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OCR for page 95
Colloquium
.
Nonhuman primate parthenogenetic stem ceils
Kent E. Vrana*t, Jason D. Hipp*, Ashley M. Goss*, Brian A. McCool*, David R. Riddlet, Stephen J. Walkert,
Peter J. Wettstein§, Lorenz P. Student, Viviane Rabat, Kerrianne Cunniffll, Karen Chapman**, Lucy Vilner**,
Michael D. West**, Kathleen A. Grant*, and Jose B. Cibellit~t
*Center for Neurobehavioral Study of Alcohol, Department of Physiology and Pharmacology and "Department of Neurobiology and Anatomy, Wake Forest
University School of Medicine, Winston-Salem, NC 27157; §Department of Microbiology and Immunology, Mayo Clinic, Rochester, MN 55905; ~SIoan-
Kettering Cancer Center, New York, NY 10021; **Advanced Cell Technology, Worcester, MA 01605; Millennium Pharmaceuticals, Cambridge
MA 02139; and subdepartment of Animal Science-Physiology, Michigan State University, East Lansing, Ml 48824
Parthenogenesis is the biological phenomenon by which embry-
onic development is initiated without male contribution. Whereas
parthenogenesis is a common mode of reproduction in lower
organisms, the mammalian parthenote fails to produce a successful
pregnancy. We herein describe in vitro parthenogenetic develop-
ment of monkey (Maraca fascicularis) eggs to the blastocyst stage,
and their use to create a pluripotent line of stem cells. These
monkey stem cells (Cyno-1 cells) are positive for telomerase activity
and are immunoreactive for alkaline phosphatase, octamer-bind-
ing transcription factor 4 (Oct-4), stage-specific embryonic antigen
4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor
rejection antigen 1-81 (TRA 1-81) (traditional markers of human
embryonic stem cells). They have a normal chromosome karyotype
(40 + 2) and can be maintained in vitro in an undifferentiated state
for extended periods of time. Cyno-1 cells can be differentiated in
vitro into dopaminergic and serotonergic neurons, contractile
cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and
adipocytes. When Cyno-1 cells were injected into severe combined
immunodeficient mice, teratomas with derivatives from all three
embryonic germ layers were obtained. When grown on fibronec-
tin/laminin-coated plates and in neural progenitor medium,
Cyno-1 cells assume a neural precursor phenotype (immunoreac-
tive for nestin). However, these cells remain proliferative and
express no functional ion channels. When transferred to differen-
tiation conditions, the nestin-positive precursors assume neuronal
and epithelial morphologies. Over time, these cells acquire elec-
trophysiological characteristics of functional neurons (appearance
of tetrodotoxin-sensitive, voltage-dependent sodium channels).
These results suggest that stem cells derived from the partheno-
genetically activated nonhuman primate egg provide a potential
source for autologous cell therapy in the female and bypass the
need for creating a competent embryo.
The use of human embryos to derive embryonic stem cells (ES
cells) is viewed by some sectors of our society as ethically
problematic. In nonhuman primates, there are currently three
methods for deriving pluripotent stem cells: from embryos
produced by in vitro fertilization (1-4), parthenogenesis (5), and
from adult tissues such as cells derived from the bone marrow
(6~. We have previously reported the creation of a line of
nonhuman primate stem cells from parthenogenetically acti-
vated eggs (54. By using this technique, ES cells were derived
without the need to create or destroy a viable embryo.
Parthenogenesis, the process by which a single egg can develop
without the presence of the male counterpart, is a common form
of reproduction in nature. Flies, ants, lizards, snakes, fish, birds
reptiles, amphibians, honeybees, and crayfish routinely repro-
duce in this manner. Eutherians (placental mammals) are not
capable of this form of reproduction. However, chimeras of
parthenogenetic cells coupled with biparentally derived embry-
onic tissues have generated apparently normal offspring, and the
parthenogenetic origin of several tissues has been confirmed in
www.pnas.org/cgi/cloi/10. ~ 073/pnas.2034195 ~ 00
such chimeric animals (7~. In a reported case of a human
parthenogenetic chimera, contribution to several tissues has
been demonstrated, including blood where 100% of the leuko-
cytes were found to be of parthenogenetic origin (8~.
