U.S. patent application number 11/393820 was filed with the patent office on 2007-02-08 for method for producing a population of homozygous stem cells having a pre-selected immunotype and/or genotype, cells suitable for transplant derived therefrom, and materials and methods using same.
Invention is credited to Steve Chien-Wen Huang, Ruchi Khanna, Jingqi Lei, Hua (Helen) Lin, Minh-Thanh Nguyen, Wen Liang Yan.
Application Number | 20070031859 11/393820 |
Document ID | / |
Family ID | 22982529 |
Filed Date | 2007-02-08 |
United States Patent
Application |
20070031859 |
Kind Code |
A1 |
Yan; Wen Liang ; et
al. |
February 8, 2007 |
Method for producing a population of homozygous stem cells having a
pre-selected immunotype and/or genotype, cells suitable for
transplant derived therefrom, and materials and methods using
same
Abstract
A method of producing a homogenous population of homozygous stem
(HS) cells pre-selected for immunotype and/or genotype from donor
cells is described herein. The invention relates to methods of
using immunohistocompatible HS cells for diagnosis, therapeutic and
cosmetic transplantation, and the treatment of various genetic
diseases, neurodegenerative diseases, traumatic injuries and
cancer. The invention further relates to methods for using
histocompatible HS stem cells pre-selected for a non-disease
genotype for prophylactic and therapeutic intervention including,
but not limited to, therapeutic and cosmetic transplantation, and
the treatment of various genetic diseases, neurodegenerative
diseases, and cancer. Furthermore, the invention relates to a
catalogued transplant depository of HS cells derived from multiple
donors, each of the HS cells being homozygous for a unique HLA
haplotype, for the purpose of having a constant, reliable,
comprehensive supply of immunohistocompatible cells for diagnosis,
treatment and/or transplantation.
Inventors: |
Yan; Wen Liang; (Potomac,
MD) ; Huang; Steve Chien-Wen; (Germantown, MD)
; Nguyen; Minh-Thanh; (Rockville, MD) ; Lin; Hua
(Helen); (N. Potomac, MD) ; Lei; Jingqi;
(Gaithersburg, MD) ; Khanna; Ruchi; (Germantown,
MD) |
Correspondence
Address: |
REED SMITH LLP
3110 FAIRVIEW PARK DRIVE
FALLS CHURCH
VA
22042
US
|
Family ID: |
22982529 |
Appl. No.: |
11/393820 |
Filed: |
March 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10032495 |
Jan 2, 2002 |
7030292 |
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11393820 |
Mar 31, 2006 |
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60258881 |
Jan 2, 2001 |
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Current U.S.
Class: |
435/6.16 ;
435/325; 435/366; 435/455 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2506/04 20130101; C12N 2506/30 20130101; C12N 5/0603 20130101;
C12N 2502/13 20130101; A61K 35/12 20130101; C12N 2501/125
20130101 |
Class at
Publication: |
435/006 ;
435/455; 435/325; 435/366 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/08 20060101 C12N005/08; C12N 5/06 20070101
C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 2, 2002 |
WO |
PCT/US02/00107 |
Claims
1-28. (canceled)
29. A method for producing a homozygous stem (HS) cell depository,
wherein each HS cell that produces a cell line has alleles that are
homozygous for a Major Histocompatibility Complex (MHC) haplotype,
comprising: (a) selecting donors; (b) determining the MHC haplotype
of each donor; (c) mitotically activating non-fertilized female
post-meiosis I diploid germ cells obtained from each donor to
develop multiple blastocyst-like masses, each of which contains an
inner cell mass (ICM) that is homozygous for a particular MHC
haplotype, wherein the non-fertilized female post-meiosis I diploid
germ cells are activated by calcium ionophore and sham
intracytoplasmic sperm injection (ICSI); (d) isolating HS cells
from the ICM obtained from each donor; (e) culturing the isolated
HS cells to obtain HS cell lines; (f) determining the MHC haplotype
of each HS cell line; and (g) cataloging the MHC haplotype of each
HS cell line obtained in (e).
30. (canceled)
31. The method of claim 29, wherein the donors are mammalian.
32. The method of claim 31, wherein the donors are human.
33. The method of claim 31, wherein the donors are non-human.
Description
[0001] The present application is a divisional application of
parent application Ser. No. 10/032,495, filed Jan. 2, 2002, now
Pat. No. 7,030,292. The present application also claims the benefit
of U.S. Provisional Application Ser. No. 60/258,881, filed, Jan. 2,
2001.
I. FIELD OF THE INVENTION
[0002] The present invention relates to methods of producing a
homogenous population of homozygous stem (HS) cells pre-selected
for immunotype and/or genotype from donor homozygous post-meiosis I
diploid germ cells. The invention further relates to methods of
using such histocompatible HS cells for diagnosis, therapeutic and
cosmetic transplantation, cell replacement and/or gene therapy, and
the treatment of various genetic diseases, neurodegenerative
diseases, traumatic injuries, and cancer.
[0003] The invention further relates to methods for using
histocompatible HS cells pre-selected for a non-disease genotype
for prophylactic and therapeutic intervention including, but not
limited to, diagnosis, therapeutic and cosmetic transplantation
(including, but not limited to, skin grafts for wound and burn
repair, replacement bone, cartilage and tendon for use in
reconstruction of broken limbs, and repair of ears, noses, lips,
and scalp), cell replacement and/or gene therapy, and the treatment
of various genetic diseases including, but not limited to,
neurodegenerative diseases, traumatic injuries, and cancer.
[0004] Furthermore, the invention relates to methods for
differentiating genotyped and/or HLA-typed ("immunotyped") HS cells
to create progenitor cells, and other differentiated cells, masses
of cells or tissue types.
[0005] Finally, the invention relates to methods for providing
catalogued transplant depository for HS cell lines from multiple
donors, each HS cell line being homozygous for a unique HLA
haploytpe, and histocompatible with any individual carrying the HLA
components of such haplotype for the purpose of having a constant,
reliable, comprehensive supply of cells for diagnosis, treatment
and/or transplantation.
[0006] Moreover, the methods described in the present application
are useful in combination with materials and methods disclosed in
U.S. patent application Ser. No. 09/997,240, entitled "Isolated
Homozygous Stem Cells, Differentiated Cells Derived Therefrom, and
Methods of Making and Using Same," filed Nov. 30, 2001, that claims
the benefit of U.S. Provisional Application Ser. No. 60/253,943,
filed Nov. 30, 2000, both of which are incorporated by reference
herein.
II. BACKGROUND OF THE INVENTION
[0007] Tissue transplants from one individual to another are at
risk of rejection. The degree of risk is proportional to the degree
of disparity between certain genetic products, antigens, expressed
on the surface of donor and recipient cells. To ensure the success
of a transplant, donor tissue must be immunohistologically
compatible with recipient tissue, that is, donor and recipient
tissue must be matched. Matching is accomplished when donor tissue
expresses "self" antigens. T-cells from the recipient will then
recognize donor tissue as "self", and will not form an immune
response against it.
[0008] In all mammalian species studied, there is a single genetic
locus that encodes the strongest transplantation antigens. This is
known as the major histocompatibility complex (MHC). Human
leukocyte antigen (HLA) is the genetic designation for the human
counterpart to the MHC. HLA restricts, and therefore regulates, the
immune response in a highly specific way. Molecules encoded by HLA
bind and present foreign microbial antigens to an individual's own
T-cells. In fact, foreign antigens can only be recognized by the
T-cells of an individual if they are presented in the context of
the individual's own HLA molecules. Therefore, HLA molecules
participate in a trimolecular complex comprised of the T-cell
receptor, the foreign microbial antigen, and the self-HLA
molecule.
[0009] In the context of transplants, donor tissue expressing
non-self HLA antigens is reliably killed by cytotoxic T-cells.
Tissue grafts can be rejected on the basis of HLA molecules that
differ from self-HLA molecules by as little as one amino acid. It
is believed that the main reason for such rejection is that
non-self HLA molecules recognize and form complexes with many
peptides not recognized by self-HLA molecules. T-cells are
intolerant of these non-self HLA-peptide complexes, and possibly
bind to distinctive features of the non-self HLA molecule.
[0010] Therefore, matching donor and recipient tissue for HLA
reduces the chances of a cytotoxic T-cell response in the
recipient, and thus raises the chances of survival of a transplant.
However, matching itself presents a problem for transplantation.
First, there are two classes of HLA molecules, class I and II.
Second, there are several genes for each HLA class, and each of
these genes has many alleles (i.e. HLA is polymorphic). While HLA
provides an individual with the capability of a diverse immune
response, it makes transplantation from one individual to another a
difficult task because it makes donor-recipient matching difficult
to achieve.
[0011] In humans, there are three genes for class I HLA molecules,
HLA-A, HLA-B, and HLA-Cw, and three genes for class II HLA
molecules, HLA-DR, -DP, and -DQ. Furthermore, HLA genes are
polymorphic. There are 22 different HLA-A alleles, 42 different -B
alleles, 9 different -Cw alleles, and 18 different -DR alleles.
Adding to the complexity of matching, an individual has two of each
A, B, Cw, and DR alleles, where one set of A, B, Cw, and DR (a
haplotype) is inherited from each parent, therefore individuals may
be homozygyous or heterozygous for the A, B, Cw, and DR
haplotypes.
[0012] Currently, transplantation using HLA-A, -B, -Cw, and -DR
matched unrelated donor tissue makes enormous demands on tissue
typing resources. This is largely due to the polymorphic nature of
the HLA genes, and the need for high stringency of matching to
minimize rejection and acute graft vs. host disease. On average,
due to HLA polymorphism, the chance of finding a donor-recipient
match for HLA-A, -B, -Cw, and -DR would range from one in 1,000 to
one in several million depending on the frequency of the patient's
tissue type in the general population. See also, Hansen, J. A.,
Anasetti, C., Peterdorf, E., Clift, R. A. and Martin, P. J.,
"Marrow tranplants from unrelated donors," Transplant Proc., June
1994; 26:1719-1712 (Review).
