Download Derivation and characterization of new human embryonic stem cell

Derivation and characterization of new human embryonic
stem cell lines in Czech Republic
Aleš Hampl 1,2,3, Stanislava Košková 1, Martina Vodinská 1, and Petr Dvořák 1,2,3
1
Department of Molecular Embryology, Institute of Experimental Medicine Academy of
Sciences of the Czech Republic, 142 20 Prague, Czech Republic;
2
Mendel University Brno, 613 00 Brno, Czech Republic;
3
Center for Cell Therapy and Tissue Repair, Charles University, 150 06 Prague, Czech
Republic
Human embryonic stem cells (hESCs) are exceptionally useful tool for studies of
human development and represent a potential source for transplantation therapies.
At present, only limited number of hESC lines representing a very small sample of
the genetic diversity of the human population is available. Here we report the
derivation and characterization of 7 new hESC lines that are maintained in the
undifferentiated state for more than 18 months.
DERIVATION, CHARACTERIZATION, AND MAINTENANCE OF hESCs
Human embryos
For derivation of hESCs, human embryos produced by in vitro fertilization for medically
assisted induction of pregnancy were used. Human embryos were obtained after
informed consent of patients and employed for hESCs derivation upon the approval from
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the ethical review board of Institute of Experimental Medicine. From the total number of
98 embryos that have been processed in this study, 10 embryos were at the morula stage
and 88 were at the blastocyst stage. Importantly, according to embryological criteria
routinely used in IVF clinic, majority of the blastocysts (n=63) was graded as quality I.
Derivation and culture of hESCs
All embryos were first treated with pronase to remove zona pellucida. Morula stage
embryos (n=10) were then immediately placed onto feeder layers of mitotically
inactivated mouse embryonic fibroblasts (MEFs). MEFs obtained from CF-1 mice were
used for all hESC derivations and cultures. Immunosurgery [1] to isolate inner cell
masses (ICMs) was then applied on early to expanded blastocysts (n=69) but not on
hatching/hatched blastocysts (n=19). Together, 64 ICMs and 19 zona pellucida-free
hatched blastocysts were plated onto feeder layers. None of the morula stage embryos
became attached to the dish whereas 44 isolated ICMs (69%) and 13 hatched blastocysts
(68%) were found firmly attached after 24 hours in culture. An initial outgrowth was
observed in 11 isolated ICMs and 3 hatched blastocysts at an average of 7.5 days after
plating (varied between 4 and 12 days). Importantly, all embryos that produced an initial
outgrowth (14 in total) were of high quality. According to clinical embryological criteria
they were graded as category I. In summary, well recognizable colonies of prospective
hESCs were obtained from 2 early blastocysts, 9 expanded blastocysts, and 3 hatched
blastocysts. Finally, from these 14 initial outgrowths, 7 independent hESC lines named
CCTL6, 8, 9, 10, 12, 13, and 14 were successfully established, expanded, and
cryopreserved. Typical progression of hESC derivation process is shown in Figure 1. It
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is of note that from 7 embryos that gave rise to hESC lines, 3 embryos were produced by
intracytoplasmic sperm injection (ICSI, see Table 1). Until now, each cell line has been
propagated in culture for at least 5 months using mechanical dissociation. Then, 3 hESC
lines (CCTL9, 12, and 14) were adapted to passaging with collagenase. Two of these
hESC lines (CCTL12 and CCTL14) were also adapted to culture on feeder layer of
mitotically inactivated human foreskin fibroblasts (hFF). Newly established line of hFFs
(SCRC-1041; Dr. Jonathan Auerbach, Stem Cell Resource, American Type Culture
Collection, Manassas, VA) was used for this experiment. Both CCTL12 and CCTL14
hESC lines cultured on hFF were expanded and cryopreserved to create separate sublines.
All 7 hESC lines were derived and are maintained in D-MEM:F-12 media supplemented
with serum replacement (15%) and basic fibroblast growth factor (bFGF, 4 ng/ml). The
population doubling time for hESCs included in our panel is approximately 20-40 hours.