Eutherian oocytes, on the other hand, can undergo parthe-
nogenesis in vitro with variable success. When mammalian
oocytes are activated (emulating the fertilization process) and
transferred to a surrogate mother, they are capable of surviving
to day 10 of development in the mouse, day 21 for sheep, day 29
in pigs, and day 11.5 in rabbit (9-12~.
The reason for this halted development is believed to be due to
genetic imprinting. It has been shown that maternal and paternal
genomes are epigenetically different, and that both sets are required
for successful development (13-15~. In parthenotes (activated
eggs), all of the genetic material is of maternal origin, and hence
lacking paternal imprinting. It is believed that parthenotes are not
capable of developing to term because they fail to develop a
trophectoderm and primitive endoderm-extraembryonic tissues
(9~. They resemble ovarian teratomas and consist of only embryonic
tissue. Androgenotes (created by the fusion of two sperm nuclei or
diplodization of one sperm in the absence of female counterpart)
are of purely paternal origin and develop into a structure consisting
of a trophoblast and yolk sac (164. These resemble hydatidiform
moles (solely trophoblastic tissue), which are formed when a sperm
fertilizes an enucleated egg (17~.
In the present report, we describe the parthenogenetic acti-
vation of cynomolgus macaque eggs in vitro and the derivation of
a pluripotent cell line (Cyno-1~. When cultured under selective
conditions, these cells have divided for >2 yr, and, on induced
differentiation, cell derivatives from all three germ layers were
obtained.
Materials and Methods
Superovulation, Oocyte Retrieval, and Ooeyte Maturation/Activation.
Monkeys were injected (i.m.) with 1,000 units of pregnant mare
serum gonadotrophin 5 days before surgery and then injected
with 500 units of human chorionic gonadotropin 24 h before
surgery. For ovary isolation, monkeys were tranquilized with
ketamine (10 mg/kg of body weight), intubated endotracheally,
and anesthetized with isoflurane (monitored to effect: no pal-
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, "Regenerative Medicine," held October 18-22, 2002, at the Arnold and Mabel
Beckman Center of the National Academies of Science and Engineering in Irvine, CA.
Abbreviations: ES cell, embryonic stem cell; Cyno-1 cell, parthenogenetically derived stem
cell line from the cynomolgus macaque; bFGF, basic fibroblast growth factor; Snrpn, small
nuclear ribonucleoprotein polypeptide N; TUJ1, ,8-tubulin lil; PBL, peripheral blood Iym-
phocytes; ICM, inner cell mass.
iTo whom correspondence should be adciressecl. E-mail: kvrana@?wfubmc.edu or
cibelli@?msu.edu.
C) 2003 by The National Academy of Sciences of the USA
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11911-11916
OCR for page 96
pebral reflex, no deep pain response). Ovaries were removed by
midline laparotomy incision.
Oocytes were manually harvested under a dissecting micro-
scope. Oocyte maturation was performed in CMRL-1066 media
(Sigma) with 20 To FCS (HyClone), 10 units/ml pregnant mare
serum (Sigma), 10 units/ml human chorionic gonadotropin
(Sigma), 0.05 mg/ml penicillin, and 0.075 mg/ml streptomycin
(Sigma). Eggs were incubated for 36 h at 37°C, in JO CO2 and
20% O2. Mature metaphase II eggs were subsequently activated
by incubation with 10 ,uM ionomycin for 8 min. followed by
culture with 2 mM 6-dimethylaminopurine for 4 h. The inner cell
masses (ICM) were isolated by immunosurgery as described (20)
and cultured on a feeder layer of mitotically inactive mouse
embryonic fibroblasts in Dulbecco's minimal essential medium
(GIBCO) with 15% FCS (HyClone).
