat. No. A8960, Sigma- Aldrich, St. Louis MO) with or without the gamma-secretase inhibitor Compound E (0.2 micromolar, Cat. No. 565790, EMD Millipore, Billerica MA). Every 2-3 days, half spent medium was removed from each wel I of the culture plates and the same vol ume of fresh medi um was added i nto each wel I .

[00071] At two weeks of NSC differentiation into neuronal cells, cells were fixed with 4% paraformaldehyde and stained with antibodies against neuronal marker MAR2 (polyclonal, Cat. No. PA5-17646, Thermo Fisher Scientific, Waltham MA) and neural stem cell marker SOX1 (polyclonal, Cat. No. AF3369, R&D Systems, Minneapolis M N) according to the manufacturers' protocols.

[00072] At the end of that two week period, cd I s without gamma-secretase i nhi bitor treatment almost reached confluence (FIG. 2A). Although a portion of cells extended neuritesand showed the morphology of neuronal cells, the majority of cellsformed cell clumps without neurite extension as shown in FIG. 2A. Immunocytochemical staining showed that cellslacking gamma-secretase inhibitor present in the differentiation medium contained a mixed population of M A P2 positive neuronal cells (FIG. 2C) and a substantial number of SOX 1 positive NSCs as shown i n Fl G. 2E. I n the culture without gamma- secretase inhibitor treatment, SOX1 positive NSCs kept dividing which led to a high density of undesired NSCs and clump formation (FIG. 2A).

[00073] In contrast, cells with gamma-secretase inhibitor present in the differentiation medium were distributed evenly without the formation of cell clumps as shown by FIG. 2B and nearly every cell extended neurites (FIG. 2D). Also, in cells differentiated in the presence of the gamma-secretase inhibitor, nearly all cellswere MAFE positive neuronal cells (FIG. 2D) with only afew SOX1 positive cells (FIG. 2F).

[00074] Also, at the end of that two week period of differentiation, the area of cell clumps was calculated using the INCUCYTE ZOOM™ System (Essen Bioscience, Ann Arbor M I ) by def i ni ng the area of cell mass□ 10,000 μm² as a cell el ump. The data of Fl G. 3 show a 90 % reduction i n cell cl umpi ng as a result of the presence of the gamma secretase inhi bitor.

Example 3 - Retarding Proliferation is not Accompanied by Cell Death

[00075] Differentiation of H9 ESC-derived NSCs was the same as for Example2. At Day 6 of differentiation, cellswere incubated with the nucleoside thymidine analog EdU for 24 h. EdU is incorporated into DNA during active DNA synthesiswith the result that proliferating cells become labeled. EdU positive cells were detected using the CLICK -IT™ EdU Alexa FLUOR™ 488 HCS Assay (Cat. No. C10350, Thermo Fisher Scientific, Waltham MA) according to the manufacturer's protocols. Cellswere then fixed with 4% paraf ormal dehyde and stai ned wi th anti bodi es agai nst the apoptoti c cell death marker caspase 9 (polyclonal, Cat. No. PA5-16358, Thermo Fisher Scientific, Waltham MA) accordi ng to the manufacturer's protocol .

[00076] Without gamma-secretase inhibitor presence in the differentiation medium, a substantial portion of EdU positive cells were present in the culture as shown by FIG. 4A. In contrast, only afew EdU positive cellswere observed in cultures differentiated in the presence of the gamma-secretase i nhi bitor (Fl G. 4B). In both cultures, where the gamma- secretase i nhi bitor was absent (Fl G. 4C) or where the i nhi bitor was present duri ng differentiation (FIG. 4D), a very low portion of cells were positive for the cell death marker caspase 9.

[00077] These data are plotted in FIG. 5A and FIG. 5B . FIG. 5A shows a significant reduction i n percentage of EdU positive cells as a result of the presence of the gamma- secretase inhibitor in the differentiation medium, that is, a significant reduction in prol iferation has occurred. The data of Fl G. 5B show a very low percentage of caspase 9 positi ve cell s with or wi thout presence of the gamma-secretase i nhi bi tor duri ng

differentiation, that is, very little cell death isoccurring in each culture.

