U.S. patent application number 10/141220 was filed with the patent office on 2003-02-27 for differentiated cells suitable for human therapy.
Invention is credited to Gold, Joseph D., Lebkowski, Jane S..
Application Number | 20030040111 10/141220 |
Document ID | / |
Family ID | 27400669 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040111 |
Kind Code |
A1 |
Gold, Joseph D. ; et
al. |
February 27, 2003 |
Differentiated cells suitable for human therapy
Abstract
This invention provides a system for producing differentiated
cells from a stem cell population for use wherever a relatively
homogenous cell population is desirable. The cells contain an
effector gene under control of a transcriptional control element
(such as the TERT promoter) that causes the gene to be expressed in
relatively undifferentiated cells in the population. Expression of
the effector gene results in depletion of undifferentiated cells,
or expression of a marker that can be used to remove them later.
Suitable effector sequences encode a toxin, a protein that induces
apoptosis, a cell-surface antigen, or an enzyme (such as thymidine
kinase) that converts a prodrug into a substance that is lethal to
the cell. The differentiated cell populations produced according to
this disclosure are suitable for use in tissue regeneration, and
non-therapeutic applications such as drug screening.
Inventors: |
Gold, Joseph D.; (San
Francisco, CA) ; Lebkowski, Jane S.; (Portola Valley,
CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
|
Family ID: |
27400669 |
Appl. No.: |
10/141220 |
Filed: |
May 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10141220 |
May 7, 2002 |
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09783203 |
Feb 13, 2001 |
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10141220 |
May 7, 2002 |
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PCT/US01/44309 |
Nov 26, 2001 |
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60253443 |
Nov 27, 2000 |
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60253357 |
Nov 27, 2000 |
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Current U.S.
Class: |
435/368 ;
435/366; 435/370 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61K 35/12 20130101; C12N 15/85 20130101; C12N
2510/00 20130101; C12N 2502/13 20130101; C12N 5/0606 20130101 |
Class at
Publication: |
435/368 ;
435/370; 435/366 |
International
Class: |
C12N 005/08 |
Claims
What is claimed as the invention is:
1. A pair of isolated cell populations, consisting of: a first cell
population comprising undifferentiated cells from a line of human
embryonic stem (hES) cells; and a second cell population that is
free of undifferentiated hES cells, but which contains progeny of
said hES cell line.
2. The cell populations of claim 1, wherein the cells contain a
nucleic acid molecule comprising the structure P-X, wherein: X is a
nucleic acid sequence encoding a product that is lethal to a cell
in which it is expressed, or renders a cell in which it is
expressed susceptible to a lethal effect of an external agent; and
P is a transcriptional control element that causes X to be
preferentially expressed in undifferentiated cells.
3. The cell populations of claim 2, wherein X encodes a toxin, or a
protein that induces or mediates apoptosis.
4. The cell populations of claim 2, wherein X encodes an enzyme
that converts a prodrug to a compound that is lethal to a cell in
which X is expressed.
5. The cell populations of claim 4, wherein X encodes a thymidine
kinase.
6. The cell populations of claim 2, wherein P-X is an introduced
heterologous molecule.
7. The cell populations of claim 2, wherein P is an endogenous
transcriptional control element.
8. The cell populations of claim 2, wherein P is an OCT-4 promoter
or a promoter of telomerase reverse transcriptase (TERT).
9. The cell populations of claim 1, wherein the second cell
population is a population of neurons or neural precursor
cells.
10. The cell populations of claim 1, wherein the second cell
population is a population of hepatocytes.
11. The cell populations of claim 1, wherein the second cell
population is a population of cardiomyocytes.
12. The cell populations of claim 1, wherein the second cell
population is formulated for tissue reconstitution or regeneration
in a human patient.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional and continuation-in-part of
U.S. patent application Ser. No. 09/783,203, filed Feb. 13, 2001,
pending. This application is also a continuation of International
Patent Application PCT/US01/44309, filed Nov. 26, 2001, designating
the U.S., to be published on .about.May 27, 2002; and claims the
priority benefit of provisional patent applications U.S. Ser. No.
60/253,443, and U.S. Ser. No. 60/253,357, both filed Nov. 27,
2000.
[0002] The aforelisted priority documents are hereby incorporated
herein by reference in their entirety.
TECHNICAL FIELD
[0003] This invention relates generally to the field of cell
biology of embryonic cells, and the molecular biology of promoter
controlled viral vectors. More specifically, it describes a
technology for removing undifferentiated cells from populations
derived from pluripotent stem cells using selectively expressed
lytic vectors.
BACKGROUND
[0004] Precursor cells have become a central interest in medical
research. Many tissues in the body have a back-up reservoir of
precursors that can replace cells that are senescent or damaged by
injury or disease. Considerable effort has been made recently to
isolate precursors of a number of different tissues for use in
regenerative medicine.
[0005] U.S. Pat. No. 5,750,397 (Tsukamoto et al., Systemix) reports
isolation and growth of human hematopoietic stem cells which are
Thy-1+, CD34+, and capable of differentiation into lymphoid,
erythroid, and myelomonocytic lineages. U.S. Pat. No. 5,736,396
(Bruder et al.) reports methods for lineage-directed
differentiation of isolated human mesenchymal stem cells, using an
appropriate bioactive factor. The derived cells can then be
introduced into a host for mesenchymal tissue regeneration or
repair.
[0006] U.S. Pat. No. 5,716,411 (Orgill et al.) proposes
regenerating skin at the site of a burn or wound, using an
epithelial autograft. U.S. Pat. No. 5,766,948 (F. Gage) reports a
method for producing neuroblasts from animal brain tissue. U.S.
Pat. No. 5,672,499 (Anderson et al.) reports obtaining neural crest
stem cells from embryonic tissue. U.S. Pat. No. 5,851,832 (Weiss et
al., Neurospheres) reports isolation of putative neural stem cells
from 8-12 week old human fetuses. U.S. Pat. No. 5,968,829 (M.
Carpenter) reports human neural stem cells derived from primary
central nervous system tissue.
[0007] U.S. Pat. No. 5,082,670 (F. Gage) reports a method for
grafting genetically modified cells to treat defects, disease or
damage of the central nervous system. Auerbach et al. (Eur. J.
Neurosci. 12:1696, 2000) report that multipotential CNS cells
implanted into animal brains form electrically active and
functionally connected neurons. Brustle et al. (Science 285:754,
1999) report that precursor cells derived from embryonic stem cells
interact with host neurons and efficiently myelinate axons in the
brain and spinal cord.
[0008] Considerable interest has been generated by the development
of embryonic stem cells, which are thought to have the potential to
differentiate into many cell types. Early work on embryonic stem
cells was done in mice. Mouse stem cells can be isolated from both
early embryonic cells and germinal tissue. Desirable
characteristics of pluripotent stem cells are that they be capable
of proliferation in vitro in an undifferentiated state, retain a
normal karyotype, and retain the potential to differentiate to
derivatives of all three embryonic germ layers (endoderm, mesoderm,
and ectoderm).
[0009] Development of human pluripotent stem cell preparations is
considerably less advanced than work with mouse cells. Thomson et
al. propagated pluripotent stem cells from lower primates (U.S.
Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995), and
then from humans (Science 282:114, 1998). Gearhart and coworkers
derived human embryonic germ (hEG) cell lines from fetal gonadal
tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,
1998; and U.S. Pat. No. 6,090,622).
[0010] Both hES and hEG cells have the long-sought characteristics
of pluripotent stem cells: they are capable of being grown in vitro
without differentiating, they have a normal karyotype, and they
remain capable of producing a number of different cell types.
Clonally derived human embryonic stem cell lines maintain
pluripotency and proliferative potential for prolonged periods in
culture (Amit et al., Dev. Biol. 227:271, 2000). These cells hold
considerable promise for use in human therapy, acting as a
reservoir for regeneration of almost any tissue compromised by
genetic abnormality, trauma, or a disease condition.
[0011] International Patent Publication WO 99/20741 (Geron Corp.)
refers to methods and materials for growing primate-derived
primordial stem cells. In one embodiment, a cell culture medium is
provided for growing primate-derived primordial stem cells in a
substantially undifferentiated state, having a low osmotic pressure
and low endotoxin levels. The basic medium is combined with a
nutrient serum effective to support the growth of primate-derived
primordial stem cells and a substrate of feeder cells or an
extracellular matrix component derived from feeder cells. The
medium can further include non-essential amino acids, an
anti-oxidant, and growth factors that are either nucleosides or a
pyruvate salt.
[0012] A significant challenge to the use of stem cells for therapy
is to control growth and differentiation into the particular type
of tissue required for treatment of each patient.
[0013] U.S. Pat. No. 4,959,313 (M. Taketo, Jackson Labs) provides a
particular enhancer sequence that causes expression of a flanking
exogenous or recombinant gene from a promoter accompanying the gene
that does not normally cause expression in undifferentiated cells.
U.S. Pat. No. 5,639,618 (D. A. Gay, Plurion Inc.) proposes a method
for isolating a lineage specific stem cell in vitro, in which a
pluripotent embryonic stem cell is transfected with a construct in
which a lineage-specific genetic element is operably linked to a
reporter gene, culturing the cell under conditions where the cell
differentiates, and then separation of cells expressing the
reporter are separated from other cells.
[0014] U.S. Pat. No. 6,087,168 (Levesque et. al., Cedars Sinai Med.
Ctr.) is directed to transdifferentiating epidermal cells into
viable neurons useful for both cell therapy and gene therapy. Skin
cells are transfected with a neurogenic transcription factor, and
cultured in a medium containing an antisense oligonucleotide
corresponding to a negative regulator of neuronal
differentiation.
[0015] International Patent Publication WO 97/32025 (McIvor et al.,
U. Minnesota) proposes a method for engrafting drug resistant
hematopoietic stem cells. The cells in the graft are augmented by a
drug resistance gene (such as methotrexate resistant dihydrofolate
reductase), under control of a promoter functional in stem cells.
The cells are administered into a mammal, which is then treated
with the drug to increase engraftment of transgenic cells relative
to nontransgenic cells.
[0016] International Patent Publication WO 98/39427 (Stein et al.,
U. Massachusetts) refers to methods for expressing exogenous genes
in differentiated cells such as skeletal tissue. Stem cells (e.g.,
from bone marrow) are contacted with a nucleic acid in which the
gene is linked to an element that controls expression in
differentiated cells. Exemplary is the rat osteocalcin promoter.
International Patent Publication WO 99/10535 (Liu et al., Yale U.)
proposes a process for studying changes in gene expression in stem
cells. A gene expression profile of a stem cell population is
prepared, and then compared a gene expression profile of
differentiated cells.
[0017] International Patent Publication WO 99/19469 (Braetscher et
al., Biotransplant) refers to a method for growing pluripotent
embryonic stem cells from the pig. A selectable marker gene is
inserted into the cells so as to be regulated by a control or
promoter sequence in the ES cells, exemplified by the porcine OCT-4
promoter.
[0018] International Patent Publication WO 00/15764 (Smith et al.,
U. Edinburgh) refers to propagation and derivation of embryonic
stem cells. The cells are cultured in the presence of a compound
that selectively inhibits propagation or survival of cells other
than ES cells by inhibiting a signaling pathway essential for the
differentiated cells to propagate. Exemplary are compounds that
inhibit SHP-2, MEK, or the ras/MAPK cascade.
[0019] Klug et al. (J. Clin. Invest. 98:216, 1996) propose a
strategy for genetically selecting cardiomyocytes from
differentiating mouse embryonic stem cells. A fusion gene
consisting of the .alpha.-cardiac myosin heavy chain promoter and a
cDNA encoding aminoglycoside phosphotransferase was stably
transfected into the ES cells. The resulting lines were
differentiated in vitro and selected using G418. The selected
cardiomyocyte cultures were reported to be highly differentiated.
When engrafted back into mice, ES-derived cardiomyocyte grafts were
detectable as long as 7 weeks after implantation.
[0020] Schuldiner et al. (Proc. Natl. Acad. Sci. USA 97:11307,
2000) report the effects of eight growth factors on the
differentiation of cells from human embryonic stem cells. After
initiating differentiation through embryoid body formation, the
cells were cultured in the presence of bFGF, TGF-.beta.1,
activin-A, BMP-4, HGF, EGF, .beta.NGF, or retinoic acid. Each
growth factor had a unique effect on the differentiation pathway,
but none of the growth factors directed differentiation exclusively
to one cell type.
[0021] There is a need for new approaches to generate populations
of differentiated cells suitable for human administration.
SUMMARY OF THE INVENTION
[0022] This invention provides a system for depleting relatively
undifferentiated cells from a heterogeneous cell population, such
as may be obtained by differentiation of stem cells. The population
is treated with a vector that puts a lethal or potentially lethal
effector gene under control of a gene element that allows the gene
to be expressed at a higher level in the undifferentiated
subpopulation. This produces a population relatively enriched for
mature cells, and suitable for use in regenerative medicine.
[0023] One embodiment of this invention is a population of cells
differentiated from stem cells cultured ex vivo, which is
essentially free of undifferentiated cells. Exemplary are
pluripotent stem cells of primate origin, such as human embryonic
stem cells.
[0024] Cells in the population can contain or be derived using a
polynucleotide comprising the structure P-X, where X is a nucleic
acid sequence that is lethal to a cell in which it is expressed, or
renders a cell in which it is expressed susceptible to a lethal
effect of an external agent; and P is a transcriptional control
element that causes X to be preferentially expressed in
undifferentiated cells. The connecting line in P-X indicates that
the genetic elements are operatively linked, whether or not they
are adjacent in the nucleic acid molecule.
[0025] X is referred to in the description that follows as an
effector sequence. X can encode a toxin, a protein that induces or
mediates apoptosis, or an enzyme (such as thymidine kinase) that
converts a prodrug (such as ganciclovir) to a compound that is
lethal to a cell in which X is expressed. Other examples are
provided later in this disclosure.
[0026] In certain embodiments, P-X is an introduced heterologous
molecule, meaning that the cell or its ancestors was genetically
altered with a vector comprising P-X. In other embodiments the cell
or its ancestors was genetically altered with a vector to place X
under control of an endogenous transcriptional control element.
Following transfection, X can be either transiently expressed in
undifferentiated cells in the population, or P-X can be inheritable
and expressed in undifferentiated progeny. Non-limiting examples
for P include the OCT-4 promoter, and the promoter of telomerase
reverse transcriptase (TERT). The cells can also contain a drug
resistance gene Y under control of P, depicted in this disclosure
as P-X-Y, indicating a functional relationship where P regulates
transcription of both X and Y, with the elements being in any
orientation in the sequence that links the functions in this
manner.
[0027] Another embodiment of the invention is a stem cell
genetically altered so as to contain a nucleic acid with the
structure P-X, as already described. The invention also provides
polynucleotide vectors adapted to genetically alter stem cells in
this fashion.
[0028] Another embodiment of the invention is a method of producing
a population of differentiated cells. A cell population comprising
undifferentiated stem cells that contain a nucleic acid molecule
comprising the structure P-X is treated to cause at least some
undifferentiated cells in the population to differentiate.
[0029] Another embodiment of the invention is a method for
depleting undifferentiated stem cells from a cell population. Stem
cells in the population are genetically altered so that they
contain a nucleic acid molecule comprising the structure P-X as
already described. In this way, a gene that is lethal to a cell in
which it is expressed, or renders it susceptible to a lethal effect
of an external agent, is placed under control of a transcriptional
control element that causes the gene to be preferentially expressed
in undifferentiated cells. The cell population can be genetically
altered when it is still predominantly undifferentiated (before
being caused to differentiate), or when it already predominantly
comprises differentiated cells.
[0030] If X is lethal to the cell, then undifferentiated stem cells
can be depleted simply by culturing the cell population under
conditions where X is expressed. If X renders the cell susceptible
to lethal effects of an external agent (such as a drug or prodrug),
then undifferentiated stem cells are depleted by combining the
cells with the external agent. This can be done by contacting the
cells in vitro with the agent in tissue culture, or administering
the cells to the subject simultaneously or sequentially with the
external agent, if not already present.
[0031] The reagents and techniques of this invention can be brought
to bear on cell populations containing any type of stem cells. They
are especially suited for application to primate pluripotent stem
cells, such as human embryonic stem cells.
[0032] Other embodiments of the invention will be apparent from the
description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 provides an analysis of OCT-4 and hTERT expression in
hES cells cultured with feeder cells (mEF) or extracellular matrix
(Matrigel.RTM. or laminin) with regular medium (RM) or conditioned
medium (CM). The upper panel is a copy of a gel showing OCT-4 and
hTERT expression at the mRNA level by RT-PCR. The lower panel is a
bar graph comparing the level of expression for cells grown on
different substrates, expressed as the ratio of OCT-4 or hTERT to
the 18s standard. hES cells grown on Laminin and Matrigel.RTM. in
conditioned medium have similar expression patterns to those of
cells grown on a feeder layer.
[0034] FIG. 2 is a half-tone reproduction of a gel showing
telomerase activity measured in cultured hES cells by TRAP activity
assay. All the culture conditions showed positive telomerase
activity after 40 days in feeder-free culture.
[0035] FIG. 3 is a half-tone reproduction showing expression of the
GFP reporter gene in hES cells transduced with retrovirus and then
differentiated. hES cells were transferred to suspension culture to
form embryoid bodies, cultured for a further 4 days, replated onto
gelatin-coated slides and cultured for a week, and then fixed and
photographed under fluorescence for GFP expression. Left panels
show bright-field illumination; right panels show fluorescence due
to GFP expression.
[0036] FIG. 4 shows the results of a study in which hES cells were
transiently genetically altered in feeder-free culture by
lipofection. Panel A is a half-tone reproduction of a light
micrograph showing morphology of hES cells on laminin after they
have been transfected. Panel B is a half-tone reproduction of a
fluorescence micrograph showing GFP expression in the same colony.
Panel C is a bar graph showing percentage of cells expressing GFP
under various conditions.
[0037] FIG. 5 is a map of TPAC vector designated pGRN376. This is
an adenovirus vector of 7185 bp comprising the herpes simplex
thymidine kinase (tk) gene under control of a promoter taken from
the upstream sequence of the human gene for telomerase reverse
transcriptase (hTERT). Expression of tk is promoted in cells
expressing hTERT, such as undifferentiated embryonic stem
cells.
[0038] FIG. 6 is a two-panel line graph, showing the effect of the
TPAC thymidine kinase vector on undifferentiated hES cells. 48 h
after replating, the cells were transduced with TPAC vector at an
MOI of 30 or 100, or mock transduced (no vector added). Four h
later, the cells were exchanged into fresh medium containing the
prodrug ganciclovir (GCV). By day 3, wells treated with TPAC
vector+GCV contained 8% as many cells as the control wells.
[0039] FIG. 7 is a bar graph showing titration of GCV in TPAC
vector treated hES cells. 4 h after transduction with the vector,
fresh medium was added containing GCV at the concentration shown.
.about.20 .mu.M GCV was optimal under the conditions tested.
[0040] FIG. 8 is a two-panel bar graph showing titration of GCV on
TPAC vector transduced and mock-transduced hES cells from two
different lines. Both lines are sensitive to GCV after treatment
with the TPAC vector.
[0041] FIG. 9 shows the effect of TPAC+GCV treatment on mixed cell
populations obtained from differentiation of hES cells. The cells
were fed daily with conditioned medium to maintain the
undifferentiated state, or with either 500 nM retinoic acid or 0.5%
DMSO, to induce differentiation into committed cells of mixed
phenotype. 7 days later, they were infected with the TPAC vector at
an MOI of 30, plus 20 .mu.M GCV.
[0042] The Upper Panel is a bar graph showing the number of cells
surviving in culture. Treatment with TPAC+GCV eliminated cells
cultured under each condition. In each instance, culture of the
surviving cells produced populations that appeared highly
differentiated and substantially free of undifferentiated
morphology. The Lower Panel is a half-tone reproduction of a gel
showing RT-PCR analysis of the surviving cells. Those cells
cultured with conditioned medium (mEF-CM) or DMSO had no detectable
OCT-4 expression, while 2 out of 4 samples treated with retinoic
acid (RA) showed amplification products consistent with very low
levels of OCT-4 expression.
[0043] FIG. 10 is a reproduced micrograph of an hES cell line that
has been transduced by combining with a control adenovirus vector
(Panel A), or pGRN376 (Panel B), which contains the tk gene under
control of the TERT promoter. Both wells of transduced cells were
cultured for 3 days in a medium containing ganciclovir.
Undifferentiated colonies typical of normal hES cell cultures were
seen in the control wells. In the wells treated with pGRN376, most
or all undifferentiated ES cell colonies were gone, and only
differentiated cells remained.
[0044] FIG. 11 is a two-panel line graph, showing drug sensitivity
of undifferentiated cells containing the telomerase promoter driven
thymidine kinase gene (TPAC). Upper and lower panels show
sensitivity to the prodrugs ganciclovir (GCV) and
(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), respectively.
Ganciclovir at a concentration as low as 2.5 .mu.M kills virtually
all of the undifferentiated TPAC ES cells within .about.4 days.
[0045] FIGS. 12(A) and (B) comprises black-and-white reproductions
of fluorescence micrographs of differentiated ES cells. Cell lines
H9-376m-18, H9-376m-62, and H9-376m-6 contain the TPAC gene;
H9-pGK-neo-1 is the control cell line transfected only with the
drug selection plasmid. The stably transfected cells were
differentiated into embryoid bodies, and plated for
immunocytochemistry analysis. A least three of the TPAC containing
stem cell lines show areas that stain for muscle specific actin,
.alpha.-fetoprotein, .beta.-tubulin, and cardiac troponin I,
representative of all three embryonic germ layers.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Stem cells of various kinds have become an extremely
attractive modality in regenerative medicine. They can be
proliferated in culture, and then differentiated in vitro or in
situ into the cell types needed for therapy. Recently, it has been
demonstrated that human embryonic stem cells continuously express a
high level of telomerase, enabling them to maintain telomere length
and grow almost indefinitely in culture.
[0047] So far, efforts to differentiate stem cells have been
directed primarily towards identifying culture conditions that
promote outgrowth of a cell population with phenotypic features of
a tissue type desirable for regenerative medicine. Schuldiner et
al. (supra) report the effects of growth factors on the
differentiation of human embryonic stem cells. In U.S. Pat. No.
5,639,613, stem cells are transfected with a lineage-specific gene
that is operably linked to a reporter gene, which is then used to
select for cells expressing the reporter. In WO 97/32025,
hematopoietic stem cells are augmented by a drug resistance gene,
and then engrafted into a subject. The cells are administered into
a mammal, which is then treated with the drug to increase
engraftment of transgenic cells. Klug et al. (supra) used a
construct in which the .alpha.-cardiac myosin heavy chain promoter
controlled expression of aminoglycoside phosphotransferase.
Transfected differentiated cells were selected using G418, which
produced lines of cardiomyocyte like cells. This is a positive
selection strategy that uses gene expression patterns of the
desired tissue type to allow preferential survival of
differentiated tissue.
[0048] It is a hypothesis of this invention that some of the
populations of differentiated cells produced using adaptive culture
and positive selection methods will be suboptimal for use in human
therapy. In some circumstances, undifferentiated cells in the
population may impair engraftment or function of the cells in vivo.
Undifferentiated cells may also increase the possibility of a
malignancy or other tumor forming at the site of the therapeutic
implant, or by migration of transplanted cells.
[0049] This invention is directed towards a strategy in which
undifferentiated cells remaining in such differentiated cell
populations can be depleted. This is effected by genetically
altering the cells, so that a gene that is lethal to a cell in
which it is expressed, or renders it susceptible to a lethal effect
of an external agent, is placed under transcriptional control of a
genetic element that causes it to be expressed preferentially in
any undifferentiated cells in the population. This is a negative
selection strategy, designed to minimize the proportion of
undifferentiated cells. It is possible to combine this technique
with positive selection techniques of various kinds, in order to
obtain relatively pure populations of the desired tissue type that
are essentially free of undifferentiated cells.
[0050] As a non-limiting validation of the invention, human
embryonic stem (hES) cells have been transduced with an adenovirus
vector (TPAC) in which a herpes virus thymidine kinase gene was
placed under control of a promoter sequence for human telomerase
reverse transcriptase (hTERT). hES cells constitutively express
hTERT, but this ability is lost upon differentiation. Example 10
(FIGS. 6-8) show that transduction of hES cells with TPAC vector
renders undifferentiated cells susceptible to lethality by the
prodrug ganciclovir, a substrate for thymidine kinase, at a
concentration of .about.20 .mu.M. Example 11 (FIG. 9) shows that
when hES cells are transduced with TPAC vector and then
differentiated with DMSO, there are no surviving cells with
detectable OCT-4 expression (a phenotype of undifferentiated
cells).
