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Vol.1, No.3, 67-70 (2011) Stem Cell Discovery
doi:10.4236/scd.2011.13007
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/SCD/
Human embryonic stem cell lines with CCR5-del32 allele
conferring resistance to HIV
Ekaterina Pomerantseva, Valeri Kukharenko, Adam Goodman, Oleg Verlinsky,
Svetlana Rechitsky, Anver Kuliev*
Reproductive Genetics Institute, Chicago, USA.
*Corresponding Author: anverkuliev@hotmail.com
Received 24 June 2011; revised 29 July 2011; accepted 12 August 2011.
ABSTRACT
A 32bp deletion in the chemokine receptor 5
(CCR5) gene (CMKBR5) was shown to be linked
to HIV resistance. Bone marrow transplantation
from the homozygous CCR5-del32 donor to a
CDC Stage 2 HIV-positive recipient was demon-
strated to confer a HIV resistance, resulting in
discontinuation of antiretroviral therapy. In se-
arch for an unlimited source of CCR5-del32 cells
for transplantation purposes, we tested 137 hu-
man embryonic stem cell (hESC) lines from the
Reproductive Genetics Institute’s hESC lines col-
lection, and report here the finding of 12 hESC
lines with the CCR5-del32 allele, one of which
represents a unique partenogenetic ESC line co-
ntaining two copies of this deletion and may be
studied for utility in stem cell transplantation
treatment of HIV.
Keywords: Human Embryonic Stem Cell Lines;
Resistance to HIV; CCR5-del32 Allele;
Parthenogenetic Stem Cell Line with Two Copies
of CCR5-del32 Allele; Stem Cells Transplantation
1. INTRODUCTION
The first HIV-positive individual has recently been
successfully cured via bone marrow transplantation from
the unrelated donor, who was chosen not only by human
leukocyte antigen (HLA) matching, but also for having a
homozygous polymorphism in the chemokine receptor 5
(CCR5) gene CMKBR5 [1]. Although CCR5del32 allele
was linked to HIV resistance long time ago, providing an
immense promise of being able to treat HIV positive
patients [2], finding acceptable donor matches homozy-
gous for the CCR5del32 presented a real challenge. Of
course, as in other conditions treatable by allogeneic bone
marrow transplantation, finding an HLA match related
donor is ideal, but probability of finding such a match is
extremely low [3]. Further, this is almost unrealistic if
there is a need for a related or even unrelated donor with
a specific allele, like CCR5-del32. Clearly, transplanta-
tion from an adult unrelated donor is limited by the ava-
ilability of fully HLA matched donors, while an increa-
sed HLA disparity provides for lower survival rates and
a higher chance of graft-versus-host disease (GVHD).
The use of unrelated umbilical cord blood (UCB), wh-
ich is a valuable source of hematopoietic stem cells, is
presently an established alternative to bone marrow tran-
splantation. Its potential advantages was expected to be
the possibility of using UCB with one or two HLA mis-
matches, reducing conditioning intensity and avoiding the
risk of severe GVHD [4]. These effects seemed to be due
to the “naïve” nature of umbilical cord lymphocytes [5].
However, the major disadvantage of UCB is a reduced
engraftment, which is due to the limited number of CD34
stem cells obtainable from a UCB sample, limiting the
application of UCB transplantation to alder children.
On the other hand, with the current progress in stem
cell research, there is presently an important potential al-
ternative source of stem cell for transplantation, residing
in human embryonic stem cell (hESC) lines, which are
readily available in a few centers around the world [6,7].
Since embryonic stem cells may be expected to have even
more “naïve” immunological features compared to UCB
stem cells, they should allow for similarly high acceptable
HLA disparities, thereby making patient-donor matching
much more permissible, to avoid aggressive conditioning
before and after transplantation, a critical advantage for
patients with serious health conditions [8,9]. Also, one of
important advantages of this option is that hESC collec-
tions provide an unlimited source, as they may be expan-
ded virtually without limit, so a single hESC line can po-
tentially be used for the transplantation treatment of any
number of recipients.