Expression of molecular markers of undifferentiated hESCs
All CCTL hESC lines grow in colonies with typical hESC morphology that is
demonstrated by round shape, compactness, high nucleocytoplasmic ratio of cells, and
the presence of several prominent nucleoli per cell. The cells show strong positivity for
molecular markers of undifferentiated hESCs, including TRA-1-60, TRA-1-81, TRA-254, SSEA-3, SSEA-4, Thy-1, alkaline phosphatase, and Oct-4, while they are negative for
SSEA-1. The example of expression analysis of undifferentiated hESCs is shown in
Figure 2A.
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In vitro differentiation
Two protocols were adopted to assess the capacity of hESCs to differentiate in vitro: a)
non-adherent culture to form embryoid bodies and b) two-step differentiation protocol
involving aggregation step followed by adherent culture of predifferentiated cells [2].
Interestingly, it was noted that under standard conditions some hESC lines were not able
to efficiently form well-organized embryoid bodies. Still, increasing the starting density
of hESCs by two-fold (from 5 x 105 to 1 x 106 per ml) was usually enough to restore this
ability. No significant differences were observed among the CCTL lines in their potential
to produce various differentiated cell types in two-step protocol. Figure 2B documents
the outcomes of applying both differentiation protocols to hESCs of the lines CCTL12
and CCTL14.
Karyotype analysis
It was determined using Giemsa banding that out of 7 hESC lines, 6 lines have normal
46XX (CCTL8, 9, and 14) or 46XY (CCTL6, 10, and 13) karyotypes. Only in CCTL12
hESC line (46XX) karyotypic change occurred after 20 passages that was characterized
by haploid karyotype in about 30% of cells. Surprisingly, this karyotypic abnormality
was no longer detectable at passage 32. However, further culture (to passage 42) of
CCTL12 line have led to trisomy of chromosome 12 in about 80% of cells. Notably, gain
of chromosome 12 was repeatedly observed also in other laboratories [3, 4] thus pointing
to the risk of use of hESCs for therapeutic purposes and to the necessity of periodical
testing. Still, in contrast to what was observed by other investigators, increased dosage of
chromosome 12 in CCTL12 is not accompanied by any obvious proliferative advantage.
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Karyotypes of hESC lines CCTL14 (46XX) and CCTL12 (47XX,+12) are shown in
Figure 3A.
HLA haplotype
As the spectrum of HLA antigens expressed on hESCs and/or their derivatives belongs to
the clinically relevant characteristics, PCR-based HLA typing was performed in some
CCTL hESC lines. As shown in Figure 3B, the results document that CCTL hESC lines
represent a range of HLA haplotypes with alleles A01 and A02 shared by 3 lines of 4
analyzed.
Microsatellite markers
Ability to identify the presence of hESCs and/or their derivatives in various experimental
or clinical settings may prove to be very useful in future. Therefore, we created
fingerprints of CCTL hESC lines by determining 16 short tandem repeat (STR) loci using
automated fluorescent PCR technology. All STRs analyzed are listed in the legend to
Table 1.
CONCLUSION
Most importantly, derivation of 7 new hESC lines described here significantly
extends the list of publicly available hESCs. Still, besides establishing this hESC
line panel, our experiments also suggest a) that morula stage embryos are not
suitable for derivation of hESCs, at least using current standard technology, b) that
blastocysts created by virtue of ICSI have no disadvantage compared to those
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created by standard in vitro insemination, and c) that hatched blastocysts may
represent an alternative source to isolated ICMs for derivation of hESC lines.
Finally, our finding of progressive karyotypic changes in one of our hESC lines
further calls for a maximal caution when manipulating hESCs to be used in cellbased therapies.
Acknowledgments
We are very grateful to Dr. Jonathan Auerbach for providing us with human foreskin
fibroblasts, to Dr. Petr Draber for providing us with antibodies, and to Dr. Vendula
Wernerova for karyotyping. This research was supported in part by the Academy of
Sciences of the Czech Republic (AV 0Z5039906) and by the Ministry of Education,
Youth, and Sports of the Czech Republic (MSM 432100001 and LN 00A065).
