CA2120730A1 - Preparation of pure cultures of post-mitotic human neurons - Google Patents

Abstract

NTera 2/cl.D1 (NT2) cells, a human teratocarcinoma cell line, were manipulated following retinoic acid (RA) treatment to yield >95 % pure cultures of neuronal cells (NT2-N cells). This culture method is capable of yielding sufficient highly differentiated post-mitotic NT2-N cells for both biochemical and molecular biological studies. NT2 cells can be transfected efficiently and the transfected gene products can be expressed in both NT2 and NT2-N
cells.

Description

W093/08266 PCT/US92/06~0 2~ 3~

PREPARATIO~ OF ~URE CULTURE~ OF PO~T-MITOTIC ~N~N N~RON~

INTROD~CTION
This invention was made in the course of research ~ponsored by the NIH grant number NS18616. The Government 5 has certain rights in this invention.
B~CRG~O~ND
Mature mammalian neurons are incapable of cell division and rannot, with the exception of olfactory neurons, be generated from stem cells in the adult nervous system. Thu~, continuous dividing clonal cell lines with neuronal characteristics have proven to be very useful to neurobiologists studying almost every aspect of the nervous systemr Such cell line~ allow the generation of large numbers of homogeneous cells and the manipul~tion of these cells through gene transfer to yield novel derivatives expressing foreign gene produ ts. These advantages have~
led to the development and characterization of a variety of neuronal cell lines, some of which have been useful for cell biolo~lcal, biochemical, and molecular biological studies. The utility of these different cell lines and their ability to approxi~ate aspects of the neuronal phenotype vary widely~ However, studies conducted over the past decade have shown that the usefulness of a cell line primarily depends on two characteristics: 1) the extent to which a particular cell line resembles post-mitotic neurons; and 2) the doublin~ time. Frequently, these two key characteristics are inversely related. Many differentiated properties of neurons are not fully articulated in vivo until the stem ce'l becomes ~0 post-mitotic. However, rapidly dividing neuronal cell W093/08266 2 1 ~ ~ rl ~ ~ PCT/US92/06~0 lines usually do not possess the phenotypic properties of terminally differentiated non-dividing neurons, instead, they often resemble in vivo neuroblasts or embryonic neurons. For example, many of these cell lines elaborate S immature neurites with an imma~ure cytoskeleton but lack most of the morphology and neuritic differentiation of post-mitotic neurons. Nevertheless, since they divide rapidly, these cell lines are useful for biochemical and transfection experiments. Naturally occurring neoplastic derivatives of many neuronal cell types of the central (CNS) and peripheral (PNS) nervous systems usually fall in this category (e.g., neuroblastomas, pheochromocytomas and medulloblastomas). At the other end of the spectrum are cell lines exemplified by HCN 1 (Ronnett et al., 1990, 15 Science, 248:603-605). These cells have many characteristics of differentiated neurons, but they divide ~; so slowly (i.e., doubling time of 72 hours when undifferentiated) that they are not amenable to many experimental manipulations. Even PC 12 cells, the classic example of a neuronal cell line, revert to their less neuronal, rapidly dividing phenotype upon removal of NGF
(Greene and Tischler, 1982, Advances_in Cellular Neurobioloav, S. Federoff and L. Hertz, eds., Academic Press, New York).
Recently, considerable effort has been expendad to immortalize specific neuronal precursors that are found transiently during developmant (for recent reviews, see Cepko, Ann. Rev. Neuro., 12:47-6S, 1989; or Lendahl and McKay, TINS, 13:132-137,1990; and, for specific examples 30 see, Bartlett et al., Proc. Natl. Acad. Sci. USA, 85:3255-3259, 1988; Fredericksen et al., N~uron, 1:439-448, 1988;
Birren et al., Neuron, 4:189-201 (1990); Hammang, et al, Neuron, 4:775-782, 1990; Ryder et al., ~. Neurobiol., 21:356-375, 1990; Lo et al., Dev. Biol., 145:139-153, 1991). This approach is particularly valuable because these cell lines seem to approximate characteristics of ; specific cell types at particular stages of development.

WO 93/08266 2 1 2 0 7 3 0 PCr/US92/06540 ..