[0013] The present invention provides a reliable source of cells or
tissue carrying two sets of identical HLA haplotypes, that
significantly increases the possibility of a histological match
between a donor and recipient, and hence, may be used for
diagnosis, transplant and/or treatment. HS cells are homozygous and
express only one HLA haplotype. Thus, a donor HS cell has only one
HLA haplotype to be matched with the recipient instead of two, as
in heterozygous stem cells. HS cells can be taken from random
donors of multiple ethnicities to provide a depository or bank of
HS cell populations that immunohistologically match at least one
haplotype carried by individuals in the population at large. The
depository needs only contain about 200 different HS cell lines,
each having a distinct HLA haplotype, to service most of the
general population (i.e., provide immunohistocompatible cells
suitable for transplant).
[0014] Further, gene therapy in humans raises ethical concerns. A
distinction has been drawn between genetic manipulations that
involve only somatic cells and manipulations that may involve germ
cells. Somatic manipulations are generally allowed as they affect
only the patient, whereas germ cell manipulations are disallowed as
they affect the patient's progeny. Moreover, research in transgenic
animals has demonstrated that gene defects can be effectively
corrected only by genetic manipulations of embryos. However, this
approach is not feasible for gene therapy in humans for practical
reasons, for example the level of insertional mutagenesis caused by
the integration of retroviral vectors into cellular DNA may be
unacceptably high.
[0015] The present invention also promises significant advances in
gene-replacement therapy. Currently, there are many obstacles to
gene therapy. Concerns such as whether a disease is suitable for
genetic intervention, whether the gene for the disorder has been
identified and cloned, whether there is an efficient way of
introducing a gene into cells, or whether the gene can be expressed
in tissues other than the affected tissue to be efficacious,
significantly impede the practicability of gene therapy.
[0016] Thus, there is clearly a need in the art for a reliable
source of histocompatible cells or tissue, which may be used for
transplant and/or treatment, including gene therapy. For example,
HS cells that are histocompatible with a patient suffering from a
genetic disease can be genetically manipulated before being
differentiated into a particular cell type for transplant.
III. SUMMARY OF THE INVENTION
[0017] The present invention relates to isolated homozygous stem
(HS) cells and the discovery that these cells may be selected for
immunotype and genotype, therefore being useful for diagnosis,
transplantation and/or treatment.
[0018] The present invention also provides a depository or bank for
HS cell lines typed for HLA-A, -B, -Cw, and -DR by serological or
molecular methods, which would reduce the demand on tissue typing
resources. Additionally, the present invention provides cells
selected for not just immunotype, but also genotype. Hence, it
provides a method for treating genetic disorders for example by
cell transplants, where HS cells pre-selected for a non-disease
genotype may be transplanted to the affected area. Thus,
effectively providing gene therapy without actual genetic
intervention.
[0019] Furthermore, the invention disclosed herein provides methods
for producing HLA-typed HS cells, and progenitor cells and
differentiated cells derived therefrom. Hence, the present
invention, is useful in combination with the teachings of U.S.
patent application Ser. No. 09/997,240, entitled "Isolated
Homozygous Stem Cells, Differentiated Cells Derived Therefrom, and
Methods of Making and Using Same," filed Nov. 30, 2001, and U.S.
Provisional Application Serial No. 60/253,943, which provides
pluripotent homozygous stem (HS) cells, methods and materials for
making same, and methods for differentiating HS cells into
progenitor cells or other desired differentiated cells, groups of
cells or tissues in vivo or in vitro.
[0020] HS cells are created upon activation of non-fertilized
post-meiosis I diploid germ cells from stemplasms when transplanted
into a live animal. It is a further object to isolate HS cells from
the various stages of development within said stemplasm. It is
another object of the invention to provide methods of selecting the
cells to be isolated from said stemplasm.
[0021] The HS cells of the present invention are pluripotent, and
raise no ethical concerns as they are isolated from cell-masses
that are non-fertilized and incapable of developing into viable
embryos. Moreover, immunohistocompatibility matching is difficult
to accomplish when heterozygous ES cell lines are employed in
tissue or cell transplantation therapy, or maintained in banks
and/or depositories. This is because the ES cell lines, including
those developed by Advanced Cell Technology and other
organizations, are derived from fertilized embryos or from nuclear
transfer techniques using adult differentiated cells, and are
genomically heterozygous (e.g., WO 01/84920A1, WO 01/79445A2 and WO
01/29206A1). Because the pluripotent stem cells of the present
invention, being derived from post-meiosis I germ cells, or
stemplasms, are genomically homozygous (with minimal heterozygosity
or uniform homozygosity) or are selected for MHC homozygosity (as
in the cases of the fusion and nuclear transfer of two haploid germ
cells), such cells may be used to overcome immunohistocompatibility
problems faced by currently available transplantation, cell
replacement, and gene therapy techniques employing ES cell lines,
or maintaining ES cell line banks and/or depositories.
[0022] During gametogenesis, heterozygous germ cells, i.e. germ
cells with both paternal and maternal chromosomes, undergo meiosis.
In the first meiotic division (meiosis I), homologous chromosomes
separate to form two homozygous daughter cells that contain either
paternal or maternal chromosomes with some heterozygosity
introduced because of the phenomenon of crossing-over. Further,
during oogenesis, the extrusion of one daughter cell (the primary
polar body) is observed. The other daughter cell is arrested at
metaphase II Such metaphase II diploid oocytes may be used to
derive homozygous stem cells with minimal heterozygosity.
[0023] Upon proper activation, a metaphase II oocyte can proceed to
complete meiosis by the extrusion of one chromatid (i.e. the
secondary polar body) and give rise to a haploid cell. Such
meiosis-completed haploid oocyte self-replicates without
cytokinesis, rendering it diploid and uniformly homozygous. Such
meiosis-completed haploid oocytes, hence, may also be used to
create the homozygous stem cells of the present invention with no
heterozygosity. See also, Kaufman M. H., Robertson E. J., Handyside
A. H., Evans M. J., "Establishment of pluripotential cell lines
from haploid mouse embryos," J. Embryol. Exp. Morphol., 73:249-61
(1983). Stem cells derived from stemplasms derived from
post-meiosis I or II germ cells also carry the above mentioned
homozygosity. Stem cells derived from the fusion or nuclear
transfer of two haploid germ cells from an individual can have a
larger degree of heterozygosity, nevertheless those homozygous for
MHC can be selected and used in cell based therapeutic
applications.
[0024] Both HS cells with minimal heterozygosity and uniform
homozygosity are superior to stem cells with heterozygous ES cells
(such as those derived from using fertilized embryos, nuclear
transfer, therapeutic cloning embryos, and adult stem cells) in
that homozygous stem cells can contain two sets of identical Major
Histocompatibility Complex (MHC) haplotypes. Therefore,
immunohistocompatibility matching between a donor and an individual
in need of transplantation therapy is easier to achieve with HS
cells. Such stem cells homozygous for one MHC haplotype are
tolerated not only by recipients carrying the identical haplotype,
but also by recipients with the same MHC components in either of
their parental haplotypes.
[0025] Human MHC loci are within 4 Mb on chromosome 6, and MHC
alleles are usually inherited en bloc. Some MHC allelic
combinations are shared in a considerably higher frequency in the
population, for example the 15 most common HLA-A, -B, -Cw, -DR
haplotypes are shared by 21.3% Caucasian Americans, and similar
observations of haplotype frequency are seen in other ethnical
backgrounds. Furthermore, common HLA haplotypes in one racial group
are shared significantly by other groups. For example, 156
haplotypes of the 184 common haplotypes in Caucasian Americans are
shared at least once by African Americans, Asian Americans, Latin
Americans, or Native Americans. The number of common haplotypes of
other racial groups and the corresponding number of shared
haplotypes are: African Americans (198/63); Asian Americans
(210/100); Latin Americans (194/147); and Native Americans
(194/173). Mori, M., et al., "HLA gene and haplotype frequencies in
the North American population: the National Marrow Donor Program
Donor Registry," Transplantation, 64(7): 1017-27 (1997).
Considering such evidence supporting such linkage disequilibrium,
the use of non-fertilized post-meiosis I diploid gamete derived HS
cells can reduce the number of immunologically different cell lines
needed to be maintained in a stem cell bank or depository for
tissue or cell transplantation.
[0026] Hence, potentially, a few hundred stem cell lines that are
homozygous for different haplotypes will be sufficient to match a
majority of the population. This number is tremendously smaller in
contrast to the number of haplotypes needed to maintain a bank or
depository for stem cell lines derived from embryonic stem cells,
adult stem cells, or therapeutic cloning stem cells. For example,
for every 200 haplotypes there are more than 20,000 heterozygous
possibilities (FIG. 1).
[0027] The present invention when used in combination with the
invention described in U.S. patent application Ser. No. 09/997,240,
and U.S. Provisional Application Ser. No. 60/253,943, supra, is
useful for, including, but not limited to, the diagnosis and
treatment of various diseases, such as genetic diseases
neurodegenerative diseases, endocrine-related disorders and cancer,
traumatic injuries, cosmetic and therapeutic transplantation, and
gene therapy and cell replacement therapy.
[0028] The present invention provides a method of producing a
population of homozygous stem cells that are selected for
immunotype and/or genotype. HS cells are immunotyped for HLA-A, -B,
-Cw, and -DR using serological and/or molecular techniques known in
the art. HS cells are further selected for genotype using molecular
techniques known in the art. Such HS cells may be then be used to
derive progenitor cells, and/or other differentiated cells, groups
of cells or tissue types using methods disclosed in U.S. patent
application Ser. No. 07/997,240, and U.S. Provisional Application
Ser. No. 60/253,943, incorporated by reference herein.
Additionally, the present invention provides methods for using
pre-HLA typed HS-derived differentiated cells and/or tissues for
therapeutic or cosmetic transplantation, and/or treatment of
genetic and neurodegenerative disorders, traumatic injuries and
cancer.
[0029] It is an object of the invention to provide a method for
producing a population of homozygous stem cells from mitotically
activated homozygous post-meiosis I diploid germ cells having a
target immunotype such that they are suitable for transplant into
an intended recipient by, (a) assaying the immunotype, that is, the
homozygous or heterozygous HLA haplotype, of the intended recipient
to determine the target immunotype, (b) producing mitotically
activated post-meiosis 1 diploid germ cell samples to give rise to
multiple blastocyst-like masses, each of which contains an inner
cell mass (ICM) homozygous for a particular HLA haplotype, (c)
assaying the immunotype of each said ICM so as to select one
carrying the target HLA haplotype, and (d) culturing HS cells
isolated from the selected ICM to create permanent HS cell lines
homozygous for the target HLA haplotype. It is a further object of
the invention to provide HS cells derived from the intended
recipient, or a close relative of the intended recipient or person
unrelated to the intended recipient carrying at least one HLA
haplotype common to the intended recipient. Known techniques are
used for assaying and harvesting HS cells carrying the target HLA
haplotype, and assaying the immunotype of the intended recipient
and donor.
