Non-virally mediated gene transfer into human central nervous system precursor cells

During the deiclopmcnt of the ccntsal nt”~vou:-a ~~wm tCNS).

multipotent

proliferate to gi\ e rise tu transiently dividing pmgenitor~ that \vill cvt’ntually differentiate into the sc’~.~‘ra1~41 type3 that ~ompo~c the” adult brain [IX]. These prccur4orh h3i.e been i!,oltited from the embryonic CNS of several mammalian species and shown to retain properties of immature cells such as Nestin expression [ 161, extended proliferative potential and the capacity to give rise to differentiated progeny [9.25]. The factors and genes that regulate CNS precursor ccl/ proliferation and progenitor differentiation have begun to be investigated thanks to the identification of cell culture methodologies enabling specific populations of immature cells tcI be maintained in vitro [3.4.18.14.20.23.26] and to advanc\ , in gene transfer techniques that allow the introduction of genes of interest into these cells [2]. Introduction of foreign genes into human CNS cells has only recently been attempted. In a recent report the Ltrt*Z gene was vehicled to human brain progenitor\ via adenoviral vectors [B]. However, the possibility to genetically modify proliferating human CNS precursor cells would be of fundamental importance for improving our knowledge

* Corraponding

precursor

author.

Fax:

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+ 39

(2)

2910-4961;

cattaneo~jisfunix.!i\rma.uniilli.ir 0169-328X/96/%

IS.00Copyright

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01’t’icnts fhsl occur in the hunam brain w&n- normal rend pathological c
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similar lipid-based reagent have been successfully applied to rodent primary cultures of hippocampal neurons. allowing to obtain a transfection efficency of approximately 3% [131. Multipotential human fetal CNS precursor cells were previously obtained from the diencephalon of a 105weekold fetus [27]. These cells have been serially subcultured and expanded for longer than I year in serum-free growth medium containing epidermal growth factor (EGF) and shown to have a duplication time of approximately 30 days (A.V., in preparation). No changes in their growth characteristics or differentiation potential (see below) were observed over this period (A.V.. not shown). Human CNS precursors, as well as their rodent homologues, maintained in EGF-containing medium, grow in suspension forming aggregates of variable sizes named “neurospheres” [23,26,27]. It has been observed that upon removal of the growth factor, these cells can differentiate into neurons, expressing multiple neuronal antigens, as well as glia [23,26,27]. The day before tral;siection the human “neurospheres” were collected and dissociated using trypsine 0.5 X in Ca’+/Mg’+-free HBSS for 10 min at 37°C. Dissociated cells were plated onto laminin-coated (20 pg/ml) 24-well dishes. Transfection was performed using the Lipofectamine method (Gibco-BRL, Life Technologies, Italy). We achieved the best working conditions by varying several parameters. In particular we varied the density of cells seeded, the amount of DNA and Lipofectamine utilized. and the time of exposure to the reagent (shown below). The day after transfection cells were rinsed 3 times with PBS (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l NaJ HPO,. n.:! g/l KH, PO,) and fresh growth medium was added. To identify the XS’ transfection conditions we utilized the CMV-P plasmId carrying the Ltr~vZE. 1~1ligene under the control of the CMV promoter: plasmid DNA was prepared using cesium chloride purification. Results shown in Fig. 1 indicate that the optimal parameters to obtain the highest percentage of histochemically reacted X-Gal positive human CNS cells are 2 X 10” cells/cm’, 1 pg of DNA. 2% Lipofectamine and 5 h of exposure to the reagent. Under these conditions the efficiency of transfection (number of blue cells over number of plated cells) reached 7.4% (Fig. IA and Fig. 2A). When different amounts of plasmid DNA or Lipofectamine were used (Fig. IA) or when exposure times to the transfectant or number of cells seeded were varied (Fig. lB), a decreased efficiency of transfection was In particular conditions (asterisks in Fig. 1) a observed. reduced cell viability was observed as evaluated by Trypan blue exclusion. I%u%hermore,we found that concentrations of Lipofectamine over 2% were toxic to the cells. This transfection procedure did not alter the ability of the cells to differentiate. Following transfection, the cultures were incubated for 6 days in serum-free medium deprived of growth factors, and thereafter cells were fixed with 6 paraformaldehyde. Transfected cultures were