[00078] Taken together, these resul ts suggest that the effect of cell cl ump reducti on by the gamma-secretase inhibitor is to prevent, arrest or retard cell proliferation without inducing significant cell death.

Example 4 - Effect of Various Gamma Secretase I nhi bitor son Differentiation of NSCs

[00079] Exampl es 1 , 2 and 3 focused on the use of Compound E duri ng neural stem cell differentiation to neuronal cells The present example compares the effect of various other gamma secretase i nhi bi tors and concentrati ons on that process versus the effect of Compound E.

[00080] The differentiation of H9 ESC-derived NSCs was the same as for Example2. Cellscultured in differentiation medium without the presence of the gamma-secretase inhibitor served asacontrol. In the test groups, cells were separately differentiated in the presence of Compound E at 0.2 micromolar, YO-01027 at concentrations of 0.3 micromolar, 1.5 micromolar and 7.5 micromolar, LY411575 at concentrations of 0.01 micromolar, 0.05 micromolar, 0.25 micromolar, 0.5 micromolar, 1.0 micromolar and 2.0 micromolar, and MK- 0752 at concentrations of 0.8 micromolar, 4 micromolar, 20 micromolar, 40 micromolar and at 80 mi cromol ar. Ei ght wel I s were tested for each concentrati on of each i nhi bi tor.

[00081] At theend of a two week period of differentiation, the area of cell clumpswas calculated for each test well using the INCUCYTE ZOOM™ System (Essen Bioscience, Ann Arbor M l) by defining the area of cell mass□ 10,000 μιη² as a cell clump and the data plotted in FIG. 6. The data of FIG. 6 show resultsfrom Compound E at 0.2 micromolar, YO- 01027 at 0.3 micromolar, LY411575 at 0.25 micromolar and M K-0752 at 20 micromolar. A significant reduction in cell clumping resulted from the presence of the gamma-secretase inhibitor during differentiation as compared with the cell clump area in the control group. The results showed similar effects on neurite length and cell cl umping area of differentiated neuronal cellsfrom hESC-derived NSCsfor Compound E at 0.2 micromolar, for YO-01027 at 0.3 micromolar, for LY411575 at 0.5 micromolar, 1 micromolar and at 2 micromolar, and for M K-0752 at a concentrati on of 40 mi cromol ar and 80 mi cromol ar. Use of M K-0752 at 160 micromolar showed cell toxicity.

[00082] Tabl e I provi des the percentage of cell s that co-stai ned posi ti ve f or H uC/D (a neuronal marker) and negative for SOX1 (a neural stem cell marker) from

immune-fluorescent image anal ysis.

Table I

[00083] Note that a 20 micromolar concentration of the inhibitor MK-0752 generated 64.7% of the cells as differentiated cellswhile O.2 micromolar Compound E generated 74.9% differentiated cell a These results suggest that, while the effect of cell clump reduction during the differentiation of hPSC-deri ved NSCs may be a common feature of the gamma-secretase inhibitors tested herein, their efficacy varies. Those compounds having an acetamido backbone appear most effective (in some commercial chemical names, the term

"acetyl ami no" is used to refer to the same backbone structure). Example 5 - Long-Term Maintenance of Differentiated Neuronal Cells

[00084] For thisstudy, the differentiation of H9 ESC-derived NSCs was the same as for Example2. Without presence of the gamma secretase inhibitor during differentiation, neuronal cells detached from the culture plate at three weeks after differenti ation as shown by the i mage of Fl G . 7A . This effect may be due to the very hi gh densi ty of prol i f erated neural stem cell s present. Cel I s cul tured i n the presence of the gamma secretase i nhi bi tor differenti ated i nto neuronal cells and were mai ntai ned at least for five weeks as shown by the image of FIG. 7B with an even distribution of differentiated neuronal cella