[0030] As described in detail in U.S. patent application Ser. No.
09/997,240, and U.S. Provisional Application Ser. No. 60/253,943,
which are incorporated by reference herein, HS cells are derived
from a blastocyst-like mass that is created by: (a) fusing two
oocytes or two spermatids; (b) preventing the extrusion of the
second polar body during oogenesis; (c) allowing the extrusion of
the second polar body and spontaneous genomic self-replication in
appropriate conditions; or, (d) transferring two haploid egg or
sperm nuclei into an enucleated oocyte, where methods (a) and (d)
require the additional for screening for homozygous stem cells by
genotyping.
[0031] It is another object of the present invention to provide a
method of isolating a population of recipient histocompatible
homozygous stem cells from stemplasms created by the process
described in detail in U.S. patent application Ser. No. 09/997,240
and U.S. Provisional Application Ser. No. 60/253,943, which are
incorporated by reference herein. Such a population of recipient
histocompatible, HS cells would be created by, (a) assaying the
immunotype and genotype of the intended recipient to determine the
target immunotype and genotype; (b) mitotically activating donor
homozygous post-meiosis I diploid germ cells to develop multiple
blastocyst-like masses, each of which contains an inner cell mass
(ICM) that is homozygous for a particular allele; (c) isolating HS
cells from the selected ICM, and culturing said HS cells to create
cell lines; (d) assaying the genotype of each said HS cell line,
and selecting one that is homozygous for the target genotype. Said
HS cells may be identified from the stemplasm created after
implantation through histological means and assayed for proper HLA
haplotype and genotype.
[0032] It is another object of the present invention to provide a
method for producing a population of recipient histocompatible,
homozygous stem cells from mitotically activated homozygous
post-meiosis I diploid germ cells having a target genotype, that
is, the non-disease allele of the target gene, such that they are
suitable for transplant into an intended recipient by: (a) assaying
the genotype and HLA-haplotype of the intended recipient to
determine the target immunotype and genotype; (b) mitotically
activating donor homozygous post-meiosis I diploid germ cells to
develop multiple blastocyst-like masses, each of which contains an
inner cell mass (ICM) that is homozygous for a particular allele,
and a particular HLA haplotype; (c) isolating HS cells from the
said ICM, and culturing said HS cells to create cell lines; and (d)
assaying the HLA haplotype and genotype of each said HS cell line
and selecting one carrying the target HLA haplotype and
genotype.
[0033] It is a further object of the invention to provide HS cells
derived from the intended recipient carrying one functional allele,
or a close relative of the intended recipient or person unrelated
to the intended recipient carrying at least one HLA haplotype
common to the intended recipient, and at least one non-disease
allele of the target gene. Known techniques are used for assaying
and harvesting HS cells carrying the target HLA haplotype and the
target genotype, and assaying the immunotype and genotype of the
intended recipient and donor.
[0034] It is another object of the invention to provide methods for
making a desired cell, group of cells, or tissue type which
comprises directing the differentiation of an isolated,
immunotyped, and/or genotyped HS cell, under suitable conditions
using factors in vivo or in vitro, so as to arrive at the desired
cell, group of cells, or tissue type. Exemplary tissues include,
but are not limited to, epithelial tissues, connective tissue,
muscle tissue or nervous tissue.
[0035] Illustrative types of epithelial cells include, but are not
limited to, keratinizing epithelial cells; wet-stratified barrier
epithelia; lining epithelial cells; exocrine-secreting epithelial
cells; endocrine-secreting epithelial cells; extracellular
matrix-secreting epithelial cells; absorptive epithelial cells,
such as those of the gut, exocrine glands, and urogenital tract;
and contractile epithelial cells. Illustrative types of connective
tissue cells include, but are not limited to, extracellular
matrix-secreting cells; cells specialized for metabolism and
storage; and circulating cells of the blood and immune systems.
Illustrative types of muscle cells include, but are not limited to,
contractile cells and ciliated cells with propulsive function.
Illustrative types of nervous or sensory cells include, but are not
limited to, sensory transducers; autonomic neurons; supporting
cells of sense organs and of peripheral neurons; and neurons and
glial cells of the central nervous system. Illustrative types of
reproductive cells include, but are not limited to, germ cells and
nurse cells.
[0036] It is another object of the invention to provide a
catalogued transplant depository or bank containing populations of
pre-HLA-typed HS cells derived from multiple donors, each
population homozygous for at least one HLA haplotype, so as to have
a reliable, constant, comprehensive supply of immunohistocompatible
cells for diagnosis transplantation and/or treatment.
[0037] It is another object of the invention to provide a method of
treating a disorder or disease state in an individual by
generating, in situ or in vitro, suitable replacement cells, groups
of cells, tissues or organs from isolated HS cells that are
immunohistologically compatible with said individual. Illustrative
disorders and disease states include, but are not limited to,
traumatic injury (e.g., post-trauma repair and reconstruction, limb
replacement, spinal cord injury, burns, and the like) and birth
defects; pathological and malignant conditions of the cells,
tissues, and organs (e.g., cancer); and degenerative and congenital
diseases of the cells and tissues of the muscles (e.g., cystic
fibrosis, muscular dystrophy, cardiac conditions), nerves (e.g.,
Alzheimer's, Parkinson's, and multiple sclerosis), epithelium
(e.g., blindness and myopathy, atherosclerosis and other stenotic
vascular conditions, enzyme deficiencies such as Crohn's disease,
and hormone deficiencies such as diabetes), and connective tissues
(e.g., immune conditions and anemia). HS-derived cells and tissues
may be grafted or transplanted to a subject in need, preferably
using the subject's own donor material.
[0038] These and further objects of the invention are fully
described by the below detailed description, examples, and claims,
although none is a limitation of the scope of the invention.
IV. BRIEF DESCRIPTION OF THE FIGURES
[0039] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0040] FIG. 1: Graph depicting the difference in the quantity of
cells needed for a non-HS cell bank versus a HS cell bank.
[0041] FIG. 2: Schematic diagram illustrating the derivation of HS
cells from post-meiosis I oocytes.
[0042] FIG. 3: Schematic representation of spermatogenesis and
oogenesis.
[0043] FIG. 4A: Table showing serological specificities of HLA
loci.
[0044] FIG. 4B: Officially recognized HLA-DR and HLA-DQ
Specificities.
[0045] FIG. 5: A list of exemplary restriction endonucleases for
genotyping of HLA class II alleles.
[0046] FIG. 6: A list of exemplary SSO probes for HLA class II
typing. SSO Probe L11 (SEQ ID NO. 1); SSO Probe D11 (SEQ ID NO. 2);
SSO Probe N77 (SEQ ID NO. 3); SSO Probe H33 (SEQ ID NO. 4); SSO
Probe E58 (SEQ ID NO. 5); SSO Probe L37 (SEQ ID NO. 6); SSO Probe
G11 (SEQ ID NO. 7); SSO Probe L74 (SEQ ID NO. 8); SSO Probe Y26
(SEQ ID NO. 9); SSO Probe V11 (SEQ ID NO. 10); SSO Probe E71 (SEQ
ID NO. 11); SSO Probe K71 (SEQ ID NO. 12); SSO Probe H60 (SEQ ID
NO. 13); SSO Probe N37 (SEQ ID NO. 14); SSO Probe D37 (SEQ ID NO.
15); SSO Probe AV86 (SEQ ID NO. 16); SSO Probe G13 (SEQ ID NO. 17);
SSO Probe H30-2 (SEQ ID NO. 18); SSO Probe N37 (SEQ ID NO. 19); SSO
Probe S37 (SEQ ID NO. 20); SSO Probe S57 (SEQ ID NO. 21); SSO Probe
H60 (SEQ ID NO. 22); SSO Probe F67 (SEQ ID NO. 23); SSO Probe 167
(SEQ ID NO. 24); SSO Probe I67-2 (SEQ ID NO. 25); SSO Probe E71
(SEQ ID NO. 26); SSO Probe K71 (SEQ ID NO. 27); SSO Probe QK71 (SEQ
ID NO. 28); SSO Probe QR71 (SEQ ID NO. 29); SSO Probe QR71-2 (SEQ
ID NO. 30); SSO Probe R71 (SEQ ID NO. 31); SSO Probe R71-2 (SEQ ID
NO. 32); SSO Probe RR71 (SEQ ID NO. 33); SSO Probe E74 (SEQ ID NO.
34); SSO Probe L74 (SEQ ID NO. 35); SSO Probe V86 (SEQ ID NO. 36);
SSO Probe G86 (SEQ ID NO. 37); SSO Probe AV86 (SEQ ID NO. 38).
[0047] FIG. 7: A list of exemplary sequences of primers used for
microsatellite typing and sequencing. D6S276 5' Primer (SEQ ID NO.
39); D6S105 5' Primer (SEQ ID NO. 40); MOGCA 5' Primer (SEQ ID NO.
41); D6S265 5' Primer (SEQ ID NO. 42); MIB 5' Primer (SEQ ID NO.
43); D6S275 5' Primer (SEQ ID NO. 44); DQCAR11 5' Primer (SEQ ID
NO.45); DQCAR 5' Primer (SEQ ID NO. 46); G51152 5' Primer (SEQ ID
NO. 47); TAP1CA 5' Primer (SEQ ID NO. 48); RING3CA 5' Primer (SEQ
ID NO. 49); D6S291 5' Primer (SEQ ID NO. 50); D6S276 3' Primer (SEQ
ID NO. 51); D6S105 3' Primer (SEQ ID NO. 52); MOGCA 3' Primer (SEQ
ID NO. 53); D6S265 3' Primer (SEQ ID NO.54); MIB 3' Primer (SEQ ID
NO. 55); D6S275 3' Primer (SEQ ID NO. 56); DQCAR11 3' Primer (SEQ
ID NO. 57); DQCAR 3' Primer (SEQ ID NO. 58); G51152 3' Primer (SEQ
ID NO. 59); TAP1CA 3' Primer (SEQ ID NO. 60); RING3CA 3' Primer
(SEQ ID NO. 61); D6S291 3' Primer (SEQ ID NO. 62).