16000 g 14000 t a! 12000 2 ; 10000 j

6000

2 0 z

6000

3

2000

4000

0

0.5 I 0 15 2.0 pga of plasmid DNA (CMVB)

0

3.0

3.5

20

15

10

5

2.5

tlmo of rrposure to LlpoFectamine (Hours)

6

4

2

0 0

S"lLr6

105

1 Bail05

21105

2.51105

number of cells re0dod/cm2

Fig. I. Efficiency of transfection into hutnan CNS precursorcells. In A. results arc provided as absolute number of blue cells out of 2 X IO5 cells/cm’ which have been exposed to Lipofectamine for 5 h. Cells were transfected with increasing amount of plasmid DNA in the presence of 1 and 2% t n 1 Lipofectamine. Percentageof blue cells in each condition is also indicated. The number of transfectedcells was estimated by counting all blue cells present in a 2 cm’ dish. Duplicate dishes were counted per each condition. In B. the number of transfected cells (expressed as percentage) is evaluated after different exposure times to the reagent or under various plating den\itie\. In B cells were Wan+ fected with I pg of plasmid DNA in the presenceof 2% Lipofectamine. Other combinations of DNA and Lipofectamine were less effective. Asterisks in the graphs indicate the conditions in which cell viability was reduced (over 30% of dead cells as judfcd by Trypan blue exclusion). D&I 4own arc from one out of three experiments that provided similar resuhs.

therefore double-stained for J3-galactosidase and for the neuron-specific microtubule-associated protein MAP2 or the glia-specific intermediate filament GFAP. For the identification of the phenotype of the transfected cells, the

were induced

to differentiate

were also

s only slightly lower ~~pprox~i~~~A~e9y 3% I. This result indicates the possibility to an ze gene function in mature human CNS cells. e were interested in whether- an immortalizing oncobe expressed in human CNS precursor cells. ment of immortal human CNS precursor cell indeed represent an invaluable experimental too9 in studies of commitment. differentiation and gene function in neura9 cells of human origin. The availability of oncogenes and the understanding of home aspects of oncogrnesis. along bvith the advent of efficient gene transfer ~~~,t~‘rns.;199ow for an approach to the estahlishmcnt ot neural cell lines that offers several advantages over the spontaneous9y transformed and tumor derived ce99 lines obtained in the past. Oncogenes such as A IIIW and Large-T antigen have been shown to exhibit immortalizing abilities without fully transformin, 0 the cells. Furthermore. the discovery of mutant lrlleles of prtrticular oncogenes has alIowed the creation of cell lines in which the state of the cells could be regulated by altering the temperature of

FIN. 7. A: hun~an C’NS prt’ctmor CL~I/\c’xprc\\ing the f.~l./: gc‘nc ~9-c idcrrlltied hk rhc prcscnic (of ;I hlw prcc’ipil;llc. Fi\ c hour\ at’lcr Iran5l~clion. ww111 10 .I 1’1ndco~i~~cr~lr;~t~~m 01’2’; \%&I\ dklc~l lo Ilka cullurc’s toi -I 11 md tlwertlier fresh gro\\th

replaced with fresh serum-free medium. thr day after W.I~ added. After 24 !I cells were fixed with

medium

glutaraldehyde 0. I % in PBS + 2 mM MgCl,

for IO min. rinsed 3 time\

u ith PBS and exposed to Triton .I. I Cl ,r. Trkfcctcd

cell\ wrt’ identitled

by exposure to the chromogenic wbstrate .4-bromo-~-chloro-?-inJolyl-~* i@acto\idr

(X-Gal:

K,Fe(CN),.

10

mg/ml)

Scale bar. lo0 pm.

B-C:

in PBS.

2.59

0.5 M I; ,Fc(CN),.

DMSO.

mM

MgClz.