Already, new molecules which may serve important developmental functions have been isolated using these novel cell lines (Johnson et al., Nature, 346:858-861, 1990; Lendahl et al., Cell, 60:585-595, 1990). However, 5 with the exception of NAH cells (Birren et al., Neuron 4:
189-201, 1990), cell lines generated using this strategy have a limited ability to undergo further neuronal differentiation. Rather, they seem to be more useful for examining specific branch points in the emergence of 10 neuronal lineages.
The ideal cell line for analysis of the processes of neuronal maturation and the intrinsic factors which affect the establishment of the neuronal phenotype would be one~that divides rapidly so that it could be grown in large 15 quantities and transfected to produce a stable population of~icells expressing exogenous gene products. Upon induction with an agent promoting differentiation, this ideal~ cell line would leave the cell cycle, undergo an irr~eversible commitment to a neuronal phenotype, and exist 20 in~a stable post-mitotic state. These cells would subsequently elaborate extensive neuritic processes and would mature to a state similar to that of primary neurons in culture.
Embryonal carcinoma cell lines satisfy some of 25 the above criteria. These cells, which have been derived from both murine and human embryonal tumors, consist of undifferentiated multipotential cells which will differentiate into one or several cell types when placed under certain conditions (usually including treatment with 30 retinoic acid [RA]). This process resembles the actual commitment to different phenotypes which are found in vivo.
These cell types frequently include neurons, glial, muscle, and/or endothelial cells at various stages of differentiation. Thus, their~ usefulness to neurobiologists Y-~ 35 ~; is~limited~ by their heterogeneity. NTera 2/Dl (NT2), a human~ teratocarcinoma cell line, has characteristics in con with its murine counterparts in that they are W093/08266 2 1~ ~ 7 ~ O PCT/US92/06~0 capable of undergoing phenotypic changes in response to RA.
However, unlike most of the murine embryonal carcinoma cell lines, the only identifiable phenotype ~ound following R~
treatment of NT2 cells are neurons (Andrews, Dev. Biol ., 103:285-293 (1984); Andrews et al., Lab. Invest., 50:147-162 (1984); Lee and Andrews, J. Neurosci., 6:514-521 (1986). Unfortunately, in all previous studies, these neurons represented only a small percentage of the cells, and they coexisted with a large unidentified population of dividing large flat cells and a residual number of undifferentiated stem cells (Andrews, 1984).
~UNMARY OF T~E INVENTION
Treatment with retinoic acid and primary culture techniques (including differential attachment to tissue culture plastic and treatment with mitotic inhibitors) are used to obtain highly purified populations of neurons from a human teratocarcinoma cell line. This culture method is capable of yielding highly differentiated post-mitotic cells. When undifferentiated cells were transfected with a ~-galactosidase (~-gal) expression plasmid, ~-gal expression was shown to be present in both undifferentiated : and post-mitotic cells. Thus, transfection of expression plasmids into undifferentiated cells allows the introduction of exogenous gene products into cells which can then be induced to become stable, post-mitotic human neurons.
DE8CRTPTION OF DRA~ING8 Figure 1 is a schematic showing the method for generation of pure cultures of NT2-N cells from RA treated NT2 cells.
Figures 2A-F are phase contrast photomicrographs showing the morphologic changes which occur during the course of the method shown in Figure 1: A, untreated NT2 cells; B, NT2 cells following Replate #1 (note the round, phase bright cells in clumps sitting above the adherent cells below); C, 1 day following Replate #2 (note that many cells have begun to elaborate rudimentary processes similar W093/08266 2 1 2 ~ PCT/US92/06~0 to a number of human neuroblastoma cell lines); D, 7 days following Replate #2 (the non-neuronal cells have begun to die off and the cultures are now dominated by neuron-like NT2-N cells); E, 30 days following Replating #2 (note that most cells exhibit the morphology of neurons and have migrated into large aggregates and that extensive process outgrowth has occurred; F, high power photomicrograph of NT2-N cells showing the typical neuronal morphology of these cells. The arrows point to the long thin untapering process resembling an axon emanating from a cell in Figure 2F. The arrowheads point to the two major processes which resemble dendrites. The bar in E applies to A-E and equals 200~m and the bar in F equals 30~m.
Figures 3A-C show the results of BrDU labeling of NT2 and NT2-N cells. A, Undifferentiated NT2 cells labeled with BrDU and stained with BU-l. B, NT2-N cells labeled with ~rDU and stained with 8U-l. C, the same field as B
labeled with an anti-NF-L antiserum. The bar in ~ is 200~m.
Figures 4A-F show immunocytochemistry results demonstrating the expression of neuronal markers in NT2-N
cells: A and B, double labeling with RM0254, a mouse anti-NF-M mAb, and anti-NF-66, a rabbit antiserum raised against NF-66; C, lWM 3G5, a mouse anti-MAPlb mAb; D, AP14, a mouse anti-M~P2 mAb; E, A2B5, a mouse mAh specific for a ganglioside found on many neurons; F, T14, a mouse anti-tau mAb. The bar in F is 30~m.
Figure 5 shows the results of immunoblot analysis of the expression of cytoskeletal markers in NT2-N cells.
Lanes 1-6 are from 6% SDS-PAGE gels and lanes 7-13 are from 10% SDS-P~GE gels. l, 20 ~g of cytoskeletal extract from NT2-N cells and 2, bovine MAPs blotted with lWM 3G5, an anti-MAPlb mAb. 3, 20~g of cytoskeletal extract and 4, bovine MAP2 blotted with AP14, an anti-MAP2 mAb. 5, lO~m of cytoskeletal extract and 6, human nerve root IF
preparation blotted with RM0254, an anti-N~-M mAb. 7, 20~g of cytoskeletal extract and 8, human nerve root WO g3/08266 Pcr/US92/o6s4O

intermediate filament preparation blotted with anti-NF-L, an antiserum raised to a human NF-L. 9, 20~g of cytoskeletal extract and 10, bovine NF-66 blotted with anti-NF-66 an antiserum raised to rat NF-66. 11, purified adult human tau; 12, purified human fetal tau and 13, 60~g of cytoskeletal extract blotted with T14, an anti-tau mAb.
MW markers are kD as indicated.
Figure 6: Confocal microscopy of NT2-N cells stained with markers for axons and dendrites. A and B show microscopic fields stained with HO14 (green) and RMdO20 (réd). HO14 is a rat mAb specific for the highly phosphorylated forms of NF-M while RMdO20 is a mouse mAb specific for the poorly phosphorylated forma of NF-M. C
shows a high power field of cells stained with HO14 and AP14, a mouse mAb specific for MAP2. These images were genen~eed~ by collecting separate data for each channel and then~merging them and imparting them with computer genèrated ~pseudocolor. The bar in C is 25 ~m when applied to C,~ and is 50 ,um when applied to A and B.
20- Figure 7: 3H-uridine labeling of N$2-N cell cultures five weeks after Replate #2. A (low power) and B
(high power) are photomicrographs of NT2-N cells produced using Hoffman Modulation Contrast optics. The labeled cells and processes appear dark grey or blaak because of 25 the silver grains from the NTB-2 emulsion autoradiography.
A æhows a field of cells containing labeled cell bodies and dendritic processes (arrowheads) with many unlabeled axonal processes (some examples are shown with arrows) arising from the clumps of cells in the field and elsewhere on the , 30 culture dish. B shows a higher power micrograph of several cells. The dendritic processes of one of these cells have been indicated using arrowheads. The bar in A is 50 ,um when applied to A, and 10Q ~m when applied to B~
Figures 8A-D are photographs showing neurite regeneration following Replate #3. A, NT2-N cells grown on Matrigel for one week following Replate #3. B, NT2-N cells grown on poly-D-lysine for one week following Replate #3.