[0048] FIG. 8: Map of HLA class I, II, and III regions showing the
relative location of the twelve microsatellites.
[0049] FIG. 9A: C57/DBA2 mouse.
[0050] FIG. 9B: Superovulated oocytes-derived blastocysts-like
masses.
[0051] FIG. 9C: Representative colony I.
[0052] FIG. 9D: Representative colony II.
[0053] FIG. 9E: Results of genotyping experiment.
[0054] FIG. 9F: Results immunotyping experiment.
[0055] FIG. 10A: Photo of an isolated inner cell mass growing on
feeder layers derived from mouse HS cells.
[0056] FIG. 10B: Photo depicting the development of a morula-like
mass derived from human homozygous post-meiosis I diploid
oocytes.
[0057] FIG. 10C: Photo of an early blastocyst-like mass derived
from human homozygous post-meiosis I diploid oocytes.
[0058] FIG. 10D: Photo of a blastocyst-like mass revealing the
inner cell mass derived from human homozygous post-meiosis I
diploid oocytes.
[0059] FIG. 11: An exemplary list of PCR primers for amplification
of HLA class H alleles. Gene DQA1, 5' Primer GH26 (SEQ ID NO. 63);
Gene DQA1, 3' Primer GH27 (SEQ ID NO. 64); Gene DQB1 5' Primer
GH28NL (SEQ ID NO. 65); Gene DQB1 3' Primer QB202 (SEQ ID NO. 66);
Gene DQB1 5' Primer GH28NL (SEQ ID NO. 67); Gene DQB1 3' Primer
QB204 (SEQ ID NO. 68); Gene DRB1 5' Primer 5'R2 (SEQ ID NO. 69);
Gene DRB1 5' Primer 5'R4 (SEQ ID NO. 70); Gene DRB1 5' Primer
5'R9-1 (SEQ ID NO. 71); Gene DRB1 5' Primer 5'R1 (SEQ ID NO. 72);
Gene DRB1 5' Primer 5'R7 (SEQ ID NO. 73); Gene DRB1 5' Primer 5'R10
(SEQ ID NO. 74); Gene DRB1 5' Primer 5'R3568 (SEQ ID NO. 75); Gene
DRB1 3' Primer 3'R (SEQ ID NO. 76); Gene DRB3 5' Primer DRBAMP-52
(SEQ ID NO. 77); Gene DRB3 3' Primer 3'R (SEQ ID NO. 78); Gene DRB5
5' Primer 5'DRB5 (SEQ ID NO. 79); Gene DRB5 3' Primer 3'R (SEQ ID
NO. 80); Gene DPA1 5' Primer PL (SEQ ID NO. 81); Gene DPA1 3'
Primer PR (SEQ ID NO. 82); Gene DPA1 5' Primer PLTM (SEQ ID NO.
83); Gene DPA1 3' Primer PRTM (SEQ ID NO. 84); Gene DPB1 5' Primer
DPB101N (SEQ ID NO. 85); Gene DPB1 3' Primer DPB201 (SEQ ID NO.
86).
V. DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE
INVENTION
[0060] All references cited herein are hereby incorporated by
reference in their entirety. Nothing herein is to be construed as
an admission that the invention is not entitled to antedate such
disclosure by virtue of prior invention. Throughout this
description, the preferred embodiments and examples shown should be
considered as exemplars, rather than as limitations on the present
invention.
[0061] A. Definitions
[0062] In the context of the present invention, a HS cell system
refers to stem cells derived from the blastocyst-like mass that
results from the mitotic activation of an unfertilized homozygous
post-meiosis I diploid germ cell, or from the fusion or nuclear
transfer of two haploid germ cells, to stem cells derived from
naturally occurring teratogenic tissues and to stem cells derived
from stemplasms as described in U.S. Patent Application Ser. No.
09/997,240, and U.S. Provisional Patent Application Ser. No.
60/253,943, incorporated by reference herein. HS cells isolated
from HS cell systems are pluripotent, and under appropriate
conditions can be differentiated to produce multi-potent progenitor
cells, somatic cell precursors, and other types of differentiated
cells.
[0063] "Differentiation" is a highly regulated process that cells
undergo as they mature into normal functional cells. Differentiated
cells have distinctive characteristics, perform specific functions
and are less likely to divide. Conversely, undifferentiated cells
are rapidly dividing immature, embryonic or primitive cells having
a nonspecific appearance with multiple nonspecific activities and
functions.
[0064] As used herein, the term "stem cell" refers to a relatively
undifferentiated cell that actively divides and cycles, giving rise
upon proper stimulation to a lineage of mature, differentiated,
functional cells. The defining properties of a stem cell include:
(a) it is not itself terminally differentiated; (b) it can divide
without limit for the lifetime of the animal; and (c) when it
divides, each daughter has a choice of remaining a stem cell or
embarking on a course that leads irreversibly to terminal
differentiation. Those stem cells that are initially unrestricted
in their capabilities (i.e., capable of giving rise to several
types of differentiated cell) are called "pluripotent". Current
sources of pluripotent cells include embryonic (ES) stem cells,
embryonic carcinoma (EC) cells, cells generated from somatic
cloning, teratomas and teratocarcinomas.
[0065] Progenitor cell lines, each capable of producing cells from
one of the three germ layers, i.e. the endoderm, mesoderm and
ectoderm, are referred to in the present application as
"multi-potent". While each progenitor cell line is not terminally
differentiated and can continue to divide for the lifetime of an
animal, it is considered to be committed to different tissues or
cells from only one type of embryonic layer. Therefore, particular
progenitor cell lines may be differentiated into bone, cartilage,
smooth muscle, striated muscle and hematopoietic cells (mesoderm);
liver, primitive gut, and respiratory epithelium (endoderm); or,
neurons, glial cells, hair follicles and tooth buds (ectoderm). The
term "progenitor cells" hence may be used synonymously with
"multi-potent stem cells" or "precursor cells". Such progenitor
cells lines, which are created by the directed differentiation of
HS cells in vivo (where the term "in vivo" includes differentiation
induced by encapsulating said HS cells in an immunocompromised
animal to generate stemplasms from such encapsulated cells) or in
vitro, can be maintained in culture as permanent cell lines.
[0066] A "teratoma" is a disorganized mass of cells containing many
types of differentiated tissue, tissues derived from all three
embryonic layers, such as bone, muscle, cartilage, nerve,
tooth-buds, glandular epithelium, and so forth, mixed with
undifferentiated stem cells that continually divide and generate
yet more of these differentiated tissues.
[0067] A "teratocarcinoma" is secondary to a teratoma. Teratomas
are largely benign; however if they become malignant, a
teratocarcinoma develops and can be deadly to the host. Compared to
other tumors, teratomas exhibit unique histological features. They
are composed of various differentiated tissues, including tissues
such as epidermis, central nervous system tissue, or mature
cartilage. They also contain nonspecific tissue types, e.g.,
lymphoid tissue or fibrous stroma.
[0068] A "stemplasm" is a newly derived term used to describe a
mass that develops upon the transplantation of HS cells into a
host. Unlike teratomas, a stemplasm exhibits controlled growth,
while still containing cells from all three embryonic germ layers.
It can therefore be used as a means for the in vivo differentiation
of the HS cells of the present invention.
[0069] A "homozygous stem cell" is an undifferentiated stem cell
arising from a non-fertilized post-meiosis I diploid germ cell.
Preferably, it is formed by preventing the extrusion of the second
polar body during oogenesis (or "activation"), or allowing the
extrusion of the second polar body and spontaneous genomic
self-replication of the haploid oocyte in appropriate conditions.
Homozygous stem (HS) cells are isolated cells generated from the
inner cell mass of blastocyst-like masses that develop upon
"mitotic activation" of non-fertilized germ cells, which can be
accomplished by: (a) fusing two oocytes or two spermatids; (b)
preventing the extrusion of the second polar body during oogenesis;
(c) allowing the extrusion of the second polar body and spontaneous
genomic self-replication in appropriate conditions; or, (d)
transferring two haploid egg or sperm nuclei into an enucleated
oocyte.
[0070] In mammalian development, cleavage produces a thin-walled
hollow sphere, the "blastocyst", with the embryo proper being
represented by a mass of cells at one side, otherwise known as the
"inner cell mass". The blastocyst is formed before implantation and
is equivalent to the "blastula". The wall of the thin-walled hollow
sphere is referred to as the "trophoblast", which is the
extra-embryonic layer of epithelium that forms around the mammalian
blastocyst, and attaches the embryo to the uterus wall. The
trophoblast forms the outer layer of the chorion, and together with
maternal tissue will form the placenta.
[0071] In the context of the present invention, a "blastocyst-like
mass" is different from a "blastocyst" (as used in the art) in that
it is the product of a mitotically activated non-fertilized
post-meiosis I germ cells.
[0072] As used herein, the term "mitotically activated" means
acquiring the ability to undergo regular cell divisions
mitotically. Mitotic activation refers to the activation of germ
cells so that meiosis II is either, (1) resumed and the haploid
gamete cells, after self-replicating their genome without
cytokinesis, then undergo mitotic division, or (2) resumed, but the
haploid gamete cells are prevented from karykinesis and
cytokinesis, and then undergo mitotic division.
[0073] The term "homozygous post-meiosis I diploid germ cells", as
used herein, means germ cells that are the stage of gametogenesis
at which the cells contain two copies of either the paternal or
maternal homologous chromosomes. And haploid germ cells means germ
cells that have accomplished meiosis and carry only one copy of the
chromosomes.
[0074] "Alleles" are alternative forms of a genetic locus. A single
allele for each locus is inherited separately from each parent. In
the context of the present invention, a cell is classified as
"homozygous" if it contains two copies of the same allele.
Conversely, a "heterozygous" cell contains two different alleles of
the same genetic locus.
[0075] In the context of the present invention, an individual's
"immunotype" is characterized by the unique array of proteins,
known as histocompatibility antigens, disposed on the surface of a
cell. In humans, these histocompatibility antigens are referred to
as human leukocyte antigens or HLAs, a complex family of
genetically inherited proteins found on the surface of cells
throughout the body.
[0076] A primary determinant of a cell's immunotype is the HLA
profile, a genetic fingerprint on white blood cells and platelets,
composed of proteins that play a critical role in activating the
body's immune system to respond to foreign organisms. The HLAs
determine the match between an intended host and a potential donor
in transplant procedures. Reactions to HLA antigens are the primary
cause of tissue graft rejection. HLA factors are inherited from the
mother and the father (one from each) so the greatest chance of
having the same HLA type is between siblings (e.g., 1 in 4).