[0051] The techniques of this invention are designed in part to
provide cell populations with improved characteristics for human
therapy. After depleting undifferentiated cells, the differentiated
population is expected to possess better functional and engraftment
characteristics, and have reduced risk of creating unwanted tissue
architecture and malignancies in the treated subject. In addition,
cell populations depleted of undifferentiated cells are more
homogeneous, which provides a distinct advantage for
non-therapeutic applications, such as producing antibody, cDNA
libraries, and screening drug candidates.
[0052] Definitions
[0053] Prototype "primate Pluripotent Stem cells" (pPS cells) are
pluripotent cells derived from pre-embryonic, embryonic, or fetal
tissue at any time after fertilization, and have the characteristic
of being capable under appropriate conditions of producing progeny
of several different cell types that are derivatives of all of the
three germinal layers (endoderm, mesoderm, and ectoderm), according
to a standard art-accepted test, such as the ability to form a
teratoma in 8-12 week old SCID mice.
[0054] Included in the definition of pPS cells are embryonic cells
of various types, exemplified by human embryonic stem (hES) cells,
described by Thomson et al. (Science 282:1145, 1998); embryonic
stem cells from other primates, such as Rhesus stem cells (Thomson
et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem
cells (Thomson et al., Biol. Reprod. 55:254, 1996) and human
embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad.
Sci. USA 95:13726, 1998). Other types of pluripotent cells are also
included in the term. Any cells of primate origin that are capable
of producing progeny that are derivatives of all three germinal
layers are included, regardless of whether they were derived from
embryonic tissue, fetal tissue, or other sources. This invention
relates to pPS cells that are not derived from a malignant source.
It is desirable (but not always necessary) that the cells be
karyotypically normal.
[0055] pPS cell cultures are described as "undifferentiated" when a
substantial proportion of stem cells and their derivatives in the
population display morphological characteristics of
undifferentiated cells, clearly distinguishing them from
differentiated cells of embryo or adult origin. Undifferentiated
pPS cells are easily recognized by those skilled in the art, and
typically appear in the two dimensions of a microscopic view in
colonies of cells with high nuclear/cytoplasmic ratios and
prominent nucleoli. It is understood that colonies of
undifferentiated cells within the population will often be
surrounded by neighboring cells that are differentiated.
Nevertheless, the undifferentiated colonies persist when the
population is cultured or passaged under appropriate conditions,
and individual undifferentiated cells constitute a substantial
proportion of the cell population. Cultures that are substantially
undifferentiated contain at least 20% undifferentiated pPS cells,
and may contain at least 40%, 60%, or 80% in order of increasing
preference. Whenever a culture or cell population is referred to in
this disclosure as proliferating "without differentiation", what is
meant is that after proliferation, the composition is substantially
undifferentiated according to the preceding definition.
[0056] "Feeder cells" or "feeders" are terms used to describe cells
of one type that are co-cultured with cells of another type, to
provide an environment in which the cells of the second type can
grow. The feeder cells are optionally from a different species as
the cells they are supporting. For example, certain types of pPS
cells can be supported by primary mouse embryonic fibroblasts,
immortalized mouse embryonic fibroblasts, or human fibroblast-like
cells differentiated from hES cells, as described later in this
disclosure. pPS cell populations are said to be "essentially free"
of feeder cells if the cells have been grown through at least one
round after splitting in which fresh feeder cells are not added to
support the growth of the pPS. Cultures essentially free of feeder
cells contain less than about 5% feeder cells. Whenever a culture
or cell population is referred to in this disclosure as
"feeder-free", what is meant is that the composition is essentially
free of feeder cells according to the preceding definition, subject
only to further constraints explicitly required.
[0057] The term "embryoid bodies" is a term of art synonymous with
"aggregate bodies". The terms refer to aggregates of differentiated
and undifferentiated cells that appear when pPS cells overgrow in
monolayer cultures, or are maintained in suspension cultures.
Embryoid bodies are a mixture of different cell types, typically
from several germ layers, distinguishable by morphological
criteria.
[0058] The terms "committed precursor cells", "lineage restricted
precursor cells" and "restricted developmental lineage cells" all
refer to cells that are capable of proliferating and
differentiating into several different cell types, with a range
that is typically more limited than pluripotent stem cells of
embryonic origin capable of giving rise to progeny of all three
germ layers. Non-limiting examples of committed precursor cells
include hematopoietic cells, which are pluripotent for various
blood cells; hepatocyte progenitors, which are pluripotent for bile
duct epithelial cells and hepatocytes; and mesenchymal stem cells.
Another example is neural restricted cells, which can generate
glial cell precursors that progress to oligodendrocytes and
astrocytes, and neuronal precursors that progress to neurons.
[0059] For the purposes of this description, the term "stem cell"
can refer to either a pluripotent stem cell, or a committed
precursor cell, both as defined above. Minimally, a stem cell has
the ability to proliferate and form cells of more than one
different phenotype, and is also capable of self renewal--either as
part of the same culture, or when cultured under different
conditions. Embryonic stem cells can be identified as positive for
the enzyme telomerase.
[0060] As used in this disclosure, "differentiated" and
"undifferentiated" are relative terms depending on the context in
which they are used. Specifically, in reference to a particular
type of self-renewing stem cell, the term "undifferentiated" refers
back to the same self-renewing stem cell, whereas the term
"differentiated" refers to one or more of the relatively mature
phenotypes the stem cell can generate--as discernable by
morphological criteria, antigenic markers, and gene transcripts
they produce. Undifferentiated pPS cells have the ability to
differentiate into all three germ layers. The cells differentiated
from them do not, and can readily be recognized by one skilled in
the art by morphological criteria.
[0061] The terms "polynucleotide" and "nucleic acid molecule" refer
to a polymer of nucleotides of any length. Included are genes and
gene fragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA and RNA, nucleic acid probes, and primers. As used in
this disclosure, the term polynucleotides refer interchangeably to
double- and single-stranded molecules. Unless otherwise specified
or required, any embodiment of the invention that is a
polynucleotide encompasses both a double-stranded form, and each of
the two complementary single-stranded forms known or predicted to
make up the double-stranded form. Included are nucleic acid analogs
such as phosporamidates and thiophosporamidates.
[0062] A cell is said to be "genetically altered", "transfected",
or "genetically transformed" when a polynucleotide has been
transferred into the cell by any suitable means of artificial
manipulation, or where the cell is a progeny of the originally
altered cell that has inherited the polynucleotide. The
polynucleotide will often comprise a transcribable sequence
encoding a protein of interest, which enables the cell to express
the protein at an elevated level. The genetic alteration is said to
be "inheritable" if progeny of the altered cell have the same
alteration.
[0063] A "control element" or "control sequence" is a nucleotide
sequence involved in an interaction of molecules that contributes
to the functional regulation of a polynucleotide, such as
replication, duplication, transcription, splicing, translation, or
degradation of the polynucleotide. Transcriptional control elements
include promoters, enhancers, and repressors.
[0064] Particular gene sequences referred to as promoters, like the
"TERT promoter", or the "OCT-4 promoter", are polynucleotide
sequences derived from the gene referred to that promote
transcription of an operatively linked gene expression product. It
is recognized that various portions of the upstream and intron
untranslated gene sequence may in some instances contribute to
promoter activity, and that all or any subset of these portions may
be present in the genetically engineered construct referred to. The
promoter may be based on the gene sequence of any species having
the gene, unless explicitly restricted, and may incorporate any
additions, substitutions or deletions desirable, as long as the
ability to promote transcription in the target tissue. Genetic
constructs designed for treatment of humans typically comprise a
segment that is at least 90% identical to a promoter sequence of a
human gene. A particular sequence can be tested for activity and
specificity, for example, by operatively linking to a reporter gene
(Example 9).
[0065] Genetic elements are said to be "operatively linked" if they
are in a structural relationship permitting them to operate in a
manner according to their expected function. For instance, if a
promoter helps initiate transcription of the coding sequence, the
coding sequence can be referred to as operatively linked to (or
under control of) the promoter. There may be intervening sequence
between the promoter and coding region so long as this functional
relationship is maintained.
[0066] In the context of encoding sequences, promoters, and other
genetic elements, the term "heterologous" indicates that the
element is derived from a genotypically distinct entity from that
of the rest of the entity to which it is being compared. For
example, a promoter or gene introduced by genetic engineering
techniques into an animal of a different species is said to be a
heterologous polynucleotide. An "endogenous" genetic element is an
element that is in the same place in the chromosome where it occurs
in nature, although other elements may be artificially introduced
into a neighboring position.
[0067] The terms "polypeptide", "peptide" and "protein" are used
interchangeably in this disclosure to refer to polymers of amino
acids of any length. The polymer may comprise modified amino acids,
it may be linear or branched, and it may be interrupted by
non-amino acids.
[0068] General Techniques
[0069] For further elaboration of general techniques useful in the
practice of this invention, the practitioner can refer to standard
textbooks and reviews in cell biology, tissue culture, and
embryology. Included are Teratocarcinomas and embryonic stem cells:
A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987);
Guide to Techniques in Mouse Development (P. M. Wasserman et al.,
eds., Academic Press 1993); Embryonic Stem Cell Differentiation in
Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and
uses of Embryonic Stem Cells: Prospects for Application to Human
Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil.
Dev. 10:31, 1998). Differentiation of stem cells is reviewed in
Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod.
Fertil. Dev. 10:31,1998.
[0070] Methods in molecular genetics and genetic engineering are
described generally in the current editions of Molecular Cloning: A
Laboratory Manual, (Sambrook et al.); Oligonucleotide Synthesis (M.
J. Gait, ed.,); Animal Cell Culture (R. I. Freshney, ed.); Gene
Transfer Vectors for Mammalian Cells (Miller & Calos, eds.);
Current Protocols in Molecular Biology and Short Protocols in
Molecular Biology, 3rd Edition (F. M. Ausubel et al., eds.); and
Recombinant DNA Methodology (R. Wu ed., Academic Press). Reagents,
cloning vectors, and kits for genetic manipulation referred to in
this disclosure are available from commercial vendors such as
BioRad, Stratagene, Invitrogen, and ClonTech.
[0071] General techniques in cell culture and media collection are
outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr.
Opin. Biotechnol. 8:148, 1997); Serum-free Media (K. Kitano,
Biotechnology 17:73, 1991); Large Scale Mammalian Cell Culture
(Curr. Opin. Biotechnol. 2:375, 1991); and Suspension Culture of
Mammalian Cells (Birch et al., Bioprocess Technol. 19:251, 1990).
Other observations about the media and their impact on the culture
environment have been made by Marshall McLuhan and Fred Allen.
[0072] Sources of Stem Cells
[0073] This invention can be practiced using stem cells of various
types, which may include the following non-limiting examples.
[0074] U.S. Pat. No. 5,851,832 reports multipotent neural stem
cells obtained from brain tissue. U.S. Pat. No. 5,766,948 reports
producing neuroblasts from newborn cerebral hemispheres. U.S. Pat.
No. 5,654,183 and 5,849,553 report the use of mammalian neural
crest stem cells. U.S. Pat. No. 6,040,180 reports in vitro
generation of differentiated neurons from cultures of mammalian
multipotential CNS stem cells. WO 98/50526 and WO 99/01159 report
generation and isolation of neuroepithelial stem cells,
oligodendrocyte-astrocyte precursors, and lineage-restricted
neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem
cells obtained from embryonic forebrain and cultured with a medium
comprising glucose, transferrin, insulin, selenium, progesterone,
and several other growth factors.
[0075] Primary liver cell cultures can be obtained from human
biopsy or surgically excised tissue by perfusion with an
appropriate combination of collagenase and hyaluronidase.
Alternatively, EP 0 953 633 A1 reports isolating liver cells by
preparing minced human liver tissue, resuspending concentrated
tissue cells in a growth medium and expanding the cells in culture.
The growth medium comprises glucose, insulin, transferrin, T.sub.3,
FCS, and various tissue extracts that allow the hepatocytes to grow
without malignant transformation. The cells in the liver are
thought to contain specialized cells including liver parenchymal
cells, Kupffer cells, sinusoidal endothelium, and bile duct
epithelium, and also precursor cells (referred to as "hepatoblasts"
or "oval cells") that have the capacity to differentiate into both
mature hepatocytes or biliary epithelial cells (L. E. Rogler, Am.
J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol.
10:710, 1998; Lazaro et al., Cancer Res. 58:514, 1998).
[0076] U.S. Pat. No. 5,192,553 reports methods for isolating human
neonatal or fetal hematopoietic stem or progenitor cells. U.S. Pat.
No. 5,716,827 reports human hematopoietic cells that are Thy-1
positive progenitors, and appropriate growth media to regenerate
them in vitro. U.S. Pat. No. 5,635,387 reports a method and device
for culturing human hematopoietic cells and their precursors. U.S.
Pat. No. 6,015,554 describes a method of reconstituting human
lymphoid and dendritic cells.
[0077] U.S. Pat. No. 5,486,359 reports homogeneous populations of
human mesenchymal stem cells that can differentiate into cells of
more than one connective tissue type, such as bone, cartilage,
tendon, ligament, and dermis. They are obtained from bone marrow or
periosteum. Also reported are culture conditions used to expand
mesenchymal stem cells. WO 99/01145 reports human mesenchymal stem
cells isolated from peripheral blood of individuals treated with
growth factors such as G-CSF or GM-CSF. WO 00/53795 reports
adipose-derived stem cells and lattices, substantially free of
adipocytes and red cells. These cells reportedly can be expanded
and cultured to produce hormones and conditioned culture media.
[0078] The invention can be practiced using stem cells of any
vertebrate species. Included are stem cells from humans; as well as
non-human primates, domestic animals, livestock, and other
non-human mammals.
[0079] Amongst the stem cells suitable for use in this invention
are primate pluripotent stem (pPS) cells derived from tissue formed
after gestation, such as a blastocyst, or fetal or embryonic tissue
taken any time during gestation. Non-limiting examples are primary
cultures or established lines of embryonic stem cells.
[0080] Media and Feeder Cells
[0081] Media for isolating and propagating pPS cells can have any
of several different formulas, as long as the cells obtained have
the desired characteristics, and can be propagated further.
Suitable sources are as follows: Dulbecco's modified Eagles medium
(DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagles
medium (KO DMEM), Gibco # 10829-018; 200 mM L-glutamine, Gibco #
15039-027; non-essential amino acid solution, Gibco 11140-050;
.beta.-mercaptoethanol, Sigma # M7522; human recombinant basic
fibroblast growth factor (bFGF), Gibco # 13256-029. Exemplary
serum-containing ES medium is made with 80% DMEM (typically KO
DMEM), 20% defined fetal bovine serum (FBS) not heat inactivated,
0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM
.beta.-mercaptoethanol. The medium is filtered and stored at
4.degree. C. for no longer than 2 weeks. Serum-free ES medium is
made with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential
amino acids, 1 mM L-glutamine, and 0.1 mM .beta.-mercaptoethanol.
An effective serum replacement is Gibco # 10828-028. The medium is
filtered and stored at 4.degree. C. for no longer than 2 weeks.
Just before use, human bFGF is added to a final concentration of 4
ng/mL (Bodnar et al., Geron Corp, International Patent Publication
WO 99/20741).
[0082] Feeder cells (where used) are propagated in mEF medium,
containing 90% DMEM (Gibco # 11965-092), 10% FBS (Hyclone #
30071-03), and 2 mM glutamine. mEFs are propagated in T150 flasks
(Corning # 430825), splitting the cells 1:2 every other day with
trypsin, keeping the cells subconfluent. To prepare the feeder cell
layer, cells are irradiated at a dose to inhibit proliferation but
permit synthesis of important factors that support hES cells
(.about.4000 rads gamma irradiation). Six-well culture plates (such
as Falcon # 304) are coated by incubation at 37.degree. C. with 1
mL 0.5% gelatin per well overnight, and plated with 375,000
irradiated mEFs per well. Feeder cell layers are typically used 5 h
to 4 days after plating. The medium is replaced with fresh hES
medium just before seeding pPS cells.
[0083] Conditions for culturing other stem cells are known, and can
be optimized appropriately according to the cell type. Media and
culture techniques for particular cell types referred to in the
previous section are provided in the references cited.
[0084] Embryonic Stem Cells
[0085] Embryonic stem cells can be isolated from blastocysts of
members of the primate species (Thomson et al., Proc. Natl. Acad.
Sci. USA 92:7844, 1995). Human embryonic stem (hES),cells can be
prepared from human blastocyst cells using the techniques described
by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145,1998;
Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature
Biotech. 18:399,2000.
[0086] Briefly, human blastocysts are obtained from human in vivo
preimplantation embryos. Alternatively, in vitro fertilized (IVF)
embryos can be used, or one cell human embryos can be expanded to
the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989).
Human embryos are cultured to the blastocyst stage in G1.2 and G2.2
medium (Gardner et al., Fertil. Steril. 69:84, 1998). Blastocysts
that develop are selected for ES cell isolation. The zona pellucida
is removed from blastocysts by brief exposure to pronase (Sigma).
The inner cell masses are isolated by immunosurgery, in which
blastocysts are exposed to a 1:50 dilution of rabbit anti-human
spleen cell antiserum for 30 minutes, then washed for 5 minutes
three times in DMEM, and exposed to a 1:5 dilution of Guinea pig
complement (Gibco) for 3 minutes (see Solter et al., Proc. Natl.
Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM,
lysed trophectoderm cells are removed from the intact inner cell
mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder
layers.
[0087] After 9 to 15 days, inner cell mass-derived outgrowths are
dissociated into clumps either by exposure to calcium and
magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by
exposure to dispase or trypsin, or by mechanical dissociation with
a micropipette; and then replated on mEF in fresh medium.
Dissociated cells are replated on mEF feeder layers in fresh ES
medium, and observed for colony formation. Colonies demonstrating
undifferentiated morphology are individually selected by
micropipette, mechanically dissociated into clumps, and replated.
ES-like morphology is characterized as compact colonies with
apparently high nucleus to cytoplasm ratio and prominent nucleoli.
Resulting ES cells are then routinely split every 1-2 weeks by
brief trypsinization, exposure to Dulbecco's PBS (without calcium
or magnesium and with 2 mM EDTA), exposure to type IV collagenase
(.about.200 U/mL; Gibco) or by selection of individual colonies by
micropipette. Clump sizes of about 50 to 100 cells are optimal.
[0088] Embryonic Germ Cells
[0089] Human Embryonic Germ (hEG) cells can be prepared from
primordial germ cells present in human fetal material taken about
8-11 weeks after the last menstrual period. Suitable preparation
methods are described in Shamblott et al., Proc. Natl. Acad. Sci.
USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.
[0090] Briefly, genital ridges are rinsed with isotonic buffer,
then placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution
(BRL) and cut into <1 mm.sup.3 chunks. The tissue is then
pipetted through a 100 .mu.L tip to further disaggregate the cells.
It is incubated at 37.degree. C. for .about.5 min, then .about.3.5
mL EG growth medium is added. EG growth medium is DMEM, 4500 mg/L
D-glucose, 2200 mg/L mM sodium bicarbonate; 15% ES qualified fetal
calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL);
1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF,
Genzyme); 1-2 ng/ml human recombinant basic fibroblast growth
factor (bFGF, Genzyme); and 10 .mu.M forskolin (in 10% DMSO). In an
alternative approach, EG cells are isolated using
hyaluronidase/collagenase/DNAse. Gonadal anlagen or genital ridges
with mesenteries are dissected from fetal material, the genital
ridges are rinsed in PBS, then placed in 0.1 ml HCD digestion
solution (0.01% hyaluronidase type V, 0.002% DNAse I, 0.1%
collagenase type IV, all from Sigma prepared in EG growth medium).
Tissue is minced and incubated 1 h or overnight at 37.degree. C.,
resuspended in 1-3 mL of EG growth medium, and plated onto a feeder
layer.
[0091] Ninety-six well tissue culture plates are prepared with a
sub-confluent layer of feeder cells cultured for 3 days in modified
EG growth medium free of LIF, bFGF or forskolin, inactivated with
5000 rad .gamma.-irradiation. Suitable feeders are STO cells (ATCC
Accession No. CRL 1503). .about.0.2 mL of primary germ cell (PGC)
suspension is added to each of the wells. The first passage is
conducted after 7-10 days in EG growth medium, transferring each
well to one well of a 24-well culture dish previously prepared with
irradiated STO mouse fibroblasts. The cells are cultured with daily
replacement of medium until cell morphology consistent with EG
cells are observed, typically after 7-30 days or 1-4 passages.
[0092] Propagation of pPS Cells in an Undifferentiated State
[0093] pPS cells can be propagated continuously in culture, using a
combination of culture conditions that promote proliferation
without promoting differentiation.
[0094] Traditionally, pPS cells are cultured on a layer of feeder
cells, typically fibroblast type cells, often derived from
embryonic or fetal tissue. The cell lines are plated to near
confluence, usually irradiated to prevent proliferation, and then
used to support pPS cell cultures.
[0095] In one illustration, pPS cells are first derived and
supported on primary embryonic fibroblasts. Mouse embryonic
fibroblasts (mEF) can be obtained from outbred CF1 mice (SASCO) or
other suitable strains. The abdomen of a mouse at 13 days of
pregnancy is swabbed with 70% ethanol, and the decidua is removed
into phosphate buffered saline (PBS). Embryos are harvested;
placenta, membranes, and soft tissues are removed; and the
carcasses are washed twice in PBS. They are then transferred to
fresh 10 cm bacterial dishes containing 2 mL trypsin/EDTA, and
finely minced. After incubating 5 min at 37.degree. C., the trypsin
is inactivated with 5 mL DMEM containing 10% bovine serum (FBS),
and the mixture is transferred to a 15 mL conical tube and
dissociated. Debris is allowed to settle for 2 min, the supernatant
is made up to a final volume of 10 mL, and plated onto a 10 cm
tissue culture plate or T75 flask. The flask is incubated
undisturbed for 24 h, after which the medium is replaced. When
flasks are confluent (.about.2-3 d), they are split 1:2 into new
flasks.
[0096] Scientists at Geron have discovered that hPS cells can be
maintained in an undifferentiated state even without feeder cells.
The environment for feeder-free cultures includes a suitable
culture substrate, particularly an extracellular matrix, such as
may be derived from basement membrane or that may form part of
adhesion molecule receptor-ligand couplings. A suitable preparation
is available from Becton Dickenson under the name Matrigel.RTM..
Other extracellular matrix components and component mixtures are
suitable as an alternative. Depending on the cell type being
proliferated, this may include laminin, fibronectin, proteoglycan,
entactin, heparan sulfate, and the like, alone or in various
combinations. Laminins are major components of all basal laminae in
vertebrates, which interact with integrin heterodimers such as
.alpha.6.beta.1 and .alpha.6.beta.4 (specific for laminins) and
other heterodimers (that cross-react with other matrices).
[0097] The pluripotent stem cells are plated onto the substrate in
a suitable distribution and in the presence of a medium that
promotes cell survival, propagation, and retention of the desirable
characteristics. It has been found that plating densities of at
least .about.15,000 cells cm.sup.-2 (typically 90,000 cm.sup.-2 to
170,000 cm.sup.-2) promote survival and limit differentiation. The
passage of pPS cells in the absence of feeders benefits from
preparing the pPS cells in small clusters. Typically, enzymatic
digestion is halted before cells become completely dispersed (say,
.about.5 min with collagenase IV). Clumps of .about.10-2000 cells
are then plated directly onto the substrate without further
dispersal. Alternatively, primate PS cells can be passaged between
feeder-free cultures as a finer cell suspension, providing that an
appropriate enzyme and medium are chosen, and the plating density
is sufficiently high. By way of illustration, confluent human
embryonic stem cells cultured in the absence of feeders are removed
from the plates by incubating with a solution of 0.05% (wt/vol)
trypsin (Gibco) and 0.053 mM EDTA for 5-15 min at 37.degree. C.
With the use of pipette, the remaining cells in the plate are
removed and the cells are triturated with the pipette until the
cells are dispersed into a suspension comprising single cells and
some small clusters. The cells are then plated at densities of
50,000-200,000 cells/cm.sup.2 to promote survival and limit
differentiation. The phenotype of ES cells passaged by this
technique is similar to what is observed when cells are harvested
as clusters by collagen digestion. As another option, the cells can
be harvested without enzymes before the plate reaches confluence.
The cells are incubated .about.5 min in a solution of 0.5 mM EDTA
alone in PBS, washed from the culture vessel, and then plated into
a new culture without further dispersal.