Example 6 - Differentiation of Primary Rodent Neuronal Cells

[00085] Cryopreserved pri mary rat cortical neuronal cells (Cat. No. A1084001 , Thermo Fisher Scientific, Waltham MA), which had been isolated from rodent embryonic brain, were thawed and plated on poly-D-lysine96-well plates (Cat. No. 08-774-255, Thermo Fisher Scientific, Waltham MA) coated with laminin (Cat. No. 23017, Thermo Fisher Scientific, Waltham MA) at a density of 8 x 104 cells/cm2. The culture medium was neuronal differentiation medium containing NEUROBASAL™ Medium (Cat. No. 21103, Thermo Fisher Scientific, Waltham MA), 2% B27 Supplement (Cat. No. 17504, Thermo Fisher Scientific, Waltham MA), 1% GLUTAMAX™ Supplement (Cat. No. 35050, Thermo Fisher Scientific, Waltham MA) without or with gamma secretase inhibitor Compound E. Every 2-3 days, half spent medium was removed from each well of the culture plates and the same vol ume of fresh medi urn was added i nto each wel I . At two weeks of differenti ati on, cells were fixed with 4% paraformaldehyde and stained with antibodies against neuronal marker HuC&D and astroglial marker glial fibrillary acidic protein (GFAP).

[00086] Without gamma secretase inhibitor presence in the differentiation medium, resultant cells contained a large number of GFAP positive astrocytes (FIG. 8A) as well as Hu C & Hu D positive neuronal cells(FIG. 8C). However, cellstreated with the gamma secretase inhibitor compound E showed that GFAP positive astrocytes were essentially eliminated (FIG. 8B) with only Hu C & Hu D positive neuronal cells present in the culture (FIG. 8D).

Example 7 - Proliferation and Electrophysiology of Differentiated Neuronal Cel Is as Compared to Neural Stem Cells [00087] The proliferation and electrophysiology of cells resulting from differentiation in the presence of Compound E were compared to NSC control cultures. NSC cultures were plated at 20,000 cells per well on Day 0.

[00088] The CYQUANT™ Di red Cel I Prol iferation Assay (M olecul ar Probes Cat. No. C35011 , Eugene OR) was used to provide quantitation of proliferation at Day 4 and Day 7 of culture. As shown by FIG. 9A, at Day 4, the relative fluorescence units (RFU) measurement for the control culture (n=8) had a value of .93 and the RFU measurement for cultures different! ated i n the presence of Compound E (n=7) had an RFU val ue of .36, an approxi mate 2.5-fold reduction in cell count. A visual inspection of the cells showed robust proliferation in the control culture with virtually no neurite growth while the cultures differentiated in the presence of Compound E had fewer cell s and those cell s had el aborated I ong processes (images not shown).

[00089] Compared to the data at Day 4, Fl G. 9B shows the same assay measurement at Day 7 for control cultures (n=8) havi ng an RFU of 2.94 (more than a 3-fol d i ncrease i n number than at Day 4) and for cultures differentiated in the presence of Compound E (n=8) having an RFU of .55 (about a 1.5-fold increase). These results indicate that the NSC control cultures continue to proliferate while the cells cultured in the presence of Compound E do not proliferate at the rate of the NSCs without Compound E. These data are consistent with that of Example 3 in that the cells differentiated in the presence of Compound E are terminally differentiated neuronal cellsand are not in active DNA synthesis.

[00090] At Day 14, the same assay measurement for control cultures (n=8) had an RFU of 18.7 (~ a 20-fold increase in number than at Day 4) and cultures differentiated in the presence of Compound E (n=8) had an RFU of .41 (a drop as compared to Day 7).

[00091] At Day 21 , the same assay measurement for control cultures (n=8) had an RFU of 21.5 and cultures differentiated in the presence of Compound E (n=8) had an RFU of .30.

[00092] The el ectrophysi ol ogy of the N SC control cul ture and the cul ture

diff erenti ated i n the presence of Compound E was studi ed usi ng dye and quencher components from the Fluo-4 Calcium Imaging Kit (Molecular Probes Cat. No. F10489, Eugene OR) which provides for detection of calcium flux by fluorescence imaging. Mature neurons express voltage gated calcium ion channels that open in proportion to the concentration of a stimulus. For example, with this kit, graded potassium additions to the medi um i nduce a graded depol ari zati on on the membrane. M ore exci tabl e cul tures have larger responses in proportion to neuronal maturity.