W093/08266 2 1 2 0 ~ 3 ~ PCT/US92/06~0 The arrow in Figure 6D points to a flat non-neuronal cell.
The bar in D refers to A, B, and D and is 200 ~m. The bar in C is 30 ~m.
Figures 9A-C are photographs showing ~-gal expression of NT2-SPUD cells visuali~ed by X-gal histochemistry using Hoffman modulation contrast microscopy. A, NT2-SPUD cells before RA treatment. B and ~, NT2-SPUD cells after Replate #2. The bar in C is lO0 ~m when applied to A and C and is 50 ~M when applied to B.
10 D~8CRIPTION OF T~B INVBNTION
A human teratocarcinoma cell line (NTera 2/Cl.Dl or NT2 cells) was manipulated following treatment with retinoic acid (RA) to yield grea~er than 95% pure cultures of neuronal cells (NT2-N cells).
The generation of these highly purified cultures of stable post-mitotic cells is schematically outlined in Figure 1. When undifferentiated NT2 cells were initially plated, they appeared as phase dark cells with a granular appearance (Figure 2A). Following four weeks of RA
treatment, the cells formed an extremely dense multi-layered culture. These cultures were then dispersed and replated at a lower density to release the NT2-N cells buried in the midst of the many layer~ of cells. Following this treatment, the NT2-N cells were seen as small phase bright cells above a layer of flat cells (Replate #l;
Figure 2B). The derivation, differentiation paradigm, and early morphological appearance of NT2 cells following treatment with RA to this point (i.e., Replate #l) were examined in detail previously (Andrews et al., 1984;
Andrews, lg84). Approximately 5% of the cells after Replate #l are neurons as judged by the presence of neuron specific markers. To further enrich for these neurons, we took advantage of the fact that the small phase bright NT2-N cells were loosely attached to the layer of flat cells after Replate #l cultures and could be mechanically dislodged and enriched. This treatment (Replate ~2), yielded a culture consisting primarily of small rounded wo g3/08266 2 1 2 ~) 7 3 0 PCr/USg2/06540 phase bright cells, some of which had short processes reminiscent of human neuroblastoma cell lines (Figure 2C).
This step led to an approximately fourfold enrichment of the neuronal cells as assayed by the measurement of S neuronal specific enzymes. These cultures also contained some flat cells, which resembled undifferentiated NT2 cells, which, when allowed to grow unchecked, gradually formed a monolayer with the small, phase bright cells sitting on top. After Replate #2, the phase bright NT2-N
cells grew numerous processes and formed large cellular aggregates within two weeks and were viable for up to five months without ever showing any phenotypic reversion as ; determined by the continuous presence of neuron specific . ~:
markers. However, these cultures were not a significant improvement over the ~ixed cultures derived from Replate #l because of the presence of the contaminating monolayer of flat cells. To eliminat:e these dividing flat cells, the ~ cultures were treated with a combination of mitotic - inhibitors following Replate #2. This treatment had no ; 20 effect on the NT2-N cells, but it completely suppressed the growth of the flat cells. Hence, nearly all of the flat cells were eliminated, leaving behind a very small number of mitotically poisoned cells with extensive cytoplasm. By two weeks of treatment, about 95% of the cells were differentiated neurons, i.e., NT2-N cells. We confirmed this observation, obtained roughly from phase contrast microscopy, by double-staining cultures of cells using monoclonal antibodies (mAb) specific for either the undifferentiated NT2 cells (Cam5.2 which reacts with keratins 8 and 18) or the NT2-N cells (rabbit anti-NF-L
which reacts exclusively with the low molecular weight neurofilament protein). These experiments showed large numbers of NT2-N cells stained with the NF-L antiserum and only very occasional flat cells~with extensive cytoplasm stained with CamS.2. When cultured on Matrigel, which servsd as a bétter substrate for these cells than poly-D-lysine or poly-D-lysine and laminin, these nearly g _ pure cultures of NT2-N cells were viable for about 10 weeks.
When these cultures were examined using phase contrast microscopy at various times (Figures 2D & E), progressive development of extensive neuritic networks covering the entire culture dish was observed. Fine untapering long processes, similar to the axonal processes described in primary neuronal cultures, were also evident.
As shown in Figure 2F, single cells in these cultures elaborated processes that were thick at the base and tapered progressively from the cell body (arrowheads in Figure 2F). Additionally, thin untapering processes arose from these cells (arrows in Figure ~F). These two morphologically distinct types of processes resembled the dendrites and axons, respectively, of primary cultures of CNS and PNS neuronæ. The identity of these two classes of ~ processes as axons and dendrites was borne out by molecular ;~ markers of axons and dendrites they expressed (See Figures 6 and 7). During this period of extensive process outgrowth, the NT2-N cells displayed growth cones typical of primary cultures of neurons from various regions of the ~ nervous system. Another consistent feature of pure NT2-N
;- cells was their motility. Initially, NT2-N cells were evenly dispersed over the entire surface of the culture ~- 25 dish (Figure 2C), but over time they migrated together to form cellular aggregates with large interconnecting fascicles of axons (Figure 2D). If the cell~ were plated on poly-D-lysine either alone or with laminin, they formed even larger aggregates that resembled explanted ganglia.
We examined the reproducibility of this new method to generate large numbers of NT2-N cells by recording the number of cells harvested from Replate #2 in a series of experiments. Over a number of trials (n = 9), beginning with a T75 culture flask seeded with 2 x 106 NT2 cells, treated with 1 x 105 M RA for four weeks and then taken through Replates #1 and #2, we recovered an average of 48.9 x 10 celIs (s.e.m. 3.3 x 10 , n = 9). Despite W093/08266 2 1 2 ~ 7 3 ~ PCT/US92/06~0 limitations in available techniques to estimate the number of NT2-N cells, our quantitative data indicate that approximately 20% of the cells recovered after Replate #2 were NT2-N cells. Thus, the average yield per T75 is about 10 x 106 cells. To date, this culture method has been reproduced at a weekly interval for almost 2 years.
In order to demonstrate that NT2-N cells were incapable of cell division, cultures of both NT2-N cells and undifferentiated NT2 cells were incubated with bromodeoxyuridine (BrDU), and labeled nuclei were visualized by indirect immunofluorescence using a mAb to BrDU incorporated into DNA. More than 50% of the nuclei from undifferentiated cells were immunostained by the mAb after three hours of BrDU labeling (Figure 3A). In contrast, there were no labeled NT2-N nuclei in pure Replate #2 cultures (Figure 38), nor was there labeling after 20 hours exposure. The same cells were also stained with an antiserum against NF-L to show that NT2-N cells are present in the field (Figure 3C). Furthermore, visual inspection of NT2-N cells, either in pure cultures after withdrawal from mitotic inhibitors, or in mixed cultures, never detected an increase in the number of neuronal cells.
In fact, we have followed single clumps of NT2-N cells over three months and we have not detected an increase in cell number despite the absence of mitotlc inhibitors and the presence of 10% fetal bovine serum in the medium for the entire culture period. Taken together, these data suggest strongly that NT2-N cells are post-mitotic.
Previous studies estimated that routine cultures of NT2-N cells (i.e., cultures derived from Replate #l -see Figure 1) make up a minor component of the total cell population (Andrews, 1984; Lee and Andrews, 1986). These cells were shown previously to express neurofilament proteins (Andrews, 1984: Lee and Andrews, 1986), the antigen recognized by A2B5 (a mAb recognizing a cell surface glycolipid characteristic of neurons and some glial cells), and to possess tetrodotoxin sensitive Na~- channels (Rendt W093/08266 2 1 2 0 7 3 0 PCT/US92/06~0 et al., 1989). We have confirmed and extended many of these findings using pure cultures of NT2-N cells in studies designed to examine the expression of several markers typical of neurons in vivo and in vitr~ (Table I).
In addition to the well known neurofilament triplet proteins (Table I: Figures 3C, 4A, and Figure 5), NT2-N
cells expressed NF-66 (also known as alpha-internexin;
Figure 4B) but not peripherin (Table I), the two more recently described members of the neuronal intexmediate ~~~ 10 filament family. NT2-N cells, therefore, may resemble CNS
neurons because NF-66 is abundant only in the CNS, and peripherin is found in virtua}ly all PNS neurons. NT2-N
cells also expressed several neuronal microtubule associated proteins (MAPs) i.e., MAPlA, MAPlB, NAP2, and tau (Table I; Figures 4C, D & F). Neuronal membrane or ~e~brane associated antigens wexe also expressed by NT2-N
cells. These included the antigen reco~nized by A2B5 (Figure 4E), NCAM (another cell surface molecule freguently expressed by neurons and neuronal neoplasms) and GAP43 (a growth associated protein concentrated in growth cones).
We also found that NT2-N cells expressed markers of secretory activity typical of neurons and neuroendocrine cells (e.g., synaptophysin and chromogranin; Tabie I).
Synaptophysin is a marker for small transparent synaptic vesicles which store and releaæe classical neurotransmitters, and chromogranin is a marker for larger dense-cored vesicles which are involved in neuropeptide and catecholamine biosynthesis.
Table I represents the results obtained when NT2-, 30 N cells were examined with a number of antibodies specific for the proteins shown in the first column. The (+~ symbol indicates that the antibody stained and/or blotted the released protein in NT-2N cells. The (-) symbol indicates -that the relevant antibody does not stain NT2-N cells.
, ~ ~

