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Background:0 0 Parkinson’s" disease" (PD)" is" the" second" most"
common"neurodegenera/ve"disease"in"the"United"States." "The"
onset"of"PD"is"characterized"by"the"death"of"dopamine"neurons"
in"the"substan/a"nigra." "In"order"to"understand"the"molecular"
mechanisms"of"PD,"it"is"desirable"to"culture"a"dopamine"cell"line"
to"serve"as"an"in#vitro"model"of"the"disease.""In"the"1990’s,"our"
laboratory"developed"a"dopaminergic"cell"line"derived"from"fetal"
rat" mesencephalic" dopamine" neurons" immortalized" with" the"
SV40"large"T"An/gen.""These"were"named"N27"cells."The"original"
N27" cell" line" was" a" heterogeneous" mixture" of" cell" types"
expressing"highly"variable"levels"of"tyrosine"hydroxylase"(TH),"a"
primary" marker" for" the" dopamine" neuron" phenotype," our"
objec/ve"was"to"culture"and"isolate"single"N27"cells"with"a"high"
level" of" expression" of" TH," and" other" markers" of" authen/c"
dopamine"neurons"to"create"a"more"homogenous"cell"line."
Fig.01:0Substan/a"nigra"of"control"(leW)"and"substan/a"nigra"of"a"Parkinson’s"pa/ent"(right)."Note"that"
the"substan/a"nigra"of"the"Parkinson’s"pa/ent"is"diminished"and"has"lost"its"dark"brown"coloring."This"
is"characterized"by"the"death"of"dopamine"neurons."
ABSTRACT0 METHODS0
N270Cell0SelecEon:0N27"cells"were"first"plated"at"a"
very"low"density"in"a"10Ycm"dish."AWer"a"few"days"
of"growth,"the"cells"began"to"form"colonies."Twelve"
of" these" colonies" were" then" selected" and" plated"
into"a"12Ywell"plate"for"expansion."AWer"expanding"
the" culture" in" duplicate," cells" were" treated" with"
differen/a/ng" agents" or" were" maintained" as""
undifferen/ated"cells.0
Immunohistochemistry:" The" cultures" were" fixed"
with" 4%" Paraformaldehyde" for" 30" minute," then"
rinsed"with"PBS."Cells"were"incubated"with"rabbit"
an/Ytyrosine"hydroxylase"an/body"(1:200"dilu/on,"
PelYFreez)" overnight" at" room" temperature," and"
with" goat" an/Yrabbit" Alexa" 488" (1:200" dilu/on,"
Invitrogen)" in" the" dark" for" two" hours" at" room"
temperature." Finally," cells" were" rinsed" with" PBS"
again," and" checked" for" immunostaining" under" a"
fluorescent"microscope."
Western0 BloJng:0 The" cells" were" dissected" and"
homogenized"in"dissocia/on"buffer"with"protease"
inhibitors." Protein" concentra/on" was" determined"
by" the" BCA" method." " 100" µg" of" protein" was"
separated"on"12%"MiniYProtean"TGX"gel"(BioYRad)"
and"transferred"to"a"nitrocellulose"membrane."Blot"
was"probed"with"an/bodies"to"TH"(1:2,000,"Sigma)"
and"βYac/n"(1:4000,"Sigma)."Blots"were"incubated"
with" HRPYconjugated" secondary" an/body"
(1:10,000;"Jackson"Immuno"Research),"followed"by"
chemiluminescent"detec/on.0
RESULTS0
SUMMARY0
• The" N27" cell" line" is" a" heterogeneous" mixture" of" cell"
types" expressing" highly" variable" levels" of" tyrosine"
hydroxylase" (TH)," a" primary" marker" for" the" dopamine"
neuron"phenotype."
• A"subclone"of"N27"cells"contains"small"clusters"of"cells"
expressing"high"levels"of"TH."Upon"differen/a/on,"these"
TH" posi/ve" N27" cells" change" their" morphology" and"
become"neuronal"shaped"with"long"neurites"
• The"process"of"enriching"and"isola/ng"single"cell"clones"
expressing"high"amounts"of"TH."
FUTURE00RESEARCH0
• Con/nue"clonal"selec/on"
• Isola/on"of"other"dopamine"markers,"DAT"
• Response"to"dopamine"neuron"toxins,"6YOHDA"and"MPP+"
• Transplant"cells"into"Parkinsonian"animal"models"
ACKNOWLEDGMENTS0
Curt0Freed0Laboratory0
Brent"Fitzwalter,"PhD"Candidate"
Breanna"Symmes,"PhD"(PostYdoc)"
Esteban"Lucero,"Graduate"Student"
Mary"Wang,"PRA"
Kim"Bjugstad,"PhD"
Lu"Gao,"PhD"
Augustana0College0
Ian"Harrington,"PhD"
Shara"Stough,"PhD"
Kevin"Geedey,"PhD"
Stephanie"Fuhr,"MS"
Wartburg0West0Program0
Bonita"and"Nelson"Bock"
This research has been made possible by support from the Wartburg West Program, and
by a grant from The Walter and Lucienne Driskill Foundation."