Cell Culture Conditions. Neural progenitor-ES cells were plated in
flasks coated with fibronectin/laminin or fibronectin/BSA/
collagen with NPMM (Clonetics, East Rutherford, NJ) and
maintained at 37°C in 5% CO2. Media were changed every 3
days. Differentiation was induced by removing basic fibroblast
growth factor (bFGF) and epidermal growth factor, with the
addition of 200 ~M ascorbic acid.
Immunohistochemical Staining. A variety of markers of stem cells
and stem cell differentiation were assessed by immunocytochem-
istry. Antibodies and staining conditions were as follows. For
surface markers, cells were incubated with primary antibodies
tstage-specific embryonic antigen-4 (1:20; Developmental Hybrid-
oma Bank), tumor rejection antigen 1-81 (1:80), and tumor rejec-
tion antigen 1-60 (a gift from P. Andrews, Sheffield, U.K.~. For
immunocytochemisrty of embryonic markers, primary antibodies
were diluted in PBS supplemented with 0.5% BSA. After washing
with PBS-BSA, cells were fixed with 2% formaldehyde for 30 min
and washed three times in PBS-BSA, followed by incubation with
10% normal goat serum in PBS at room temperature. Subse-
quently, primary antibody was added for 30 min at room temper-
ature; cells were then washed with PBS-BSA three times, followed
by incubation with secondary antibody for 30 min. washed, stained
with 4',6-diamidino-2-phenylindole (DAPI), and mounted. For
immunocytochemistry of differentiated cells, cells were fixed in 4%
paraformaldehyde at room temperature for 20 min. followed by
permeabilization for 2 min in 100% ethanol. After fixation, cells
were washed with PBS, blocked with 10% normal goat serum in
PBS at room temperature for 2 h, followed by incubation at room
temperature for 2 h with nestin antibody (1:200, Chemicon), TH
polyclonal 1:200 (Pel-Freez Biologicals) or TH monoclonal 1:1000
(Sigma) ,B-tubulin type III (TuJ1) monoclonal (1:500, Babco,
Richmond, CA), in PBS. After washing, cells were incubated with
a rabbit secondary antibody in PBS-BSA at room temperature for
30 min. Cells were then washed in PBS and mounted.
Alkaline Phosphatase. Alkaline phosphatase was determined as
described (54. Briefly, culture medium was removed from the
plates, and cells were fixed with 4% paraformaldehyde for 20
min. Cells were washed three times in Tris-maleate buffer t(3.6
g of Trizma base (Sigma), in 1 liter of water, pH raised to 9.0 with
1 M maleic acid)] for 10 min each wash. The last wash was
removed, and the staining solution [(Tris-maleate buffer: 200 ~l
of a 5 mM MgCl naphthol AS-MX phosphate (Sigma), 0.4
mg/ml; Fast red (Sigma), 1 mg/ml)] was added to the cells for
15 to 20 min. Once red colonies were detected, the reaction was
stopped by adding PBS and bringing the pH to 7.4.
Antigen Profiling. Peripheral blood lymphocytes (PBLs) were
isolated from whole blood by flotation on Ficoll-Hypaque. Cells
were harvested from the medium:Ficoll-Hypaque interface, and
the remaining red cells were hypotonically lysed. After addi-
11912 1 www.pnas.org/cgi/doi/10.1073/pnas.2034195100
tional washes, cells were labeled with FITC-conjugated anti-
HLA-A,-B,-C tclone G46 52.6, Pharmingen) and phycoerythrin
(PE)-conjugated anti-HLA-DR (clone G46.6, Pharmingen).-Iso-
type-matched control antibodies were included as negative con-
trols (shaded curves in Fig. 7~. Cyno-1-derived neural cells were
cultured, harvested, and stained with FITC-labeled anti-HLA-
A,-B,-C and PE-labeled anti-HLA-DR as above and compared
with cells stained with isotype-matched control antibodies (shad-
ed curves in Fig. 7~. Treatment of Cyno-1-derived neural cells
with IFN-y involved overnight incubation with human IFN-y (40
ng/ml). Stained cells were analyzed with a FACScan flow
cytometer and CEL~QUEST software (Becton Dickinson).