W 0 93/08266 ~ a~3o - Pc~r/US92~06540 T~i3LE I
M~r~er~ of th~ neuronal phe~otype of NT2-N cells R~erence or Protein Antibo~y Re~ult 80urc0 o~ Antibody 5 NF-L rabbit anti-NF-~ + Trojanowski et al., Am. J. Pathol.
135:~47-758 (1989) NF-N RM0254 + Lee et al., J.
Neurosci., 7: 3474-3488 ~1987) NF-H RM024 + Lee et al., J.
Neurosci., 7:3474-3488 (1987) NF-66 rabbit anti-NF-66 + Chui et al ., Neuron , 2:1435-1445 (1989) vimentin rabbit anti-vimentin +* Pleasure et al., J.
Neurosci ., 10:2428-2437 (1990) 20 peripherin xabbit anti-peripherin - Parysek ~t al., J.
Neuroscl., 8:55-63 ( lg88 ) GFAP 2.2B10 - Lee et al., J.
Neurochem ., 42:25~32 (198~) Keratins 8 and 18 Cam5.2 - Becton Dickinson MAPlA HMl + Huber and Matus, J.
Neurosci ., 4:151-160 (19~4~
MAPlB 1WM3&5 + L. Binder, unpublished MAP2 AP14 + Geisert et al., Proc.
Natl. Acad. Sci. USA, 87:39~7-3971 (1990) tau T14 + Trojanowski et al., J.
~listochem. Cytochem., 37:209-215 (1~89) wo g3,08266 2 1 2 ~ 7 3 0 PCTtUS92~06540 synap-tophysin SY-38 + Boehringer Mannheim c~romo-granin LKHllO + Boehringer Mannheim 5 GAP-43 9-lElO + Goslin et al., J.
Neurosci ., 10:588-602 (1990) gang-lioside 10 GT3 A2B5 + Dubois et al., J.
Biol. Chem., 265:2797-2803 (199O) N-CAM ERIC-l ~ Patel et al., Biochem.
Soc. Trans., 18:264 (199O) PSA-NCAM MenB + Theodosis et al., Proc. Natl. Acad. Sci U.S.A. (1991) Ll(NILE) Guinea pig anti-NILE ~ Lee et al., Neurosci., 6:2773-2786 tl~81) ~ Rabbit anti-vimentin is positive on Western blots of NT2-N cell extracts but does not stain these cells by indirect immunofluorescence. This antiserum is capable of staining vimentin in other cells (including the undifferentiated NT2 cells) therefore, it seems likely that the large amount of NF proteins assembled in the same filaments with vimentin masks the reactivity of vimentin in NT2-N cells.