Div."of"Clinical"Pharmacology,"Dept"of"Medicine,"Neuroscience"Program,"Gates"Center"for"Stem"Cell"Biology,"and"
Human"Medical"Gene/cs"and"Genomics"Program,"University"of"Colorado"Denver,"Aurora,"CO"80045"
*Neuroscience"and"Biology"Programs,"Augustana"College,"Rock"Island,"IL"61201"
Marcela"Fitzpatrick*,"Megan"Petersohn*,"Wenbo"Zhou,"PhD.,"Curt"Freed,"MD."
Purifying0a0dopamine0cell0line0to0improve0a0cell0culture0model0of0Parkinson’s0disease"
Fig.04:0(a)"N27"cells"five"day"postY
differen/a/on," showing" uniform"
neuronal" morphology" with" phase"
contrast"(b)"This"image"represents"
a" good" dopamine" phenotype"
(elongated," spindle" shape)" and"
posi/ve" TH" staining." Although" the"
well" is" confluent," there" is" only" a"
‘hot" spot’" of" TH" fluorescence." (c)"
Although" these" cells" were"
differen/ated," they" were" s/ll"
dividing"slowly"into"what"appeared"
to"be"sister"pairs."Small"numbers"of"
these"sister"pairs"show"very"bright"
TH"staining"of"green"fluorescence."
It" is" expected" that" once" cells" are"
differen/ated" they" should" stop"
dividing," however" since" this" cell"
line"is"immortalized"differen/a/ng"
agents" do" not" inhibit" the" process"
of"cellular"division."
Low"density"
pla/ng"
Forma/on"of"
colonies"
Culture"
expansion"
Differen/ate/
Freeze"
Immunostain"for"
tyrosine"
hydroxylase"(TH)"
Fig02:00(a)"Original"
heterogeneous"
N27"cell"line"In#
Vitro,"1994."
"(b)"Rat"embryo"
aWer"15"days"of"
gesta/on."N27"
cells"are"derived"
from"an"E15"fetal"
rat"midbrain,"as"
shown"below."
4a)0DifferenEated0Cell0
Morphology0
3a)0UndifferenEated0Cell0
Morphology0
4b)0DifferenEated0TH0Staining0
Fig.0 3:0 (a)" Undifferen/ated" N27"
cells"aWer"five"days"of"growth."(b)"
We"iden/fied"Clone"3"which"had"
small" clusters" of" cells" exhibi/ng"
very" bright" TH" posi/ve" staining,"
even"in"undifferen/ated"cultures,"
as" demonstrated" by" green"
fluorescence.""
N270CELL0SELECTION0
The" star/ng" N27" cell" line" was" a" mixed"
popula/on" of" cells," and" immunostaining" for"
tyrosine" hydroxylase" (TH)" revealed" a" few"
clones"of"dividing"cells"that"had"high"levels"of"
TH"and"a"good"neuronal"phenotype.""
Western"Blosng"has"also"revealed"posi/ve"TH"
presence;"these"results"were"faint"due"to"low"
protein" concentra/on" (results" not" shown)."
Further"immunohistochemical"experiments"are"
underway.""
4c)0DifferenEated0TH0Staining0
3b)0UndifferenEated0TH0Staining0
Clone030
Clone030
N270Cell0Line0
N270Cell0Line0
Clone030
htp://jackkruse.com/theYdopamineYrxYgoodYchoicesYorYbad/"
2a)0Original0N270cell0line0
2b)0E150Rat0Embryo0
htp://www.cram.com/flashcards/neuroanatomyYlabY4YnotesYonYstructuresYinYmesencephalonY2342316"
Upon" observa/on" of" TH" staining"
under"a"fluorescent"microscope,"we"
discovered" that" Clone" 3" expressed"
the" highest" dopamine" neuron"
marker," TH." Bright" TH" fluorescence"
and" elongated," neuronalYshaped"
cells" can" be" observed" in" figures" 3"
and"4."
We" performed" a" series" of"
experiments"trying"to"enrich"the"TH"
posi/ve" cells" from" Clone" 3." This"
enrichment"and"isola/on"process"is"
s/ll"ongoing.""
CONCLUSION0
Clonal"selec/on"can"improve"the"N27"dopamine"cell"line."
*Photos"of"cells"only"represent"
only"about"1%"of"culture."
ISOLATION0OF0CLONE030