(Magnification:

0.5

M

IO0 x ‘j.

transfected human CNS cells differentiate inro

neurons (B) and glial cells (Cl. After transfwtion in warn-free

2

the cclI\ were incubattzd

medium and altowed to diffcrentilrtc

cultures were reacted with X-Gal

for 6 day%. Fixed

and subsequently exposed IO a mow-

cionai antibody ag;Lin\t MAP5 (Boehringer Munnhtzim). The presence ot the antigen \vas revealed by incubatin,*I the cells with a srcondary HKP anti-mouw antibody followed by DAB reaction. The arrowhead\ indicate MAP? staining in ;1 &Gal of the blue precipitate). proce3w4.

expressing cell (arrow indicate\ the prcsenw

Inwt. .

iI

tran4’wted

bipolar ct~ll with clalwratt’d

ln C is ;1 doublt~ irnmunohistclc.hemlc;tl rwctton

clonal antibody

against

@-Gal (left

against GFAP !right panel) followed by FITC-anti-mouse [irabbit antibodies (Vector.

DBA.

v.ith monci-

panel) and a polyclonal antibody and TRITC-an-

!tcdy). A small cluster of transfrcrcd

( /3-Gal positive) cells that probably had undcrsone it few round\ of ceil divisions is visible. Arrowhead: untransfected cells. Right panel: GFAP i\ expressed in a subwt of P-Gal positive cells and (ilrrt)whead) in untran+ fected cells. A GFAP Scale bar. IO pm.

negative transfected cell is alw

visible (iu-row).

Fig. 1\. Ex B). 6 iC i 18 h with antitnDdy for

nin at 4 ( A and d inc1.M .ed for ated sectmdary for nuclear staining. Coverslips were mounted using

I h. After several rinses with PBS, cells were incubated with Hoechst 33258 (5 pg/ml) Permafluor Wscientifica, Italy). A. C and E: immunofluorescent staining. The arrows indicate Large-T antigen expressing cells. E. D and F show Hoechst staining. Scale bar. IO gm.

rocedures

identi-

C‘cpho. c’.. Retroviru\ \cctor\

and tljeir applications in neurohio!og>.

.vcwnu?.I ( !9XX)345-35.i. P~jpSV~SAS~~ t 9

PlXGillid. CXrj btlg the ~et~per~~~urc-sensiallele of SV3Q under the ntrol of 8 retrowiral LT into human CPJS precursor Is. Cells ~~w-e fiwd a oints after transfection an mistry utilizin g the mot~oclonal antibody the Large-T antigen onccprotein [ 1 11. As shown in Fig. 3, 4 days after transfection. sin& hbe cells could be identified (Fig. 3A and Fig. 3 pressed the oncoprotein at high levels in the After 6 days. Large-T antigen expressing cells gone duplication (Fig. 3C tend Fig. doublets of cells that were still positive after transfection a proportion (up to 1W) of these doublets of cells were stiii slowly but regressively expandi and colonies of immuno-labeled cells could be identified (Fig. 3E and Fig. 3F). To our knowledge. these data provide the first indications on the possibility to target exogenous genes into immature human CNS cells using an efficient gene transfer approach alternative to viral-based methodologies. Furthermore. we found that cells transiently transfected with an immortalizing oncogene show high !c, .;‘I:; off q~:.:.~~~~ (71‘ the oncoprotein which is mainLned for sc’i,craI days after transfection. This argueb in fll\‘or of the possibility ot obtaining human CNS precursor cell line\ uirL :\I; increased and unlimited proliferative ability which ma) provide a plentiful source of neurons and gIia suitable for transplantation as well as for in vitro studies on the physiology of human brain cells.

tive

Crilri.

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Pmti.

FxaLclli.

E..

Ckna.

L.. 3loraswttl.

L.. Frolicl~~thal. D.. Roicn.

~1Ultlpi~tcnllal stem cell\ from Illc ,clf‘-rtwu

in

I6 (IWO)

lO‘~l-I

iialob.

rt’\pon\c’

t..

m

P..

Galli.

R.. Wdnhc. E..

F.. Xichc!. D. and \‘:;co\ ;du!I

I. A..

hrafn prolrlc‘rati’ and

wotiw

ha\ic 1’1hrohl~\t growth tutor.

./. .~‘w~-ouY.,

100.

CrrrI\ttorJ.

L.t’..