Immunochemical studies were conducted to confirm the expression of several of the markers mentioned above (Figure 5) as well as to demonstrate the feasibility of using NT2-N cells for biochemical studies. Using gel replicas of NT2-N cytoskeletal extracts, we confirmed that MAPlB, MAP2, tau, NF-L, NF-M and NF-66 were indeed present in these cells. Coomassie blue stained gels, together with the immunoblot in Figure 5, shows that MAP2 in NT2-N cells comigrates with the lower of the two isoforms of bovine MAP2. Because of its sensitivity to proteolysis, MAP2 has never been identified biochemically from human tissue.
Nevertheless, we believe that the MAP2 from NT2-N cells is W093/08266 2 1 2 0 7 ~ O PCT/US92/06~0 likely to be MAP2b, a less phosphorylated form of MAP2, which predominates during development and corresponds to the more rapidly migrating of the two MAP2 polypeptides in the bovine MAP2 preparations. Figure S also shows that the S tau found in NT2-N cells corresponds to the fetal forms rather than the adult forms of tau. Fetal tau is translated from differentially spliced forms of tau mRNA
~ which are predominant during embryonic life. MAPlB (also - known as MAP5) is also an embryonic MAP which persists into adulthood but at greatly reduced levels. NT2-N cells express low levels of MAPlA and NF-H (Table I), two proteins which are up regulated in their expression during develop~ent, but which only achieve their highest levels in adult nervous systems, including that of humans. This f inding, together with the expression of three embryonic MAPs~and NF-66, implies that the cytoskeleton of NT2-N
cells resembles that of embryonic CNS neurons.
A~ong the most identifiable and highly differèntiated features of neurons is their hiqhly 20~polarized phenotype. Neurons typically have a single axon and~multiple dendrites which can be distinguished by their morphology, by the organelles they co~ltain and by several differentially distributed molecular markers. For example, the cell body and dendrites tsomato-dendritic domain) have ribosomes (and thus contain RNA) while axons (axonal domain) lack ribosomes. Further, the microtubules in dendrites are oriented in both directions while those in axons have their (+) end oriented distally. Finally, axons are rich in highly phosphorylated NF proteins and tau, ~, 30 while cell bodies and dendrites have primarily hypophosphorylated variants of NF proteins. These differences are all likely to contribute to the distinct functions of dendrites as primarily post-synaptic processes and of axons as projecting pre-synaptic processes.
~ ~ ~Because of~the highly polarized morphology of NT2-N cells after 3-4 weeks in culture, we decided to exàmine whether the processes of NT2-N cells can be ,. ": , , W093/08266 2 1 2 Q 7 ~ O PCT/USg2/~0 differentiated into axons and dendrites. Indeed, when we examined the distribution of phospho-isoforms of NF-M in NT2-N cells, using confocal microscopy, we found that highly phosphorylated NF-M is found preferentially in the lonq thin processes emanating from NT2-N cells while hypophosphorylated NF-M is found in the cell body and short taperinq processes (Figures 6A and 6B). MAP2 is also localized exclusively in the cell soma and the short processes of NT2-N cells (Figure 6C). The non-overlapping distribution of these proteins is very clearly seen using the computer generated superimposed pseudocolor images in Figure 6. The somato-dendritic domains of the cells were stained (in red) by the mAbs against hypophosphorylated NF-M (RMdO20-Figures 6A and 6B) and MAP2 (AP14 - Figure 6C) but were unstained by HO14 (in green), a mAb specific for ~` highly phosphorylated NF-M. Conversely, the axons passing through the fields shown in Figure 6 were only stained with HO14.
A more directly functional feature of the dendrites was examined to determine if NT2-N cells possessed functioning dendrites. As mentioned above, dendrites accumulate ribosomes so that they can respond rapidly to synaptic signals with changes in protein synthesis. This makes it possible to use 3H-uridine to label dendritic processes in neurons in culture. This technique was used to label NT2-N cells and found that, following emulsion autoradiography, processes resembling dendrites are indeed darkly labeled with silver grains while long projecting untapered processes remain unlabeled (Figure 7). This demonstrates that NTi-N cells do indeed possess identifiable dendrites and axons as determined by several criteria. One interesting point is the localization of tau throughout the cell body, dendrites and axons in NT2-N cell (Figure 4F). This is distinct from the finding in sympathetic and cerebral neurons in culture but is quite similar to what has been observed in hippocampal neurons. Thus, while it is clear that tau is necessary for WOg3/08266 2~ a PCT/US92/06~0 axonal elongation in some neurons in culture (perhaps only in those where tau is restricted to axons) additional studies will be necessary to determine the functional role of tau in axons. This peculiarity o~ NT2-N aells and hippocampal neurons may be related to their expression of fetal tau rather than adult tau.
Since NT2-N cells may be a model for immature CNS
neurons, studies were conducted to assess their plasticity.
When pure cultures of NT2-N cells (cells grown for 1-3 weeks following Replate #2) were enzymatically detached and replated again (Replate #3) they rapidly re-extended neurites. These prccesses continued to elongate until a dence network of neurites was formed by one week following replating (Figure ~A). The rapid elaboration of neurites and the eventual formation of the network of processes was found to be highly dependent on the substratum used. For example, rapid outgr~wth oc~urred on cells grown on poly-D-lysine plus laminin or Matrigel substrates but not on~poly-D-lysine alone. Figure 8B illustrates cell clumps grown on poly-D-lysine alone. Even after seven days, these cells had very short processes which ended in flat broad growth cones. By twenty hours, the NT2-N cells on Matrigel ` began to elaborate their processes (Figure 8C). These differential effects of Matrigel (or laminin) and poly-D-lysine on neurite regrowth in NT2-N cells are consistent with the well known neurite-outgrowth promoting effects of laminin on a variety of PNS and CNS neuronal cells in culture. The ability of NT2-N cells to regenerate neurites after multiple replating shows that they retain the plasticity of immature neurons. Furthermore, Replate #3 affords us a means to obtain more pure cultures of NT2-N
cells (at least 99%) which will allow us to pursue experiments in the future which depend on the availability of purer cultures.
In addition to the substratum, non-neuronal cells played a role in promoting neurite outgrowth; this is most evident in Replate #3 cultures. We observed that NT2-N