Telomerase Activity Measurement. Telomerase activity was mea-
sured by using the TRAPeze kit (Intergen, Purchase, NY) as
recommended by the manufacturer. Control template, buffer,
and control extract were supplied by the TRAPeze kit. Extracts
from the mouse feeder cells, the Cyno-1 cells (maintained on
mouse feeder layer), and the differentiated Cyno-1 cells (grown
for 14 days without mouse feeder layer) were normalized to the
protein concentration. Heat inactivated extracts were boiled for
3 min before the assay.
Electrophysiology. The whole cell patch clamp technique was
performed on differentiated stem cells that were continuously
perfused with a Hepes-buffered saline (HBS) solution (contain-
ing, in mM: 150 NaCl, 10 Hepes, 2.5 KCl, 2.5 CaCl2, 1.0 MgCl2,
10 D-glucose, pH 7.4, with NaOH, osmolality 320 mmol/kg
adjusted with sucrose). Tetrodotoxin (Calbiochem) was diluted
from concentrated stocks into HBS and applied within 100 ,um
of the cell by using a linear array of fused-silica tubes (150 mm
I.D., Hewlett-Packard) mounted on a manipulator. A Cs+-based
internal solution (in mM: 130 CsCl, 10 Hepes, 10 EGTA, 1
CaCl2, 4 Mg-ATP, pH 7.2 with CsOH, osmolality 305 mmol/kg
adjusted with sucrose) was used in the patch electrode.
Recordings were performed at room temperature according
to published procedures by using an Axopatch ID amplifier
(Axon Instruments, Foster City, CA) in voltage-clamp mode as
described (18, 194. Whole-cell capacitance and series resistance
were determined by fits of the capacitive transients during
square-wave voltage steps by using standard software procedures
contained within PCLAMP 7.0 software (^on Instruments) and
monitored throughout the recordings. Resting membrane po-
tentials were -70 mV. Voltage-gated currents were elicited by
square-wave membrane depolarizations to 0 mV.
Results
Creation and Characterization of Monkey Parthenogenetic Stem Cells.
Stem cells were created via parthenogenetic activation of eggs as
described (5~. Briefly, 77 eggs were isolated from the ovaries of
three different cynomolgus monkeys (Macaca fascicularis, ~18
yr of age) after hormone-induced superovulation. The oocytes
were then maintained in maturation medium for 36 h. Twenty-
eight eggs reached metaphase II stage and were subsequently
activated by incubation with 10 ,u M ionomycin for 8 min,
followed by culture with 2 mM 6-dimethylaminopurine for 4 h.
Four embryos developed to the blastocyst stage after 8 days in
culture (14%) (Fig. 1A). Immunosurgically isolated ICMs (20)
were cultured on a feeder layer of mitotically inactive mouse
embryonic fibroblasts in Dulbecco's minimal essential medium
with 15% FCS (HyClone). Three ICM showed outgrowth within
1 week of plating, and one stable cell line (Cyno-1) was success-
fully derived (Fig. 1B). Cyno-1 cells displayed many features that
are typical for ES cells: cytoplasmic lipid bodies, small cytoplas-
mic/nuclear ratio, and clearly distinguishable nucleoli. These
cells were immunoreactively positive for alkaline phosphatase,
stage-specific embryonic antigen 4, tumor rejection antigen 1-60,
and tumor rejection antigen 1-81 and were positive for octamer-
Vrana et a/.