References
1.
Solter D, Knowles BB. Immunosurgery of mouse blastocysts. PROC NATL
ACAD SCI USA 1975;72:5099-5102.
2.
Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors
on the differentiation of cells derived from human embryonic stem cells. PROC NATL
ACAD SCI USA 2000;97:11307-11312.
3.
Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosome 17q and 12
in cultured human embryonic stem cells. NAT BIOTECHNOL 2004;22:53-54.
4.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell
lines from human blastocysts. N ENGL J MED 2004;350:1354-1356.
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Figures
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Figure legends
Figure 1. Establishment of hESC lines
The same standard derivation technology has been used in all hESC lines.
Briefly, human blastocyst (A) was freed of zona pellucida (B) by pronase (Protease, cat.
no. P 8811; Sigma, St. Louis, MO) and then it was, except for more advanced hatching
blastocysts, subjected to immunosurgery (Complement sera from guinea pig, cat. no. S
1639; Anti-Human Serum antibody produced in rabbit, cat. no. H 3383; both Sigma) to
release ICM. Isolated ICM (or whole embryo in case of more advanced hatching/hatched
blastocyst) was immediately placed onto feeder layer of MEFs. Twenty-four hours later,
ICM was inspected for its attachment (C), and then in daily intervals for occurrence of an
initial outgrowth (D – day 7). After reaching appropriate size (E – day 10), the initial cell
colony was subjected to the first splitting using glass needle. This first passage gave rise
to several colonies (usually 2 to 4) of various sizes (F – day 3 after passage). Typical
example of derivation process is shown. Scale bar = 25 mm.
Figure 2. Determination of undifferentiated state of hESCs and their ability to
differentiate in vitro
A. Human ES cells were plated onto Permanox chambers (Nunc, Inc., Naperville,
IL) and grown in standard D-MEM:F-12 media supplemented by 15% serum replacement
and 4 ng/ml bFGF. At day 3 after passage the cells were processed for determination of
markers of undifferentiated state by cytochemistry, immunocytochemistry, and Western
blotting. For immunocytochemical analysis the cells were fixed in 4% paraformaldehyde
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for 30 minutes at 4°C and blocked in 5% normal goat serum and 0.01% Tween in PBS
pH 7.4 for 1 hour at RT. Then the cells were incubated overnight at 4°C with primary
antibody from the following selection: TRA-1-60 (MAB4360; CHEMICON
International, Inc., Temecula, CA), TRA-1-81 (MAB4381; CHEMICON), TRA-2-54
(MAB4354; CHEMICON), SSEA-1(TEC-1; from Dr. Petr Draber, Institute of Molecular
Genetics, Academy of Sciences of the Czech Republic, Prague), SSEA-3 (MAB4303;
CHEMICON), SSEA-4 (MAB4304; CHEMICON), and Thy-1 (CBL 415; CHEMICON).
Antibody binding was visualized by incubation for 1 hour at RT with appropriate FITClabeled secondary antibody. Nuclei were visualized by staining with propidium iodide.
Microscopical analysis was performed using an upright Olympus BX60 microscope
equipped with a Fluoview confocal laser scanning unit (Olympus C&S Ltd., Prague,
Czech Republic). Cytochemical analysis of the activity of alkaline phosphatase (AP) was
accomplished by substrate kit (Alkaline Phosphatase Substrate Kit I, Vector Laboratories,
Inc., Burlingame, CA) according to the manufacturer’s instructions. For determination of
expression of Oct-4 by Western blot analysis the cells were lysed in Laemmli sample
buffer and boiled for 5 minutes. After being separated on 10% SDS PAGE, the proteins
were electrotransferred onto Hybond-P membrane (Amersham, Aylesbury, UK),
immunodetected using primary antibody against Oct-4 (sc-9081; Santa Cruz
Biotechnology, Santa Cruz, CA) and appropriate secondary antibody, and visualized by
ECL+Plus reagent (Amersham) according to the manufacturer’s instructions.