Pllll.

dlld

11.c.

M

Illldlllson.

NM.

Monoclona! anlibodit3 5pecitic for Simian Virus 40 tumor ;fntifcn4.

(lY8I 1X6!-WI.

1. hlr,l. 1.’ 14.. 39

ht. P.S.~trmcl Sharp. \itt\c

P.A.. Ccl1 line ~~t;Lh!i~hct_l hy ;Ltrmptwturc-\cn-

Simian Virw 40 Large-T-antigen

the nonpcrmc~si\

gent arc groivth restricted rrt

i2lol. Ctal!. Hiol.. Y

c tcnipcr;wcc.

( li)W) !673-

IhXl. tiacch.

S.. Bum Kim. J.. Cwola.

lipid-mediated

,2foi. Hrtrin f!c.~, 25

nwron3. Kilpatrich.

cultuw

ticIn. ,V~,/II~v’ Irl

rc’quirt’mt’nt4 for prolifcr~twn

E.J. and PCrrkdudct.Al.

,Umn

1ru4 and aJcnc)-a~4ol.i,:t~J

HI-. ,ZIctl. HII//.. 5 I

!.cnd.~h!. C.. %~mmcrm;u~. !_.B. and !%.Ka). ;I IIC\L

and diffcrcn1i.k

!!YY3) 255-265

\ iruk ;rwdratcd gcnc trxbl>r. c\prc\4

of hippocampal

( !Wi,) 3-W-31X.

T. and Bartlett. P.. Clonin, 0 and frob tli of multipotcnti~rl

iicuronal prc’curwrx Kwmcr.

M. and Ral~ron. E.. !mpro\cd

gcntt transfer into primq

cia\4

01

inrcrnidi;W

(IYY513 I -1-l.

R.D.G.. CNS stcill cc!l\

t‘ii:uncnl prokin. (‘cl/. h0

( IWO)

5x5 -.i95. Major. O.E..

Miller,

A.E..

Mourrain.

P.. Triluh. R.G.. De Widt.

E.

and Sever. J.. Establirhmcnt of a line of human fetal glia! ce!!s that wpports viru\ multiplication. I257-

Prcri.. ~G’trrl.~Iw~I. St*i. USA, 82 ( I9XS)

I Xl.

I\;lcK;~y.R.D.(;..The origin\

of cellular divrr\ity

in the m;rmmu!iim

central nrrvou~ sj stem. Cc//. 58 ( 10891 X 1542 I. Mt$ler.

Acknowledgements

M.F..

3.. Citohine

Rorenta!. R.. Dougherty.

M.. Sp’ay. D. itnd Kc\\!cr.

regulation of neuronal differentiation

of hippocampa!

propcnilor cells. Mirwt~. 362 ( !c)c)3~ 63-65.

This work was supported by the Alzheimer’s Association/The Hearst Corporation Pilot Research Grant 94-057 to E.C. and partially by funds from The Ministry of Health, Italy. (ICS49.2/RS93.42) to A.V.

in Ray. J. and Gage. F.H.. Spinal cord ncxroblasts prolikrate responw to basic fibrohlmt gro\+ th factor. J. Nwru~.\c~i.. I-43( ! 99-l)

5-M-%4. Rcdic\. C.. Lend&!. (1. mJ McKay. R.D.G.. heterogeneity

in T-antisen

mouse cerebellum.

implatation AlmuLan. G. and McKay.

R.D.G..

An oligodendrocyle prwurw

line from optic nerve. Rlain hks.. 579 (1992) [z] Breakefield. system.

.I. Ntwwscx

Ka..

30

X.0.

and Geller.

A.I.,

cc!!

23-t-215.

Gene tran4t’r

into the nc’rvou\

Mol.Nmtwhiol.. I ( 1087) WI-37 I.

[J] But-Caron.M.H..

Neuroepithelia!

progenitor cell\ ~xplanld

from

i:99!)

Oiffertznlialion and

pwcurwr

ccl1 lint\

from

i I99I 160I-6 ! 5.

Rcnfranx.P.J.. Clmmnghumm, M.G. and spcciilc &ffcrcnt:iion of the hippocampal

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