OCR for page 97
-
! ~ ' ,t, ~ ~
OCT4 | ~ l
Fig. 1. Characterization of parthenogenetic embryos and derived cell lines.
(A) Parthenogenetically activated eggs at day 8 of development before ICM
isolation. (B) Phase contrast of Cyno-1 stem cells growing on top of mitotically
inactivated mouse feeder layer (met). (C) Alkaline phosphatase staining. (D)
Stage-specific embryonic antigen 4. (E) Tumor rejection antigen 1-60. (F)
Tumor rejection antigen 1-81 staining. (G) RT-PCR octamer-binding transcrip-
tion factor 4 expression in undifferentiated Cyno-1 cells. (Scale bars = 50 Am
in A, 10 Am in B and D-F, and 4 mm in C.) [A reprinted with permission from
ref. 5 (Copyright 2002, AAAS, www.sciencemag.org).]
binding transcription factor 4 mRNA (Fig. 1 C-G) and negative
for stage-specific embryonic antigen-1 and -3 (data not shown).
These cells have been propagated for >2 yr maintaining their
undifferentiated state. Karyotype analysis revealed 40 + 2
chromosomes, in accordance with the species of origin, M.
fascicularis (data not shown).
Parthenogenetic Stem Cell Differentiation. In vitro differentiation
was induced by isolating the cells from the mouse feeder layer
and culturing them in the presence of Dulbecco's minimal
essential medium with 15% FCS; in some instances, 1,000 units
of leukemia inhibitory factor was added to the media. A large
variety of specialized cell types could be generated in vitro, such
as spontaneously beating cardiomyocyte-like cells and ciliated
epithelium, smooth muscle cells and cytokeratin-positive cells, as
well as neuronal cells (data not shown).
To assess the differentiation capacity of Cyno-1 cells, we
injected them into the peritoneal cavity of immunocompromised
severe combined immunodeficient mice. Eight to 15 weeks after
injection, teratomas were isolated and histologically analyzed.
Microscopic observations revealed the presence of mature tis-
sues and low frequency of mitotic figures, indicating their benign
nature. Furthermore, derivatives of all three germ layers were
observed, including cartilage, neurons, skin, and hair follicles
(ectoderm), intestinal epithelia (endoderm), and muscle and
bone (mesoderm) (Fig. 24.
Telomerase activity is often correlated with replicative im-
mortality and is typically expressed in germ cells, cancer cells,
and a variety of stem cells, including ES cells, but absent in most
somatic cell types (21-23~. Undifferentiated Cyno-1 cells dis-
played high levels of telomerase activity as detected by the
TRAP assay (TRAPeze kit). However, no telomerase activity
Vrana et a/.
Fig. 2. In viva differentiation of Cyno-1 cells. Cells were injected i.p. in severe
combined immunodeficient mice. Eight and 15 weeks after injection, teratomas
12 and 30 mm in diameter, respectively, were isolated, fixed with 10% para-
formaldehyde, and paraffin-embedded. Sections were stained with
hematoxylin/eosin. The following complex structures were observed: gut (A),
intestinal epithelium with typical goblet cells (gc) and smooth muscle (sm) (B),
neuronal tissue with melanocytes (C), hair follicle complex with evident hair (h)
and sebaceous gland (sg) (D), skin (E), cartilage (F), ganglion cells (G), and bone
(H). (Scale bars = 40 ,um in A, 10 ,um in B and D-H, and 20 ,um in C.) [A, D, and H
reprinted with permission from ref. 5 (Copyright 2002, AAAS).]
could be detected in differentiated progeny of Cyno-1 cells (Fig.
3A). These data indicate a physiologically normal control of
telomerase activity in Cyno-1 cells.