[00093] Control and test cul tures were depolarized with 0, 5, 15, and 30 mM added KCI from an i sotoni c sti mul us. Representati ve traces of f I uorescent response to the addi ti on of the 30 mM KCI sti mul us to control NSC culturesand to cultures differentiated in the presence of Compound E to neuronal cell s are provi ded by Fl G. 9C whi ch i s a pi ot of si gnal vs ti me for measuri ng cal ci um f I ux (average of 8 wel I s) . The si gnal is measured at 1 hZ and plotted in a running average of multiple wel Is as fold increase, designated as (signal max - si gnal mi n)/si gnal mi n. Peak responses were averaged +/- two seconds for each wel I .

[00094] The data are shown i n Fl G. 9C for Day 4 of differentiation; the basal signal i s unchanged i n al I wel I s at or near a val ue of 1 , unti I the potassi um chl ori de sti mul us i s i nj ected i nto the sol uti on above the cell s. The depol ari zi ng i nf I uence to the cell ul ar membrane elicited an increase in cytosolic calcium in proportion to the expression of voltage gated calcium ion channels opened on the cells. Without Compound E in the differentiation medium, the NSCs provided an about 23% increase in response over baseline. With Compound E in the differentiation medium, the differentiated cells provi ded an about 57% i ncrease i n response over the basel i ne. Gi ven that a much I arger cal ci um response i s comi ng from a smal I er number of cell s, these data are i nterpreted to mean that the cell s express significantly higher copy numbers of calcium ion channels, which are an important marker for neural maturity and excitability.

[00095] FIG. 10A provi des tabul ar data of these el ectrophysi ol ogi cal resul ts at Day 4. An i ncreased cytosol i c cal ci um response to the potassi um chl ori de sti mul us can be seen across the enti re range of concentrati ons of KCI when compari ng the cul tures wi thout compound E treatment with those cultures differentiated in the presence of Compound E, indicating an increased expression of voltage gated calcium ion channels in the differentiated cultures as a result of Compound E presence. [00096] Tabular results from Day 7 are provided in FIG. 10B. Without Compound E in the differentiation medium and in response to the addition of the 30 mM KCI stimulus, the NSCs provided an about 37% increase in response over baseline. With Compound E in the different! ati on medi urn at Day 7, the response to the additi on of the 30 mM KCI sti mul us was an about 128% increase over the baseline. Again, there is a much greater response from a smal I er number of cell s i n the cul tures differenti ated i n the presence of Compound E as compared to the number of cell s cul tured i n the absence of Compound E. The 7 day differentiated cells (those treated with Compound E) as measured by this assay demonstrate greater neural maturity and excitability in response to a sti mul us as compared to those not treated with Compound E.

[00097] Data from Day 14 from cultures differentiated without Compound E i n the differenti ati on medi urn and i n response to the addi ti on of the 30 mM KCI sti mul us show that the NSCs provided an about 72% i ncrease i n calci um response over basel i ne. With

Compound E in the differenti ati on medium at Day 14, the calcium response to the addition of the 30 mM KCI sti mul us was an about 117% i ncrease over the basel i ne.

[00098] Data from Day 21 from cultures differentiated without Compound E i n the differenti ati on medi um and i n response to the addi ti on of the 30 mM KCI sti mul us show that the NSCs provided an about 112.4% increase in calcium response over baseline. With Compound E in the differenti ati on medium at Day 21, the calcium response to the addition of the 30 mM KCI sti mul us was an about 286% i ncrease over the basel i ne.

[00099] In summary, the control culture provided an increase in signal of 23%, 37%, 72% and 112.4% for Day 4, 7, 14, and 21, respectively, while the test culture differenti ated in the presence of Compound E provided an increase in signal of 57%, 128%, 116.9% and 286.6% for Day 4, 7, 14, and 21, respectively. The data demonstrate that cells differenti ated i n the presence of Compound E have greater exci tability in response to a sti mul us as compared to cells i n the same medi um without Compound E, demonstrati ng that the maturity of the neuronal cells is accelerated.

[000100] The composi ti ons, methods, and ki ts of the current teachi ngs have been descri bed broadl y and generi cal I y herei n. Each of the narrower sped es and sub-generi c groupi ngs fal I i ng withi n the generi c di scl osure also form part of the current teachi ngs. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material isspecif ically recited herein.

