- The document describes efforts to purify a dopamine cell line to improve a cell culture model of Parkinson's disease. Specifically, it involves selecting a subclone of N27 cells that express high levels of tyrosine hydroxylase (TH), a marker for dopamine neurons. - Through clonal selection and expansion of colonies that express TH, the researchers identified Clone 3, which shows bright TH fluorescence and elongated neuronal morphology. Experiments are ongoing to further enrich the TH-positive cells from this clone.

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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