Genomic imprinting is initiated at gametogenesis and further
modified during development. The small nuclear ribonucleopro-
tein polypeptide N (~Snrpn) gene is an example of an imprinted
gene that is expressed solely from the paternal allele (24, 25~. It
is monoallelically expressed from the onset of its expression at
the four-cell stage (25~. RT-PCR analysis confirmed the absence
of Snrpn expression in Cyno-1 cells, whereas it is readily detected
in heterozygous fibroblast cell cultures from the same species
(Fig. 3B). Whereas biparental ES cells from M. fascicularis were
unavailable to us for analysis, the Snrpn gene was readily
detectable in biparental mouse ES cells under these same
conditions (data not shown). These results indicate that the
imprinting profile of at least one gene is consistent with the
parthenogenetic origin of the Cyno-1 cells.
Nestin-Positive Neural Precursors. Cyno-1 ES cells were plated in
flasks coated with fibronectin/laminin and NPMM (supple-
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11913
OCR for page 98
: I:
: :: ~ I: :~
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B
Snrpn 1, :3
Gapdh ~
Fig. 3. Telomerase activity. (A) Cyno-1 cells, maintained in the undifferentiated
state on mouse feeder layers, express telomerase activity that diminishes to
undetectable levels in differentiated Cyno-1 cells. (B) RT-PCR to detect expression
of the paternally expressed imprinted gene Snrpn in Cyno-1 cells (lane 1) and in
adult fibroblasts (lane 2) from the same species. The housekeeping gene Gapdh
is used as a control to demonstrate that equal amounts of mRNA were used.
mented with bFGF, epidermal gowth factor, and neural survival
factor-14. These cells begin to differentiate into a neuronal-like
morphology. Within 10 days, they do not differentiate further
and proliferate at a rate of 5- to 8-fold increase over a 10-day
period (Figs. 4 and 5 A and B). Their stage of development seems
to be similar to those described by Ying et al. (264. Specifically,
these cells express a high amount of nestin, which is an inter-
mediate filament found in the developing CNS, mesenchymal
tissue of the developing pancreas, and immature skeletal muscle
(National Center for Biotechnology Information Locus Link).
With the removal of bFGF and epidermal growth factor and
the addition of ascorbic acid, we are able to generate a high
percentage of dopaminergic-like neurons (25% of TUJ+), glial,
and epithelial cells. Immunocytochemistry stained positive for
TUJ1, dopamine transporter (DAT), and microtubule associ-
~_
A_
DA
8 ~ ~ ~ I c: - r
a, 4a
~ 0~
-
Q 1I Elution time
5 10 15
F
Fig. 5. Neural differentiation of Cyno ES cells in vitro. (A) Phase contrast
microscopy of proliferating Cyno1-derived neural precursors at day 1 in vitro
(DIV1). (B) Same clusterof precursors shown at DIV9. Thetotal cell number has
increased by 5- to 8-fold over a 9-day period. (Inset) One of many mitotic
figures. Immunohistochemical analyses after 5 days of neural differentiation
in the absence of bFGF and epidermal growth factor and the presence of
ascorbic acid revealed positive staining for glial fibrillary acidic protein (GFAP),
an astrocytic marker seen in C, and TUJ1, a neuronal marker seen in D. (E)
Sequential exposure to sonic hedgehog, FGF8b, and ascorbic acid yielded an
average of 25% TUJ1+ neurons coexpressing tyrosine-hydroxylase (TH), a
marker for dopamine neurons. (F) HPLC revealing the release of dopamine
(DA) and serotonin (Ser). [F reprinted with permission from ref. 5 (Copyright
2002, AAAS).]
ated protein-2, and negative for acetylcholine transferase, dopa-
beta-hydroxylase, and NeuN (Fig. 5 C-E and data not shown).
Cyno-1-derived neurons exhibited both basal and KCl-evoked
synaptic release of dopamine and serotonin (Fig. 5F). Single-cell
Fig. 4. Nestin-positive neural precursors derived from Cyno-1 cells. (A) Neural precursors stained for nestin (green). The nuclei were stained with 4',6-diamidino-
2-phenylindole (DAPI) (blue). (B) Phase contrast of nestin precursors.