Representative data are shown. Scale bar = 25 mm.
B. For assessment of their differentiation potential, hESCs were collagenased and
plated in small clusters onto non-adherent 24-well dishes (Costar 3473; Corning Inc.,
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Corning, NY) in D-MEM:F-12 media supplemented with 20% fetal calf serum. After 15
days of culture, hESCs produced cavitated embryoid bodies (EBs) documenting their
ability to properly differentiate into outer endodermal and inner ectodermal layers.
Simple compact EBs at day 8 of culture (8 days) and cavitated EBs at day15 of culture
(15 days) originating from hESCs of CCTL12 and CCTL14 lines are shown. There were
differences between CCTL hESC lines in their ability to form morphologically well
organized EBs. White bordered box (CCTL14, 8 days) distinguishes EBs with improved
morphology upon increasing the density of hECs. Two step differentiation protocol that
includes formation of EBs (5 days of non-adherent culture) followed by further 10 days
of differentiation as an adherent culture was also employed and representative examples
are shown (5+10 days). Scale bar = 300 mm.
Figure 3. Karyotype and HLA haplotype analysis
A. For karyotyping, hESCs grown to subconfluency were exposed for 1 hour to
0.1 mg/ml colchicine (SERVA Electrophoresis GmbH, Heidelberg, Germany) in culture
media, trypsinized, pelleted by centrifugation, and swollen by 10 minute treatment at RT
with hypotonic solution of 75 mM KCl in water. After pelleting again, 10 ml of cold
fixative (methanol and acetic acid, 3:1) was added dropwise to cells and let stand at
–20°C for 10 minutes. After final pelleting, cells were resuspended in fresh fixative to a
concentration of about 5x105 cells/ml and then they were used to make chromosomal
spreads to be Giemsa banded. A minimum of 50 metaphases was analyzed for all
karyotypes. Normal karyotype of line CCTL14 (Normal) and karyotype of line CCTL12
with trisomy for chromosome 12 (Trisomic) are shown.
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B. For analysis of HLA class I and class II antigens, DNA samples were isolated
from hESCs grown under standard undifferentiated conditions using Blood & Cell
Culture DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). PCR-based kits INNOLiPA HLA-A Update, HLA-B Update, HLA-C, HLA-DRB1, and HLA-DQB1 Update
(INNOGENETICS, Ghent, Belgium) were used according to the manufacturer’s
instructions to determine HLA haplotypes. All currently available data are shown.
Table 1. Summary of current status and available characteristics of hESC lines
Seven independent hESC lines (CCTL) are currently available in Brno, Czech Republic
(Department of Molecular Embryology), with 3 lines originating from blastocysts
prepared by ICSI. Staining for markers of undifferentiated hESCs (1) and formation of
embryoid bodies (2) are described in legend to Figure 2. Experiments toward
determining the potential of hESCs to produce teratomas in mice are under progression
(3). Karyotyping (4) and determination of HLA haplotypes (5) are described in legend to
Figure 3. For microsatellite characterization of hESC lines (6) the following short
tandem repeat loci have been analyzed by PCR combined with capillary electrophoresis:
D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338,
D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA. Freezing of hESCs
in standard cryotubes in DMSO-containing media has been successfully achieved. All
hESC lines were shown to resume their undifferentiated growth after being frozen stored
(7). The number of cryotubes stored in liquid nitrogen is given for each hESC line (8).
The number of passages achieved with each hESC line by the end of August 2004 is
provided (9).
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Correspondence:
Petr Dvořák
Aleš Hampl
Department of Molecular Embryology
Department of Molecular Embryology
Institute of Experimental Medicine AS CR
Institute of Experimental Medicine AS CR
& Mendel University Brno
& Mendel University Brno
Zemědělská 1, 613 00 Brno
Zemědělská 1, 613 00 Brno
Czech Republic
Czech Republic
Tel: +420-545133298
Tel: +420-545133297
Fax: +420-545133357
Fax: +420-545133357
E-mail: [email protected]
E-mail: [email protected]
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