[000101] Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein. Theforegoing exampl es are provi ded to better i 11 ustrate the present teachi ngs and are not i ntended to limit the scope of the teachi ngs herei n. Certai n aspects of the present teachi ngs can be further understood in light of the foil owing claima

Claims

WHAT IS CLAIM ED IS:

1. A method for accelerating differentiation of at least one neural stem cell to at least one neuronal cell and concomitantly retarding neural stem cell proliferation, comprising: cul turi ng the at I east one neural stem cell i n a d i f f erenti ati on medi urn f or a ti me and under conditions to form the at least one neuronal cell,

wherein the different! ati on medium comprises a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, w herei n exci tabi I i ty of the at I east one neuronal cell i s accel erated as compared to culturing the at least one neural stem cell in the differentiation medium lacking the at least one gamma secretase inhibitor.

2. The method of Claim 1 wherein excitability of the at least one neuronal cell is accelerated by at least 100% at Day 7 of differentiation.

3. The method of Claim 1, wherein the at least one neural stem cell is derived from an induced pi uri potent stem cell.

4. The method of Claim 1, wherein the at least one neural stem cell is derived from an embryonic stem cell.

5. The method of Claim 1 wherein the at I east one neural stem cell isa SOXI positive neural stem cell and the at least one neuronal cell isa MAP2 positive neuronal cell.

6. The method of Claim 1, wherein the serum-free supplement of the different! ati on medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof.

7. The method of Claim 6, wherein the serum-free supplement of the differentiation medi um compri ses a gamma secretase i nhi bi tor sel ected from the group consi sti ng of Compound E, YO-01027, LY411575, asalt thereof, and a combination thereof.

8. The method of Claim 6, wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.1 micromolar to 40 micromolar.

9. The method of CI ai m 7, wherei n the gamma secretase i nhi bi tor i s present i n the differentiation medium at a concentration of 0.2 micromolar to 10 micromolar.

10. The method of CI ai m 7, wherei n the gamma secretase i nhi bi tor i s present i n the differentiation medium at a concentration of 0.2 micromolar to 2.0 micromolar.

11. The method of CI ai m 1 wherei n the at I east one neuronal cell i s mai ntai ned i n cul ture for at least a period of five weeks.

12. A method of reducing cell clumping during differentiation of neural stem ceJIsto neuronal cells, comprising:

culturing the neural stem cells in a different! ati on medium for atimeand under conditions to form neuronal cells,

wherein the differentiation medium comprises a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor, wherein cell clumping isreduced at least 50% when compared to culturing the neural stem cells in the differentiation medium lacking the at least one gamma secretase inhibitor.

13. The method of Claim 12, wherei n the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof.

14. The method of CI ai m 13, wherei n the gamma secretase i nhi bi tor i s present i n the differentiation medium at a concentration of 0.2 micromolar to 2.0 micromolar.

15. The method of Claim 12 wherein cell clumping isreduced at least 75% when compared to culturing the neural stem cells in the differentiation medium lacking the at least one gamma secretase inhibitor.

16. A method of differentiation of primary ceJIsto Hu C & Hu D positive neuronal cells and concomitantly retarding differentiation of the primary cells to GFAP positive astrocytes, comprising:

culturing the primary cells in a differentiation medium for a time and under conditions to form Hu C & Hu D positive neuronal cells, wherein the different! ati on medium comprises at I east one serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secret ase inhi bitor,

wherein differentiation of the primary celIsto GFAP positive astrocytes is reduced when compared to cul turi ng the pri mary cell s i n the di f f erenti ati on medi um I acki ng the at least one gamma secretase i nhi bitor.

17. The method of Claim 16, wherei n the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof.

18. A kit for accelerating differentiation of at least one neural stem cell to at least one neuronal cell and concomitantly retarding neural stem cell proliferation, the kit comprising: at least one serum-free neural stem cell culture medium, and

a serum-free supplement comprising at least one gamma secretase inhibitor, and opti onal I y , reagents and i nstructi ons pertai ni ng to use of the ki t.

19. The kit of Claim 18, wherein the serum-free supplement comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK- 0752, a salt thereof, and a combination thereof.

20. A method of treating a subject having a neurodegenerative condition, comprising administering to the subject a pharmaceutical composition comprising neuronal cells made by the method of Claim 1.