[0098] pPS cells plated in the absence of fresh feeder cells
benefit from being cultured in a nutrient medium. The medium will
generally contain the usual components to enhance cell survival,
including isotonic buffer, essential minerals, and either serum or
a serum replacement of some kind. Conditioned medium can be
prepared by culturing irradiated primary mouse embryonic
fibroblasts (or another suitable cell preparation) at a density of
.about.5-6.times.10.sup.4 cm.sup.-2 in a serum free medium such as
KO DMEM supplemented with 20% serum replacement and 4 ng/mL basic
fibroblast growth factor (bFGF). The culture supernatant is
harvested after .about.1 day at 37.degree. C.
[0099] As an alternative to primary mouse fibroblast cultures,
conditioned medium can be prepared from an embryonic fibroblast
cell line tested for its ability to condition medium appropriately.
Such lines can optionally be transfected with telomerase reverse
transcriptase to increase their replicative capacity. Another
possible source is differentiated pPS cells with the morphological
features of fibroblasts. pPS cells are suspension cultured as
aggregates in differentiation medium using non-adherent cell
culture plates (.about.2.times.10.sup.6 cells/9.6 cm.sup.2). After
2 days the aggregates are transferred into gelatin-coated plates,
and fibroblast-like cells appear in clusters of 100-1000 cells in
the mixed population after .about.11 days. After brief collagenase
treatment, the fibroblast-like cells can be collected under a
microscope, passaged in mEF medium, and tested for their ability to
condition ES medium.
[0100] Medium that has been conditioned for 1-2 days is typically
used to support pPS cell culture for 1-2 days, and then exchanged.
If desired, conditioned medium can be supplemented before use with
additional growth factors that benefit pPS cell culture. For hES, a
growth factor like bFGF or FGF-4 can be used. For hEG, culture
medium may be supplemented with a growth factor like bFGF, an
inducer of gp130, such as LIF or Oncostatin-M, and perhaps a factor
that elevates cyclic AMP levels, such as forskolin.
[0101] Characteristics of Undifferentiated pPS Cells
[0102] In the two dimensions of a standard microscopic image, hES
cells have high nuclear/cytoplasmic ratios in the plane of the
image, prominent nucleoli, and compact colony formation with poorly
discernable cell junctions. Cell lines can be karyotyped using a
standard G-banding technique (available at many clinical
diagnostics labs that provides routine karyotyping services, such
as the Cytogenetics Lab at Oakland Calif.) and compared to
published human karyotypes.
[0103] hES and hEG cells can also be characterized by expressed
cell markers. In general, the tissue-specific markers discussed in
this disclosure can be detected using a suitable immunological
technique--such as flow cytometry for membrane-bound markers,
immunohistochemistry for intracellular markers, and enzyme-linked
immunoassay, for markers secreted into the medium. The expression
of protein markers can also be detected at the mRNA level by
reverse transcriptase-PCR using marker-specific primers. See U.S.
Pat. No. 5,843,780 for further details.
[0104] Stage-specific embryonic antigens (SSEA) are characteristic
of certain embryonic cell types. Antibodies for SSEA markers are
available from the Developmental Studies Hybridoma Bank (Bethesda
Md.). Other useful markers are detectable using antibodies
designated Tra-1-60 and Tra-1-81 (Andrews et al., Cell Lines from
Human Germ Cell Tumors, in E. J. Robertson, 1987, supra). hES cells
are typically SSEA-1 negative and SSEA-4 positive. hEG cells are
typically SSEA-1 positive. Differentiation of pPS cells in vitro
results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression
and increased expression of SSEA-1. pPS cells can also be
characterized by the presence of alkaline phosphatase activity,
which can be detected by fixing the cells with 4% paraformaldehyde,
and then developing with Vector Red as a substrate, as described by
the manufacturer (Vector Laboratories, Burlingame Calif.).
[0105] Embryonic stem cells are also typically telomerase positive
and OCT-4 positive. Telomerase activity can be determined using
TRAP activity assay (Kim et al., Science 266:2011, 1997), using a
commercially available kit (TRAPeze.RTM. XK Telomerase Detection
Kit, Cat. s7707; Intergen Co., Purchase N.Y.; or TeloTAGGG.TM.
Telomerase PCR ELISAplus, Cat. 2,013,89; Roche Diagnostics,
Indianapolis). hTERT expression can also be evaluated at the mRNA
level by RT-PCR. The LightCycler TeloTAGGG.TM. hTERT quantification
kit (Cat. 3,012,344; Roche Diagnostics) is available commercially
for research purposes.
[0106] Differentiating pPS Cells
[0107] Differentiation of the pPS can be initiated by first forming
embryoid bodies. General principles in culturing embryoid bodies
are reported in O'Shea, Anat. Rec. (New Anat. 257:323, 1999). pPS
cells are cultured in a manner that permits aggregates to form, for
which many options are available: for example, by overgrowth of a
donor pPS cell culture, or by culturing pPS cells in culture
vessels having a substrate with low adhesion properties which
allows EB formation. Embryoid bodies can also be made in suspension
culture. pPS cells are harvested by brief collagenase digestion,
dissociated into clusters, and plated in non-adherent cell culture
plates. The aggregates are fed every few days, and then harvested
after a suitable period, typically 4-8 days. The cells can then be
cultured in a medium and/or on a substrate that promotes enrichment
of cells of a particular lineage. The substrate can comprise matrix
components such as Matrigel.RTM. (Becton Dickenson), laminin,
collagen, gelatin, or matrix produced by first culturing a
matrix-producing cell line (such as a fibroblast or endothelial
cell line), and then lysing and washing in such a way that the
matrix remains attached to the surface of the vessel. Embryoid
bodies comprise a heterogeneous cell population, potentially having
an endoderm exterior, and a mesoderm and ectoderm interior.
[0108] Scientists at Geron Corporation have discovered that pPS
cells can be differentiated into committed precursor cells or
terminally differentiated cells without forming embryoid bodies or
aggregates as an intermediate step. Briefly, a suspension of
undifferentiated pPS cells is prepared, and then plated onto a
solid surface that promotes differentiation. Suitable substrates
include glass or plastic surfaces that are adherent. For example,
glass coverslips can be coated with a polycationic substance, such
as a polyamines like poly-lysine, poly-ornithine, or other
homogeneous or mixed polypeptides or other polymers with a
predominant positive charge. The cells are then cultured in a
suitable nutrient medium that is adapted to promote differentiation
towards the desired cell lineage.
[0109] In some circumstances, differentiation is further promoted
by withdrawing serum or serum replacement from the culture medium.
This can be achieved by substituting a medium devoid of serum and
serum replacement, for example, at the time of replating. In
certain embodiments of the invention, differentiation is promoted
by withdrawing one or more medium component(s) that promote(s)
growth of undifferentiated cells, or act(s) as an inhibitor of
differentiation. Examples of such components include certain growth
factors, mitogens, leukocyte inhibitory factor (LIF), and basic
fibroblast growth factor (bFGF). Differentiation may also be
promoted by adding a medium component that promotes differentiation
towards the desired cell lineage, or inhibits the growth of cells
with undesired characteristics. For example, to generate cells
committed to neural or glial lineages, the medium can include any
of the following factors or medium constituents in an effective
combination: Brain derived neurotrophic factor (BDNF),
neutrotrophin-3 (NT-3), NT-4, epidermal growth factor (EGF),
ciliary neurotrophic factor (CNTF), nerve growth factor (NGF),
retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid,
forskolin, fetal bovine serum (FBS), and bone morphogenic proteins
(BMPs).
[0110] General principals for obtaining tissue cells from
pluripotent stem cells are reviewed in Pedersen (Reprod. Fertil.
Dev. 6:543, 1994), and U.S. Pat. No. 6,090,622. Other publications
of interest include the following: For neural progenitors, neural
restrictive cells and glial cell precursors, see Bain et al.,
Biochem. Biophys. Res. Commun. 200:1252, 1994; Trojanowski et al.,
Exp. Neurol. 144:92, 1997; Wojcik et al., Proc. Natl. Acad. Sci.
USA 90:1305-130; and U.S. Pat. Nos. 5,851,832, 5,928,947,
5,766,948, and 5,849,553. For cardiac muscle and cardiomyocytes see
Chen et al., Dev. Dynamics 197:217, 1993 and Wobus et al.,
Differentiation 48:173, 1991. For hematopoietic progenitors, see
Burkert et al., New Biol. 3:698, 1991 and Biesecker et al., Exp.
Hematol. 21:774, 1993. U.S. Pat. No. 5,773,255 relates to
glucose-responsive insulin secreting pancreatic beta cell lines.
U.S. Pat. No. 5,789,246 relates to hepatocyte precursor cells.
Other progenitors of interest include but are not limited to
chondrocytes, osteoblasts, retinal pigment epithelial cells,
fibroblasts, skin cells such as keratinocytes, dendritic cells,
hair follicle cells, renal duct epithelial cells, smooth and
skeletal muscle cells, testicular progenitors, and vascular
endothelial cells.
[0111] Scientists at Geron Corporation have discovered that
culturing pPS cells or embryoid body cells in the presence of
ligands that bind growth factor receptors promotes enrichment for
neural precursor cells. The growth environment may contain a neural
cell supportive extracellular matrix, such as fibronectin. Suitable
growth factors include but are not limited to EGF, bFGF, PDGF,
IGF-1, and antibodies to receptors for these ligands. The cultured
cells may then be optionally separated by whether they express a
marker such as A2B5. Under the appropriate circumstances,
populations of cells enriched for expression of the A2B5 marker may
have the capacity to generate both neuronal cells (including mature
neurons), and glial cells (including astrocytes and
oligodendrocytes). Optionally, the cell populations are further
differentiated, for example, by culturing in a medium containing an
activator of cAMP.
[0112] Scientists at Geron Corporation have discovered that
culturing pPS cells or embryoid body cells in the presence of a
hepatocyte differentiation agent promotes enrichment for
hepatocyte-like cells. The growth environment may contain a
hepatocyte supportive extracellular matrix, such as collagen or
Matrigel.RTM.. Suitable differentiation agents include various
isomers of butyrate and their analogs, exemplified by n-butyrate.
The cultured cells are optionally cultured simultaneously or
sequentially with a hepatocyte maturation factor, such as an
organic solvent like dimethyl sulfoxide (DMSO); a maturation
cofactor such as retinoic acid; or a cytokine or hormone such as a
glucocorticoid, epidermal growth factor (EGF), insulin,
TGF-.alpha., TGF-.beta., fibroblast growth factor (FGF), heparin,
hepatocyte growth factor (HGF), IL-1, IL-6, IGF-I, IGF-II, and
HBGF-1.
[0113] Scientists at Geron Corporation have discovered that it is
also possible to differentiate hPS cells into a highly enriched
population comprising cardiomyocytes or cardiomyocyte precursors.
The cardiomyocyte lineage cells can be obtained, for example, by
differentiating hES cells in a growth environment comprising a
cardiotrophic factor that affects DNA-methylation, exemplified by
5-azacytidine. Spontaneously contracting cells can then be
separated from other cells in the population, for example, by
density centrifugation. Further process steps can include culturing
the cells in a medium containing creatine, carnitine, or taurine.
Alternatively, it is possible to differentiate hPS cells into a
highly enriched population comprising osteoprogenitors or
osteoblasts expressing osteocalcin and collagen-1. The cells can be
obtained by taking pPS-derived mesenchymal cells and
differentiating them in a medium containing a bone morphogenic
protein (particularly BMP-4), a ligand for a human TGF-.beta.
receptor, or a ligand for a human vitamin D receptor.
[0114] Characteristics of differentiated cells
[0115] Cells can be characterized according to a number of
phenotypic criteria. The criteria include but are not limited to
characterization of morphological features, detection or
quantitation of expressed cell markers and enzymatic activity, and
determination of the functional properties of the cells in
vivo.
[0116] Markers of interest for neural cells include .beta.-tubulin
III or neurofilament, characteristic of neurons; glial fibrillary
acidic protein (GFAP), present in astrocytes; galactocerebroside
(GalC) or myelin basic protein (MBP); characteristic of
oligodendrocytes; OCT-4, characteristic of undifferentiated hES
cells; nestin, characteristic of neural precursors and other cells.
A2B5 and NCAM are characteristic of glial progenitors and neural
progenitors, respectively. Cells can also be tested for secretion
of characteristic biologically active substances. For example,
GABA-secreting neurons can be identified by production of glutamic
acid decarboxylase or GABA. Dopaminergic neurons can be identified
by production of dopa decarboxylase, dopamine, or tyrosine
hydroxylase.
[0117] Markers of interest for liver cells include
.alpha.-fetoprotein (liver progenitors); albumin,
.alpha..sub.1-antitrypsin, glucose-6-phosphatase, cytochrome p450
activity, transferrin, asialoglycoprotein receptor, and glycogen
storage (hepatocytes); CK7, CK19, and .gamma.-glutamyl transferase
(bile epithelium). It has been reported that hepatocyte
differentiation requires the transcription factor HNF-4.alpha. (Li
et al., Genes Dev. 14:464, 2000). Markers independent of
HNF-4.alpha. expression include .alpha..sub.1-antitrypsin,
.alpha.-fetoprotein, apoE, glucokinase, insulin growth factors 1
and 2, IGF-1 receptor, insulin receptor, and leptin. Markers
dependent on HNF-4.alpha. expression include albumin, apoAl,
apoAll, apoB, apoCIII, apoCII, aldolase B, phenylalanine
hydroxylase, L-type fatty acid binding protein, transferrin,
retinol binding protein, and erythropoietin (EPO).
[0118] Cell types in mixed cell populations derived from pPS cells
can be recognized by characteristic morphology and the markers they
express. For skeletal muscle: myoD, myogenin, and myf-5. For
endothelial cells: PECAM (platelet endothelial cell adhesion
molecule), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin,
MECA-32, and MEC-14.7. For smooth muscle cells: specific myosin
heavy chain. For cardiomyocytes: GATA-4, Nkx2.5, cardiac troponin
1, .alpha.-myosin heavy chain, and ANF. For pancreatic cells, pdx
and insulin secretion. For hematopoietic cells and their
progenitors: GATA-1, CD34, AC133, .beta.-major globulin, and
.beta.-major globulin like gene .beta.H1.
[0119] Certain tissue-specific markers listed in this disclosure or
known in the art can be detected by immunological techniques--such
as flow immunocytochemistry for cell-surface markers,
immunohistochemistry (for example, of fixed cells or tissue
sections) for intracellular or cell-surface markers, Western blot
analysis of cellular extracts, and enzyme-linked immunoassay, for
cellular extracts or products secreted into the medium. The
expression of tissue-specific gene products can also be detected at
the mRNA level by Northern blot analysis, dot-blot hybridization
analysis, or by reverse transcriptase initiated polymerase chain
reaction (RT-PCR) using sequence-specific primers in standard
amplification methods. Sequence data for the particular markers
listed in this disclosure can be obtained from public databases
such as GenBank (URL www.ncbi.nlm.nih.gov:80/entrez).
[0120] Preparing Cell Populations Essentially Free of
Undifferentiated Cells
[0121] In accordance with this invention, populations of
differentiated cells are depleted of relatively undifferentiated
cells by expressing a gene that is lethal to cells or renders them
susceptible to a lethal effect of an external agent, under control
of a transcriptional control element that causes the gene to be
preferentially expressed in the undifferentiated cells.
[0122] To accomplish this, the cells are genetically altered either
before or after the process used to differentiate the cells into
the desired lineage for therapy, in a way that puts an effector
gene suitable for negative selection of undifferentiated cells,
under control of a transcriptional control element with the desired
properties.
[0123] Transcriptional Control Elements for Driving Negative
Selection
[0124] The control element is selected with a view to the protein
expression patterns of the undifferentiated and differentiated
cells in the population.
[0125] Genes with desirable expression patterns can be identified
by comparing expression at the transcription, translation, or
functional level in two different cell populations--one relatively
enriched for differentiated cells, the other relatively enriched
for undifferentiated cells. Suitable methods of comparison include
subtractive hybridization of cDNA libraries, and microarray
analysis of mRNA levels. Once a transcript is identified with an
appropriate expression pattern, the promoter or enhancer of the
corresponding gene can be used for construction of the negative
selection vector.
[0126] A suitable microarray analysis is conducted using a Genetic
Microsystems array generator, and an Axon GenePix.TM. Scanner.
Microarrays are prepared by amplifying cDNA fragments in a 96 or
384 well format, and then spotted directly onto glass slides. To
compare mRNA preparations from two cell populations, one
preparation is converted into Cy3-labeled cDNA, while the other is
converted into Cy5-labeled cDNA. The two cDNA preparations are
hybridized simultaneously to the microarray slide, and then washed
to eliminate non-specific binding. Any given spot on the array will
bind each of the cDNA products in proportion to abundance of the
transcript in the two original mRNA preparations. The slide is then
scanned at wavelengths appropriate for each of the labels, and the
relative abundance of mRNA is determined. Preferably, the level of
expression of the effector gene will be at least 5-fold or even
25-fold higher in the undifferentiated cells relative to the
differentiated cells.
[0127] For the depletion of pluripotent embryonic cells, an
exemplary control element is the promoter for telomerase reverse
transcriptase (TERT). Sequence of the human TERT gene (including
upstream promoter sequence) is provided below. The reader is also
referred to U.K. Patent GB 2321642 B (Cech et al., Geron
Corporation and U. Colorado), International Patent Publications WO
00/46355 (Morin et al., Geron Corporation), WO 99/33998 (Hagen et
al., Bayer Aktiengesellschaft), and Horikawa, I., et al. (Cancer
Res., 59:826, 1999). Sequence of the mouse TERT gene is provided in
WO 99/27113 (Morin et al., Geron Corporation). A lambda phage clone
designated .lambda.G.PHI.5, containing .about.13,500 bases upstream
from the hTERT encoding sequence is available from the ATCC under
Accession No. 98505. Example 9 illustrates the testing and use of
TERT promoter sequences (SEQ. ID NO:1) in vector expression
systems.
[0128] Another exemplary control element is a promoter sequence for
Octamer binding transcription factor 4 (OCT-4), a member of the POU
family of transcription factors. OCT-4 transcription is activated
between the 4 and 80 cell stage in the developing embryo, and it is
highly expressed in the expanding blastocyst and then in the
pluripotent cells of the egg cylinder. Transcription is
down-regulated as the primitive ectoderm differentiates to form
mesoderm, and by 8.5 days post coitum is restricted to migrating
primordial germ cells. High-level OCT-4 gene expression is also
observed in pluripotent embryo carcinoma and embryonic stem cell
lines, and is down-regulated when these cells are induced to
differentiate. Pig, mouse, and human OCT-4 promoter sequences are
provided in International Patent Publication WO 9919469
(Biotransplant Inc.).
[0129] Other suitable control elements can be obtained from genes
causing expression of markers characteristic of undifferentiated
cells in the population but not of the differentiated cells. For
example, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 are characteristic
of various types of undifferentiated pluripotent embryonic stem
cells. The enzyme responsible for synthesis of SSEA-4 may have
transcriptional control elements with the desirable expression
specificity. A more recent example is the promoter for Rexl
protein, a retanoic acid regulated zinc finger protein that is
expressed in preimplantation embryos. The mouse Rex1 promoter has
been shown to act as an effective transcription marker for
undifferentiated embryonic stem cells (Eiges et al., Current Biol.
11:514, 2001.
[0130] Suitability of particular elements can be estimated by
analysis of gene transcript expression, for example, by microarray
analysis. Reporter constructs can then be tested in differentiated
and undifferentiated cells for the appropriate specificity, using a
promoter or enhancer sequence from the identified cell-specific
gene to control transcription of a reporter gene, such as green
fluorescence protein, secreted alkaline phosphatase,
.beta.-glucuronidase, or .beta.-galactosidase. Use of reporter
constructs to test promoter specificity is illustrated below in
Example 9.
[0131] Effector Genes for Achieving Negative Selection
[0132] A transcriptional regulatory element with appropriate
specificity is operatively linked to an encoding region for a
product that will provide elimination of cells in which it is
expressed--either directly, or by rendering the cell susceptible to
an otherwise innocuous external agent.
[0133] Suitable effector genes include those that encode a peptide
toxin--such as ricin, abrin, diphtheria, gelonin, Pseudomonas
exotoxin A, Crotalus durissus terrificus toxin, Crotalus adamenteus
toxin, Naja naja toxin, and Naja mocambique toxin. Hughes et al.,
Hum. Exp. Toxicol. 15:443, 1996; Rosenblum et al., Cancer Immunol.
Immunother. 42:115, 1996; Rodriguez et al., Prostate 34:259, 1998;
Mauceri et al., Cancer Res. 56:4311; 1996.
[0134] Also suitable are genes that induce or mediate
apoptosis--such as the ICE-family of cysteine proteases, the Bcl-2
family of proteins, Bax, bclXs and caspases (Favrot et al., Gene
Ther. 5:728, 1998; McGill et al., Front. Biosci. 2:D353, 1997;
McDonnell et al., Semin. Cancer Biol. 6:53, 1995). Another
potential anti-tumor agent is apoptin, a protein that induces
apoptosis even where small drug chemotherapeutics fail (Pietersen
et al., Adv. Exp. Med. Biol. 465:153, 2000). Koga et al. (Hu. Gene
Ther. 11:1397, 2000) propose a telomerase-specific gene therapy
using the hTERT gene promoter linked to the apoptosis gene
Caspase-8 (FLICE). Gu et al. (Cancer Res. 60:5359, 2000) reported a
binary adenoviral system that induced Bax expression via the hTERT
promoter. They found that it elicited tumor-specific apoptosis in
vitro and suppressed tumor growth in nude mice.
[0135] Also of interest are enzymes present in the lytic package
that cytotoxic T lymphocytes or LAK cells deliver to their targets.
Perforin, a pore-forming protein, and Fas ligand are major
cytolytic molecules in these cells (Brandau et al., Clin. Cancer
Res. 6:3729, 2000;Cruz et al., Br. J. Cancer 81:881, 1999). CTLs
also express a family of at least 11 serine proteases termed
granzymes, which have four primary substrate specificities (Kam et
al., Biochim. Biophys. Acta 1477:307, 2000). Low concentrations of
streptolysin O and pneumolysin facilitate granzyme B-dependent
apoptosis (Browne et al., Mol. Cell Biol. 19:8604, 1999).
[0136] Other suitable effectors encode polypeptides having activity
that is not itself toxic to a cell, but renders the cell sensitive
to an otherwise nontoxic compound--either by metabolically altering
the cell, or by changing a non-toxic prodrug into a lethal drug.
Lethality to progeny with an undifferentiated phenotype only occurs
when the prodrug is present. Thus, the prodrug can be combined with
the cells while they are being differentiated, expanded, or
maintained in vitro, to minimize the proportion of cells with
undifferentiated phenotype. The reader will readily appreciate that
the prodrug can also be given to a patient being treated with the
cells, either simultaneously with the treatment, or at a subsequent
time, in order to minimize the emergence of progeny with an
undifferentiated phenotype in vivo.
[0137] Exemplary effector genes with this property encode thymidine
kinase (tk), such as may be derived from a herpes simplex virus,
and catalytically equivalent variants. The HSV tk converts the
anti-herpetic agent ganciclovir (GCV) to a toxic product that
interferes with DNA replication in proliferating cells.
[0138] U.S. Pat. No. 5,631,236 (Baylor College of Medicine)
outlines adenoviral vectors containing an HSV tk gene operatively
linked to a promoter that expresses tk in cancer cells. U.S. Pat.
No. 5,997,859 and EP 702084 B1 (Chiron) pertain to
replication-defective recombinant retrovirus, carrying a vector
construct directing expression of HSV tk gene for converting an
otherwise inert compound into a cytotoxic form. EP 415731 A1, EP
657540 A1, and EP 657541 A1 (Wellcome Foundation) propose
retroviral vectors encoding an enzyme such as VZV tk,
carboxypeptidase G2, alkaline phosphatase, penicillin-V amidase,
and cytosine deaminase, for converting a prodrug into an agent
toxic to a cancer cell. International Patent Publications WO
98/14593 and WO 00/46355 (Geron Corporation) describe constructs
comprising HSV tk under control of hTERT promoter sequences.
[0139] The human HSV tk gene sequence is provided below (SEQ. ID
NOS:2 & 3), along with illustrations of its use to target cells
expressing TERT. Simultaneously or following expression of the gene
in target cells, a convertible prodrug such as ganciclovir is added
to the environment to effect depletion of the targets.
[0140] Another type of effector that renders the cell susceptible
to an otherwise non-toxic agent is a gene that causes presentation
of a foreign antigen on the cell membrane. The presented substance
may be an alloantigen, a xenoantigen, or an antigen from a
non-mammalian species for which specific antibody is readily
available. Expression of the gene leads to presentation of the
antigen on undifferentiated cells, which then can be used to effect
depletion by a suitable immunological separation--such as
immunoaffinity (e.g., panning), fluorescence-activated cell
sorting, or complement-mediated lysis.