11914 1 www.pnas.org/cgi/doi/10.1073/pnas.2034195100
Vrana et al.
OCR for page 99
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Fig. 6. Single-cell electrophysiology. (A) Neurons derived from Cyno-1 ex-
press voltage-dependent inward currents that are blocked by tetrodotoxin.
Currents were elicited by membrane depolarizations to O mV every 15 s from
a holding potential of -70 mV. Application of 0.5 ,uM tetrodotoxin inhibited
>90% of these currents. (B) Inhibition was complete within 30 s of tetrodo-
toxin application and washed completely in <1 min.
electrophysiology was also performed on these differentiated
neurons. At around day 20, they begin to express voltage-
dependent sodium channels. The identities of these channels
were confirmed by blocking with Tetrodotoxin. By day SO, 50%
of neuron-like cells express these channels although none have
yet to be identified in the neural precursors (Fig. 64. The
differentiating cells assume morphological characteristics of
neurons before functional ion channels become evident. This
result suggests that there is a cascade of differentiation signals
and/or precise coordination of differentiation events.
DNA Profiling of Cyno-1 Cells. To confirm their autologous origin,
DNA profiling was performed. Total genomic DNA from the
cynomolgus monkey oocyte donor (no. 5571) and from a prep-
aration of cultured stem cells (Cyno-1; derived from no. 5571)
were genotyped and compared by using seven simple sequence
repeat (SSR) human markers (Research Genetics, Huntsville,
AL) that had been shown previously to amplify monkey DNA
and to discriminate between two individuals. The markers
represent seven different chromosomes (nos. 3, 6, 7, 10, 11, 16,
and 17) and in all cases except one (marker D16S403), alleles for
no. 5571 were identical in number and size to the alleles for the
Cyno-1 cells. An additional test was performed on DNA from no.
5571 and from the Cyno-1 cells (as well as two control animals).
Micro SSPTM Generic HLA Class II DNA typing was per-
formed in a 96-well tray format through the Wake Forest
University-Baptist Medical Center Histocompatibility Labora-
tory. The data demonstrated that Cyno-1 stem cells and somatic
cells from no. 5571 were indistinguishable from each other and
therefore should be considered autologous (data not shown).
Histocompatibility Antigen Profile of Cyno-1. The histocompatibility
antigen profile of Cyno-1-derived neurons was investigated and
compared with lymphocytes from the oocyte donor by investi-
gating polymorphic genes within the MHC that encode class I
and class II cell surface proteins. These proteins present immu-
nogenic peptides to CD8+ and CD4+ T cells, respectively. We
vrana et a/.
~-
_ ~
All. 1.
4 ~ - .
=, ~
1 ~ 101 102 103 1 ~
1'''' 1
oats
10 10 10 10 10
o-
J
_. ~ tee
' _
1 ~ 101 102 103 1
c'
3~ ~_<
~ ° 4
101U 1D' 10~ 10~ 10' 10 10 10 10 10
FlTC-anti-Class I FlTC-anti-Class I I
Hl~m~n PRI
Cyno-1 neurons
Cyno-1 neurons + IFFY
Monkey PBL
Fig. 7. Immunological profile of Cyno-1 cells. PBLs and Cyno-1-derived
neural cells were analyzed by flow cytometry to quantitate expression of M.
fasicularis class I (anti-HLA-A,-B,-C) and class 11 (anti-HLA-DR) antigens and
compared with cells stained with isotype-matched control antibodies (shaded
curves). PBLs express both class I and class 11 (DR) antigens whereas differen-
tiated Cyno-1-derived neurons do not express either class of antigen unless
treated with IFN-~.