The Reproductive Genetics Institute has currently de-
veloped the world’s largest collection of hESC lines, [6,7]
which has been screened for the presence of the CCR5-
E. Pomerantseva et al. / Stem Cell Discovery 1 (2011) 67-70
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/SCD/
68
del32 allele. This paper presents the first results of the
study, which revealed 12 hESC lines containing this ge-
ne, of which one is unique parthenogenetic line with two
copies of CCR5-del32.
2. MATERIAL AND METHODS
Using our ongoing practice of preimplantation genetic
diagnosis (PGD), we initiated the development of the
hESC lines with normal and abnormal genotypes [6-7],
which were characterized by cytogenetic analysis, and a
set of ESC criteria, including the presence of octamer-
binding transcription factor-4 (Oct-4), tumor rejection
antigen-2-39 (TRA-2-39), high molecular weight glyco-
proteins (antibodies TRA-1-60, TRA-1-81), and stage
specific embryonic antigens (SSEA-3, SSEA-4). Overall,
this collection is presently contains a total of 327 hESC
lines, including 240 normal and 87 with genetic and
chromosomal disorders (www.stemride.com) [6-7]. The
lines were maintained in vitro for up to over a dozen
passages before freezing in sufficient amounts.
To screen these hESC lines for the CCR5del32 dele-
tion, the cells were removed from cryogenic storage, wa-
shed in PBS, and were placed directly into a lysis solu-
tion, consisting of 0.5 µL of 10 x PCR buffer, 0.5 µL of
1% Tween 20, 0.5 µL of 1% Triton X-100, 3.5 µL of
water, and 0.05 µL of Proteinase K (20 mg/mL in 0.5 mL
PCR tube). After spinning down, the samples were cov-
ered with 1 drop of mineral oil and incubated at 45˚C for
15 minutes in a thermal cycler. Proteinase K was then
inactivated at 96˚C for 20 minutes. Hot start PCR was
performed at 72˚C for 10 minutes, followed by denatura-
tion at 95˚C for 3 minutes. Round 1 PCR master mix
consisted of dNTP 400 µM, 1 × PCR buffer, Taq poly-
merase (2 U), 1.5 mM magnesium dichloride, 6% di-
methylsulfoxide, and 0.5 µM of outside upstream and
downstream primers for the mutation in a final volume
of 50 µL. The cycling conditions for the first round of
PCR were as follows: 95˚C for 30 seconds, 55˚C for 1
minute 30 seconds, 72˚C for 45 seconds, for 5 cycles;
during the following 23 cycles, the annealing tempera-
ture was gradually decreased from 55 to 45 degrees;
with a final incubation at 72˚C for 10 minutes.
We designed the outer primers CCR5-1 GCGTCTCT-
CCCAGGAATCATC and CCR5-2 GATTCCCGAGTA-
GCAGATGACC for performing the first round of am-
plification, and inner primer CCR5-3 GCGTCTCTCC-
CAGGAATCATC for second round of hemi-nested (CC-
R5-1 and CCR5-3) PCR. The cycling conditions for the
second round of PCR were as follows: 92˚C for 30 sec-
onds, 55˚C for 30 seconds, 72˚C for 30 seconds, for a
total of 30 cycles; and followed by a final 10-minute in-
cubation at 72˚C.
Primers for chromosome specific microsatellite mark-
ers were added to identify the copy number and parental
origin of the polymorphic markers, used for aneuploidy
testing of chromosomes 13, 16, 18, 21, 22 and X [10].
The PCR product was assessed by gel electrophoresis
and Ethidium Bromide staining. As shown in the Figure
1, the normal allele corresponds to the 141 bp band while
the CCR5-del32 allele corresponds to 109 bp band.