[0141] When the transducing agent is a viral vector, the effector
can be a viral gene required for replication of the virus.
Essential genes for replication of adenovirus include the E4, E1a,
E1b, and E2 regions. Essential genes for replication of HSV-1
include ICP6 and ICP4. These genes are placed under control of the
specific promoter, and used to transduce cells in the
differentiated cell population. The viruses then replicate
specifically in any undifferentiated cells present, causing them to
rupture. See International Patent Publication WO 00/46355 (Morin et
al., Geron Corporation) for a description of lytic vectors that
replicate in cells expressing TERT.
[0142] Another type of effector sequence encodes a membrane protein
that contains the epitope recognized by the specific antibody. The
membrane protein may be a protein expressed in the same species on
other types of cells, but more typically is obtained from another
species, or is an artificial sequence. In this case, the antigen
will be foreign to the species from which the stem cells are
derived, and antibodies made in the same species will not
cross-react with other antigens on the cell.
[0143] Alternatively, the target antigen can be a cell-surface
carbohydrate or lipid component. In this case, the effector
sequence will encode an enzyme involved in antigen synthesis. Of
particular interest are glycosyl transferases of mammalian or
non-mammalian origin that synthesize carbohydrate differentiation
antigen, alloantigen, xenoantigen, or novel determinants detectable
by antibody. Examples include the marker SSEA-1, for which the
effector sequence encodes the corresponding fucosyltransferase; the
Gal.alpha.(1,3)Gal linkage present on endothelial tissue of most
mammals except for humans and old-world monkeys, formed by an
.alpha.(1,3)galactosyltransferase (.alpha.1,3GT);and the ABO histo
blood group antigens present on most human cells, for which the
encoding sequence is the corresponding ABO transferase. See GenBank
Accession Nos. S71333, J05175, and AF134414. The Gal.alpha.(1,3)Gal
and ABO determinants are all susceptible to lysis mediated by
antibodies that naturally occur in subjects that do not have the
determinants as a self-antigen.
[0144] Another possible effector sequence is based on
RNA-interference (RNAi) technology. Double-stranded or hairpin RNA
corresponding to a portion of mature mRNA in a cell (such as a gene
transcript) cause the target mRNA to be destroyed (Sharp et al.,
Genes Dev. 13:139, 1999; Wianny et al., Nat. Cell Biol. 2:70,
2000). For example, a plasmid containing a promoter that drives
expression of a hairpin RNA (a transcript consisting of inverted
repeats taken from the coding region of a cellular gene, separated
by a short linker sequence, thus generating a synthetic RNA
containing a double-stranded region) has been used to induce stable
and inheritable RNAi effects in C. elegans (Tavernarakis et al.;
Nat. Genet. 24:180, 2000).
[0145] In certain embodiments of this invention, a control element
driving transcription in an undifferentiated cell (or a cell
expressing TERT) is operatively linked to an encoding sequence for
hairpin RNAi that targets a particular gene transcript. The target
gene is chosen to be essential for cell viability: for example, a
basic transcription or translation factor, a tRNA gene, a ribosomal
RNA subunit, or DNA or RNA polymerase. In one illustration, the
hTERT promoter drives RNAi that inactivates a gene in the purine
salvage pathway. In the presence of drugs such as aminopterin, de
novo synthesis of purines is prevented, because the activity of the
enzymes hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and
thymidine kinase (TK) is inhibited. Cells that possess HGPRT and TK
survive in the presence of aminopterin, as long as the medium is
supplemented with hypoxanthine and thymidine (HAT medium). Residual
undifferentiated cells can be removed from the population by
incubation with HAT medium. Transcripts of essential genes such as
HGPRT and TK can also be targeted using other types of effector
sequences--for example, antisense polynucleotides, ribozymes, or
encoding sequences for dominant negative analogs that lack a
functional catalytic domain.
[0146] The effector region used in the vectors of this invention
can be constructed so as to be functionally controlled by a
molecular switch. Fusions between the ligand-binding domain of
receptors result in molecules in which the normal function of the
native protein is inhibited in the absence of the hormone
recognized by the ligand-binding domain. A fusion protein is
constructed comprising a switch molecule binding domain coupled to
an effector domain, in such a way that binding of a ligand (such as
a small molecule hapten) to the binding domain unmasks or activates
the effector domain. Suitable ligand binding domains can be taken
from a receptor (such the estrogen receptor) or an antibody. The
effector domain can be any of the proteins already listed that are
lethal to the cell, such as peptide toxins, endonucleases
(meganucleases such as I-Sce -1 or humanized versions of standard
restriction endonucleases), or mediators of apoptosis. The lethal
function of the effector gene is quiescent, unless the ligand is
present. This provides another system where the effector gene
renders the cell susceptible to toxic effects of an external agent
(in this case the ligand), which can be administered at will to
control the depletion of undifferentiated cells, either in culture
or in vivo.
[0147] The vector constructs for use in this invention can also
contain a positive selection marker, such as an antibiotic
resistance gene, that is also under control of the specific
promoter. Exemplary is a vector having the configuration hTERT
promoter--tk gene--IRES--neo. This is designed so that both the
suicide effector and drug resistance gene are expressed under
control of the TERT promoter. The internal ribosome entry site
(IRES) sequence allows both the tk gene and the neo gene to be
under transcriptional control of the hTERT promoter. A
post-translational cleavage site, such as 2A sequences (Felipe et
al., Gene Ther. 6:198, 1999( can be used to similar effect.
Generation and selection of hES lines that have stably integrated
such a construct is facilitated by the activity of the drug
resistance gene in the undifferentiated cells. This has an
advantage over co-transfection methods using a drug resistant gene
under control of a different promoter, because the drug resistance
gene will not be expressed in differentiated cells. This should
avoid an unwanted immunological response by the host against the
gene product in transplanted cells.
[0148] Selection Techniques to Eliminate Undifferentiated Cells
[0149] To deplete differentiated cell populations of
undifferentiated cells, the effector gene is selectively expressed
in the undifferentiated cells.
[0150] This can be accomplished in several ways. In one embodiment,
the population is genetically altered using a vector in which a
transcriptional control element of the appropriate specificity is
operatively linked to the effector gene. The genetic alteration may
be transient (for example, using an adenovirus vector), meaning
that the level of expression diminishes as the cells divide. This
is suitable for generating differentiated cell populations that
will be free of heterologous genes at the time of therapy. The
genetic alteration may also be permanent (for example, using a
retroviral vector), meaning that the alteration is inheritable by
progeny of the initially altered cell. This is suitable for
generating differentiated cell populations that will have an
ongoing corrective function as they proliferate in vitro or in
vivo, to eliminate any undifferentiated or dedifferentiated cells
that arise in the population.
[0151] Any suitable expression vector can be used. Suitable viral
vector systems for producing stem cells altered according to this
invention can be prepared using commercially available virus
components. Viral vectors comprising effector genes are generally
described in the publications referenced in the last section.
Alternatively, vector plasmids can be introduced into cells by
electroporation, or using lipid/DNA complexes, such as those
described in U.S. Patent Nos. 5,578,475; 5,627,175; 5,705,308;
5,744,335; 5,976,567; 6,020,202; and 6,051,429. Exemplary is the
formulation Lipofectamine 2000.TM., available from Gibco/Life
Technologies. Another exemplary reagent is FuGENE.TM. 6
Transfection Reagent, a blend of lipids in non-liposomal form and
other compounds in 80% ethanol, obtainable from Roche Diagnostics
Corporation.
[0152] In another embodiment, the effector gene is placed under
control of an endogenous transcriptional control element, such as
the hTERT or OCT-4 promoter. This can be effected, for example, by
homologous recombination, using a vector comprising the effector
encoding sequence, flanked on one side by the transcriptional
control element and other upstream genomic sequence, and flanked on
the other side by downstream genomic sequence for the targeted
gene. U.S. Pat. Nos. 5,464,764 and 5,631,153 describe a
double-selection strategy, in which two sequences homologous to the
gene target flank a positive selection marker, and a negative
selection marker is attached to the 3' terminal of the second
flanking region. U.S. Pat. No. 5,789,215 reports the use of
homologous recombination targeting vectors for modifying the cell
genome of mouse embryonic stem cells. Other information of interest
for homologous recombination targeting can be found in U.S. Pat.
Nos. 5,589,369, 5,776,774, and 5,789,215.
[0153] If the effector gene directly causes cell lysis or
apoptosis, then the population will be depleted of undifferentiated
cells upon culturing the cells under conditions where the control
element is expected to cause transcription of the gene. However, if
the effector gene is not directly lethal, but renders the cell
susceptible to the lethal effects of an external agent, then
depletion will be postponed until the external agent is provided.
For example, where the gene is a prodrug converting enzyme, then
depletion is effected upon placing the cells in an environment
containing the prodrug. Where the gene is an antibody target, then
depletion is effected upon placing the cells in an environment
containing specific antibody, plus complement. The environment can
be a culture vessel, in which case the agent can just be added to
the culture medium at the requisite concentration. Alternatively or
in addition, depletion can be performed in vivo, by administering
the cell population to a subject, and simultaneously or
sequentially administering the agent, if not already present.
[0154] Cell populations in which the majority of cells are
differentiated can be genetically modified according to these
procedures to deplete undifferentiated cells. Alternatively, a
precursor population of relatively undifferentiated cells can be
genetically modified according to these procedures, and then
differentiated. In this situation, it is more typical to use an
effector gene that does not kill the cells immediately upon
expression, but renders the cells susceptible to the lethal effect
of some external agent. In one illustration, undifferentiated pPS
cells grown in culture are transduced with a retrovirus vector in
which the herpes thymidine kinase gene is under control of the
hTERT promoter. The cells are optionally selected for positive
transduction, either by incorporating a selectable marker in the
construct, or by measuring expression of the transduced gene, and
proliferated in culture. When differentiated cells are desired, the
population is taken through a differentiation procedure (for
example, to make hepatocyte or neuron precursors, as described
earlier). They are then cultured under conditions that permit
expression of the tk gene in the presence of ganciclovir.
[0155] As an illustration where RNAi is the effector sequence, hES
cells are stably transfected (e.g., by lipofection) with a
construct consisting of 2 cassettes: one in which the PGK promoter
drives the neomycin phosphotransferase gene (resulting in
resistance to toxic neomycin analogs such as geneticin); the other
in which the hTERT promoter drives an encoding region for RNAi that
contains double-stranded regions from HGPRT or TK. Stably modified
clones are isolated in a medium containing both geneticin and
azaguanine or 6-mercaptopurine (to select for HGPRT negative
cells), or in medium containing both geneticin and
5-bromodeoxyuridine (to select for TK negative cells).
Alternatively, the transfection could be done with just the RNAi
kit, in which case geneticin is omitted from the medium. After
isolation of the surviving clones, these lines are induced to
differentiate to the desired cell type, and exposed to HAT medium
to kill any residual stem cells.
[0156] Cell populations may be obtained using these techniques that
are "depleted" of undifferentiated cells, which indicates any
significant reduction in the proportion of undifferentiated cells
present. After the procedure is effected, the proportion of
undifferentiated cells may be decreased by 50% or even 90%.
Depending on the control element and effector chosen, it may be
possible to achieve differentiated cell populations that are
"essentially free" of undifferentiated cells. This means that the
population as a whole contains less than 1% of cells with the
undifferentiated phenotype. Populations containing less than 0.2%,
0.05%, 0.01% , 20 ppm or 5 ppm undifferentiated cells are
increasingly more preferred. For pPS cells, the presence of
undifferentiated cells can be determined by counting cells
expressing SSEA-4 by FACS analysis, or by counting cells expressing
TERT or OCT-4 by fluorescence in-situ hybridization.
[0157] Use of Differentiated Cells
[0158] Cells prepared according to this invention can be used for a
variety of commercially important research, diagnostic, and
therapeutic purposes.
[0159] Because the cell populations of this invention are depleted
of undifferentiated cells, they can be used to prepare antibodies
and cDNA libraries that are specific for the differentiated
phenotype. General techniques used in raising, purifying and
modifying antibodies, and their use in immunoassays and
immunoisolation methods are described in Handbook of Experimental
Immunology (Weir & Blackwell, eds.); Current Protocols in
Immunology (Coligan et al., eds.); and Methods of Immunological
Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags GmbH).
General techniques involved in preparation of mRNA and cDNA
libraries are described in RNA Methodologies: A Laboratory Guide
for Isolation and Characterization (R. E. Farrell, Academic Press,
1998); cDNA Library Protocols (Cowell & Austin, eds., Humana
Press); and Functional Genomics (Hunt & Livesey, eds.,
2000).
[0160] Relatively homogeneous cell populations are particularly
suited for use in drug screening and therapeutic applications.
[0161] Drug Screening
[0162] Differentiated pPS cells of this invention can be used to
screen for factors (such as solvents, small molecule drugs,
peptides, polynucleotides, and the like) or environmental
conditions (such as culture conditions or manipulation) that affect
the characteristics of differentiated cells.
[0163] In some applications, differentiated cells are used to
screen factors that promote maturation, or promote proliferation
and maintenance of such cells in long-term culture. For example,
candidate maturation factors or growth factors are tested by adding
them to pPS cells in different wells, and then determining any
phenotypic change that results, according to desirable criteria for
further culture and use of the cells.
[0164] Particular screening applications of this invention relate
to the testing of pharmaceutical compounds in drug research. The
reader is referred generally to the standard textbook "In vitro
Methods in Pharmaceutical Research", Academic Press, 1997, and U.S.
Pat. No. 5,030,015). Assessment of the activity of candidate
pharmaceutical compounds generally involves combining the
differentiated cells of this invention with the candidate compound,
determining any change in the morphology, marker phenotype, or
metabolic activity of the cells that is attributable to the
compound (compared with untreated cells or cells treated with an
inert compound), and then correlating the effect of the compound
with the observed change.
[0165] The screening may be done, for example, either because the
compound is designed to have a pharmacological effect on certain
cell types, or because a compound designed to have effects
elsewhere may have unintended side effects. Two or more drugs can
be tested in combination (by combining with the cells either
simultaneously or sequentially), to detect possible drug-drug
interaction effects. In some applications, compounds are screened
initially for potential toxicity (Castell et al., pp. 375-410 in
"In vitro Methods in Pharmaceutical Research," Academic Press,
1997). Cytotoxicity can be determined in the first instance by the
effect on cell viability, survival, morphology, and expression or
release of certain markers, receptors or enzymes. Effects of a drug
on chromosomal DNA can be determined by measuring DNA synthesis or
repair. [.sup.3H]thymidine or BrdU incorporation, especially at
unscheduled times in the cell cycle, or above the level required
for cell replication, is consistent with a drug effect. Unwanted
effects can also include unusual rates of sister chromatid
exchange, determined by metaphase spread. The reader is referred to
A. Vickers (PP 375-410 in "In vitro Methods in Pharmaceutical
Research," Academic Press, 1997) for further elaboration.
[0166] Therapeutic Use
[0167] Differentiated cells of this invention can also be used for
tissue reconstitution or regeneration in a human patient in need
thereof. The cells are administered in a manner that permits them
to graft to the intended tissue site and reconstitute or regenerate
the functionally deficient area.
[0168] In one example, neural stem cells are transplanted directly
into parenchymal or intrathecal sites of the central nervous
system, according to the disease being treated. Grafts are done
using single cell suspension or small aggregates at a density of
25,000-500,000 cells per .mu.L (U.S. Pat. No. 5,968,829). The
efficacy of neural cell transplants can be assessed in a rat model
for acutely injured spinal cord as described by McDonald et al.
(Nat. Med. 5:1410, 1999. A successful transplant will show
transplant-derived cells present in the lesion 2-5 weeks later,
differentiated into astrocytes, oligodendrocytes, and/or neurons,
and migrating along the cord from the lesioned end, and an
improvement in gate, coordination, and weight-bearing.
[0169] Certain neural progenitor cells embodied in this invention
are designed for treatment of acute or chronic damage to the
nervous system. For example, excitotoxicity has been implicated in
a variety of conditions including epilepsy, stroke, ischemia,
Huntington's disease, Parkinson's disease and Alzheimer's disease.
Certain differentiated cells of this invention may also be
appropriate for treating dysmyelinating disorders, such as
Pelizaeus-Merzbacher disease, multiple sclerosis, leukodystrophies,
neuritis and neuropathies. Appropriate for these purposes are cell
cultures enriched in oligodendrocytes or oligodendrocyte precursors
to promote remyelination.
[0170] Hepatocytes and hepatocyte precursors prepared according to
this invention can be assessed in animal models for ability to
repair liver damage. One such example is damage caused by
intraperitoneal injection of D-galactosamine (Dabeva et al., Am. J.
Pathol. 143:1606, 1993). Efficacy of treatment can be determined by
immunohistochemical staining for liver cell markers, microscopic
determination of whether canalicular structures form in growing
tissue, and the ability of the treatment to restore synthesis of
liver-specific proteins. Liver cells can be used in therapy by
direct administration, or as part of a bioassist device that
provides temporary liver function while the subject's liver tissue
regenerates itself following fulminant hepatic failure.
[0171] The efficacy of cardiomyocytes prepared according to this
invention can be assessed in animal models for cardiac cryoinjury,
which causes 55% of the left ventricular wall tissue to become scar
tissue without treatment (Li et al., Ann. Thorac. Surg. 62:654,
1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai et al.,
J. Thorac. Cardiovasc. Surg. 118:715, 1999). Successful treatment
will reduce the area of the scar, limit scar expansion, and improve
heart function as determined by systolic, diastolic, and developed
pressure. Cardiac injury can also be modeled using an embolization
coil in the distal portion of the left anterior descending artery
(Watanabe et al., Cell Transplant. 7:239, 1998), and efficacy of
treatment can be evaluated by histology and cardiac function.
Cardiomyocyte preparations embodied in this invention can be used
in therapy to regenerate cardiac muscle and treat insufficient
cardiac function (U.S. Pat. No. 5,919,449 and WO 99/03973).
[0172] The examples that follow are provided by way of further
illustration, and are not meant to imply any limitation in
practicing the claimed invention.
EXAMPLES
Example 1
[0173] Feeder-free passage of hES cells
[0174] In this experiment, undifferentiated hES cells that had been
maintained on primary mouse embryonic feeder cells were harvested,
and then maintained in the absence of feeders. The culture wells
were coated with Matrigel.RTM., and the cells were cultured in the
presence of conditioned nutrient medium obtained from a culture of
irradiated primary fibroblasts.
[0175] Preparation of conditioned media (CM) from primary mouse
embryonic fibroblasts (mEF):
[0176] Fibroblasts were harvested from T150 flasks by washing one
time with Ca.sup.++/Mg.sup.++free PBS and incubating in 1.5-2 mL
trypsin/EDTA (Gibco) for about 5 min. After the fibroblasts
detached from the flask, they were collected in mEF media (DMEM+10%
FBS). The cells were irradiated at 4000 rad (508 sec at 140 kV:
shelf setting 6 in a Torrex.TM. generator), counted and seeded at
about 55,000 cells cm.sup.-2 in mEF media (525,000 cells/well of a
6 well plate). After at least 4 hours the media were exchanged with
SR containing ES media (containing bFGF), using 3-4 mL per 9.6 cm
well of a 6 well plate. Conditioned media was collected daily for
feeding of hES cultures. Alternatively, medium was prepared using
mEF plated in culture flasks, exchanging medium daily at 0.3-0.4 mL
cm.sup.-2. Before addition to the hES cultures, the conditioned
medium was supplemented with 4 ng/mL of human bFGF (Gibco).
Fibroblasts cultures were used in this system for about 1 week,
before replacing with newly prepared cells.
[0177] Matrigel.RTM. coating:
[0178] Growth Factor Reduced Matrigel.RTM. or regular Matrigel.RTM.
(Becton-Dickinson, Bedford Mass.) was thawed at 4.degree. C. The
Matrigel.RTM. was diluted 1:10 to 1:500 (typically 1:30) in cold KO
DMEM. 0.75-1.0 mL of solution was added to each 9.6 cm.sup.2 well,
and incubated at room temp for 1 h. The coated wells were washed
once with cold KO DMEM before adding cells. Plates were used within
2 h after coating, or stored in DMEM at 4.degree. C. and used
within .about.1 week.
[0179] Human ES culture:
[0180] Undifferentiated hES colonies were harvested from hES
cultures on feeders as follows. Cultures were incubated in
.about.200 U/mL collagenase IV for about 5 minutes at 37.degree. C.
Colonies were harvested by picking individual colonies up with a 20
.mu.L pipet tip under a microscope or by scraping and dissociating
into small clusters in conditioned medium (CM). These cells were
then seeded onto Matrigel.RTM. in conditioned media at 15 colonies
to each 9.6 cm.sup.2 well (if 1 colony is .about.10,000 cells, then
the plating density is .about.15,000 cells cm.sup.-2).
[0181] The day after seeding on Matrigel.RTM., hES cells were
visible as small colonies (.about.100-2,000 cells) and there were
single cells in-between the colonies that appeared to be
differentiating or dying. As the hES cells proliferated, the
colonies became quite large and very compact, representing the
majority of surface area of the culture dish. The hES cells in the
colonies had a high nucleus to cytoplasm ratio and had prominent
nucleoli, similar to hES cells maintained on feeder cells. At
confluence, the differentiated cells in between the colonies
represented less than 10% of the cells in the culture.
[0182] Six days after seeding, the cultures had become almost
confluent. The cultures were split by incubating with 1 mL
.about.200 U/mL Collagenase IV solution in KO DMEM for .about.5
minutes at 37.degree. C. The collagenase solution was aspirated, 2
mL hES medium was added per well, and the hES cells were scraped
from the dish with a pipette. The cell suspension was transferred
to a 15 mL conical tube, brought up to a volume of 6 mL, and gently
triturated to dissociate the cells into small clusters of 10-2000
cells. The cells were then re-seeded on Matrigel.RTM. coated plates
in CM, as above. Cells were seeded at a 1:3 or 1:6 ratio,
approximately 90,000 to 170,000 cells cm.sup.-2, making up the
volume in each well to 3 mL. Medium was changed daily, and the
cells were split and passaged again at 13 d and again at 19 d after
initial seeding.
[0183] On day 19 after initial seeding, cells were harvested and
evaluated for surface marker expression by immunofluorescence cell
cytometry, using labeled antibodies specific for cell surface
markers. For the hES cells maintained in the absence of feeders, a
high percentage express SSEA-4, Tra-1-60 or Tra-1-81. These 3
markers are expressed on undifferentiated human ES cells that are
maintained on feeders (Thomson et al., 1998). In addition, there is
very little expression of SSEA-1, a glycolipid that is not
expressed(or expressed at low levels) on undifferentiated ES cells.
Immunohistochemical evaluation of SSEA-4, Tra-1-60 and Tra-1-81
indicates that the expression of these markers is localized to the
ES colonies, not the differentiated cells in between the
colonies.
[0184] Cultures of hES cells have been grown in the absence of
feeder cells for over 180 days after initial seeding, with no
apparent change in the proliferative capacity or phenotype. Human
ES cells maintained on Matrigel.RTM. in mEF conditioned medium have
a doubling time of about 31-33 hours, similar to the proliferation
rate for hES cells grown on mEF feeder cells. H1 cells after 64
days of feeder-free culture showed a normal karyotype.
Example 2
[0185] Phenotypic markers of hES cells in feeder-free culture
[0186] Undifferentiated hES cells express SSEA-4, Tra-1-60,
Tra-1-81, OCT-4, and hTERT. The expression of these markers
decreases upon differentiation. In order to assess whether the
cells maintained in feeder-free conditions retained these markers,
cells were evaluated by immunostaining, reverse transcriptase PCR
amplification, and assay for telomerase activity.
[0187] For analysis by fluorescence-activated cell sorting (FACS),
the hES cells were dissociated in 0.5 mM EDTA in PBS and
resuspended to about 5.times.10.sup.5 cells in 50 .mu.L diluent
containing 0.1% BSA in PBS. For analyzing surface marker
expression, cells were incubated in the primary antibodies,
including IgG isotype control (0.5 .mu.g/test), IgM isotype control
(1:10), SSEA-1 (1:10), SSEA-4 (1:20), Tra-1-60 (1:40) and Tra-1-81
(1:80), diluted in the diluent at 4.degree. C. for 30 min. After
washing with the diluent, cells were incubated with rat anti-mouse
kappa chain antibodies conjugated with PE (Becton Dickinson, San
Jose, Calif.) at 4.degree. C. for 30 min. Cells were washed and
analyzed on FACScalibur.TM. Flow Cytometer (Becton Dickinson, San
Jose, Calif.) using CellQuest.TM. software.