- wo g3/08266 2 1 2 0 7 3 0 PCT/US92/~ ~0 - ~7 -cells replated on poly-D-lysine alone were clustered around the extremely rare non-neuronal cells found in these cultures, and that many neurites from the clump of NT2-N
cells extended directly toward the non-neuronal cells. A
dramatic example of this phenomena is shown in Figure 8D in which several processes appear to have changed direction dramatically in order to grow toward the non-neuronal cell.
~` Repeated observation of the cells in Figure 8D revealed that the neuriteæ rètracted completely following the death of the non-neuronal cell. This result implies that residual non-neuronal cells may release a diffusible substance which is chemotropic for NT2-N cells. Similarly, - the~presence of non-neuronal cells may explain the extended viabiIity of NT2-N on a non-neuronal cell monolayer (up to five months) compared to the pure NT2-N cell cultures (eight to~ten weeks). Taken together, these studies suggest a role for non-neuronal cells in promoting both the survival of NT2-N cells and neurite extension.
To assess the utility of NT~-N cells for gene transfer experiments, we stably transfected undifferentiated NT2 cells with SPUD1, a ~-galactosidase gal)~ expression plasmid. These studies were designed to determine whether NT2-N cells would continue to express the exogenous protein product following differentiation with RA~ When SPUDl was cotransfected with pSV2neo (used as a selectable marker) into undifferentiated NT2 cells, we derived a G418 resistant population of cells which expressed ~-gal as assayed by histochemical staining (Figure 9A). Upon stimulation of these NT2-SPUD cells with RA, following the same protocol as described in Figure 1, we were able to derive pure cultures of ~-gal positive NT2-N cells (Figures 9B & C). The blue reaction product indicating the preæence of ~-gal protein was concentrated in the~cell somà and only extended into the processes of some~ of~the~NT2-N cells (see arrows in Figure 9B). The -gal reaction product appeared to be concentrated in granular aggregates throughout the cell body (Figure 9B).

This finding suggests that NT2-N cells will continue to express exogenous gene products introduced into NT2 cells.
We have shown that NT2 cells closaly approximate the research criteria of an ideal neuronal cell line. Our observations on the NT2-N cells and the novel methods developed for their culture show that it is possible to generate pure cultures of post-mitotic, human neurons from rapidly dividing NT2 teratocarcinoma cells following RA
treatment. These non-dividing NT2-N cells have a stable neuronal phenotype and they can survive in culture for eight to ten weeks without the continuous presence of any exogenous differentiation promoting or trophic factors other than those in serum. Indeed, using the method of the invention, we have consistently produced NT2-N cells at weekly intervals for almost 2 years, and sufficient numbers of these ce}ls were read~ly generated for biochemical experi ents. It is also highly significant that the ~post q itotic, neuron-like NT2-N cells continue to express ns encoded by plasmids transfected into the undif~ferentiated NT2 cells. This combination of features is unique since, for the first time, cultured human cells with a fully differentiated neuronal phenotype are ava~i}able in large numbers for the study of basic questions in neurobiology including timed neurite outgrowth, the basis and development of neurite polarity, cytoskeletal maturation and neuronal plasticity.
Besides the potential of NT2-N cells for studying the differentiated neuronal phenotype, NT2 cells are an important system for studying the mechanisms whereby RA can induce the differentiation of stem cells into committed neurons without the need for any other exogenous influences. Retinoids (especially RA) are known to have a teratogenic effect during embryogenesis, leading to neural crest and CNS defects. Additionally it has recently become clear~that RA, and~possibly other retinoids, have important in ~vivo physiologic roles in development outside of the nervous system. Stud~ies of retinoid action during neuronal wo g3/08266 2 1 2 0 7 3 0 PCT/US92/06~0 development have been hindered by the relative inaccessibility of the developing nervous system for experimentation. NT2 cells will provide a valuable in vitro system to study the cellular effects of retinoids and their role in neural induction and differentiation.
NT2-N cells expressed all of the key neuronal markers we examined. Further, they have specific characteristic~ which indicate that they are CNS (and not PNS) neurons (i.e., they express the 66 kD NF protein and do not express peripherin [Table I]). Like primary neurons in culture, NT2-N cells have a cytoskeleton dominated by the immature forms of MAPs and NF proteins (e.g., they express primarily fetal tau, MAPlb, MAP2b), but they do synthesize and maintain lower quantities of MAP la and NF-H. Given the stable neuronal phenotype assumed by NT2-N
-~ ~ cells and the fact that they can be genetically engineered to express the`products of transfected genes, these cells will~be extremely useful for examining the cell biology and the~functions of neuronal proteins in human neurons. The prompt neurite regeneration following replating (see Figure 8) and the expression of well aharacterized cell adhesion molecules (see Table I) will allow NT2-N cells to be uti~lized for studying factors which regulate neurite outgrowth. Since NT2-N cells do not divide even in the presence of serum and they represent a reproducible source of highly purified human neuronal cultures, they may be useful cells for transplantation studies into nude mice and other mammals to determine their ability to integrate into the host environment. Indeed, the transfection of trophic factors or other proteins into NT2 cells that are then induced to differentiate into stable, post-mitotic neurons, may be useful as a novel delivery system for bioactive molecules in human neurodegenerative diseases.
The present invention provides methods for preparing a pure culture of post-mitotic human neurons comprising culturing undifferentiated human teratocarcinoma celIs with retinoic acid to obtain a multi-layered culture;