Foreign cells having the same immunotype or HLA profile as those of
a host's native cells will be accepted by the host as "self" and
not rejected by the host's immune system.
[0077] HLA antigens are the products of multiple, closely linked
genes on a single chromosome usually inherited as an intact unit.
This histocompatibility complex, or HLA region, also contains genes
important to complement and immune responses.
[0078] The HLA gene complex contains at least four loci known as
HLA-A, -B, -Cw, and -R. There are two classes of HLA antigens.
Class I is comprised of HLA-A, -B, and -Cw. There are 22 different
HLA-A antigens, 42 different -B antigens, 9 different -Cw antigens,
and 18 different -DR antigens. HLA-A, -B, and -C antigens are
expressed on nearly all human cells. An individual's class I HLA
profile can be used to determine the degree of compatibility or
genetic relationship between individuals, such as potential donors
and recipients in transplantation of kidneys, bone marrow, etc.,
infusions of platelets or leukocytes, or for genealogical studies.
Class I antigens may be identified by serologic methods. For
example, an individual's Class I HLA profile may be determined by
incubating sample lymphocytes with a battery of antisera having
known specificities for certain A-, B-, or C-locus antigens.
Positive cell/serum interactions characterize the HLA antigens of
the cells.
[0079] Class II is comprised of HLA-DR and -DQ. There are 18
different DR antigens. These antigens are normally expressed on B
lymphocytes, monocytes, macrophages, dendritic cells and
endothelial cells, though they are induced on other cells by immune
stimuli. Class II antigens (e.g. the DR.beta.1, DR.beta.3,
DR.beta.4, DR.beta.5, and DQ.beta.1 genes) can be detected by
serologic and/or molecular analysis of patient DNA. If only
mid-resolution typing is required, one may utilize Sequence
Specific Oligonucleotide Probes (SSOP) for all regions of the gene.
If high resolution (allelic level) is required, a combination of
SSOP testing and DNA sequencing can be used. High resolution
testing is usually performed only on bone marrow recipients and
their prospective donors where a much greater degree of matching is
essential.
[0080] Cells expressing one or more of the HLA proteins expressed
by the host cells are perceived by the host's immune system as
"self" whereas those expressing other HLA proteins are perceived as
"foreign" or "non-self". The host's immune system gears up a
response to the "foreign" or "non-self" proteins and kills off the
foreign tissue. If the foreign tissue is a transplanted organ, this
response is called rejection.
[0081] In the context of the present invention, donor cells are
characterized "suitable for transplant" to an intended host if they
may be stably grafted onto a host without rejection. To be suitable
for transplant, donor cells must be "histocompatible" with the
intended host, i.e., HLA "matched". If the HLA are substantially
matched, the immune system is much less likely to respond. Humans
inherit HLA proteins from their parents on the 6th chromosome.
Since one gets two chromosome 6s (normally, one from the mother and
one from the father), half of one's HLA proteins will match each of
our parents. The more HLA proteins that match, the less likely a
grafted organ will be rejected by the recipient.
[0082] An individual has two of each A, B, Cw, and DR alleles,
where one set of A, B, Cw, and DR (a "haplotype") is inherited from
each parent. Further, individuals may be homozygous or heterozygous
for the A, B, Cw, and DR haplotypes. In the context of the present
invention, a donor cell is considered to be HLA "matched" or
"histocompatible" to an intended recipient, provided the donor
cells do not express HLA products that are foreign to the
recipient. For example, a donor cell that is homozygous for a
haplotype such as HLA-A1, -Cw7,-B8 or HLA-A29,-Cw7,-B8, will match
a recipient having a heterozygous HLA profile with both HLA-A1,
-Cw7,-B8, and HLA-A29,-Cw8,-B65 haplotypes.
[0083] As used herein, the term "genotype" refers to the genetic
constitution of an organism or a group of organisms. In the context
of the present invention, the "target" genotype will be the normal
or wild type sequence, i.e., that genetic sequence is not
associated with a particular pathological condition. Such
pathological conditions are often referred to as "genetic
disorders", meaning they are linked to the expression of a mutant
gene or genes. Genetic disorders often arise from specific gene
mutations. In the context of the present invention, the genetic
mutation linked to pathology can either be inherited (e.g.,
hereditary genetic disorders) or induced (e.g., exposure to
carcinogens).
[0084] Exemplary inherited genetic disorders include but are not
limited to Alzheimer's disease, diabetes, Graves disease,
hemophilia, Huntington's disease, muscular dystrophy, Parkinson's
disease, sickle cell anemia, and various metabolic disorders (i.e.,
enzyme deficiencies such as Phenylketonuria (PKU) and Severe
Combined Immune Deficiency (SCID)). Exemplary induced genetic
disorders include but are not limited to cancer and multiple
sclerosis.
[0085] A catalog of genetic disorders and the human genes linked
thereto is available through the Online Mendelian Inheritance in
Man (Online Mendelian Inheritance in Man, OMIM (TM).
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins
University (Baltimore, Md.) and National Center for Biotechnology
Information, National Library of Medicine (Bethesda, Md.), 2000.
World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). OMIM is a
large searchable database of genetic diseases, disorders, and
traits and their different manifestations maintained by the
National Center for Biotechnology Information (NCBI). OMIM is
available on the world wide web.
[0086] B. Creation Of Homozygous Stem Cells Of The Present
Invention
[0087] As described in detail in U.S. application Ser. No.
09/997,240, an HS cell is isolated from a blastocyst-like mass that
develops upon the mitotic activation of a non-fertilized
post-meiosis I diploid germ cell. FIG. 2 provides a flow chart,
showing a preferred method of developing HS cells from a
non-fertilized post-meiosis I diploid germ cell.
[0088] Germ cells develop into non-fertilized post-meiosis I
diploid germ cells that, upon activation, produce blastocyst-like
masses from which the HS cells of the present invention are
derived. HS cells, and/or differentiated cells, of the present
invention find utility in the diagnosis and/or treatment of
diseases, include, but are not limited to implantation or
transplantation to an affected individual in need of such
therapy.
[0089] While homozygous post-meiosis I diploid germ cells may be
obtained from the same individual or from an immunocompatible
donor, in certain situations self-donors are preferred. However, in
cases where the affected individual selected for therapy suffers
from a genetic disease (i.e., a disease characterized by a lack of
a crucial gene, either due to mutation or improper expression), it
may be preferable to utilize a non-self donor. Alternatively, one
skilled in the art of selection procedures may choose those self
germ cells that display the desired genotype (e.g., cells lacking a
flawed or mutated gene), those cells capable of expressing the
deficient gene. Such selection techniques may also be used to avoid
an immuno-incompatible genotype or phenotype for tissue
transplant.
[0090] HS cells homozygous for a HLA haplotype and a target gene
can be derived and selected from: (a) fusing two oocytes or two
spermatids; (b) preventing the extrusion of the second polar body
during oogenesis; (c) allowing the extrusion of the second polar
body and spontaneous genomic self-replication in appropriate
conditions; or, (d) transferring two haploid egg or sperm nuclei
into an enucleated oocyte. FIG. 3 provides a schematic
representation of spermatogenesis and oogenesis, showing the
difference in phases of mitosis and meiosis in males and
females.
[0091] Oocytes useful in the context of the present invention may
be obtained using any suitable method known in the art, or yet to
be discovered. Human oocytes are typically harvested from the
ovarian follicles of a donor individual and isolated from
surrounding or adhering cells. To maximize yield, superovulation is
induced in the donor individual. Superovulation may be induced by
the administration of appropriate gonadotropins or gonadotropin
analogues, administered either alone or in combination with
clomiphene citrate (Barriere et al., Rev. Prat., 40(29):2689-93
(1990), incorporated by reference herein). In mice, an exemplary
method involves the administration of pregnant mare's serum (PMS)
to mimic follicle-stimulating hormone (FSH) and human chorionic
gonadotropin (hCG) to mimic luteinizing hormone (LH). See Hogan et
al., Manipulating the mouse embryo: A Laboratory Manual, 2nd ed.
Cold Spring Harbor Laboratory Press, 1994). Efficient induction of
superovulation depends on several variables including, but not
limited to, the age and weight of the female, the dose of
gonadotropin, the time of administration, and the strain used.
[0092] Superinduction of ovulation and harvesting of oocytes are
known in the art. For exemplary detailed mouse protocols, see Hogan
et al., supra, pp. 130-132, the entire contents of which are hereby
incorporated by reference. For example, Hogan describes the
intraperitoneal administration of PMS and hCG, both resuspended
from lyophilized powder in sterile 0.9% NaCl, to induce
superovulation. Both PMS and hCG should be administered prior to
the release of endogenous LH. The Hogan protocols, directed to the
harvesting of oocytes from mice, can be routinely adapted for
humans without undue experimentation.
[0093] Polyethylene glycol has also been shown to induce fusion of
ovulated oocytes (see, e.g., Sekirina, G. G., Ontogenez.
16(6):583-8 (1985), and Gulyas, B. J., Dev. Biol. 101(1):246-50
(1984), incorporated by reference herein). Alternatively, Nogues et
al., Zygote, 2(l):15-28 (1994), (incorporated by reference herein)
describes the induction of oocyte fusion by inactivated Sendai
virus, resulting in the production of "zygotes" or "oocyte fusion
products (OFP)" that are able to undergo the first stages of
embryonic development. For a review of oocyte fusion techniques,
see Gulyas, B. J., Dev. Biol., 4:57-80 (1986), incorporated by
reference herein.
[0094] Alternatively, preventing the extrusion of the second polar
body from oocytes can generate HS cells. A detailed protocol for
activation of mouse oocytes is described in Hogan et al. supra, pp.