[0188] Similar to the hES cells on feeders, cells on Matrigel.RTM.,
laminin, fibronectin or collagen IV expressed SSEA-4, Tra-1-60 and
Tra-1-81. There was very little expression of SSEA-1, a glycolipid
that is not expressed by undifferentiated hES cells.
[0189] For analysis by immunocytochemistry, cells were incubated
with primary antibodies, including SSEA-1 (1:10), SSEA-4 (1:20),
Tra-1-60 (1:40) and Tra-1-81 (1:80), diluted in knockout DMEM at
37.degree. C. for 30 min. Cells were then washed with warm knockout
DMEM and fixed in 2% paraformaldehyde for 15 min. After washing
with PBS, cells were incubated with 5% goat serum in PBS at room
temp for 30 min, followed by incubation with the FITC-conjugated
goat anti-mouse antibodies (1:125) (Sigma) at room temp for 30 min.
Cells were washed, stained with DAPI and mounted. The staining was
typically performed .about.2 days after passaging. Cells were also
examined for expression of alkaline phosphatase, a marker for
undifferentiated ES cells. This was performed by culturing the
cells on chamber slides, fixing with 4% paraformaldehyde for 15
min, and then washing with PBS. Cells were then incubated with
alkaline phosphatase substrate (Vector Laboratories, Inc.,
Burlingame, Calif.) at room temperature in the dark for 1 h. Slides
were rinsed for 2-5 min in 100% ethanol before mounting.
[0190] The results showed that SSEA-4, Tra-1-60, Tra-1-81, and
alkaline phosphatase were expressed by the hES colonies on
Matrigel.RTM. or laminin, as seen for the cells on feeders--but not
by the differentiated cells in between the colonies.
[0191] FIG. 1 shows OCT-4 and hTERT expression of H1 cells on
feeders and off feeders, as detected by reverse-transcriptase PCR
amplification. For radioactive relative quantification of
individual gene products, QuantumRNA.TM. Alternate18S Internal
Standard primers (Ambion, Austin Tex., USA) were employed according
to the manufacturer's instructions. Briefly, the linear range of
amplification of a particular primer pair was determined, then
coamplified with the appropriate mixture of alternate18S
primers:competimers to yield PCR products with coinciding linear
ranges. Before addition of AmpliTaq.TM. (Roche) to PCR reactions,
the enzyme was pre-incubated with the TaqStart.TM. antibody
(ProMega) according to manufacturer's instructions. Radioactive PCR
reactions were analyzed on 5% non-denaturing polyacrylamide gels,
dried, and exposed to phosphoimage screens (Molecular Dynamics) for
1 hour. Screens were scanned with a Molecular Dynamics Storm 860
and band intensities were quantified using ImageQuant.TM. software.
Results are expressed as the ratio of radioactivity incorporated
into the hTERT or OCT-4 band, standardized to the radioactivity
incorporated into the 18s band.
[0192] Primers and amplification conditions for particular markers
are as follows. OCT-4: Sense (SEQ. ID NO:4) 5'-CTTGCTGCAG
AAGTGGGTGG AGGAA-3'AntiSense (SEQ. ID NO:5) 5'-CTGCAGTGTG
GGTTTCGGGC A-3'; alternate18:competimers 1:4; 19 cycles (94.degree.
30 sec; 60.degree. 30 sec; 72.degree. 30 sec). hTERT: Sense (SEQ.
ID NO:6) 5'-CGGAAGAGTG TCTGGAGCAA-3'AntiSense (SEQ. ID NO:7)
5'-GGATGAAGCG GAGTCTGGA-3'; alternate18:competimers 1:12; 34 cycles
(94.degree. 30 sec; 60.degree. 30 sec; 72.degree. 30 sec).
[0193] hTERT and OCT-4 expression was seen in all the culture
conditions except Matrigel.RTM. and regular medium. Furthermore,
after exposure of cells to retinoic acid (RA) or dimethyl sulfoxide
(DMSO), factors that promote cell differentiation, the expression
of hTERT was markedly decreased.
[0194] FIG. 2 shows telomerase activity measured by TRAP activity
assay (Kim et al., Science 266:2011, 1997; Weinrich et al., Nature
Genetics 17:498, 1997). All the cultures conditions showed positive
telomerase activity after 40 days on Matrigel.RTM., laminin,
fibronectin or collagen IV in mEF conditioned medium.
Example 3
[0195] Differentiation of hES cells
[0196] In this experiment, differentiation using standard methods
of aggregate formation was compared with a direct differentiation
technique.
[0197] For the aggregate differentiation technique, monolayer
cultures of rhesus and human ES lines were harvested by incubating
in Collagenase IV for 5-20 min, and the cells were scraped from the
plate. The cells were then dissociated and plated in non-adherent
cell culture plates in FBS-containing medium. The plates were
placed into a 37.degree. C. incubator, and in some instances, a
rocker was used to facilitate maintaining aggregates in suspension.
After 4-8 days in suspension, aggregate bodies formed and were
plated onto a substrate to allow for further differentiation.
[0198] For the direct differentiation technique, suspensions of
rhesus and human ES cells were prepared in a similar fashion. The
cells were then dissociated by trituration to clusters of
.about.50-100 cells, and plated onto glass coverslips treated with
poly-ornithine. The cells were maintained in serum containing
medium, or defined medium for 7-10 days before analysis.
[0199] Cells from both preparations were fixed and tested by
immunoreactivity for .beta.-tubulin III and MAP-2, which is
characteristic of neurons, and glial fibrillary acidic protein
(GFAP), which is characteristic of astrocytes. Results are shown in
Table 1.
1TABLE 1 Comparison of hPS Differentiation Methods Differentiation
via Direct ES Cell Line Aggregate Bodies Differentiation used for
differentiation Neurons Astrocytes Neurons Astrocytes R366.4
(Rhesus line) + + + + R278.5 (Rhesus line) + + + + R456 (Rhesus
line) + + + + H9 (Human line) + + + + H9.1 (Clone of H9) (Not Done)
(Not + + Done) H9.2 (Clone of H9) + + + +
[0200] Rhesus and human ES lines differentiated into cells bearing
markers for neurons and astrocytes, using either the aggregate or
direct differentiation technique. In the rhesus cultures,
percentage of aggregates that contained neurons ranged from 49% to
93%. In the human lines examined, the percentage of aggregates
containing neurons ranged from 60% to 80%. Double labeling for GABA
and -tubulin indicated that a sub-population of the neurons express
the inhibitory neurotransmitter GABA. In addition, astrocytes and
oligodendrocytes were identified with GFAP immune reactivity and
GalC immune reactivity, respectively. Therefore, the human and
rhesus ES cells have the capacity to form all three major cell
phenotypes in the central nervous system.
[0201] The effect of several members of the neurotrophin growth
factor family was examined. hES cells were differentiated by
harvesting with collagenase, dissociating, and reseeding onto
poly-ornithine coated cover slips. The cells were plated into
DMEM/F12+N2+10% FBS overnight. The following day, the serum was
removed from the medium and replaced with 10 ng/mL human bFGF and
the growth factor being tested. After 24 hours, bFGF was removed
from the medium. These cultures were fed every other day. They were
fixed after 7 days of differentiation and immunostained for
analysis. The number of neurons was evaluated by counting cells
positive for .beta.-tubulin. Cultures maintained in the presence of
10 ng/mL brain derived neurotrophic factor (BDNF) formed
approximately 3-fold more neurons than the control cultures.
Cultures maintained in neurotrophin-3 (1 ng/mL) formed
approximately 2-fold more neurons than control cultures.
[0202] In a subsequent experiment, suspensions of human ES cells
were prepared from parental line H9 and two subcloned lines. The
cells were harvested using collagenase IV, and then replated onto
poly-ornithine coated glass slides in medium containing 20% FBS.
The cultures were then fed every other day for 7-10 days, then
fixed for immunostaining. From each of these lines, a number of
differentiated cells stained positively for muscle-specific actin
(antibody from Dako), but were negative for cardiac troponin I.
Several patches of cells stained positively for
.alpha.-fetoprotein, indicating the presence of endoderm cells.
Example 4
[0203] Comparison of direct differentiation with differentiation
through embryoid bodies
[0204] To induce direct differentiation, undifferentiated hES cells
were harvested and re-plated directly into differentiating
conditions. Considerable cell death was apparent upon plating, but
many cells adhered and began to proliferate and/or differentiate.
In cultures differentiated using serum containing conditions, the
cultures continued to proliferate and reached confluence within
5-10 days. At this time, the cultures contained a heterogeneous
population that displayed many different morphologies.
Immunocytochemistry revealed ectoderm, mesoderm and endoderm
lineages using antibodies against .beta.-tubulin III, muscle
specific actin and .alpha.-fetoprotein, respectively. The positive
staining for all of these cell types appeared in patches that were
sometimes quite dense, therefore it was difficult to accurately
quantify the percentages of each cell type.
[0205] In order to increase the percentage of neurons, the hES
cells were plated onto poly-ornithine coated glass coverslips and
cultures in defined media. Although these data indicate that cells
from all three germ layers can be derived without the production of
EBs, cardiomyocytes were not identified.
[0206] By way of comparison, hES cells were induced to
differentiate by generating embryoid bodies (EBs). In these
experiments, ES cells were harvested and replated in suspension
cultures. Although initially a marked amount of cell death was
observed, after 2-3 days the remaining cells formed aggregates. EBs
were maintained for as many as 16 days in culture and were still
viable and formed many structures after subsequent plating. Later
stage human EBs often showed a cystic morphology and sometimes gave
rise to beating EBs.
[0207] To assess cardiomyocyte formation, EBs were transferred to
gelatin-coated plates or chamber slides after 4 days in the
suspension cultures. The EBs attached to the surface after seeding,
proliferated and differentiated into different types of cells.
Spontaneously contracting cells were observed in various regions of
the culture at differentiation day 8 and the number of beating
regions increased until about day 10. In some cases, more than 75%
of the EBs had contracting regions. Beating cells were
morphologically similar to mouse ES cell-derived beating
cardiomyocytes. In addition, the expression of the cardiac specific
marker cardiac troponin I was examined at differentiation day 15
using immunocytochemistry. Individual contracting foci in the
differentiated cultures were photographed to record the contracting
area before the culture was fixed. The culture was then evaluated
for cardiac cTnl expression and matched to the original photographs
to determine the percentage of contracting areas that were positive
for cTnl staining. As a control, cells adjacent to the contracting
foci were also examined for cTnl staining. In these cultures 100%
of the contracting areas showed positive immunoreactivity, while
minimal immunoreactivity was observed in the non-beating cells.
[0208] Cultures of differentiated EBs were subjected to Western
blot analysis using monoclonal antibody against cTnl. This assay
gave a strong 31 kDa protein signal, corresponding to the size of
the purified native human cardiac Tn I. cTnl was detected in
differentiated human ES cells containing contracting cells but not
in undifferentiated ES cells or differentiated cultures with no
evidence of contracting cells, suggesting the specific detection of
cardiomyocytes. As a control, the blot was reprobed with
.beta.-actin specific antibody, confirming the presence of similar
amounts of proteins in all samples.
[0209] In other experiments, EBs were cultured for 8 or 16 days and
maintained as adherent cultures for an additional 10 days. RNA was
prepared from the differentiated human ES cells and
semiquantitative RT-PCR was performed to detect the relative
expression of the endoderm-specific products
.alpha..sub.1-anti-trypsin, AFP, and albumin. Low levels of
.alpha..sub.1-anti-trypsin and AFP were detected in the
undifferentiated cultures; little or no albumin was detected in the
same cultures. All 3 markers were detected at significantly higher
levels after differentiation. Expression of all 3 endoderm markers
was higher in cultures derived from 8 day embryoid bodies than 16
day embryoid bodies.
Example 5
[0210] Transfection and transduction of hES cells maintained on
primary mEF feeder layers
[0211] hES cultures were maintained in a growth medium composed of
80% KO DMEM (Gibco) and 20% Serum Replacement (Gibco) supplemented
with 1% non-essential amino acids, 1 mM glutamine, 0.1 mM
.beta.-mercaptoethanol and 4 ng/mL hbFGF (Gibco).
[0212] Plates were coated with a solution of 0.5% gelatin (Sigma)
at 37.degree. overnight before the addition of cells. Primary mEFs
were cultured in standard mEF medium, and split 1:2 every 2 days
for up to 5 splits. Subconfluent cultures of mEFs were detached
with trypsin, resuspended in 10 mL medium, and irradiated with a
cumulative dose of 3500-4000 rads with a Torrex.TM. 150D X-ray
generator. Irradiated cells were pelleted at 400.times.g for 5 min
and resuspended at 1.25.times.10.sup.5 cells per mL in standard mEF
medium. Individual wells of a 6-well plate were seeded with
3.75.times.10.sup.5 irradiated mEFs per well; individual wells of a
24-well plate were seeded with 75,000 irradiated mEFs per well.
[0213] Transfection was performed as follows. hES cells plated in 6
well plates were removed from the feeder layer with collagenase
(.about.200 units/mL) at 37.degree. for 7-10 min. When colonies
began to detach, the collagenase from each well was aspirated and
replaced with 2 mL of standard hES growth medium/well. The hES
cells were removed by scraping the surface of a single well with a
5 mL pipet and transferred to a 50 mL conical tube. Additional hES
growth medium was added to a final volume of 10 mL. The cell
suspension was triturated 10-12 times with a 10 mL pipet, and an
additional 8 mL of standard hES growth medium added. Three mL of
the cell suspension were added to each well of 6 well plates that
were pre-coated with gelatin and mEF feeder layers as described
above (i.e., 1 well of a 6 well plate was sufficient to seed 6
wells of a new plate).
[0214] Replated hES cells were tested with a number of different
transfection systems to determine whether genetic alteration of hES
cells could be achieved without causing differentiation. Systems
tested included the following: Mammalian Transfection Kit (CaPO4
and DEAE reagents), Stratagene cat # 200285; TransIT-LT1 Mirus.TM.
(Panvera), cat # MIR 2310; Polybrene (Sigma); Poly-L-Lysine
(Sigma); Superfect.TM. (Qiagen); Effectene.TM. (Qiagen);
Lipofectin.TM. (Life Technologies); Lipofectamine (differs from
Lipofectamine 2000.TM.) (Life Technologies); Cellfectin.TM. (Life
Technologies); DMRIE-C (Life Technologies); Lipofectamine 2000
(Life Technologies); and electroporation using BioRad.TM. Gene
pulser.
[0215] Under the conditions used, Lipofectamine 2000.TM. (Gibco
Life Technologies cat # 11668019, patent pending) and FuGENE.TM.
(trademark of Fugent L.L.C.; a proprietary blend of lipids and
other components, purchased from Roche Diagnostic Corporation cat #
1 814 443) both resulted in good transfection efficiency. The
efficiency was generally best if these reagents were contacted with
replated hES cells .about.48 h after the replating.
[0216] Transfection using Lipofectamine 2000.TM. was conducted as
follows: The plasmid DNA (3-5 .mu.g of pEGFP-C1, ClonTech cat. #
6084-1) was diluted in water to a final volume of 100 .mu.l. In
pilot experiments, 5 to 30 .mu.L of Lipofectamine 2000.TM. (Gibco,
cat # 11668-019) were diluted in OptiMEM.TM. (Gibco, cat #
11-58-021) to a final volume of 100 .mu.L. The DNA solution was
then added slowly to the Lipofectamine2000.TM. solution and mixed
gently. The mixture was incubated at room temperature for 20-30 min
before being supplemented with 800 .mu.l of OptiMEM.TM.. Cells were
washed with 3 mL of pre-warmed OptiMEM.TM. and incubated in 0.5-1
mL of the DNA/lipid mixture solution at 37.degree. C. for 4 h, per
well (9.6 cm.sup.2). In some experiments, at 4 h the complex was
removed before the addition of 4 mL of mEF-conditioned medium; in
others sufficient mEF-conditioned medium was added to the wells to
reach a final volume of 3.5 mL and the mixture was left on the
cells overnight. In other experiments the DNA/lipid mixture was
added to wells containing sufficient mEF-conditioned medium such
that the final volume was 3.5 mL, and the cells were incubated in
this mixture overnight.
[0217] Transfection using FuGENE.TM. was conducted as follows. Each
well was transfected with 10 .mu.g DNA using FuGENE.TM. 6 (Roche
Diagnostics Corp.), at a ratio of 3:2 FuGENE.TM. reagent to DNA as
described by the manufacturer's directions. OptiMEM.TM. serum-free
medium was used in the transfections. In the "old protocol", 4 h
after the addition of the FuGENE.TM.-DNA complex, 2.5 mL of
standard hES growth medium was added to each transfected well. In
the revised protocol ("3:2 L"), transfected wells were not re-fed
with standard hES growth medium. Twenty-four hours after
transfection, GFP-expression was assessed by flow cytometry.
[0218] Forty-eight hours before transfection, hES cells were seeded
onto 6 well plates that had been coated with gelatin and mEF feeder
layers as described above. hES cells were transfected using
FuGENE.TM. 6 (Roche) or Lipofectamine 2000.TM. (Gibco) according to
the manufacturers' instructions. Twenty-four hours after
transfection, cells were assessed for GFP expression by inspection
under a fluorescent microscope or flow cytometry. In the experiment
shown in FIG. 1, three methods were compared: the standard
Lipofectamine 2000.TM. protocol, the standard FuGENE.TM. protocol,
and a variant FuGENE.TM. protocol in which the DNA/lipid mix was
left on the cells overnight. The results demonstrated that while
Lipofectamine 2000.TM. consistently yielded a higher percentage of
GFP-expressing cells, the variant FuGENE.TM. protocol resulted in
GFP-expressing cells with a higher mean fluorescence intensity.
[0219] Transient transductions using adenoviral vectors were
conducted as follows. The vector Ad5CMV5-GFP (referred to here as
Ad5GFP) contains the green fluorescent protein encoding region
under control of the CMV promoter, and was purchased from Quantum
Biotechnologies, cat # ADV0030. Seventy-two hours before
transduction, hES cells were seeded onto 24 well plates that had
been coated with gelatin and mEF feeder layers as described above.
Before transduction, 3 wells of hES cells were detached with a
solution of 0.05% trypsin/5mM EDTA (Sigma) at 37.degree.,
resuspended in 500 .mu.L of standard mEF growth medium, and counted
with a hemocytometer (the 75,000 mEF feeder cells were subtracted
from each well) to establish the cell number before transfection.
The adenovirus stock was thawed on ice immediately prior to
use.
[0220] For infection with Ad5GFP, growth media was aspirated from
the wells containing hES cells and replaced with 1 mL of hES growth
medium plus 9 .mu.L of Ad5 GFP stock (MOI of 40). Two hours later,
the virus-containing medium was replaced with 1 mL of hES growth
medium per well. Each transduced well was refed with 1 mL of fresh
hES growth medium every 24 hours. GFP expression was assessed by
flow cytometry. The results from a typical experiment indicated
that expression was highest at 24 hr after transduction but
persisted for at least 8 days at low levels (by the later time
points, extensive differentiation had occurred due to overgrowth of
the hES cells).
Example 6
[0221] Preparation of the immortalized feeder cell line NH190
[0222] In this example, a permanent mouse cell line was established
that is suitable for conditioning medium for the culture of primate
pluripotent stem (pPS) cells. The NHG190 line is a mouse embryonic
fibroblast cell line immortalized with telomerase that is triple
drug resistant, and expresses green fluorescent protein (GFP).
[0223] Two mouse strains were obtained from Jackson Laboratory (Bar
Harbor, Me.) that have a transgene for resistance to the
antibiotics neomycin or hygromycin. The C57BL/6J
TgN(pPGKneobpA)3Ems mice and C57BL/6J-TgN(pPWL512hyg)1Ems mice from
Jackson Labs were cross-bred. Embryos that were both neomycin- and
hygromycin-resistant were dissected at day 13.5 post conception
according to standard protocols for preparing mouse embryonic
fibroblasts (mEF) for feeder layers (E. J. Robertson, pp. 71-112 in
Teratocarcinoma and Embryonic Stem Cell Lines, ed. E. J. Robertson,
Oxford: IRL Press, 1987). The derived mEF cells were stored
frozen.
[0224] The mEFs were thawed in growth medium containing 20% fetal
calf serum (HyClone), 2 mM L-glutamine (Gibco/BRL), 80% DMEM
(Gibco/BRL). The cells were expanded using 1:2 split ratios for 4
passages. Two flasks that had reached .about.75% confluence were
fed with fresh medium 4 h before electroporation. Cells were
removed from the flasks with 0.5% trypsin/500 mM EDTA (Gibco/BRL),
pelleted at 400.times.g for 5 min at room temperature, and
resuspended in the growth medium at a concentration of
4.times.10.sup.6 cells/mL.
[0225] The cell suspension was divided into two 500 .mu.L aliquots
and transferred to two 0.4 cm gap electroporation cuvettes
(BioRad). One cuvette received 5 .mu.g of the control plasmid
(pBS212; puromycin-resistance gene driven by the SV40 early
enhancer/promoter); the other received 5 .mu.g of pGRN190,
comprising the murine telomerase reverse transcriptase (mTERT)
coding region driven by MPSV promoter plus puromycin resistance
gene driven by the SV40 early enhancer/promoter. The cells and DNA
were mixed by hand, and electroporated using a BioRad gene Pulser
with a BioRad capacitance extender at a setting of 300V, 960
.mu.F.
[0226] Each aliquot of cells was transferred to an individual 150
cm plate containing 25 mL of growth medium. The medium on the
plates was exchanged on the following day, and on the next day,
growth medium was replaced by growth medium plus 0.5 .mu.g/mL
puromycin. The medium on the plates was exchanged for fresh
puromycin-containing medium every 48 hrs until 29 days after
electroporation. At this time, large individual colonies of
puromycin-resistant cells were evident in both the pBS212- and
pGRN190- electroporated plates. Ten colonies from the control plate
and 12 from the pGRN190-electroporated plate were isolated with
cloning cylinders and each colony was transferred to 1 well of a
48-well plate (1 well per colony).
[0227] One week later, all surviving colonies that had expanded to
reach confluence in the 48 well plate (three control colonies, 1
pGRN190-electroporated colony) were transferred individually to
wells of a 24 well plate. Six days later, the only colony that had
continued to expand was derived from the pGRN190-electroporated
plate, and was subsequently designated NH190. The cells were
maintained in growth medium plus 0.5 .mu.g/mL puromycin. Analysis
for telomerase activity by TRAP assay (Kim et al., Nucleic Acids
Res. 25:2595, 1997) demonstrated that NH190 cells express
functional telomerase activity.
[0228] To facilitate monitoring of the cells in mixed culture
populations and in vivo, NH190 cells were further infected with a
retroviral construct conferring expression of green fluorescent
protein (GFP). The enhanced GFP sequence from plasmid pEGFP-1 is
one of the Living Colors.TM. fluorescent protein vectors, available
from ClonTech. It contains an enhanced GFP encoding region, with
changes that alter restriction nuclease cleavage sites, and shift
the excitation and emission wavelengths of the encoded protein. The
EGFP-1 sequence was cloned into the vector pMSCV.neo, ClonTech cat
# K1062-1. NH190 cells were transduced with the engineered vector,
and GFP positive cells were separated by FACS sorting. The GFP
expressing cell line was designated NHG190. These cells have been
carried in culture for over 3 months.
Example 7
[0229] Genetic modification of hES cells maintained on NHG190
feeder cells
[0230] NHG190 cells were cultured in DMEM (Gibco) plus 20% fetal
bovine serum (HyClone) and 5 mM glutamine. Cells were split 1:10
every 3 d. Subconfluent cultures were detached with trypsin,
suspended in 10 mL medium, and irradiated with a cumulative dose of
3500 rads with a Torrex.TM. 150D X-ray generator. Irradiated cells
were pelleted at 400.times.g for 5 min and resuspended at
1.25.times.10.sup.5 cells per mL in either NHG190 medium or
standard hES medium.
[0231] Conditioned medium was prepared by plating NHG190 cells at
4.08.times.10.sup.4 cm.sup.-1 on gelatin-coated plates. At 18-24 h
after plating, medium was exchanged for standard hES medium with 4
ng/mL added bFGF. The medium was conditioned by the cells for 18-24
h, harvested, and an additional 4 ng/mL bFGF was added. The medium
was used to support hES cell cultures the same day as it was
collected. Irradiated NHG190 cells could be used for preparing
conditioned medium for 7-10 days.
