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Human embryonic stem cell lines with ccr5-del32 allele conferring resistance to HIV


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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 demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.
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Vol.1, No.3, 67-70 (2011) Stem Cell Discovery
opyright © 2011 SciRes. Openly accessible at
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:
Received 24 June 2011; revised 29 July 2011; accepted 12 August 2011.
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
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
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.
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 ( [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-
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
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
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Preimplantation Genetic Diagnosis (PGD) provides unlimited source for obtaining human embryonic stem cell (hESC) lines. As PGD involves the pre-selection of the genetic disease – free embryos for transfer back to uterus the affected embryos are either discarded, or used for confirmation of diagnosis. Based on our busy PGD practice, which presently includes over 10,000 cases, we have had a plenty of affected embryos detected, which were used for derivation of the genetic disease specific hESC lines, which presently contains 87 hESC lines obtained from embryos with single-gene and chromosomal disorders. Fourteen lines have different chromosomal abnormalities, including 4 with chromosomal rearrangements, 24 autosomal recessive, 14 X-linked and 35 autosomal dominant disorders. In addition, screening of 137 normal hESC lines for polymorphism in the chemokine receptor 5 (CCR5) CMKBR5 gene resulted in detection of 12 hESC lines with CCR5-del32 allele, including one with two copies of the gene, conferring resistance to HIV. These cell lines are currently used for research purposes in many countries around the world.
As described in Sect. 7.4, PGD provides an exciting possibility for obtaining HLA matched stem cells for treatment of siblings with bone marrow disorders. This involves preselection of HLA matched embryos in couples with affected siblings requiring the compatible stem cell transplantation, obviating the need for therapeutic cloning, which is highly controversial at the present time. However, in emergency situations, it may be too late to wait until the HLA matched child is born, so other possible approaches need to be developed. For example, some of the tested embryos fail to reach the developmental stage to be considered for transfer, so they might be used for establishment of human embryonic stem cell (hESC) lines. Alternatively, partially matched embryos may be also considered for this purpose, provided the patients provide the consent. Although, in principal a single cell removed from cleaving embryo together with the embryo biopsy may allow establishing of hESC from this single cell for using as a stem cell source for transplantation before the birth of HLA matched child, this does not seem practical, as removing additional material from the embryos at this stage may totally compromise the viability of the embryo.
Full-text available
Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.
Full-text available
A mutant allele of the beta-chemokine receptor gene CCR5 bearing a 32-basepair (bp) deletion (denoted delta ccr5) which prevents cell invasion by the primary transmitting strain of HIV-1 has recently been characterized. Homozygotes for the mutation are resistant to infection, even after repeated high-risk exposures, but this resistance appears not to be total, as isolated cases of HIV-positive deletion homozygotes are now emerging. The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals. A gene frequency of approximately 10% was found for delta ccr5 in populations of European descent, but no mutant alleles were reported in indigenous non-European populations. As the total number of non-European samples surveyed was small in comparison with the Europeans the global distribution of this mutation is far from clear. We have devised a rapid PCR assay for delta ccr5 and used it to screen 3,342 individuals from a globally-distributed range of populations. We find that delta ccr5 is not confined to people of European descent but is found at frequencies of 2-5% throughout Europe, the Middle East and the Indian subcontinent (Fig. 1). Isolated occurrences are seen elsewhere throughout the world, but these most likely represent recent European gene flow into the indigenous populations. The inter-population differences in delta ccr5 frequency may influence the pattern of HIV transmission and so will need to be incorporated into future predictions of HIV levels.
Full-text available
Although human embryonic stem (ES) cells may one day provide a renewable source of tissues for cell replacement therapy (CRT), histoincompatibility remains a significant barrier to their clinical application. Current estimates suggest that surprisingly few cell lines may be required to facilitate rudimentary tissue matching. Nevertheless, the degree of disparity between donor and recipient that may prove acceptable, and the extent of matching that is therefore required, remain unknown. To address this issue using a mouse model of CRT, we have derived a panel of ES cell lines that differ from CBA/Ca recipients at defined genetic loci. Here, we show that even expression of minor histocompatibility (mH) antigens is sufficient to provoke acute rejection of tissues differentiated from ES cells. Nevertheless, despite their immunogenicity in vivo, transplantation tolerance may be readily established by using minimal host conditioning with nondepleting monoclonal antibodies specific for the T cell coreceptors, CD4 and CD8. This propensity for tolerance could be attributed to the paucity of professional antigen-presenting cells and the expression of transforming growth factor (TGF)-β2. Together, these factors contribute to a state of acquired immune privilege that favors the polarization of infiltrating T cells toward a regulatory phenotype. Although the natural privileged status of ES cell-derived tissues is, therefore, insufficient to overcome even mH barriers, our findings suggest it may be harnessed effectively for the induction of dominant tolerance with minimal therapeutic intervention. • cell replacement therapy • acquired immune privilege • regulatory T cell
Although treatment is the major goal in the control of genetic disease, this is not yet a reality for most of inherited conditions. Even with the dramatic advancement in field of gene therapy, there are unfortunately not enough success stories to allow us to predict its impact in the near future. Preimplantation genetic diagnosis (PGD) offers the answer to the control of these inherited conditions by predicting reproductive outcome. It is no longer an experimental procedure, having become an integral part of genetic practices and assisted reproduction technology. This practical book is a vital new addition to the literature for fertility clinicians providing a comprehensive description of available experience, indications, methods and clinical outcomes. The authors of this book, from the Reproductive Genetics Institute in Chicago, USA, are among the leaders in this break-through field. Within this book, they instruct readers of the core features of the practice of PGD, while also providing incredibly detailed instructions of the genetics of a wide variety of common and not so common disorders that can be identified with the technique. This thorough and illuminating coverage ensures that this is the definitive reference book for those fertility specialists wishing to set up or already run a PGD clinic.
Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.
Rare individuals have been multiply exposed to HIV-1 but remain uninfected. The CD4+ T-cells of two of these individuals, designated EU2 and EU3, are highly resistant in vitro to the entry of primary macrophagetropic virus but are readily infectable with transformed T-cell line adapted viruses. We report here on the genetic basis of this resistance. We found that EU2 and EU3 have a homozygous defect in CKR-5, the gene encoding the recently described coreceptor for primary HIV-1 isolates. These individuals appear to have inherited a defective CKR-5 allele that contains an internal 32 base pair deletion. The encoded protein is severely truncated and cannot be detected at the cell surface. Surprisingly, this defect has no obvious phenotype in the affected individuals. Thus, a CKR-5 allele present in the human population appears to protect homozygous individuals from sexual transmission of HIV-1. Heterozygous individuals are quite common (approximately 20%) in some populations. These findings indicate the importance of CKR-5 in HIV-1 transmission and suggest that targeting the HIV-1-CKR-5 interaction may provide a means of preventing or slowing disease progression.
Given recent improvements in the technology of transplantation and histocompatibility testing, it is now possible to contemplate using related or unrelated allogeneic hematologic stem cell donors with high degrees of HLA disparity. This paper is a follow-up of an earlier publication on the probability of finding a matched donor (Transplantation 60:778-783, 1995) and addresses the probability of finding a partially mismatched donor. Assuming that a four of six antigen HLA-A, -B, -DR match is acceptable, it is possible to find unrelated donors for patients of any race from a putative registry with fewer than 10,000 potential donors. Further, storing cord blood from newborns in families with a known genetic disease would yield an acceptable future stem cell transplant product in nearly 40% of cases. These results show the potential impact of cord blood donors and emphasize the importance of improvements in transplantation using partially mismatched donors.
Lower incidence and severity of acute graft versus host disease (GVHD) has been observed in leukemia patients receiving HLA-mismatched umbilical cord (UCB) transplants. However, despite the increased use of UCB in stem cell transplantation, the mechanisms underlying these favorable outcomes are not well delineated. We analyzed antigen specific lymphocyte responses after transplant to determine whether the decreased allogeneic responsiveness of UCB lymphocytes is attributable to pan-unresponsiveness, lymphocyte repressive or recipient-specific tolerance. Circulating lymphocytes collected early (3 months) after UCB transplant demonstrate a less naïve phenotype compared with that in the infused graft. Additionally, after transplant, circulating peripheral blood UCB-derived lymphocytes produced normal levels of interferon-gamma and proliferated normally when stimulated with mitogen or third party alloantigen. In contrast, when stimulated with recipient antigen, circulating lymphocytes emerging posttransplant did not proliferate nor produce interferon-gamma. Moreover, analysis of interleukin-4 production revealed a Th2 response to recipient antigens. These data indicate early induction of immune tolerance of naïve UCB graft lymphocytes with skewing of transplant recipient-specific immune response towards Th2 cytokine profile. UCB graft lymphocyte immune naivety and observed early tolerance induction may contribute to the observed favorable GVHD incidence, despite infusion of HLA mismatch grafts in the unrelated allogeneic setting.
A human embryonic stem cell (HESC) line repository has been established, containing HESC lines with normal and abnormal genotypes, providing the source for studying the primary mechanisms of genetic disorders at the cellular level. Because the outcome of HESC transplantation treatment depends on access to human leukocyte antigen identical stem cells, the development of individual specific HESC was initiated, using the original stembrid technology, which is based on the hybridization of adult somatic cells with cytoplast of HESC lines. The data presented here demonstrate feasibility of this approach in the future development of HESC transplantation treatment of genetic and acquired disorders. The established HESC repository presently contains 166 HESC lines, including 127 with normal genotype and 39 with genetic and chromosomal disorders.