have analyzed the Cyno-1-derived neural cells by flow cytometry
for the expression of Mafa (MHC of M. fascicularis) class I and
class II antigens. PBLs from the original cell donor expressed
class I and class II antigens detected by antibodies specific for
monomorphic HLA-A, -B. -C and HLA-DR antigens, respec-
lively (Fig. 7). Seventy-five percent of PBLs were positive for
class I, and 14% of PBLs were positive for class II. However,
Cyno-1-derived neural cells were negative for both Mafa class I
and class II antigens (Fig. 7), consistent with observations that
these CNS cell types are class I- and class II-negative in normal
murine CNS (27, 28~. Viral infection or treatment with IFN-y
stimulates up-regulation of class I and class II expression in
murine CNS cells. The ability of IFN-^y to up-regulate class I and
class II expression by Cyno-1-derived neural precursor cells was
investigated by preculturing these cells with IFN-y (40 ng/ml)
overnight before staining and flow cytometry. As shown in Fig.
7, pretreatment of Cyno-1 derived cells resulted in class I-specific
staining with an intensity that was comparable to staining of
normal human PBLs. However, IFN-^y treatment did not in-
crease class II expression. These results support the prediction
that an in viva inflammatory response, expectedly involving
IFN-^y expression, would up-regulate class I expression on
transplanted Cyno-1-derived neural cells. Accordingly, in
the event of transplantation into a nonisogenic animal, these
cells should not escape surveillance by CD8+ cytotoxic T
lymphocytes.
. —
Dlscusslon
We have generated a primate parthenogenetic cell line (Cyno-1)
with ES cell-like properties that can be propagated in vitro in an
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11915
OCR for page 100
undifferentiated state for at least 2 yr. These cells express telom-
erase activity consistent with their extended lifespan property. The
in vitro derivation of large numbers of specific cell lineages from
Cyno-1 cells, including the generation of unlimited numbers of
dopaminergic neurons, is of particular interest. In the present
context, we have demonstrated that these cells (i) express TH, (ii')
release neurotransmitter (dopamine and serotonin), and (iii) are
electrophysiologically active. For these reasons, we believe that
neurons, differentiated from parthenogenetic stem cells, may pro-
vide an important source of therapeutic treatments. Clinical trans-
plantation of specific fetal neurons has shown promise in the
treatment of Parkinson's disease (29) and Huntington's disease
(30), but obtaining such cells from animals or human fetal brain
remains problematic. Neurons derived in vitro from a renewable
source, such as CNS precursors (31), ES cells (32, 33), or stem cells
of parthenogenetic origin, could alleviate some of the ethical and
technical concerns of human cell therapy.
Although the Cyno-1 parthenogenetic stem cell seems, in all
respects, to be similar to traditional ES cells, it is reasonable to
question their viability and utility. They are, after all, exclusively
derived from maternal DNA. When trying to understand how these
parthenogenetic stem cells are capable of developing into func-
tional tissue, it is important to remember the following character-
istics of genetic imprinting. First, ES cells are isolated from the
blastocyst stage, this stage exhibits low DNA methylation levels, and
the effects of imprinting could be minimized (34~. Second, imprint-
ing, in some cases, has been shown to not be completely silent: there
are reports of mRNA expressed from imprinted genes that should
not have been transcribed (244. Third, Surani and Barton (9)
suggest that parthenogenetic embryos do not develop to term
because of a high frequency of errors in X chromosome inactivation
that occurs in extraembryonic tissues when both X chromosomes
are derived only from the female. One might conclude that the
effects of imprinting have a significant effect on extraembryonic
tissue, and not the ICM from which our stem cells are derived.
One could speculate that these parthenogenetically derived
stem cells are capable of differentiating into a high percentage
12.
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11916 1 www.pnas.org/cgi/doi/10.1073/pnas.2034195100
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effects of genetic imprinting. It is believed, for example, that
0.1-1% of all mammalian genes are imprinted (35~. However,
only ~50 imprinted genes have been identified in mice, some of
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Further data analysis of functional genomic studies at the
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Representative terms from entire chapter:
telomerase activity