[0232] hES cells were transfected as follows. The cells were
removed from the feeder layer using collagenase (.about.200 U/mL)
at 37.degree. C. for 7-10 min, and transferred to a 50 mL conical
tube. hES growth medium was added to a final volume of 10 mL; the
suspension was triturated 10-12 times with a 10 mL pipet, and
another 8 mL hES medium was added. Three mL of cell suspension was
added to each well in a 6-well plate precoated with Matrigel.RTM.
and NHG190 feeder cells.
[0233] Forty-eight hours after seeding, the hES were transfected
with 10 .mu.g DNA per well using FuGENE.TM. 6 (Roche) according to
manufacturer's protocol in OptiMEM.TM. serum-free medium. The DNA
was a plasmid containing the PGK promoter driving neo.sup.r. Four h
later, 3 mL of NHG190-conditioned medium was added to each
transfected well. Cells were re-fed daily with 3 mL conditioned
medium. Forty-eight h after transfection, the cells were layered
with NHG190 conditioned medium containing 200 .mu.g/mL added
geneticin (Sigma), which was replaced daily thereafter. After 3
days of selection, additional irradiated NHG190 feeder cells were
added (1.25.times.10.sup.5 cells/well in hES medium). Twenty-four h
later, the medium was again replaced with NHG190-conditioned medium
containing 200 .mu.g/mL geneticin, replaced daily.
[0234] Individual colonies were isolated and expanded through
another round of selection. After a further 5 days, individual
colonies were identified by microscope and marked on the outside of
the dish. Medium was removed, and replaced with collagenase
(.about.200 U/mL). Individual colonies were picked using a p20
pipet tip, and transferred to individual tubes containing 2 mL
NHG190 conditioned medium (without geneticin). The suspension was
triturated 5 times to disaggregate colonies, and the contents of
each tube were transferred to a well of a 12-well plate coated with
gelatin and irradiated NHG190 cells (1.875.times.10.sup.5
cells/well). Cells were fed 24 h later with 2 mL fresh conditioned
medium. Two days after seeding, cells were layered with 2 mL
conditioned medium containing 200 .mu.g/mL geneticin, replaced
daily for 5 days. As each well became 50-75% confluent, the cells
were detached with collagenase, transferred to 6 mL conditioned
medium, and triturated 10-12 times. 3 mL cell suspension was added
to each of 2 wells of a 6-well plated coated with gelatin and
irradiated NHG190 cells (3.75.times.10.sup.5 cells/well); the cells
were refed with 3 mL conditioned medium at 24 h. The cells were
then selected for 5 days using 3 mL conditioned medium containing
geneticin, and split 1:6 as before.
[0235] Stable transduction using retrovirus was conducted as
follows. Retroviral vector designated GRN354 was constructed at
Geron Corp. using PMSCVneo vector purchased from ClonTech (cat #
K1062-1). The eGFP encoding region was inserted downstream from the
MSCV LTR. The LTR drives expression of GFP and the vector also
contains the neo.sup.r gene driven by the murine PGK promoter.
Plates were coated with 0.5% gelatin and NHG190 feeder cells
(7.5.times.10.sup.4 in 1 mL NHG190 medium for 24 well plates;
3.75.times.10.sup.5 in 3 mL medium for 6 well plates). The hES line
H7 was seeded onto a 24 well prepared plate in hES medium (1
mL/well). Forty-eight h later, 3 wells of hES cells were detached
using 0.05% trypsin/5 mM EDTA (Sigma) at 37.degree. C., resuspended
in 500 .mu.L NHG190 medium, and counted. Stock of retrovirus
construct pGRN354 was thawed on ice immediately prior to use.
Growth medium was aspirated from the wells and replaced with 400
.mu.L hES medium plus 8 .mu.L retrovirus (MOI of 10) and 4 .mu.L of
8 mg/mL polybrene solution (Sigma). Two h later, 800 .mu.L hES
growth medium were added per well. Each transduced well was refed
with 1 mL fresh hES medium every 24 h.
[0236] Four days after transduction, medium was replaced with 1 mL
hES growth medium containing 200 .mu.g/mL geneticin. After 3 days
of geneticin selection, the cells were detached with collagenase,
triturated, resuspended in 3 mL hES medium, reseeded into one well
of a 6-well plate coated with gelatin and NHG190 feeders, and refed
with hES medium after 24 h. The medium was then again replaced with
hES medium containing geneticin and refed every 24 h.
Undifferentiated colonies survived the selection, and have been
maintained for over 3 months. FACS analysis showed that 50-65% of
the selected cells express GFP, albeit at low levels. The karyotype
of the cells was normal.
[0237] FIG. 3 shows GFP expression of hES cells transduced with
retrovirus and then differentiated. The hES cell line H7 was plated
on drug resistant (NHG190) feeder layers, infected with GRN354 and
selected for resistance to the drug G418. Transduced cells were
expanded and maintained under G418 selection for multiple passages.
The cells were transferred to suspension culture to form embryoid
bodies, allowed to differentiate for 4 days, and then plated in 20%
FBS medium for 1 week. After extensive differentiation occurred,
cultures were fixed in 4% paraformaldehyde and photographed under
fluorescence for GFP expression. Many of the differentiated cells
express higher levels of GFP than the undifferentiated transfected
hES line, consistent with differential activation of the MESV-LTR
in different cell types.
Example 8
[0238] Transfection of feeder-free hES cells
[0239] In this example, hES cells maintained in feeder-free culture
on laminin in conditioned medium were genetically modified by
transfecting with a plasmid carrying green fluorescent protein
(GFP) driven by the CMV promoter.
[0240] mEF conditioned medium was prepared as described earlier.
mEFs were irradiated and seeded at about 5.7.times.10.sup.4
cells/cm.sup.2. After at least 16 hours the medium was exchanged
with hES medium including 4 ng/mL added hbFGF. Conditioned medium
was collected daily for feeding of hES cultures. Before addition to
the hES cultures, this medium was supplemented with an additional 4
ng/mL of hbFGF. Where needed for selection of stable transfectants,
the mEF-conditioned medium was supplemented with 200 .mu.g/mL
geneticin (Sigma cat. # G5013).
[0241] H9 hES cells maintained on mEF feeder layers were harvested
from cultures by incubation with .about.200 units/mL collagenase IV
at 37.degree. C. for 10 min. Cells were dissociated and resuspended
in regular hES culture medium or mEF-conditioned medium. Cells in
the regular medium were then re-seeded onto mEF feeder layers and
cells in the mEF-conditioned medium were plated onto Matrigel.RTM.
or laminin. Seeding density for all cultures was approximately
4.times.10.sup.4 cells/cm.sup.2. Cells on feeder layers were
maintained in regular medium while cells on matrices were
maintained in mEF-conditioned medium for 1 or 2 days before the
transfection. Conditioned medium was replaced every 24 h.
[0242] hES cell cultures were transfected with Lipofectamine
2000.TM. as described above. FACS analysis of GFP expression was
conducted as follows. hES cells were harvested using 0.5 mM EDTA in
PBS and resuspended at approximately 1.times.10.sup.6 cells/test.
Cells were washed in a solution containing PBS plus 2% FBS, 0.1%
sodium azide, and 2 mM EDTA. SSEA-4 staining was performed in the
same buffer using antibody obtained from the Developmental Studies
Hybridoma Bank (University of Iowa, Iowa City) at 1:15 dilution.
Isotype matched controls were obtained from Sigma, (St. Louis Mo.,
USA). Cells were incubated with antibodies in a final volume of 100
.mu.l for 30 min at 4.degree. C., washed and incubated with rat
anti-mouse .kappa. chain antibodies conjugated with PE (Becton
Dickinson, San Jose, Calif.) at 4.degree. C. for 30 min. Samples
were washed as before and analyzed for GFP and SSEA-4 expression on
FACScalibur.TM. flow cytometer (Becton Dickinson, San Jose, Calif.)
using CellQuest.TM. software.
[0243] hES cells of the H9 line maintained on laminin in
mEF-conditioned medium were transfected with a plasmid carrying GFP
driven by the CMV promoter at 24 or 48 h after plating. Initial
experiments used a mixture of 5 .mu.g of plasmid and 12 .mu.L of
Lipofectamine 2000.TM.. Cells received 1 mL of DNA/lipid complex
and were incubated for 4 h at 37.degree. before the addition of 3
mL of mEF-conditioned medium, and then monitored for GFP expression
24 h after transfection.
[0244] FIG. 4 shows the results of this experiment. Panel A:
morphology of H9 cells maintained on laminin. Panel B: GFP-positive
cells observed in the same colony shown in A. Panel C: FACS
analysis of % GFP-positive cells in SSEA-4 high
population(undifferentiated cells). Cells were transfected 24 (bar
1 and 2) or 48 h (bar 3 and 4) after the seeding and analyzed 24
(bar 1 and 3) or 48 h (bar 2 and 4) after the transfection. Bright
green cells were observed in compact areas of undifferentiated ES
colonies on laminin 24 h after transfection (Panels A & B).
Transfection at 48 h after initial seeding gave the highest
efficiency: 38% of the cells were GFP-positive as determined by
FACS analysis 24 h after the transfection (Panel C).
[0245] The next experiment compared the transfection efficiency of
H9 cells maintained on Matrigel.RTM. or laminin-coated plates in
mEF-conditioned medium with cells maintained on mEF feeders. Cells
on feeder layers maintained in regular medium were used as a
control. Morphological differences between cells on feeders and
cells off feeders were observed 1 or 2 days after seeding. Colonies
on feeders were more compact than cells maintained off feeder
layers; individual hES cells in feeder-free cultures were less
compact and flatter. There was no significant difference in cell or
colony morphology between cells on laminin and cells on Matrigel.
These cells were transfected with a plasmid expressing GFP driven
by the CMV promoter 2 days after seeding. Twenty-four hours after
the transfection, cells were examined for GFP expression under a
fluorescence microscope.
[0246] The cells were maintained on mEF feeders in regular medium
(mEF/RM), on laminin in medium conditioned by mEF (Laminin/CM) or
on Matrigel.RTM. in the conditioned medium (Matrigel/CM). Bright
green cells were observed in undifferentiated hES colonies of
feeder-free cultures. In contrast, very few green cells were found
in colonies on feeders. FACS analysis showed that 16% of cells on
Matrigel.RTM. and 14% of cells on laminin were GFP positive in
SSEA-4 high population while only 5% of cells on feeders were
positive. These results indicate that transfection efficiency is
significantly increased by using feeder-free conditions.
[0247] The next experiments evaluated the effects of 1) the ratio
of DNA:lipid; 2) adding the DNA/lipid complex to cells 4 h prior to
the addition of mEF-conditioned medium vs. addition of the complex
to cells in the presence of mEF-conditioned medium; and 3) use of
Lipofectamine 2000.TM. vs. FuGENE.TM..
[0248] Transfection using Lipofectamine2000.TM. is described above.
Transfection with FuGENE.TM. was conducted as follows. The plasmid
DNA (5-10 .mu.g of pEGFP-C1, ClonTech cat. # 6084-1) was diluted in
water to a final volume of 100 .mu.l. In pilot experiments, 5-30
.mu.L of FuGENE.TM. were added to sufficient OptiMEM.TM. to achieve
a final volume of 100 .mu.L. The DNA solution was then added slowly
to the FuGENE.TM. solution and mixed gently. The mixture was
incubated at room temperature for 30 min before being supplemented
with 800 .mu.l of OptiMEM.TM.. Cells were washed with 3 mL of
pre-warmed OptiMEM.TM. and incubated in 1 mL of the DNA/lipid
mixture solution at 37.degree. C. for 4 h. In some experiments, at
4 h the wells received an additional 2 mL of mEF-conditioned
medium; in others the DNA/lipid mixture was added to wells
containing 2 mL of mEF-conditioned medium and the cells were
incubated in this mixture overnight.
[0249] Highest efficiencies were obtained under the following
conditions: Bar 1=a mixture of 5 .mu.g plasmid plus 12 .mu.l of
Lipofectamine 2000.TM., adding 1 mL of the DNA/lipid mixture to
wells containing 2.5 mL of mEF-conditioned medium and incubating
the cells in this mixture overnight. Bars 2 & 3=a mixture of 10
.mu.g plasmid plus 15 .mu.l of FuGENE.TM. and incubating the cells
in 1 mL of the DNA/lipid mixture for 4 h before adding 2.5 mL of
mEF-conditioned medium. L=Lipofectamine2000.mu.; F=FuGENE.TM..
[0250] To investigate whether the feeder-free hES cells undergo
stable genetic modification, H1 hES cells maintained on
Matrigel.RTM. were cotransfected with a mixture of 7.5 .mu.g
plasmid carrying .beta.-galactosidase driven by the EF1a promoter,
and 2.5 .mu.g of plasmid carrying the PGK promoter driving the
neophosphotransferase gene. The cells were transfected 48 h after
plating them on Matrigel.RTM. in mEF-conditioned medium. 10 .mu.g
of plasmid plus 15 .mu.l of FuGENE.TM. were incubated with the
cells in 1 mL for 4 h before adding 2.5 mL of mEF-conditioned
medium. After 48 h, medium was exchanged for mEF-conditioned medium
supplemented with 200 .mu.g/mL geneticin. Cultures were maintained
in this geneticin-containing medium with daily medium exchange for
over 21 days. All mock-transfected cultures (i.e., those that
received FuGENE.TM. mixed with water rather than plasmid) died
within 48-72 h. Drug resistant colonies arose in the wells
transfected with both FuGENE.TM. and plasmid at a frequency of
about 1 in to 10.sup.5 originally transfected cells. The colonies
were maintained in geneticin-containing mEF-conditioned medium and
expanded.
Example 9
[0251] Preparation of vectors in which a thymidine kinase gene is
under control of an hTERT promoter sequence
[0252] The lambda clone designated .lambda.G.PHI.5 containing the
hTERT promoter is deposited with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110
U.S.A., under Accession No. 98505. .lambda.G.PHI.5 contains a 15.3
kbp insert including approximately 13,500 bases upstream from the
hTERT coding sequence.
[0253] A Not1 fragment containing the hTERT promoter sequences was
subcloned into the Not1 site of pUC derived plasmid, which was
designated pGRN142. A subclone (plasmid "pGRN140") containing a 9
kb Ncol fragment (with hTERT gene sequence and about 4 to 5 kb of
lambda vector sequence) was partially sequenced to determine the
orientation of the insert. pGRN140 was digested using Sall to
remove lambda vector sequences, the resulting plasmid (with removed
lambda sequences) designated pGRN144. The pGRN144 insert was then
sequenced.
[0254] SEQ. ID NO:1 is a listing of the sequence data obtained.
Nucleotides 1-43 and 15376-15418 are plasmid sequence. Thus, the
genomic insert begins at residue 44 and ends at residue 15375. The
beginning of the cloned cDNA fragment corresponds to residue 13490.
There are Alu sequence elements located .about.1700 base pairs
upstream. The sequence of the hTERT insert of pGRN142 can now be
obtained from GenBank (http://www.ncbi.nlm.nih.gov/) under
Accession PGRN142.INS AF121948. Numbering of hTERT residues for
plasmids in the following description begins from the translation
initiation codon, according to standard practice in the field. The
hTERT ATG codon (the translation initiation site) begins at residue
13545 of SEQ. ID NO:1. Thus, position -1, the first upstream
residue, corresponds to nucleotide 13544 in SEQ. ID NO:1.
[0255] Expression studies were conducted with reporter constructs
comprising various hTERT upstream and intron sequences. A
BgIII-Eco47III fragment from pGRN144 (described above) was digested
and cloned into the BgIII-NruI site of pSEAP2Basic (ClonTech, San
Diego, Calif.) to produce plasmid designated pGRN148. A second
reporter-promoter, plasmid pGRN150 was made by inserting the
BgIII-Fspl fragment from pGRN144 into the BgIII-NruI sites of
pSEAP2. Plasmid pGRN173 was constructed by using the EcoRV-StuI
(from +445 to -2482) fragment from pGRN144. This makes a promoter
reporter plasmid that contains the promoter region of hTERT from
approximately 2.5 kb upstream from the start of the hTERT open
reading frame to just after the first intron within the coding
region, with the initiating Met codon of the hTERT open reading
frame changed to Leu. Plasmid pGRN175 was made by APA1(Klenow
blunt)-SRF1 digestion and religation of pGRN150 to delete most of
the Genomic sequence upstream of hTERT. This makes a
promoter/reporter plasmid that uses 204 nucleotides of hTERT
upstream sequences (from position -36 to -117). Plasmid pGRN176 was
made by PML1-SRF1 religation of pGRN150 to delete most of the hTERT
upstream sequences. This makes a promoter/reporter plasmid that
uses 204 nucleotides of hTERT upstream sequences (from position -36
to -239).
[0256] Levels of secreted placental alkaline phosphatase (SEAP)
activity were detected using the chemiluminescent substrate
CSPD.TM. (ClonTech). SEAP activity detected in the culture medium
was found to be directly proportional to changes in intracellular
concentrations of SEAP mRNA. The pGRN148 and pGRN150 plasmids
(hTERT promoter-reporter) and the pSEAP2 plasmid (positive control,
containing the SV40 early promoter and enhancer) were transfected
into test cell lines. pGRN148 and pGRN150 constructs drove SEAP
expression as efficiently as the pSEAP2 in immortal (tumor-derived)
cell lines. Only the pSEAP2 control gave detectable activity in
mortal cells.
[0257] The ability of the hTERT promoter to specifically drive the
expression of the thymidine kinase (tk) gene in tumor cells was
tested using a variety of constructs: One construct, designated
pGRN266, contains an EcoRI-Fsel PCR fragment with the tk gene
cloned into the EcoRI-Fsel sites of pGRN263. pGRN263, containing
approximately 2.5 kb of hTERT promoter sequence, is similar to
pGRN150, but contains a neomycin gene as selection marker. pGRN267
contains an EcoRI-Fsel PCR fragment with the tk gene cloned into
the EcoRI-Fsel sites of pGRN264. pGRN264, containing approximately
210 bp of hTERT promoter sequence, is similar to pGRN176, but
contains a neomycin gene as selection marker. pGRN268 contains an
EcoRI-XbaI PCR fragment with the tk gene cloned into the EcoRI-XbaI
(unmethylated) sites of pGRN265. pGRN265, containing approximately
90 bp of hTERT promoter sequence, is similar to pGRN175, but
contains a neomycin gene as selection marker.
[0258] These hTERT promoter/tk constructs, pGRN266, pGRN267 and
pGRN268, were re-introduced into mammalian cells and tkl+stable
clones (and/or mass populations) were selected. Ganciclovir
treatment in vitro of the tkl+cells resulted in selective
destruction of all tumor lines tested, including 143B, 293, HT1080,
Bxpc-3', DAOY and NIH3T3. Ganciclovir treatment had no effect on
normal BJ cells.
[0259] FIG. 5 is a map of the TPAC adenovector pGRN376. It was made
by cloning the NOT1-BAMH1 fragment from pGRN267 into the NOT1-BGL2
sites of pAdBN (Quantum Biotech). The 7185 bp vector comprises the
herpes simplex thymidine kinase (TK) gene under control of the
medium-length hTERT promoter sequence.
Example 10
[0260] Transduction of hES cells with a thymidine kinase
construct
[0261] These experiments test the effect of the pGRN376 vector
described in the preceding Example on hES cells. The vector
contains the herpes virus thymidine kinase gene under control of
the telomerase reverse transcriptase promoter. Expression of the
thymidine kinase gene in cells should render them susceptible to
toxicity from the prodrug ganciclovir.
[0262] Undifferentiated H1 cells were plated into 24 well plates (1
confluent well of a 6 well plate split into 24 wells of a 24 well
plate). After 48 h, some wells were infected with the TPAC vector
at an MOI of 30 or 100. Four h after addition of the viral vector,
medium was exchanged for new mouse embryonic fibroblast conditioned
medium (mEF-CM); some wells received medium supplemented with 30
.mu.M ganciclovir (GCV). Cells exposed to GCV were re-fed with
mEF-CM containing 30 .mu.M GCV daily for 4 days. On days 2, 3, and
4 after the initiation of GCV treatment, wells were harvested and
analyzed by flow cytometry to assess changes in 1) total cell
number and 2) cell viability (measured by PI exclusion).
[0263] FIG. 6 shows the results of this experiment. No change in
total cell number was detected at MOI of 30 in the absence of GCV;
but there was some decrease at MOI of 100 in absence of GCV
starting at 48 h. Evidence for toxicity of GCV alone was detected:
wells receiving GCV alone contained approximately 55% as many cells
as the control wells on day 2, diminishing to 40% by day 4. Wells
receiving GRN376 at MOIs of 30 or 100 cultured in the presence of
GCV showed identical results: by day 2, these wells contained 18%
of the cells contained in the control wells, while at days 3 and 4
these wells contained 6% and 8% of the cells in the control
wells.
[0264] Slight toxicity was seen at MOI of 100 at day 4 in the
absence of GCV (50% cells in ES gate vs. 83% for the control
cells). Some toxicity of GCV alone was observed at d2, 75% cells in
ES gate (vs. 85% control); at day 3, 68% (vs. 82% control); at day
4, 50% (vs. 65% control). Wells receiving GRN376 at MOIs of 30 or
100 cultured in the presence of GCV showed similar results: by day
2, these wells contained 24-28% cells in the ES gate, at day 3 they
contained 19-22% cells in the ES gate, and at d4 these wells
contained 12% cells in the ES gate. Thus, GRN376 plus GCV is
effective at killing undifferentiated hES cells at an MOI as low as
30.
[0265] Titration Experiment
[0266] Undifferentiated H1 cells were plated into 24 well plates (1
confluent well of a 6 well plate split into 24 wells of a 24 well
plate). After 48 h, some wells were infected with pGRN376 at an MOI
of 30. Four h after addition of the viral vector, medium was
exchanged for new mEF-CM; some wells received medium supplemented
with 5, 10, 20, 30, or 40 .mu.M ganciclovir (GCV). Cells exposed to
GCV were re-fed with mEF-CM containing GCV daily for 2 days. On day
2 after the initiation of GCV treatment, wells were harvested and
analyzed by flow cytometry to assess changes in total cell number,
and cell viability (measured by PI exclusion).
[0267] FIG. 7 shows the results of this experiment. .about.20 .mu.M
GCV was optimal under the conditions tested.
[0268] Comparison of different hES lines
[0269] Undifferentiated hES of lines designated H1 and H7 cells
were plated into 24 well plates (1 confluent well of a 6 well plate
split into 24 wells of a 24 well plate). After 48 h, some wells
were infected with pGRN376 at an MOI of 30. Four h after addition
of the viral vector, medium was exchanged for new mEF-CM; some
wells received medium supplemented with 20 .mu.M GCV. Cells exposed
to GCV were re-fed with mEF-CM containing GCV daily for 3 days. On
day 4 after the initiation of GCV treatment, wells were harvested
and analyzed by flow cytometry to assess changes in total cell
number.
[0270] FIG. 8 shows the results. The total cell number demonstrated
decreases in cell number for both lines after TPAC vector
treatment. H7 showed less toxicity induced by GCV alone than did
H1. Thus, different hES cell lines respond to the TPAC vector. In
subsequent studies, the H9 cell line was also found to be highly
sensitive to GCV after TPAC vector treatment.
Example 11
[0271] Selection of differentiated cells
[0272] In this experiment, hES cells were treated with retinoic
acid (RA) or dimethyl sulfoxide (DMSO), and then analyzed for hTERT
and OCT-4 expression after treating with TPAC.
[0273] Undifferentiated H1 cells were plated into 24 well plates (1
confluent well of a 6 well plate split into 24 wells of a 24 well
plate). 24 h later, some wells were re-fed with mEF-CM containing
either 500 nM RA or 0.5% DMSO; wells were re-fed with medium
supplemented with RA or DMSO for the remainder of the experiment.
After 7 days of treatment with RA or DMSO, cells were infected with
GRN376 at an MOI of 30.
[0274] Four h after addition of the viral vector, medium was
exchanged for new mEF-CM (plus RA or DMSO where appropriate); some
wells also received medium supplemented with 20 .mu.M ganciclovir
(GCV). Cells exposed to GCV were re-fed with mEF-CM containing GCV
daily for 3 days. On day 3 after the initiation of GCV treatment,
wells were harvested and analyzed by flow cytometry to assess
changes in total cell number. Additional wells were used in an
effort to culture out any remaining undifferentiated stem cells;
the medium of these wells was changed to mEF-CM (without RA, DMSO,
or GCV). Cells were refed with mEF-CM every day for 7 days, then
harvested for isolation of RNA. These samples were analyzed by
quantitative RT-PCR for the expression of hTERT and OCT-4.