148-150, wherein harvested eggs with their cumulus cells attached
are maintained in a solution of 7% ethanol in Dulbecco's PBS for 5
minutes, washed with medium, and incubated at 37.degree. C. for 5
hours. The cumulus cells are subsequently removed by treatment with
hyaluronidase. In nature, following the first meiotic division and
separation of the first polar body, oocytes are arrested at
metaphase II and can only undergo the second meiotic division when
stimulated by a sperm. In vitro, the stimulation from the sperm can
be mimicked by exposing oocytes to agents such as Ca.sup.++
ionophore (A23187) or ethanol to trigger the continuation of
meiosis II. Before the extrusion of the second polar body,
karyokinesis (separation of chromosomes) and cytokinesis (division
of cells) can be inhibited by agents including, but not limited to,
6-dimethylaminopurine (6-DMAP), or cytochalasin D, resulting in the
activation of such diploid oocytes and subsequent formation of
blastocyst-like masses. FIG. 2 depicts the products of
activation.
[0095] In another embodiment, allowing the extrusion of the second
polar body can be accomplished by exposing oocytes to Ca.sup.++
ionophore (A23187) alone or followed by puromycin. The haploid
oocytes can further undergo genomic self-replication without
division, and the resulting diploid oocytes when incubated under
appropriate conditions can form blastocyst like masses and may be
used to derive HS cells. See, Taylor, A. S., et al., Hum. Reprod.,
9(12):2389-97 (1994); and Kaufman, M. H. et al., J. Embryol. Exp.
Morphol., 73:249-61 (1983).
[0096] Spermatids useful in the context of the present invention
can be obtained using any suitable method known in the art or yet
to be discovered, particularly those conventional in the field of
in vitro fertilization. To create HS cells for use in a male,
spermatids (meiosis II completed) are harvested and then induced to
fuse. Spermatid fusion can be achieved using well-established
standard techniques. For example, Asakura S, et al., Exp. Cell.
Res., 181(2):566-73 (1989), incorporated by reference herein,
teaches the use of a hypotonic medium to induce the fusion of a
pair of spermatids and the eventual formation of a single acrosome
(synacrosome). Alternately, secondary spermatocytes (meiosis I
completed) can be activated using methods that are known in the
art.
[0097] Homozygous post-meiosis I diploid germ cells can be
harvested from a donor using conventional technology, particularly
those techniques commonly used in the field of in vitro
fertilization. See, for example, Jones et al., Fertil. Steril.,
37(l):26-29 (1982), describing techniques for aspirating oocytes
from human ovarian follicles; Lisek et al., Tech. Urol., 3(2):81-85
(1997), describing techniques for collecting sperm from the
epididymis and testicle; Stice et al., Mol. Reprod. Dev.,
38(1):61-8 (1994), and Takeuchi et al., Hum. Reprod., 14(5):1312-7
(1999), describing techniques for transplanting nuclear material of
one donor to an enucleated oocyte of another. The entire contents
of these references are hereby incorporated by reference
herein.
[0098] Finally, as noted above, the isolated HS cell can be created
from transferring two haploid germ cell nuclei to an enucleated
oocyte. Specifically, two sperm or haploid egg nuclei can be
transferred into an enucleated oocyte to create a non-fertilized
diploid oocyte bearing the nuclear genetic information of the donor
male or female in the oocyte cytoplasm. The donor nuclear material
can be harvested and/or isolated using standard techniques
conventional in the art. Likewise, the transfer step can be
performed using techniques conventional in the art of in vitro
fertilization (see U.S. Pat. No. 5,945,577, WO 98/07841, and see
also, Stice and Takeuchi discussed above, as well as Wobus et al.,
Cells Tissues Organs, 166:1-5 (2000) incorporated by reference
herein.
[0099] Genetic modifications may be introduced into HS cells by
polynucleotide transfection techniques, including but not limited
to, viral vector transfer, bacterial vector transfer, and synthetic
vector transfer (e.g., via plasmids, liposomes and colloid
complexes).
[0100] Methods for isolating ES cells from the inner cell mass of
fertilized blastocysts are known in the art. Such methods may be
adapted for isolating HS cells from the inner cell mass of
blastocyst-like masses. For example, see Gardner et al., Curr. Op.
Obst. Gyn., 11:307-311 (1999), U.S. Pat. No. 5,843,780 (Thomson et
al.) and U.S. Pat. No. 5,905,042 (Stice et al.), the contents of
which are incorporated by reference herein.
[0101] HS cells can be produced from any animal donor material and
used in any animal system. Both human and non-human HS cells are
contemplated by the present invention. Suitable veterinary
applications include the generation of HS cells from and use in
mammals, fish, reptiles, birds, and amphibians.
[0102] C. Method For Selecting Immunotype
[0103] Harvested homozygous post-meiosis I diploid germ cells,
produced by the method noted above, are used to obtain HS cells
and, ultimately, HS-derived progenitor cells, and other
differentiated cells. The present invention encompasses further
selecting HS cells for the immunotype and genotype of a target gene
such that they may be used in the treatment of diseases, for
example, by implantation or transplantation to an affected
individual in need of such treatment.
[0104] Homozygous post-meiosis I diploid germ cells may be
initially procured from an affected individual, in which case there
would be no need for matching immunotype. However, in the case
where such germ cells are taken from a donor who is related, or
unrelated to the recipient, there is a need for selecting for
immunotype. Furthermore, it is an object of the present invention
to create a bank for stem cells that have been previously
immunohistologically typed. In certain situations, a self-donor is
preferred. However, in cases where an affected individual suffers
from a genetic disease (i.e., a disease characterized by the lack
of a crucial gene either due to mutation or improper expression),
it may be preferable to utilize a non-self donor. Alternatively, it
is another object of this invention that one skilled in the art of
selection procedures may choose those self germ cells, which
display the desired genotype (e.g., cells lacking a flawed or
mutated gene), those cells capable of expressing the deficient
gene. Such selection techniques may also be used to avoid an
immuno-incompatible genotype or phenotype for tissue
transplant.
[0105] The method for producing a homozygous population of cells
suitable for transplant as contemplated by the present invention,
requires assaying the immunotype (`tissue typing`) of the recipient
and then selecting immunocompatible stem cell populations. There
are two techniques used currently for tissue typing, serological
(the lymphocytotoxicity test), and molecular (DNA based)
techniques. These may be used to select stem cell populations as
well, and are described below.
[0106] 1. Serological Techniques
[0107] For serological typing of the recipient, in the present
invention, two samples of approximately 20ml of anti-coagulated
blood are required from the recipient for a full class I and class
II tissue type. For class I typing, one sample of blood is treated
with sodium citrate, and then defibrinated so as to remove
platelets as they may result in false negative reactions. The
sample is then layered onto a density gradient medium and
centrifuged so as to separate red blood cell and polymorphs from
lymphocytes. The red cell and polymorphs sediment, and a band of
lymphocytes is removed to another tube and washed. The lymphocytes
are then counted and adjusted to a standardized cell count, such as
1.5-2.0 million cells per ml, for a lymphocytotoxicity test. The
counted cells and typing sera are incubated in wells of a test
plate, such that the HLA anti-sera bind to specific cell membrane
target antigens on the surface of the lymphocytes. See FIG. 4A for
serological specificities of class I alleles. Rabbit serum, or
complement, is then added to lyse the cells. The cells will lyse,
only when there is an antibody-antigen complex formed on the cell
membranes of the lymphocytes, which then activates complement
leading to cell lysis. Lysed cells are detected by the entry of a
stain into the dead cells--a positive reaction. If there is no
lysis, cells do not stain indicating a negative reaction.
[0108] For class II typing, the second sample of blood is treated
with EDTA. The sample is then incubated with immunomagnetic beads,
(superparamagnetic monodisperse polymer particles coated with
monoclonal antibodies) and placed in a magnetic field. As class II
antigens are expressed on B cells, the immunomagnetic beads are
coated with a monoclonal antibody with a specificity for B cells.
The B cells and the beads are captured by the magnetic field,
washed, and counted using standard techniques. A standard volume,
such as 60 .mu.l, is then used for a lymphocytotoxicity test as
described above. See FIG. 4B for serological specifications of
class II alleles. The lyphocytotoxicity assay is further described
by Hui et al, Handbook of HLA Typing Techniques, p. 194 (1993).
[0109] The lymphocytotoxicity test may be used to type stem cells
as well. A panel of immunologically well-characterized anti-sera
are added to a sample of stem cells, from a related or unrelated
donor, and incubated. Rabbit serum, a source of complement, is then
added to the mixture, which is activated if the anti-sera bind the
HLA antigens on the stem cell surfaces. Upon activation, complement
will lyse the cells allowing them to absorb a standard stain, and
be detected.
[0110] Because it is unclear whether HS cells might not have
carried the proper HLA molecules, at this early stage of
development, molecular testing (see section 2 infra) should also be
used.
[0111] In an alternative embodiment, a mixed leukocyte culture test
(MLC) may also be used for recipient and stem cell class II
molecules. This test, described in Bach et al. (1964), Lymphocyte
Interaction: A Potential Histocompatibility Test In Vitro, Science
143:813-814, measures the response of a potential re-;`rent`s
T-cells to alloantigens present on the cells of a potential donor,
and is incorporated by reference herein.
[0112] 2. Molecular Techniques
[0113] Genomic DNA from a blood sample of the recipient, or stem
cells is required for these techniques. Genomic DNA is first
amplified using a polymerase chain reaction (PCR) procedure known
to one skilled in the art. The PCR amplified blood DNA can then be
typed using two techniques, digestion with restriction enzymes
(Restriction Fragment Length Polymorphism analysis), or
hybridization with multiple sequence specific oligonucleotide
probes (SSOP). The RFLP analysis is preferred as a reliable and
more convenient method as it does not require radioactive labeling.
It incorporates enzymes that recognize specific sites in some
alleles, therefore a specific HLA-haplotype may be detected by
simply checking whether the enzyme digests the amplified DNAs. FIG.
5 lists restriction endonucleases that may be used for classII
typing. Detailed methodology may be obtained from Hui et al,
Handbook, at 9.
[0114] The SSOP analysis requires the amplified DNA to be
immobilized on a nylon membrane, and then hybridized with selected
radioactive oligonucleotide probes directed to specific
HLA-alleles. FIG. 6 shows a list of SSOPs that may be used for
class II typing. A modification of this technique, the sequence
specific primer typing, (SSP), may also be used. Here, the
specificity of the typing system is part of the PCR reaction.
Oligonucleotide primers are used to amplify specific HLA-alleles,
hence completely matched primers will be more efficiently used in
the PCR reaction and may then be detected using agarose gel
electrophoresis and transillumination.
[0115] Other PCR methods such as class II matching by PCR
fingerprinting, Hui et al, Handbook, at 99, PCR characterization of
polymorphic microsatellite loci of HLA, Martin et al.,
Immunogenetics 47:131-138, (1998), are also contemplated by the
present invention.