~-"'"~

WO93/0826b 2 1 2 0 7 3 0 PCT/US92/~ ~o dispersing said cultured cells; and culturing said dispersed cells with a mitotic inhibitor or a combination of mitotic inhibitors. The present invention further contemplates that undifferentiated cells of the above S method comprise NTera2/D1 cells. Moreover, the present invention includes this method wherein a combination of mitotic inhibitors comprises cytosine arabinoside, fluorodeoxyuridine and uridine. Additionally, dispersed cells of the above method can be cultured on Matrigel.
The present invention also provides methods for producing a stable population of post-mitotic human neurons expressing exogenous gene products comprising transfectinq one or more plasmids (including a selectable marker) into cultured undifferentiated human teratocarcinoma cells;
culturing said undifferentiated human teratocarcinoma cells with retinoic acid to obtain multi-layered culture;
dispersing said cultured cells; and culturing said dîspersed cells with a mitotic inhibitor or a combination of mitotic inhibitors. It is contemplated that a single selected expression vector may be transfected and expressed in the stable population of post-mitotic cells. It is further contemplated that more than one selected expression vector may be transfected and expressed in the stable population of post-mitotic cells. It is also contemplated that the selected expression plasmid comprises a ~-galactosidase expression plasmid. The present invention contemplates that the undifferentiated cells of this transfection method comprise NTera2/Dl cells. It is further provided by the present invention that the , 30 combination of mitotic inhibitors of this transfection method comprise cytosine arabinoside, fluorodeoxyuridine and uridine. Additionally, the dispersed cells of this transfection method can be cultured on Matrigel.
The present invention also provides stable post-~` 35 mitotic hu~an neuron cells produced in accordance with the methods described above, including but not limited to stable post-mitotic human neuron cells substantially all of , ~ .

, W093/08266 2 ~ ~ ~ 7 ~ ~ PCT/US92/06~0 which comprise at least one transfected exogenous gene or stable post-mitotic human neuron cells which are not transfected.
The present invention is further illustrated by the following examples, which are not intended to be limiting in any way.
EXANPL~8 E~AMPLE 1 Cell Culture:
NT2 cells were maintained in DNEM HG including 10% fetal bovine serum and penicillin/streptomycin as previously described (Andrews, 1984). For differentiation, 2 x 106 cells were seeded in a 75cm2 flask and treated with 1 X lo 2 M RA (a 1 x 10 2 M stock dissolved in DMSO was prepared fresh monthly) twice a week for four weeks.
Following RA treatment, the cells were replated 1:6 ~Replate #l; see Figure 1). On the following two days, cells were mechanically dislodged, i. e., culture flasks were struck ten times on each side and the floating cells were washed with 5 ml of medium and replated again (Replate #2; see Figure 1) on Matrigel (Collaborative Research) diluted 1:20 (for coverslips) or 1:60 (for dishes) following the manufacturer's instructions (the dilutions used for the Matrigel Yaried somewhat from lot to lot).
Cells were seeded at a density of 0.2 x 106 cells per 12 mm coverslip or 7.5 x 106 cells per 100mm dish in ~MEM HG with 10% serum and penicillin/streptomycin supplemented with 1 ~M Cytosine arabinoside, 10 ~M fluorodeoxyuridine, and 10 ~M uridine. Cytosine arabinoside was continued for the first week of culture and fluorodeoxyuridine and uridine for the first four weeks. For neurite regeneration experiments, three week old cultures were enzymatically removed with 0.025% dispase or 0.025% trypsin and replated (Replate #3; Figure 1) on Matrigel, poly-D-lysine (10 ~g/ml), or poly-D-lysine (10 ~g/ml) plus laminin tl0 ~g~ml). Matrigel is a basement membrane extract containing collagen, laminin, and nidogen (Kleinman et al., 1986).

W093/08266 2 ~ ~ ~ 7 ~ ~ PCT/US92/06~0 This method as outlined in Figure 1 and described in detail here, was applied to three different NTera2 subclones (NT2/Dl, NT2/03 and NT2/B9) with similar results. All results presented here were obtained from NT2/Dl. During the course of the experiments it was observed that NT2 cells prefer opti-NEM (GIBC0) with 5% fetal bovine serum (F8S) and this medium was then used for maintaining undifferentiated NT2 cells.
~XAHPLE 2 BrD~ l~bel~ng:
Undifferentiated NT2 cells and differentiated NT2-N cells were gr~wn free of mitotic inhibitors for four days and then labeled with 3 mg/ml BrDU for three hours (or up to 20 hours, in some cases). The cells were then washed, fixed and processed for indirect immunofluorescence as described below using BU- 1, a mab which recognizes BrDU
~ ~ "
incorporated DNA without denaturation.
A~PLB 3 : ~ :
~n~irect Imcunofluor-~cence ~n~ Confocal Microscopy:
Cells`were washed with Hank's Buffered Salt Solution and fixed with 70% ethanol containing 0.15 N NaCl for ten minutes at room temperature. The cells were incubated with primary antibodies for one hour at room temperature, washed four times with PBS for one hour, incubated for one hour with secondary antibodies ~donkey anti-mouse IgG coupled to rhodamine, donkey anti-rat coupled to fluorescein, and donkey anti-rabbit coupled to fluorescein; Jackson Immunoresearch), and finally washed four times in PBS for one hour before mounting in Aquamount (Lerner Labs). For confocal microscopy the procedure was essentially the same except that Texas Red conjugated secondary antibody was used instead of rhodamine and the coverslips were mounted using 5% DABC0 to prevent bleaching. The coverslips were examined using a krypton laser on a Bio-Rad MRC-600 Iaser scanning confocal :: , microscope.