[0275] FIG. 9 shows that the cell number decreased after TPAC
treatment. After 7 days of drug pretreatment followed by TPAC plus
GCV, all wells contained similar cell numbers. During the attempt
to culture out surviving stem cells, the wells became confluent
with highly differentiated appearing cells; no undifferentiated hES
cells were obvious. Wells containing cells that had been
pre-treated with RA were distinct in appearance from the cells
either pre-treated with unadulterated mEF-CM or treated with mEF-CM
plus DMSO. RT-PCR analysis (Lower Panel, non-quantitative, 35
cycles) showed that the surviving cells from the mEF-CM or
DMSO-treated wells had no detectable OCT-4 expression, while 2 out
of 4 RA-pre-treated samples presented very weak OCT-4 PCR
products.
[0276] Thus, no detectable undifferentiated cells survive TPAC
treatment followed by subsequent culture of wells grown in mEF-CM
or mEF-CM plus DMSO. RA pre-treatment leads to detection of low
levels of OCT-4 in the surviving cells. It is not clear whether
this reflects persistence of undifferentiated stem cells or
induction of another cell type that expresses OCT-4.
Example 12
[0277] Stable stem cell lines containing the TPAC construct:
[0278] To facilitate creation of stable cell lines genetically
altered with the hTERT promoter/thymidine kinase construct, plasmid
pGRN376 (Example 9) was modified to delete most of the adenovirus
sequence. The plasmid was digested with StuI and Not I, followed by
blunting and re-ligation. The remaining vector contained the hTERT
promoter/thymidine kinase construct, preceded by the transcription
blocking sequence to prevent promotion of tk gene expression by
promoter sequences upstream from the integration site in the
genome. The modified vector was designated pGRN376mod.
2 pGRN376 - adenoviral T.sub.PAC vector Ad5 TRM pTERT HSV TK SV40
216 bp polyA pGRN376mod - T.sub.PAC plasmid vector TRM pTERT HSV TK
SV40 216 bp polyA
[0279] Cultures of the H9 hES cell line were split 1:4 using PBS
containing 0.5 mM EDTA, as described earlier, and plated in
feeder-free culture in 6-well plates. Twenty-four hrs after
plating, cells were cotransfected with 2 .mu.g GRN376mod plus 0.5
.mu.g of a plasmid encoding neomycin phosphotransferase, using
FuGENE.TM.-6 as described above. Medium containing 200 .mu.g/mL
geneticin was added 48 h after transfection to select out
transfected cells. After 7-10 days in geneticin-containing medium,
103 individual colonies were picked from the wells and individually
plated in 24-well plates, which were then expanded by culturing for
.about.7 days. Ninety individual clones were screened for
sensitivity to the pro-drug ganciclovir at a concentration of 30
.mu.M. Ten clones were identified in which essentially all the
undifferentiated cells died within 1-3 days of culture with
ganciclovir, leaving only remnant cells that had differentiated
outside the main colony.
[0280] In another experiment, the H1 line of hES cells (passage 59)
was split 1:6 into Matrigel.RTM.-coated 24 well plates in standard
hES cell medium (day 0; 1 ml/well, MEF conditioned medium with 4
ng/mL bFGF). The medium was exchanged with new standard medium on
the following day. On day 3, the medium was removed and replaced
with either 200 .mu.L/well standard medium (mock transfection), or
pGRN376 virus at MOI 100, diluted in 200 .mu.L/well standard hES
medium. After 4 h, the medium was removed from all wells and
replaced with 1 mg/mL standard hES medium with or without 30 .mu.M
ganciclovir. The wells were exchanged with fresh medium (with or
without 30 .mu.M ganciclovir) on each of the next three days.
[0281] FIG. 10 is a micrograph of the cells on day 6. In wells
transduced with control vector (Panel A), hES colonies formed
colonies with normal characteristics. In wells transduced with
pGRN376 virus and then treated with ganciclovir (Panel B), most or
all ES cell colonies are gone and only differentiated cells remain.
The TPAC-treated wells contained 8-fold fewer cells than the
control wells.
Example 13
[0282] Further Characterization of Stable TPAC Embryonic Stem Cell
Lines
[0283] TPAC stable clones were derived by co-transfection of
pGRN376mod and pGK-neo plasmids into the hES cell line H9 grown in
mEF conditioned media with basic FGF on growth factor reduced
Matrigel.RTM. coated plates.
[0284] In one experiment, the H9 line (p 80) was passaged 6 times
using 0.5 mM EDTA prior to transfection. 2 .mu.g of pGRN376m
plasmid and 0.5 .mu.g of pGK-neo plasmid were used for transfection
of each well of a 6 well plate of ES cells, using a 3:2 ratio of
Fugene.TM. (a lipid-based transfection facilitator) to DNA. The
cells were transfected 24 hours after seeding, and G418 selection
was initiated 24 h post-transfection. 101 G418-resistant colonies
were picked using collagenase. Clones from cells transfected only
with pGK-neo were isolated as controls for co-transfection.
[0285] Individual colonies were expanded using 0.5 mM EDTA for
primary and secondary GCV screening. Primary screen with 30 .mu.M
GCV (weeks 1 and 2) identified 10 GCV-sensitive clones and 20
partially GCV-sensitive clones. Secondary screen with 30 .mu.M GCV
(weeks 3 and 4) confirmed 9 GCV-sensitive clones, including those
designated H9-376m-18, H9-376m-77, and H9-376m-62. Following
secondary GCV screening, cells were expanded using 0.5 mM EDTA into
larger cultures and then passaged using collagenase.
[0286] In a second experiment, the H9 line (p 24) was again
initially passed using 0.5 mM EDTA, and then transfected as before.
Sixty two G418-resistant co-transfected colonies were picked using
collagenase on day 0. Primary screen with 30 .mu.M GCV (days 20,
22, and 26) identified one GCV-sensitive clone and four partially
GCV-sensitive clones. Following primary GCV screening, cells were
expanded using 0.5 mM EDTA into larger cultures and gradually
returned to standard collagenase passaging. Secondary screen with
30 .mu.M GCV (day 57) confirmed one GCV-sensitive clones
(designated H9-376m-6).
[0287] The GCV-sensitive clones from each of these experiments were
subject to further selection in culture with G418, to determine
their stability. Clones designated H9-376m-18 and H9-376m-77 showed
G418 resistance that was not stable. Clones designated H9-376m-62,
H9-376m-6, and H9-pGK-neo-1 were G418 resistant.
[0288] FIG. 11 (top) shows sensitivity of the stable TPAC lines to
ganciclovir (GCV). Each cell line was exposed to decreasing amount
of GCV to determine lowest concentration of GCV required to produce
complete killing of undifferentiated ES cells with the least amount
of toxicity. Concentrations as low as 0.5 .mu.M were determined to
kill undifferentiated ES cells similar to 30 .mu.M, but with a
significantly lower toxicity.
[0289] FIG. 11 (bottom) shows sensitivity of the TPAC lines to a
different prodrug, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU).
Although a significant decrease on cell counts was observed with
all BVDU concentrations tested, ES cell killing was not actually
observed. The number of cells present even at high concentrations
of BVDU is not reduced to the level of the blank control.
[0290] To verify expression of HSV-TK in stable TPAC ES cell lines,
RT PCR was performed on undifferentiated cells from H9-376m-6,
H9-376m-62 and H9-pGK-neo-1 cultures using HSV-TK or OCT-4 specific
primers. TK expression was detected in H9-376m-6 and H9-376m-62
samples and not in H9-pGK-neo samples at high concentrations of
RNA, while OCT-4 expression was detected in all samples.
Example 14
[0291] Promoter specificity for undifferentiated cells
[0292] To determine hTERT promoter specificity on in the stable
TPAC clones, GCV sensitivity was determined after differentiation
of each TPAC clone and the pGK-neo control clone.
[0293] Undifferentiated cells were seeded in growth factor reduced
Matrigel.RTM. coated plates with KO DMEM plus 20% Hyclone FBS
without bFGF (differentiation conditions). Cultures were maintained
in differentiation conditions for 7 days, passaged and placed in
the differentiation conditions for additional 4 days. Following
this treatment, cultures contained few if any undifferentiated
cells (assessed by morphology). At this point, 30 .mu.M GCV was
added to the differentiated cultures for 4 days.
[0294] There was no sensitivity to GCV observed in differentiated
cultures obtained from the TPAC cell lines H9-376m-6, H9-376m-62,
H9-376m-18, or H9-376m-77; or from the transfection control line
H9-pGKneo. Thus, the hTERT promoter in the integrated construct has
the appropriate specificity for turning on the TK effector gene
only in undifferentiated cells.
Example 15
[0295] ES marker expression in undifferentiated TPAC cell lines
[0296] Expression of ES markers on stable TPAC ES cell lines was
determined by flow cytometry. Cells were harvested from confluent
cultures using 0.5 mM EDTA and incubated with monoclonal antibodies
against human SSEA-4, SSEA-1, Tra-1-60, Tra-1-81, CD9, AC133, and
appropriate fluorochrome-conjugated secondary antibodies. Level of
expression was determined using FACSCalibur.TM.. Appropriate
isotype matched negative controls and viability assessment were
used to determine the level of non-specific binding.
3TABLE 2 Markers on Parental hES cells and Stable T.sub.PAC Lines
Pheno- H9-376m- H9-pGK- H9-376m- H9-376m- typic H9 62 H9 neo 6 62
marker p 35 p 80 + 15 p 25 p 24 + 16 p 24 + 19 p 80 + 27 SSEA4+ 92*
100 64 37 91 98 SSEA1+ 1 0 0 0 0 0 Tra-1-60+ 98 99 89 89 86 90
Tra-1-81+ 96 99 82 88 77 97 CD9+ 88 95 81 98 91 99 AC133+ 90 81 72
78 68 70 *% of live cells expressing markers. H9-376m-6, H9-376m-62
and H9-pGK-neo-1 cell lines have expression of all tested ES
markers at the levels comparable to those of untransfected H9 cell
line.
Example 16
[0297] Ability of TPAC cell lines to undergo appropriate
differentiation
[0298] H9-376m-6, H9-376m-62, H9-376m-77, H9-376m-18 and
H9-pGK-neo-1 were tested for their ability to generate cells from
three embryonic germ lineages. Embryoid bodies (EBs) were
established from each cell line and cultured in suspension with KO
DMEM and 20% Hyclone FBS. Following 4 day culture, EBs were plated
on gelatin-coated chamber slides and allowed to grow for additional
8-10 days. Cultures were scored for the presence of beating cells
and stained for the presence of .beta.-tubulin, AFP,
muscle-specific actin and cardiac troponin I positive cells.
[0299] FIG. 12 shows representative fluorescence micrographs of
immunocytochemistry. Results were as follows.
[0300] H9-376m-18 p 80+20 was able to generate large embryoid
bodies (1 experiment). Beating areas (consistent with
cardiomyocyte-lineage cells) were observed. Muscle-Specific
Actin+++; .alpha.-fetoprotein +++; .beta.-tubulin +++; cardiac
troponin I +.
[0301] H9-376m-77 p 80+16 generated only small embryoid bodies (1
experiment). No beating areas were observed. Muscle-Specific Actin
+; .alpha.-fetoprotein +.beta.---tubulin -(none detected); cardiac
troponin I-.
[0302] H9-376m-62 p 80+16, p 80+20, p 80+21, p 80+24 (4
experiments) were capable of generating only small embryoid bodies.
No beating areas were observed. Muscle-Specific Actin ++;
.alpha.-fetoprotein +; .beta.-tubulin +; cardiac troponin I -.
[0303] H9-376m-6 p 24+12, p 24+13, p 24+21 (3 experiments) were
able to generate large embryoid bodies. Many beating areas were
observed in all experiments. Muscle-Specific Actin ++;
.alpha.-fetoprotein ++; .beta.-tubulin ++++; cardiac troponin I
+.
[0304] H9-pGK-neo-1 p 24+9, p 24+10, p 24+13 (3 experiments) were
able to generate large embryoid bodies. Beating areas were observed
in 2 out of 3 experiments. .beta.-tubulin +++; cardiac troponin I
+; Muscle-Specific Actin ++++; .alpha.-fetoprotein ++.
[0305] The results of Examples 13-16 show that at least three of
the stem cell lines containing the telomerase promoter driven
thymidine kinase gene are capable of differentiating into cells of
each of the three germ layers. Differentiated cells from these
lines contain an important stop-gap against residual or reemerging
undifferentiated cells. Ganciclovir at a concentration as low as
2.5 .mu.M kills virtually all such modified undifferentiated ES
cells within .about.4 days.
4TABLE 3 SEQUENCE DATA Sequences listed in this Disclosure SEQ. ID.
NO: Designation Reference 1 Lambda clone designated .lambda.G.phi.5
GenBank Accession AF121948 (ATCC Accession No. 98505) International
Patent Publication Contains human Telomerase WO 00/46355. Reverse
Transcriptase (hTERT) genomic insert (residues 44-15375). The ATG
translation initiation site begins at residue 13545. 2 Herpes
simplex virus type 1 GenBank Accession J02224 thymidine kinase and
3 KBL See also McKnight et al., gene sequence Nucleic Acids Res.
8:5949 (1980); Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441
(1981) 3 Herpes simplex virus type 1 (supra) thymidine kinase and 3
KBL amino acid sequence 4-7 Probes and Primers (Artificial
Sequences)
[0306] It will be recognized that the compositions and procedures
provided in the description can be effectively modified by those
skilled in the art without departing from the spirit of the
invention embodied in the claims that follow.
Sequence CWU 1
1
7 1 15418 DNA Homo sapiens 1 gcggccgcga gctctaatac gactcactat
agggcgtcga ctcgatcaat ggaagatgag 60 gcattgccga agaaaagatt
aatggatttg aacacacagc aacagaaact acatgaagtg 120 aaacacagga
aaaaaaagat aaagaaacga aaagaaaagg gcatcagtga gcttcagcag 180
aagttccatc ggccttacat atgtgtaagc agaggccctg taggagcaga ggcaggggga
240 aaatacttta agaaataatg tctaaaagtt tttcaaatat gaggaaaaac
ataaaaccac 300 agatccaaga agctcaacaa aacaaagcac aagaaacagg
aagaaattaa aagttatatc 360 acagtcaaat tgctgaaaac cagcaacaaa
gagaatatct taagagtatc agaggaaaag 420 agattaatga caggccaaga
aacaatgaaa acaatacaga tttcttgtag gaaacacaag 480 acaaaagaca
ttttttaaaa ccaaaaggaa aaaaaatgct acattaaaat gttttttacc 540
cactgaaagt atatttcaaa acatatttta ggccaggctt ggtggctcac acctgtaatc
600 ccagcacttt gggaggccaa ggtgggtgga tcgcttaagg tcaggagttc
gagaccagcc 660 tggccaatat agcgaaaccc catctgtact aaaaacacaa
aaattagctg ggtgtggtga 720 cacatgcctg taatcccagg tactcaggag
gctaaggcag gagaattgct tgaactggga 780 ggcagaggtg gtgagccaag
attgcaccag tgcactccag ccttggtgac agagtgaaac 840 tccatctcaa
aaacaaacaa acaaaataca tatacataaa tatatatgca catatatata 900
catatataaa tatatataca catatataaa tctatataca tatatacata tatacacata
960 tataaatcta tatacatata tatacatata taatatattt acatatataa
atatatacat 1020 atataaatat acatatataa atacatatat aaatatacat
atataaatat acatatataa 1080 atatacatat ataaatatat acatatataa
atatacatat ataaatatat atacatatat 1140 aaatatataa atatacaagt
atatacaaat atatacatat ataaatgtat atacgtatat 1200 acatatatat
ataaatatat aaaaaaactt ttggctgggc acctttccaa atctcatggc 1260
acatataagt ctcatggtaa cctcaaataa aaaaacatat aacagataca ccaaaaataa
1320 aaaccaataa attaaatcat gccaccagaa gaaattacct tcactaaaag
gaacacagga 1380 aggaaagaaa gaaggaagag aagaccatga aacaaccaga
aaacaaacaa caaaacagca 1440 ggagtaattc ctgacttatc aataataatg
ctgggtgtaa atggactaaa ctctccaatc 1500 aaaagacata gagtggctga
atggacgaaa aaaacaagac tcaataatct gttgcctaca 1560 agaatatact
tcacctataa agggacacat agactgaaaa taaaaggaag gaaaaatatt 1620
ctatgcaaat ggaaaccaaa aaaagaacag aactagctac acttatatca gacaaaatag
1680 atttcaagac aaaaagtaca aaaagagaca aagtaattat ataataataa
agcaaaaaga 1740 tataacaatt gtgaatttat atgcgcccaa cactgggaca
cccagatata tacagcaaat 1800 attattagaa ctaaggagag agagagatcc
ccatacaata atagctggag acttcacccc 1860 gcttttagca ttggacagat
catccagaca gaaaatcaac caaaaaattg gacttaatct 1920 ataatataga
acaaatgtac ctaattgatg tttacaagac atttcatcca gtagttgcag 1980
aatatgcatt ttttcctcag catatggatc attctcaagg atagaccata tattaggcca
2040 cagaacaagc cattaaaaat tcaaaaaaat tgagccaggc atgatggctt
atgcttgtaa 2100 ttacagcact ttggggaggg tgaggtggga ggatgtcttg
agtacaggag tttgagacca 2160 gcctgggcaa aatagtgaga ccctgtctct
acaaactttt ttttttaatt agccaggcat 2220 agtggtgtgt gcctgtagtc
ccagctactt aggaggctga agtgggagga tcacttgagc 2280 ccaagagttc
aaggctacgg tgagccatga ttgcaacacc acacaccagc cttggtgaca 2340
gaatgagacc ctgtctcaaa aaaaaaaaaa aaaattgaaa taatataaag catcttctct
2400 ggccacagtg gaacaaaacc agaaatcaac aacaagagga attttgaaaa
ctatacaaac 2460 acatgaaaat taaacaatat acttctgaat aaccagtgag
tcaatgaaga aattaaaaag 2520 gaaattgaaa aatttattta agcaaatgat
aacggaaaca taacctctca aaacccacgg 2580 tatacagcaa aagcagtgct
aagaaggaag tttatagcta taagcagcta catcaaaaaa 2640 gtagaaaagc
caggcgcagt ggctcatgcc tgtaatccca gcactttggg aggccaaggc 2700
gggcagatcg cctgaggtca ggagttcgag accagcctga ccaacacaga gaaaccttgt
2760 cgctactaaa aatacaaaat tagctgggca tggtggcaca tgcctgtaat
cccagctact 2820 cgggaggctg aggcaggata accgcttgaa cccaggaggt
ggaggttgcg gtgagccggg 2880 attgcgccat tggactccag cctgggtaac
aagagtgaaa ccctgtctca agaaaaaaaa 2940 aaaagtagaa aaacttaaaa
atacaaccta atgatgcacc ttaaagaact agaaaagcaa 3000 gagcaaacta
aacctaaaat tggtaaaaga aaagaaataa taaagatcag agcagaaata 3060
aatgaaactg aaagataaca atacaaaaga tcaacaaaat taaaagttgg ttttttgaaa
3120 agataaacaa aattgacaaa cctttgccca gactaagaaa aaaggaaaga
agacctaaat 3180 aaataaagtc agagatgaaa aaagagacat tacaactgat
accacagaaa ttcaaaggat 3240 cactagaggc tactatgagc aactgtacac
taataaattg aaaaacctag aaaaaataga 3300 taaattccta gatgcataca
acctaccaag attgaaccat gaagaaatcc aaagcccaaa 3360 cagaccaata
acaataatgg gattaaagcc ataataaaaa gtctcctagc aaagagaagc 3420
ccaggaccca atggcttccc tgctggattt taccaatcat ttaaagaaga atgaattcca
3480 atcctactca aactattctg aaaaatagag gaaagaatac ttccaaactc
attctacatg 3540 gccagtatta ccctgattcc aaaaccagac aaaaacacat
caaaaacaaa caaacaaaaa 3600 aacagaaaga aagaaaacta caggccaata
tccctgatga atactgatac aaaaatcctc 3660 aacaaaacac tagcaaacca
aattaaacaa caccttcgaa agatcattca ttgtgatcaa 3720 gtgggattta
ttccagggat ggaaggatgg ttcaacatat gcaaatcaat caatgtgata 3780
catcatccca acaaaatgaa gtacaaaaac tatatgatta tttcacttta tgcagaaaaa
3840 gcatttgata aaattctgca cccttcatga taaaaaccct caaaaaacca
ggtatacaag 3900 aaacatacag gccaggcaca gtggctcaca cctgcgatcc
cagcactctg ggaggccaag 3960 gtgggatgat tgcttgggcc caggagtttg
agactagcct gggcaacaaa atgagacctg 4020 gtctacaaaa aactttttta
aaaaattagc caggcatgat ggcatatgcc tgtagtccca 4080 gctagtctgg
aggctgaggt gggagaatca cttaagccta ggaggtcgag gctgcagtga 4140
gccatgaaca tgtcactgta ctccagccta gacaacagaa caagacccca ctgaataaga
4200 agaaggagaa ggagaaggga gaaaggaggg agaagggagg aggaggagaa
ggaggaggtg 4260 gaggagaagt ggaaggggaa ggggaaggga aagaggaaga