[0116] Characterization of polymorphic microsatellite loci of HLA
is a preferred technique that predicts the HLA haplotype of a
recipient and a donor, and is especially useful when the donor and
recipient are related. As described by Martin et al., supra, large
numbers of microsatellite repeats are found interspersed in the
human genome. These microsatellites are highly polymorphic and are
inherited in a Mendelian fashion, thus giving them utility as
genetic markers. Twelve microsatellites within or near the HLA were
typed and sequenced by Martin et al. See FIG. 7 for PCR primers
used to sequence microsatellites. Further, alleles were designated
according to size. Linkage disequilibrium between these
microsatellites and certain HLA alleles suggests that these
microsatellites may predict HLA typing, and may be used as markers.
FIG. 8 shows a map of the HLA-I, II and III regions and the
relative position of 12 microsatellites.
[0117] D. Method For Selecting Genotype
[0118] In certain instances, it is necessary to select for the
presence of a target genotype. As noted above, the "target"
genotype in the context of the present invention will be that
genotype that is the non-mutant or "normal" form, i.e., the genetic
sequence that is not linked to pathology. Thus, in those instances
wherein the germ cell donor is affected with or is a carrier for a
genetically linked disease or disorder, it is necessary to assay
the genotype of each HS cell system arising from each donor
homozygous post-meiosis I diploid germ cell and select only those
populations carrying the desired target genotype. Those populations
carrying the mutant genotype should be eliminated from further
development. Recall, each donor germ cells is diploid and
homozygous for a particular haplotype. Thus, each population of
cells arising therefrom (i.e., the mitotic activation of
post-meoisis I diploid germ cell system and the stemplasm system)
will homogeneously carry one haplotype or the other, not both.
[0119] There are numerous techniques for assaying the genetic
constitution or "genotype" of a cell population. Most begin with
the extraction of a sample of genetic material (e.g. genomic DNA or
mRNA) from which cDNA may be derived, from a representative cell.
The sample is typically amplified, using techniques such as PCR,
and cloned to derive a partial or complete genetic "library". The
library can then be screened for the presence of the normal or
mutant sequence. As noted previously, those populations containing
the normal sequence are selected for further development; those
containing the mutant sequence (i.e., that which is associated with
pathology) will be eliminated. The genetic mutation may be detected
by a number of methods utilizing techniques including, but not
limited to, gel-based sequencing, non gel-base sequencing, and
genetic markers. The resulting sequence can be automatically
scanned for the presence of the mutant or target (i.e., normal)
sequence.
[0120] 1. Gel-Based Sequencing
[0121] The most common nucleic acid sequencing method involves the
separation of the DNA fragments, typically from genomic DNA or cDNA
cut by specific cleavages or synthesized by DNA polymerases, using
a gel, such as a polyacrylamide gel. Examples of gel-based
sequencing techniques include, but are not limited to,
Maxam-Gilbert sequencing (also called the chemical degradation
method) and Sanger sequencing (also called the chain termination or
dideoxy method).
[0122] Maxam-Gilbert sequencing (Maxam et. al., Proc. Natl. Acad.
Sci. USA 74:560-564, 1977, incorporated by reference herein) uses
chemicals to cleave DNA at specific bases, resulting in fragments
of different lengths. A refinement to the Maxam-Gilbert method
known as multiplex sequencing enables investigators to analyze
about 40 clones on a single DNA sequencing gel.
[0123] Sanger sequencing (Sanger et. al., Proc. Natl. Acad. Sci.
USA 74 5463-5467, 1977, incorporated by reference herein) involves
using an enzymatic procedure to synthesize DNA chains of varying
length in four different reactions, stopping the DNA replication at
positions occupied by one of the four bases, and then determining
the resulting fragment lengths.
[0124] Maxam-Gilbert and Sanger, differ primarily in the way the
nested DNA fragments are produced. Both methods work because gel
electrophoresis produces very high resolution separations of DNA
molecules; even fragments that differ in size by only a single
nucleotide can be resolved. Almost all steps in these sequencing
methods are now automated. Certain known disease genes can be
sequenced with technologies as high-voltage capillary and ultrathin
electrophoresis to increase fragment separation rate and use of
resonance ionization spectroscopy to detect stable isotope
labels.
[0125] 2. Non Gel-Based Sequencing
[0126] Non gel-based sequencing technologies, which aim to increase
efficiency by several orders of magnitude, can also be used. These
technologies include but are not limited to (a) enhanced
fluorescence detection of individual labeled bases in flow
cytometry; (b) direct reading of the base sequence on a DNA strand
with the use of scanning tunneling or atomic force microscopies;
(c) enhanced mass spectrometric analysis of DNA sequence, and (d)
sequencing by hybridization to short panels of nucleotides of known
sequence.
[0127] Sequencing by hybridization (SBH) can be applied to the
efficient identification and sequencing one or more DNA samples in
a short period of time. The procedure has many applications in DNA
diagnostics, forensics, and gene mapping. It also may be used to
identify mutations responsible for genetic disorders and other
traits, to assess biodiversity and to produce many other types of
data dependent on DNA sequence.
[0128] There are several approaches for sequencing by hybridization
(SBH). In SBH Format 1, DNA samples are arrayed and labeled probes
are hybridized with the samples. Replica membranes with the same
sets of sample DNAs may be used for parallel scoring of several
probes and/or probes may be multiplexed. Techniques for arraying
and hybridization of DNA samples on the nylon membranes are well
developed. Each array may be reused many times. Format 1 is
especially efficient for batch processing large numbers of
samples.
[0129] In SBH Format 2, probes are arrayed and a labeled DNA sample
fragment is hybridized to the arrayed probes. In this case, the
complete sequence of one fragment may be determined from
simultaneous hybridization reactions with the arrayed probes. For
sequencing other DNA fragments, the same oligonucleotide array may
be reused. In a variant of Format 2, DNA anchors are arrayed and
ligation is used to determine oligosequences present at the end of
the target DNA.
[0130] In Format 3, two sets of probes are used. One set may be in
the form of arrays and another, labeled set is stored in multiwell
plates. In this case, target DNA need not be labeled. Target DNA
and one labeled probe are added to the arrayed set of probes. If
one attached probe and one labeled probe both hybridize
contiguously on the target DNA, they are covalently ligated,
producing a sequence twice as long to be scored. The process allows
for sequencing long DNA fragments, e.g. a complete bacterial
genome, without DNA subcloning in smaller pieces.
[0131] For additional details on SBH, see U.S. Pat. Nos. 5,503,980,
5,683,881, and 6,025,136, incorporated by reference herein. See
also Bainst, W., et al., J. Theoret. Biol., 135: 303-307 1988;
Matthews, J. A., et al., Anal. Bicchom., 169, 1-25, 1988; Syvanen,
A. C., Medical Biology, 64, 313-324 1986; and Lehrach, H., et al.,
Genome Analysis, 1, 39-71, 1990, the contents of which are all
incorporated by reference herein.
[0132] 3. Genetic Markers
[0133] Genetic markers, short sequences that correlate to the
presence of a particular gene sequence, can be used to detect the
presence of a mutant or normal gene. Examples of genetic markers
useful in the context of the present invention include, but are not
limited to, sequence tagged sites (STSs), short sequences that are
standard markers for physical mapping, and expressed sequence tags
(ESTs), unique markers for identifying expressed genes. These
markers provide a means of rapidly identifying most human genes.
Other applications of the EST approach include determining
locations of genes along chromosomes and identifying coding regions
in genomic sequences.
[0134] Genetic markers have the greatest utility when they are
highly heritable, multi-allelic, and numerous. Most genetic markers
are highly heritable because their alleles are determined by the
nucleotide sequence of DNA, which is highly conserved from one
generation to the next, and the detection of their alleles is
unaffected by the natural environment. Markers have multiple
alleles because, in the evolutionary process, rare,
genetically-stable mutations in DNA sequences defining marker loci
arose and were disseminated through the generations along with
other existing alleles. The highly conserved nature of DNA combined
with the rare occurrence of stable mutations allows genetic markers
to be both predictable and discerning of different genotypes.
[0135] DNA fingerprinting is a broad term used to designate methods
for assessing sequence differences in DNA isolated from various
sources, e.g., by comparing the presence of marker DNA in samples
of isolated DNA. Typically, DNA fingerprinting is used to analyze
and compare DNA from different species of organisms or DNA from
different individuals of the same species. DNA sequence differences
detected by fingerprinting are referred to as DNA polymorphisms.
The presence of a DNA polymorphism in an organism's DNA can serve
to indicate that the genetic origin of such an organism is
different from the genetic origin of organisms whose DNA does not
have the polymorphism. Such polymorphisms can result, e.g., from
insertion, deletion, and/or mutation events in the genome.
[0136] Many genetic-marker technologies are adaptable to
fingerprinting, including restriction-fragment-length polymorphism
(RFLP) (Bostein et al., Am. J. Hum. Genet. 32:314-331, 1980);
single strand conformation polymorphism (SSCP) (Fischer et al.,
Proc. Natl. Acad. Sci. USA 80:1579-1583,1983; Orita et al.,
Genomics 5:874-8791989); amplified fragment-length polymorphism
(AFLP) (Vos et al., Nucleic Acids Res. 23:4407-4414, 1995);
microsatellite or single-sequence repeat (SSR) (Weber, J. L. et
al., Am. J. Hum. Genet. 44:388-3961989); rapid-amplified
polymorphic DNA (RAPD) (Williams et al., Nucleic Acids Res.
18:6531-6535 1990); sequence tagged site (STS) (Olson et al.,
Science 245:1434-1435, 1989); genetic-bit analysis (GBA) (Nikiforov
et al., Nucleic Acids Res. 22:4167-4175, 1994); allele-specific
polymerase chain reaction (ASPCR) (Gibbs et al., Nucleic Acids Res.
17:2437-2448, 1989;, Newton et al., Nucleic Acids Res.
17:2503-2516, 1989); nick-translation PCR (e.g., TaqMan..TM..) (Lee
et al., Nucleic Acids Res. 21:3761-3766, 1993); and allele-specific
hybridization (ASH) (Wallace et al., Nucleic Acids Res.
6:3543-3557, 1979); Sheldon et al., Clin. Chem. 39(4):718-719
1993). Each of the above-noted articles are incorporated by
reference herein.