W093/08~ 2 1 2 0 7 3 0 PCT/US92/06~0 EXAMPLB ~
Peroxi~so-Anti-Peroxid~se (PAP) Im~unocytochemistry:
PAP immunocytochemistry was performed to visualize low levels of fetal tau because of the great sensitivity of this technique when compared to indirect immunofluorescence. The coverslips were fixed as above and blocked for 30 minutes with O.lM Tris pH 7.0, including 2%
calf serum and 0.25~ cold water fish gelatin. Following this, the coverslips were processed as described previously in our laboratory for PAP immunocytochemistry (Carden et ~; al., J. Neurosci., 7:3489-3504 (1987).
~ S
Ivch_i--try:
NAP-enriched cytoskeletal samples were prepared by extracting the cells at room temperature for 15 minutes with~O.~lM~NES pH6.8 conta~ning 0.5mM MgS0~, l~M EGTA, 2mM
DTT,~2mM~GTP, 20~M Taxol, I% Triton X-100 and a cocktail of eàse ~inhibitors. The pellets were recovered by centrifugation at 30,000 rpm in a TL100 ultracentrifuge for 30 minutes, solubilized in sample~buffer without dye and the~protein concentrations of the samples were determined using a Coomassie blue dye binding assay (Pierce). These samples were run on SDS-PAGE gels and ~hen electroblotted to~nitroaèllulose membranes for probing with antibodies using methods described previously in our laboratory (Lee et al., 1987).
~XANPLE 6 H-Uridine ~abelling of NT2-N Cells:
60mm dishes of NT2-N cells were incubated with 30 ~50~Ci of ~5,6-3H]uridine (1.70 TBq/mmol) for 16-24 hours.
These dishes were then washed with PBS containing lO~M
unlabelled uridine and fixed with ~ouin's fixative. The dishes were then coated with NT~-2 emulsion diluted 1:1 with water, dried overnight and stored at 4-C for four :
days. ~ The dishes were~developed for one minute in Kodak Dl9~and fixed with Kodak Rapid-Fix.

W093/08t66 2 1~ ;~ 7 3 ~ PCT/US92/06~0 - 2~ -Transfection aDd 8taining for ~-gal~ctosi~ase:
Undifferentiated NT2 cells were transfected with loo ~g SPUDl and lo ~g of pSV2neo by lipofection using Lipofectin (Bethesda Research Laboratories). After two days in complete medium, the transfectants were selected with 200 ~g/ml G418 (Gibco) for seven days. Cells were stained fo~ ~-galactosidase activity with 1 mg/ml X-gal, 5 mM potassium ferrocyanide, S mM potassium ferricyanide, 2 mM MgCl2 in PBS after fixation in 2% paraformaldehyde, 0.2%
glutaraldehyde in phosphate buffered saline pH7.4. ~-gal positive cultures were subcloned twice and the subclones were used for further studies. SPUDl (kindly provided by Dr. C. Cepko) is a ~-galactosidase expression vector which utilizes the SV40 promoter and has Moloney murine leukemia virus long terminal repeats upstream and downstream. The cells were photographed using Hoffman modulation contra~st to allow the simultaneous visualization of the blue reaction product and the processes.

.

Claims (13)

What is claimed:

1. A method for preparing a pure culture of post-mitotic human neurons comprising:
culturing undifferentiated human teratocarcinoma cells with retinoic acid to obtain a multi-layered culture;
dispersing said cultured cells, culturing said dispersed cells with a mitotic inhibitor or a combination of mitotic inhibitors.

2. The method of claim 1 wherein said undifferentiated cells comprise NTera2/D1 cells.

3. The method of claim 1 wherein said combination of mitotic inhibitors comprises cytosine arabinoside, fluorodeoxyuridine and uridine.

4. The method of claim 1 wherein said dispersed cells are cultured on Matrigel.

5. A method for producing stable population of post-mitotic human neurons expressing exogenous gene products comprising:
transfecting at least one plasmid including a selectable marker into cultured undifferentiated human teratocarcinoma cells;
culturing said undifferentiated human teratocarcinoma cells with retinoic acid to obtain multi-layered culture;
dispersing said cultured cells; and culturing said dispersed cells with a mitotic inhibitor or a combination of mitotic inhibitors.

6. The method of claim 5 wherein said undifferentiated cells comprise NTera2/D1 cells.

7. The method of claim 5 wherein said combination of mitotic inhibitors comprises cytosine arabinoside, fluorodeoxyuridine and uridine.

8. The method of claim 5 wherein said dispersed cells are cultured on Matrigel.

9. Stable post-mitotic human neuron cells produced in accordance with the method of claim 1.

10. Stable post-mitotic human neuron cells produced in accordance with the method of claim 5.

11. The method of claim 5 wherein said plasmid comprises a .beta.-galactosidase expression plasmid.

12. Stable post-mitotic human neuron cells produced in accordance with the method of claim 11.

13. Stable post-mitotic human neuron cells substantially all of which comprise at least one transfected exogenous gene.