Stem cell samples from the lines containing the mutant
gene were then expanded. Frozen samples were briefly
thawed and then delicately spun down at 500 Gs for 10
minutes. After removing the cryo-preservation media, the
cells were resuspended in a pre-warmed serum free cul-
ture media. The cells were then plated onto mitotically
blocked human derived feeder layers set in a gelatin coa-
ting. Typically the culture medium was changed every
other day, carefully evaluating changes in the growth
rate and level of differentiation in the stem cell colonies.
The passage intervals were based on the expert observa-
tion and varied from 4 - 15 days between passages. Pas-
sages where conducted with the use of 1 mM EDTA with
the incubation times adjusted to increase the proportion
of healthy undifferentiated colonies passed on to new
cultures. During passages the cultures were also split to
maintain an appropriate colony density based on the
growth rate of the individual cell line. The stem cell
lines were expanded until well established and cryopre-
served. The genetic test of the cells from the expanded
lines was then repeated to confirm the presence of CCR5-
del32 deletion.
PCR product was assessed by gel electrophoresis and Ethidium Bromide
staining. The normal allele corresponds to the 141 bp band while the
CCR5-del32 allele corresponds to 109 bp band: N/Del genotype was found
in 11 lines heterozygous for deleted allele; Del/Del is the genotype of the
parthenogenetic line.
Figure 1. Testing for the CCR5-del32 allele in hESC lines.
E. Pomerantseva et al. / Stem Cell Discovery 1 (2011) 67-70
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/SCD/
6969
3. RESULTS AND DISCUSSION
Of a total of 137 hESC lines tested, 125 were without
deletion, 11 were heterozygous for the deletion, and 1
was with two copies of the genes with the deletion (Fig-
ure 1). The latter was established from the blastocyst
deriving from partenogenetic embryo (46, XX), with two
copies of all maternally derived genes, as demonstrated
by polymorphic markers for X-chromosome, and chro-
mosomes 3, 6, 11, 13, 18 and 21.
The frequency of the CCR5-del32 allele in the studied
material (4.7%) is comparable to data published in ear-
lier studies [11], making it realistic to predict the possi-
bility of identification of potentially useful hESC lines
conferring resistance to HIV even in smaller collections.
Assuming that more than 327 hESC lines are now
available in our collection, the testing of the whole ma-
terial could have led to finding of more than two dozens
of hESC lines containing the CCR5-del32 allele confer-
ring resistant to HIV infection.
It may be expected that with the establishment of lar-
ger repositories of hESC lines, there may be a possibility
to perform a search for finding HLA match for HIV pa-
tients. It was predicted that a bank of 150 donor cell
lines may already provide a chance of finding full match
of HLA-A, HLA-B, and HLA-DR for up to 20% recipi-
ents [12]. With the present progress in the differentiation
of hESC into hemopoietic stem cells [13], this material
may appear a readily available source of bone marrow
transplantation. Clinical implication of the resistant
hESC lines will further be widened with the progress in
transplantation treatment with unrelated stem cells hav-
ing significant HLA disparity following the sophisticated
immuno-suppression therapy and conditioning of the
recipients, which may soon appear routine.
The availability of a hESC lines containing CCR5-32-bp
deletion, and particularly a parthenogenetic lines with two
copies of this allele, may have particular potential for
research into the mechanisms of conferring resistance to
HIV, the results of which could lead to new treatments to
this most devastating disease. Moreover, the prospective
advantages of clinical therapies derived from hESC lines,
will likely hold true for many other congenital and ac-
quired diseases. Our repository has a large collection of
hESC lines, which provides a unique opportunity to screen
available hESC lines for polymorphisms associated with
susceptibility and/or resistance to diseases in humans. So
this study provides the first evidence that such screening
is productive for finding hESC lines with rare mutations
which may prove invaluable to the future stem cell ther-
apy of severe disorders for which there is no available
treatment.
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