agaagaaaca tatttcaaca 4320 taataaaagc cctatatgac agaccgaggt
agtattatga ggaaaaactg aaagcctttc 4380 ctctaagatc tggaaaatga
caagggccca ctttcaccac tgtgattcaa catagtacta 4440 gaagtcctag
ctagagcaat cagataagag aaagaaataa aaggcatcca aactggaaag 4500
gaagaagtca aattatcctg tttgcagatg atatgatctt atatctggaa aagacttaag
4560 acaccactaa aaaactatta gagctgaaat ttggtacagc aggatacaaa
atcaatgtac 4620 aaaaatcagt agtatttcta tattccaaca gcaaacaatc
tgaaaaagaa accaaaaaag 4680 cagctacaaa taaaattaaa cagctaggaa
ttaaccaaag aagtgaaaga tctctacaat 4740 gaaaactata aaatattgat
aaaagaaatt gaagagggca caaaaaaaga aaagatattc 4800 catgttcata
gattggaaga ataaatactg ttaaaatgtc catactaccc aaagcaattt 4860
acaaattcaa tgcaatccct attaaaatac taatgacgtt cttcacagaa atagaagaaa
4920 caattctaag atttgtacag aaccacaaaa gacccagaat agccaaagct
atcctgacca 4980 aaaagaacaa aactggaagc atcacattac ctgacttcaa
attatactac aaagctatag 5040 taacccaaac tacatggtac tggcataaaa
acagatgaga catggaccag aggaacagaa 5100 tagagaatcc agaaacaaat
ccatgcatct acagtgaact catttttgac aaaggtgcca 5160 agaacatact
ttggggaaaa gataatctct tcaataaatg gtgctggagg aactggatat 5220
ccatatgcaa aataacaata ctagaactct gtctctcacc atatacaaaa gcaaatcaaa
5280 atggatgaaa ggcttaaatc taaaacctca aactttgcaa ctactaaaag
aaaacaccgg 5340 agaaactctc caggacattg gagtgggcaa agacttcttg
agtaattccc tgcaggcaca 5400 ggcaaccaaa gcaaaaacag acaaatggga
tcatatcaag ttaaaaagct tctgcccagc 5460 aaaggaaaca atcaacaaag
agaagagaca acccacagaa tgggagaata tatttgcaaa 5520 ctattcatct
aacaaggaat taataaccag tatatataag gagctcaaac tactctataa 5580
gaaaaacacc taataagctg attttcaaaa ataagcaaaa gatctgggta gacatttctc
5640 aaaataagtc atacaaatgg caaacaggca tctgaaaatg tgctcaacac
cactgatcat 5700 cagagaaatg caaatcaaaa ctactatgag agatcatctc
accccagtta aaatggcttt 5760 tattcaaaag acaggcaata acaaatgcca
gtgaggatgt ggataaaagg aaacccttgg 5820 acactgttgg tgggaatgga
aattgctacc actatggaga acagtttgaa agttcctcaa 5880 aaaactaaaa
ataaagctac catacagcaa tcccattgct aggtatatac tccaaaaaag 5940
ggaatcagtg tatcaacaag ctatctccac tcccacattt actgcagcac tgttcatagc
6000 agccaaggtt tggaagcaac ctcagtgtcc atcaacagac gaatggaaaa
agaaaatgtg 6060 gtgcacatac acaatggagt actacgcagc cataaaaaag
aatgagatcc tgtcagttgc 6120 aacagcatgg ggggcactgg tcagtatgtt
aagtgaaata agccaggcac agaaagacaa 6180 acttttcatg ttctccctta
cttgtgggag caaaaattaa aacaattgac atagaaatag 6240 aggagaatgg
tggttctaga ggggtggggg acagggtgac tagagtcaac aataatttat 6300
tgtatgtttt aaaataacta aaagagtata attgggttgt ttgtaacaca aagaaaggat
6360 aaatgcttga aggtgacaga taccccattt accctgatgt gattattaca
cattgtatgc 6420 ctgtatcaaa atatctcatg tatgctatag atataaaccc
tactatatta aaaattaaaa 6480 ttttaatggc caggcacggt ggctcatgtc
cataatccca gcactttggg aggccgaggc 6540 ggtggatcac ctgaggtcag
gagtttgaaa ccagtctggc caccatgatg aaaccctgtc 6600 tctactaaag
atacaaaaat tagccaggcg tggtggcaca tacctgtagt cccaactact 6660
caggaggctg agacaggaga attgcttgaa cctgggaggc ggaggttgca gtgagccgag
6720 atcatgccac tgcactgcag cctgggtgac agagcaagac tccatctcaa
aacaaaaaca 6780 aaaaaaagaa gattaaaatt gtaattttta tgtaccgtat
aaatatatac tctactatat 6840 tagaagttaa aaattaaaac aattataaaa
ggtaattaac cacttaatct aaaataagaa 6900 caatgtatgt ggggtttcta
gcttctgaag aagtaaaagt tatggccacg atggcagaaa 6960 tgtgaggagg
gaacagtgga agttactgtt gttagacgct catactctct gtaagtgact 7020
taattttaac caaagacagg ctgggagaag ttaaagaggc attctataag ccctaaaaca
7080 actgctaata atggtgaaag gtaatctcta ttaattacca ataattacag
atatctctaa 7140 aatcgagctg cagaattggc acgtctgatc acaccgtcct
ctcattcacg gtgctttttt 7200 tcttgtgtgc ttggagattt tcgattgtgt
gttcgtgttt ggttaaactt aatctgtatg 7260 aatcctgaaa cgaaaaatgg
tggtgatttc ctccagaaga attagagtac ctggcaggaa 7320 gcaggtggct
ctgtggacct gagccacttc aatcttcaag ggtctctggc caagacccag 7380
gtgcaaggca gaggcctgat gacccgagga caggaaagct cggatgggaa ggggcgatga
7440 gaagcctgcc tcgttggtga gcagcgcatg aagtgccctt atttacgctt
tgcaaagatt 7500 gctctggata ccatctggaa aaggcggcca gcgggaatgc
aaggagtcag aagcctcctg 7560 ctcaaaccca ggccagcagc tatggcgccc
acccgggcgt gtgccagagg gagaggagtc 7620 aaggcacctc gaagtatggc
ttaaatcttt ttttcacctg aagcagtgac caaggtgtat 7680 tctgagggaa
gcttgagtta ggtgccttct ttaaaacaga aagtcatgga agcacccttc 7740
tcaagggaaa accagacgcc cgctctgcgg tcatttacct ctttcctctc tccctctctt
7800 gccctcgcgg tttctgatcg ggacagagtg acccccgtgg agcttctccg
agcccgtgct 7860 gaggaccctc ttgcaaaggg ctccacagac ccccgccctg
gagagaggag tctgagcctg 7920 gcttaataac aaactgggat gtggctgggg
gcggacagcg acggcgggat tcaaagactt 7980 aattccatga gtaaattcaa
cctttccaca tccgaatgga tttggatttt atcttaatat 8040 tttcttaaat
ttcatcaaat aacattcagg agtgcagaaa tccaaaggcg taaaacagga 8100
actgagctat gtttgccaag gtccaaggac ttaataacca tgttcagagg gatttttcgc
8160 cctaagtact ttttattggt tttcataagg tggcttaggg tgcaagggaa
agtacacgag 8220 gagaggactg ggcggcaggg ctatgagcac ggcaaggcca
ccggggagag agtccccggc 8280 ctgggaggct gacagcagga ccactgaccg
tcctccctgg gagctgccac attgggcaac 8340 gcgaaggcgg ccacgctgcg
tgtgactcag gaccccatac cggcttcctg ggcccaccca 8400 cactaaccca
ggaagtcacg gagctctgaa cccgtggaaa cgaacatgac ccttgcctgc 8460
ctgcttccct gggtgggtca agggtaatga agtggtgtgc aggaaatggc catgtaaatt
8520 acacgactct gctgatgggg accgttcctt ccatcattat tcatcttcac
ccccaaggac 8580 tgaatgattc cagcaacttc ttcgggtgtg acaagccatg
acaacactca gtacaaacac 8640 cactctttta ctaggcccac agagcacggc
ccacacccct gatatattaa gagtccagga 8700 gagatgaggc tgctttcagc
caccaggctg gggtgacaac agcggctgaa cagtctgttc 8760 ctctagacta
gtagaccctg gcaggcactc ccccagattc tagggcctgg ttgctgcttc 8820
ccgagggcgc catctgccct ggagactcag cctggggtgc cacactgagg ccagccctgt
8880 ctccacaccc tccgcctcca ggcctcagct tctccagcag cttcctaaac
cctgggtggg 8940 ccgtgttcca gcgctactgt ctcacctgtc ccactgtgtc
ttgtctcagc gacgtagctc 9000 gcacggttcc tcctcacatg gggtgtctgt
ctccttcccc aacactcaca tgcgttgaag 9060 ggaggagatt ctgcgcctcc
cagactggct cctctgagcc tgaacctggc tcgtggcccc 9120 cgatgcaggt
tcctggcgtc cggctgcacg ctgacctcca tttccaggcg ctccccgtct 9180
cctgtcatct gccggggcct gccggtgtgt tcttctgttt ctgtgctcct ttccacgtcc
9240 agctgcgtgt gtctctgtcc gctagggtct cggggttttt ataggcatag
gacgggggcg 9300 tggtgggcca gggcgctctt gggaaatgca acatttgggt
gtgaaagtag gagtgcctgt 9360 cctcacctag gtccacgggc acaggcctgg
ggatggagcc cccgccaggg acccgccctt 9420 ctctgcccag cacttttctg
cccccctccc tctggaacac agagtggcag tttccacaag 9480 cactaagcat
cctcttccca aaagacccag cattggcacc cctggacatt tgccccacag 9540
ccctgggaat tcacgtgact acgcacatca tgtacacact cccgtccacg accgaccccc
9600 gctgttttat tttaatagct acaaagcagg gaaatccctg ctaaaatgtc
ctttaacaaa 9660 ctggttaaac aaacgggtcc atccgcacgg tggacagttc
ctcacagtga agaggaacat 9720 gccgtttata aagcctgcag gcatctcaag
ggaattacgc tgagtcaaaa ctgccacctc 9780 catgggatac gtacgcaaca
tgctcaaaaa gaaagaattt caccccatgg caggggagtg 9840 gttggggggt
taaggacggt gggggcagca gctgggggct actgcacgca ccttttacta 9900
aagccagttt cctggttctg atggtattgg ctcagttatg ggagactaac cataggggag
9960 tggggatggg ggaacccgga ggctgtgcca tctttgccat gcccgagtgt
cctgggcagg 10020 ataatgctct agagatgccc acgtcctgat tcccccaaac
ctgtggacag aacccgcccg 10080 gccccagggc ctttgcaggt gtgatctccg
tgaggaccct gaggtctggg atccttcggg 10140 actacctgca ggcccgaaaa
gtaatccagg ggttctggga agaggcgggc aggagggtca 10200 gaggggggca
gcctcaggac gatggaggca gtcagtctga ggctgaaaag ggagggaggg 10260
cctcgagccc aggcctgcaa gcgcctccag aagctggaaa aagcggggaa gggaccctcc
10320 acggagcctg cagcaggaag gcacggctgg cccttagccc accagggccc
atcgtggacc 10380 tccggcctcc gtgccatagg agggcactcg cgctgccctt
ctagcatgaa gtgtgtgggg 10440 atttgcagaa gcaacaggaa acccatgcac
tgtgaatcta ggattatttc aaaacaaagg 10500 tttacagaaa catccaagga
cagggctgaa gtgcctccgg gcaagggcag ggcaggcacg 10560 agtgatttta
tttagctatt ttattttatt tacttacttt ctgagacaga gttatgctct 10620
tgttgcccag gctggagtgc agcggcatga tcttggctca ctgcaacctc cgtctcctgg
10680 gttcaagcaa ttctcgtgcc tcagcctccc aagtagctgg gatttcaggc
gtgcaccacc 10740 acacccggct aattttgtat ttttagtaga gatgggcttt
caccatgttg gtcaggctga 10800 tctcaaaatc ctgacctcag gtgatccgcc
cacctcagcc tcccaaagtg ctgggattac 10860 aggcatgagc cactgcacct
ggcctattta accattttaa aacttccctg ggctcaagtc 10920 acacccactg
gtaaggagtt catggagttc aatttcccct ttactcagga gttaccctcc 10980
tttgatattt tctgtaattc ttcgtagact ggggatacac cgtctcttga catattcaca
11040 gtttctgtga ccacctgtta tcccatggga cccactgcag gggcagctgg
gaggctgcag 11100 gcttcaggtc ccagtggggt tgccatctgc cagtagaaac
ctgatgtaga atcagggcgc 11160 gagtgtggac actgtcctga atctcaatgt
ctcagtgtgt gctgaaacat gtagaaatta 11220 aagtccatcc ctcctactct
actgggattg agccccttcc ctatcccccc ccaggggcag 11280 aggagttcct
ctcactcctg tggaggaagg aatgatactt tgttattttt cactgctggt 11340
actgaatcca ctgtttcatt tgttggtttg tttgttttgt tttgagaggc ggtttcactc
11400 ttgttgctca ggctggaggg agtgcaatgg cgcgatcttg gcttactgca
gcctctgcct 11460 cccaggttca agtgattctc ctgcttccgc ctcccatttg
gctgggatta caggcacccg 11520 ccaccatgcc cagctaattt tttgtatttt
tagtagagac gggggtgggg gtggggttca 11580 ccatgttggc caggctggtc
tcgaacttct gacctcagat gatccacctg cctctgcctc 11640 ctaaagtgct
gggattacag gtgtgagcca ccatgcccag ctcagaattt actctgttta 11700
gaaacatctg ggtctgaggt aggaagctca ccccactcaa gtgttgtggt gttttaagcc
11760 aatgatagaa tttttttatt gttgttagaa cactcttgat gttttacact
gtgatgacta 11820 agacatcatc agcttttcaa agacacacta actgcaccca
taatactggg gtgtcttctg 11880 ggtatcagcg atcttcattg aatgccggga
ggcgtttcct cgccatgcac atggtgttaa 11940 ttactccagc ataatcttct
gcttccattt cttctcttcc ctcttttaaa attgtgtttt 12000 ctatgttggc
ttctctgcag agaaccagtg taagctacaa cttaactttt gttggaacaa 12060
attttccaaa ccgccccttt gccctagtgg cagagacaat tcacaaacac agccctttaa
12120 aaaggcttag ggatcactaa ggggatttct agaagagcga cccgtaatcc
taagtattta 12180 caagacgagg ctaacctcca gcgagcgtga cagcccaggg
agggtgcgag gcctgttcaa 12240 atgctagctc cataaataaa gcaatttcct
ccggcagttt ctgaaagtag gaaaggttac 12300 atttaaggtt gcgtttgtta
gcatttcagt gtttgccgac ctcagctaca gcatccctgc 12360 aaggcctcgg
gagacccaga agtttctcgc cccttagatc caaacttgag caacccggag 12420
tctggattcc tgggaagtcc tcagctgtcc tgcggttgtg ccggggcccc aggtctggag
12480 gggaccagtg gccgtgtggc ttctactgct gggctggaag tcgggcctcc
tagctctgca 12540 gtccgaggct tggagccagg tgcctggacc ccgaggctgc
cctccaccct gtgcgggcgg 12600 gatgtgacca gatgttggcc tcatctgcca
gacagagtgc cggggcccag ggtcaaggcc 12660 gttgtggctg gtgtgaggcg
cccggtgcgc ggccagcagg agcgcctggc tccatttccc 12720 accctttctc
gacgggaccg ccccggtggg tgattaacag atttggggtg gtttgctcat 12780
ggtggggacc cctcgccgcc tgagaacctg caaagagaaa tgacgggcct gtgtcaagga
12840 gcccaagtcg cggggaagtg ttgcagggag gcactccggg aggtcccgcg
tgcccgtcca 12900 gggagcaatg cgtcctcggg ttcgtcccca gccgcgtcta
cgcgcctccg tcctcccctt 12960 cacgtccggc attcgtggtg cccggagccc
gacgccccgc gtccggacct ggaggcagcc 13020 ctgggtctcc ggatcaggcc
agcggccaaa gggtcgccgc acgcacctgt tcccagggcc 13080 tccacatcat
ggcccctccc tcgggttacc ccacagccta ggccgattcg acctctctcc 13140
gctggggccc tcgctggcgt ccctgcaccc tgggagcgcg agcggcgcgc gggcggggaa
13200 gcgcggccca gacccccggg tccgcccgga gcagctgcgc tgtcggggcc
aggccgggct 13260 cccagtggat tcgcgggcac agacgcccag gaccgcgctt
cccacgtggc ggagggactg 13320 gggacccggg cacccgtcct gccccttcac
cttccagctc cgcctcctcc gcgcggaccc 13380 cgccccgtcc cgacccctcc
cgggtccccg gcccagcccc ctccgggccc tcccagcccc 13440 tccccttcct
ttccgcggcc ccgccctctc ctcgcggcgc gagtttcagg cagcgctgcg 13500
tcctgctgcg cacgtgggaa gccctggccc cggccacccc cgcgatgccg cgcgctcccc
13560 gctgccgagc cgtgcgctcc ctgctgcgca gccactaccg cgaggtgctg
ccgctggcca 13620 cgttcgtgcg gcgcctgggg ccccagggct ggcggctggt
gcagcgcggg gacccggcgg 13680 ctttccgcgc gctggtggcc cagtgcctgg
tgtgcgtgcc ctgggacgca cggccgcccc 13740 ccgccgcccc ctccttccgc
caggtgggcc tccccggggt cggcgtccgg ctggggttga 13800 gggcggccgg
ggggaaccag cgacatgcgg agagcagcgc aggcgactca gggcgcttcc 13860
cccgcaggtg tcctgcctga aggagctggt ggcccgagtg ctgcagaggc tgtgcgagcg
13920 cggcgcgaag aacgtgctgg ccttcggctt cgcgctgctg gacggggccc
gcgggggccc 13980 ccccgaggcc ttcaccacca gcgtgcgcag ctacctgccc
aacacggtga ccgacgcact 14040 gcgggggagc ggggcgtggg ggctgctgct
gcgccgcgtg ggcgacgacg tgctggttca 14100 cctgctggca cgctgcgcgc
tctttgtgct ggtggctccc agctgcgcct accaggtgtg 14160 cgggccgccg
ctgtaccagc tcggcgctgc cactcaggcc cggcccccgc cacacgctag 14220
tggaccccga aggcgtctgg gatgcgaacg ggcctggaac catagcgtca gggaggccgg
14280 ggtccccctg ggcctgccag ccccgggtgc gaggaggcgc gggggcagtg
ccagccgaag 14340 tctgccgttg cccaagaggc ccaggcgtgg cgctgcccct
gagccggagc ggacgcccgt 14400 tgggcagggg tcctgggccc acccgggcag
gacgcgtgga ccgagtgacc gtggtttctg 14460 tgtggtgtca cctgccagac
ccgccgaaga agccacctct ttggagggtg cgctctctgg 14520 cacgcgccac
tcccacccat ccgtgggccg ccagcaccac gcgggccccc catccacatc 14580
gcggccacca cgtccctggg acacgccttg tcccccggtg tacgccgaga ccaagcactt
14640 cctctactcc tcaggcgaca aggagcagct gcggccctcc ttcctactca
gctctctgag 14700 gcccagcctg actggcgctc ggaggctcgt ggagaccatc
tttctgggtt ccaggccctg 14760 gatgccaggg actccccgca ggttgccccg
cctgccccag cgctactggc aaatgcggcc 14820 cctgtttctg gagctgcttg
ggaaccacgc gcagtgcccc tacggggtgc tcctcaagac 14880 gcactgcccg
ctgcgagctg cggtcacccc agcagccggt gtctgtgccc gggagaagcc 14940
ccagggctct gtggcggccc ccgaggagga ggacacagac ccccgtcgcc tggtgcagct
15000 gctccgccag cacagcagcc cctggcaggt gtacggcttc
gtgcgggcct gcctgcgccg 15060 gctggtgccc ccaggcctct ggggctccag
gcacaacgaa cgccgcttcc tcaggaacac 15120 caagaagttc atctccctgg
ggaagcatgc caagctctcg ctgcaggagc tgacgtggaa 15180 gatgagcgtg
cgggactgcg cttggctgcg caggagccca ggtgaggagg tggtggccgt 15240
cgagggccca ggccccagag ctgaatgcag taggggctca gaaaaggggg caggcagagc
15300 cctggtcctc ctgtctccat cgtcacgtgg gcacacgtgg cttttcgctc
aggacgtcga 15360 gtggacacgg tgatcgagtc gactcccttt agtgagggtt
aattgagctc gcggccgc 15418 2 2403 DNA Herpes simplex virus type 1 2
gggtcctagg ctccatgggg accgtatacg tggacaggct ctggagcatc gcacgactgc
60 gtgatattac cggagacctt ctgcgggacg agccgggtca cgcggctgac
ggagcgtccg 120 ttgggcgaca aacaccagga cggggcacag gtacactatc
ttgtcacccg gagcgcgagg 180 gactgcagga gcttcaggga gtggcgcagc
tgcttcatcc ccgtggcccg ttgctcgcgt 240 ttgctggcgg tgtccccgga
agaaatatat ttgcatgtct ttagttctat gatgacacaa 300 accccgccca
gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc ggggcggcgc 360
ggtcccaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac cgagcgaccc
420 tgcagcgacc cgcttaacag cgtcaacagc gtgccgcaga tcttggtggc
gtgaaactcc 480 cgcacctctt tggcaagcgc cttgtagaag cgcgtatggc
ttcgtacccc tgccatcaac 540 acgcgtctgc gttcgaccag gctgcgcgtt
ctcgcggcca tagcaaccga cgtacggcgt 600 tgcgccctcg ccggcagcaa
gaagccacgg aagtccgcct ggagcagaaa atgcccacgc 660 tactgcgggt
ttatatagac ggtcctcacg ggatggggaa aaccaccacc acgcaactgc 720
tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg acttactggc
780 aggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac
cgcctcgacc 840 agggtgagat atcggccggg gacgcggcgg tggtaatgac
aagcgcccag ataacaatgg 900 gcatgcctta tgccgtgacc gacgccgttc
tggctcctca tgtcgggggg gaggctggga 960 gttcacatgc cccgcccccg
gccctcaccc tcatcttcga ccgccatccc atcgccgccc 1020 tcctgtgcta
cccggccgcg cgatacctta tgggcagcat gaccccccag gccgtgctgg 1080
cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc
1140 cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag
cggcttgacc 1200 tggctatgct ggccgcgatt cgccgcgttt acgggctgct
tgccaatacg gtgcggtatc 1260 tgcagggcgg cgggtcgtgg tgggaggatt
ggggacagct ttcggggacg gccgtgccgc 1320 cccagggtgc cgagccccag
agcaacgcgg gcccacgacc ccatatcggg gacacgttat 1380 ttaccctgtt
tcgggccccc gagttgctgg cccccaacgg cgacctgtat aacgtgtttg 1440
cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat gcacgtcttt atcctggatt
1500 acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc
gggatggtcc 1560 agacccacgt caccacccca ggctccatac cgacgatctg
cgacctggcg cgcacgtttg 1620 cccgggagat gggggaggct aactgaaaca
cggaaggaga caataccgga aggaacccgc 1680 gctatgacgg caataaaaag
acagaataaa acgcacgggt gttgggtcgt ttgttcataa 1740 acgcggggtt
cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg 1800
ccaatacgcc cgcgtttctt ccttttcccc accccaaccc ccaagttcgg gtgaaggccc
1860 agggctcgca gccaacgtcg gggcggcaag cccgccatag ccacgggccc
cgtgggttag 1920 ggacggggtc ccccatgggg aatggtttat ggttcgtggg
ggttattctt ttgggcgttg 1980 cgtggggtca ggtccacgac tggactgagc
agacagaccc atggtttttg gatggcctgg 2040 gcatggaccg catgtactgg
cgcgacacga acaccgggcg tctgtggctg ccaaacaccc 2100 ccgaccccca
aaaaccaccg cgcggatttc tggcgccgcc ggacgaacta aacctgacta 2160
cggcatctct gccccttctt cgctggtacg aggagcgctt ttgttttgta ttggtcacca
2220 cggccgagtt tccgcgggac cccggccagc tgctttacat ctcgaagacc
tacctactcg 2280 gccggccccc gaacgcgagc ctgcccgccc ccatcacggt
cgagccgacc gcccagcctc 2340 cccccgcggt cgcccccctt aagggtctct
tgcacaatcc aaccgcctcc gtgttgctgc 2400 gtt 2403 3 376 PRT Herpes
simplex virus type 1 3 Met Ala Ser Tyr Pro Cys His Gln His Ala Ser
Ala Phe Asp Gln Ala 1 5 10 15 Ala Arg Ser Arg Gly His Ser Asn Arg
Arg Thr Ala Leu Arg Pro Arg 20 25 30 Arg Gln Gln Glu Ala Thr Glu
Val Arg Leu Glu Gln Lys Met Pro Thr 35 40 45 Leu Leu Arg Val Tyr
Ile Asp Gly Pro His Gly Met Gly Lys Thr Thr 50 55 60 Thr Thr Gln
Leu Leu Val Ala Leu Gly Ser Arg Asp Asp Ile Val Tyr 65 70 75 80 Val
Pro Glu Pro Met Thr Tyr Trp Gln Val Leu Gly Ala Ser Glu Thr 85 90
95 Ile Ala Asn Ile Tyr Thr Thr Gln His Arg Leu Asp Gln Gly Glu Ile
100 105 110 Ser Ala Gly Asp Ala Ala Val Val Met Thr Ser Ala Gln Ile
Thr Met 115 120 125 Gly Met Pro Tyr Ala Val Thr Asp Ala Val Leu Ala
Pro His Val Gly 130 135 140 Gly Glu Ala Gly Ser Ser His Ala Pro Pro
Pro Ala Leu Thr Leu Ile 145 150 155 160 Phe Asp Arg His Pro Ile Ala
Ala Leu Leu Cys Tyr Pro Ala Ala Arg 165 170 175 Tyr Leu Met Gly Ser
Met Thr Pro Gln Ala Val Leu Ala Phe Val Ala 180 185 190 Leu Ile Pro
Pro Thr Leu Pro Gly Thr Asn Ile Val Leu Gly Ala Leu 195 200 205 Pro
Glu Asp Arg His Ile Asp Arg Leu Ala Lys Arg Gln Arg Pro Gly 210 215
220 Glu Arg Leu Asp Leu Ala Met Leu Ala Ala Ile Arg Arg Val Tyr Gly
225 230 235 240 Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln Gly Gly Gly
Ser Trp Trp 245 250 255 Glu Asp Trp Gly Gln Leu Ser Gly Thr Ala Val
Pro Pro Gln Gly Ala 260 265 270 Glu Pro Gln Ser Asn Ala Gly Pro Arg
Pro His Ile Gly Asp Thr Leu 275 280 285 Phe Thr Leu Phe Arg Ala Pro
Glu Leu Leu Ala Pro Asn Gly Asp Leu 290 295 300 Tyr Asn Val Phe Ala
Trp Ala Leu Asp Val Leu Ala Lys Arg Leu Arg 305 310 315 320 Pro Met
His Val Phe Ile Leu Asp Tyr Asp Gln Ser Pro Ala Gly Cys 325 330 335
Arg Asp Ala Leu Leu Gln Leu Thr Ser Gly Met Val Gln Thr His Val 340
345 350 Thr Thr Pro Gly Ser Ile Pro Thr Ile Cys Asp Leu Ala Arg Thr
Phe 355 360 365 Ala Arg Glu Met Gly Glu Ala Asn 370 375 4 25 DNA
Artificial PCR primer sequence 4 cttgctgcag aagtgggtgg aggaa 25 5
21 DNA Artificial PCR primer sequence 5 ctgcagtgtg ggtttcgggc a 21
6 20 DNA Artificial PCR primer sequence 6 cggaagagtg tctggagcaa 20
7 19 DNA Artificial PCR primer sequence 7 ggatgaagcg gagtctgga
19
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References