[0137] Kits for RAPD and AFLP analyses are commercially available,
e.g., from Perkin Elmer Applied Biosystems (Foster City, Calif.).
For example, the restriction fragment length polymorphism (RFLP)
technique employs restriction enzyme digestion of DNA, followed by
size separation of the digested DNA by gel electrophoresis, and
hybridization of the size-separated DNA with a specific
polynucleotide fragment. Differences in the size of the restriction
fragments to which the polynucleotide probe binds reflect sequence
differences in DNA samples, or DNA polymorphisms. See Tanksley,
Biotechnology 7:257-264 (1988), incorporated by reference
herein.
[0138] PCR-based fingerprinting methods result in the generation of
a large number of reproducible DNA fragments of specific size that
can be separated, typically by gel electrophoresis. These fragments
are visualized to produce a "fingerprint" of the amplified DNA.
Visualization of the size-separated fragments is effected either by
direct visualization, e.g., with a fluorescent dye, by
hybridization with a polynucleotide probe, or by labeling the
amplification products during PCR (radioactively or fluorescently),
followed by detection of the labeled products in the gel. These
fingerprints have a variety of uses: parentage analysis; linkage
analysis of specific traits; analysis of the degree of generic
relationship between individuals within a species; and analysis of
phylogenetic relationships between species. This has considerable
commercial use in agriculture for marker assisted selection of
genetic traits specific to particular genotypes (e.g., in crops or
animals), identification and mapping of quantitative trait loci
(QTLs) and the like.
[0139] For additional details on genetic markers and techniques for
detecting genes without sequencing, see U.S. Pat. Nos. 6,141,657,
5,683,880, and 6,100,030, all incorporated by reference herein.
[0140] E. Methods For Directed Differentiation of HS Cells
[0141] HS cells can be induced to differentiate into various types
of tissues originating from all three germ layers (endoderm,
mesoderm, and ectoderm) including, but not limited to, skin, hair,
nervous tissue, pancreatic islet cells, bone, bone marrow,
pituitary gland, liver, bladder, and other tissues having
diagnostic or therapeutic utility in animals, including humans as
described in U.S. patent application Ser. No.: 09/997,240 and U.S.
Provisional Application Ser. No. 60/253,943, fully incorporated by
reference herein.
[0142] One skilled in the art of differentiation techniques,
particularly those developed for differentiation of ES cells and
embryonic carcinoma (teratocarcinoma) cells, can induce a
pluripotent HS cell to differentiate into a desired type/of tissue
without undue experimentation.
[0143] The pluripotent isolated HS cells of the present invention
can be differentiated into selected tissues for a variety of
therapeutic uses including the in vitro culture of differentiated
tissues for purposes of study, diagnostics, or for implantation
into an individual. Preferably, HS cells will be used
therapeutically in the individual that provided the donor material
for HS formation. In females, this may be achieved by activation of
post-meiosis I diploid oocytes, or by fusion two haploid oocytes;
in males, this may be achieved by activation of secondary
spermatocytes or fusion of spermatids, or by transferring sperm
nuclei to enucleated oocytes.
[0144] Well known methods used for isolating and culturing ES cells
can be adapted for use with HS cells. Exemplary procedures are
provided by Hogan et al., supra, pp. 254-262, 265-272. For example,
Hogan et al. describe optimal culture media as a 7.2-7.4 pH
buffered bicarbonate media, such as Dulbecco's modified Eagle's
medium, containing glucose and sodium pyruvate, further
supplemented with glutamine (2 mM), nonessential amino acids (0.1
mM), mercaptoethanol (0.1 mM) or monothioglycerol (0.15 mM),
gentamycin (50 .mu.g/ml), 15% serum (e.g. fetal bovine serum), and
leukemia inhibitory factor (LIF). A mixture of trypsin and EDTA in
Ca++/Mg++--free phosphate buffered saline can be used to detach
cells from tissue culture dishes and dissociate them from one
another. Stem cells are preferably cultured on a feeder cell layer
in medium supplemented with LIF to provide factors that enhance the
proliferation and maintain the undifferentiated state of stem
cells. Fibroblasts, particularly mouse embryo fibroblasts (MEF) and
STO mouse fibroblasts, are the preferred feeder cells. Feeder cells
should be mitotically inactivated, by treatment with mitomycin C,
or gamma radiation. Additional types of feeder cells may used or a
feeder-free system may also be used. Detailed protocols for each
step in the preparation and maintenance of viable stem cell
cultures are provided by Hogan et al., supra.
[0145] Similarly, methods for isolating individual colonies of ES
cells and expanding them to a sufficient number to allow isolation
and screening of DNA can be applied to HS cells. For example, Hogan
et al., supra, at pp. 279-281, describe stem cell isolation
techniques including: the extraction and transfer of a trypsinized
colony to single wells of a culture dish using an automatic
pipettor and extraction and transfer of a nontrypsizinized colony
to a microdrop of trypsin/EDTA using a sterile, glass Pasteur
pipette attached to a tubing and a mouthpiece. Preferred clones can
be identified and assayed using rapid techniques and PCR.
[0146] To minimize the danger of accumulating chromosomal
anomalies, vials of stock should be frozen and stored as soon as
possible. Methods for proper freezing and storage of cultures are
well known for ES cells, and may be applied to HS cells. See, for
example, Hogan, et al., supra, pp. 283, setting forth a detailed
protocol for freezing, trypsinizing, pelleting and resuspending
samples from cell cultures. Methods for differentiation of
pluripotent cells are discussed below. These methods are designed
to be an illustrative not an exhaustive, list of methods for
differentiating pluripotent cells including the HS cells of the
present invention. The present invention can be practiced using
differentiation methods known in the art, including techniques not
recited here, or not yet discovered.
[0147] For example, Hole (Cells Tissues Organs 165:181-189, 1999,
and incorporated by reference herein) describes methods for
directing the differentiation of hematopoietic cells from embryonic
stem cells in vitro that may be adapted to differentiate HS cells.
Furthermore, Doetschman et al., Embryol. Exp. Morphol. 87:27-45,
1985, incorporated by reference herein, suggests that the
withdrawal of leukemia inhibitory factor (LIF) from ES cells grown
in suspended culture results in the formation of cystic embryoid
bodies containing blood islands made up of erythrocytes and
macrophages. The production of other hematopoietic cells, including
neutrophils, mast cells, macrophages and erythroid cells, from stem
cells has also been described (See, e.g., Wiles and Keller, Devel.
111 :259-267,1991; Keller et al, Mol. Cell. Biol. 13:473-486,
1993a; and Lieschke and Dunn, Exp. Hematol. 23:328-334, 1995; each
of which are hereby incorporated by reference herein in their
entirety). These methods may be adapted for use with HS cells of
the present invention without undue experimentation.
[0148] The techniques described by Cho et al., Proc. Natl. Acad.
Sci. USA 96:9797-9802, 1999, incorporated by reference herein, for
efficiently differentiating ES cells into mature Ig-secreting B
lymphocytes can also be adapted for use with the HS cells of the
present invention. Likewise, Dani, Cells Tissues Organs 165:
173-180,1999 incorporated by reference herein, describes a method
for differentiating ES cells into adipocytes. For example, the
treatment of embryoid bodies at an early stage of their
differentiation with retinoic acid (RA) for a short period of time
appears to be linked to adipogenesis.
[0149] Techniques for eliciting the differentiation of stem cells
into a variety of neuronal cells and neurons are described by Okabe
et al., Mech. Dev. 59: 89-102, 1996, incorporated by reference
herein. Likewise, stem cell-derived oligodendrocytes and neurons
having particular use in treating injured spinal cords are
described by McDonald et al., Nature Medicine 5:1410-1412, 1999
incorporated by reference herein. These techniques can be adapted
for use with HS cells of the present invention without undue
experimentation.
[0150] The use of accessory cell lines, such as OP9, to derive
particular cell lineages is also contemplated. See, for example,
Nakano el al., Science 265:1098-1101, 1994, (incorporated by
reference herein) and Nakayama el al., Blood 91:2283-2295, 1998
(incorporated by reference herein) that relate to erythroid,
myeloid and lymphoid lineages.
[0151] Techniques for eliciting the differentiation of HS cells of
the invention into follicular cells, as well as epidermal cells are
also contemplated. See, for example, Taylor et al., Cell 102:
451-361, 2000 (incorporated by reference herein). The expression of
particular regulatory genes may also be used to direct
differentiation. See, for example, Hole et al., Blood 90:1266-1276
1996a, and Battieres Clin. Hematol. 3:467-483, 1997 (incorporated
by reference herein), relating to hematopoietic genes. Likewise,
nuclear regulatory factors involved in lipid metabolism, including
but not limited to PPARs (PPAR.delta. and PPAR.gamma.) and
C/EBP.delta. (C/EBP.beta., C/EBP.delta. and C/EBP.alpha.), may also
be triggers of terminal differentiation of preadipocytes into
adipocytes. Such factors would find utility in the context of the
differentiation methods of the present invention.
[0152] Depending on the function needed, differentiation may be
assessed by detecting expression of a gene specific for
differentiation, by detecting tissue-specific antigens, by
examining cell or tissue morphology, by detecting functional
expression such as ion channel function; or by any means suitable
for detecting the differentiation of HS cells. Methods for inducing
differentiation of embryonal carcinoma (EC) cells into a variety of
embryonic and extraembryonic cell types can be used to induce
differentiation of HS cells (Andrews, APMIS 106:158-168 1998). HS
cells can undergo directed differentiation in vitro by exposure to
various factors known to trigger cell commitment and
differentiation into a desired cell type or tissue. Many in vitro
differentiation schemes involve the removal of growth factors known
to favor initiation of differentiation. Once these factors are
removed from the medium, the stem cells, growing in suspension
without feeder cells, form clusters, known as embryoid bodies,
within which descendants of all three embryonic germ layers can be
found. The presence of certain cell lineages within the embryoid
body can be enhanced through supplementation of the medium with
additional growth factors and chemicals. The resulting cell
population will then contain an increased proportion of a desired
cell type, which then can be selectively isolated. See Edwards et
al., Modern Trend., 74(1): 1-7, 2000 (incorporated by reference
herein) for a discussion of pluripotent stem cells and their use in
medicine.
[0153] Illustrative examples of such differentiation control
factors include but are not limited to cytokines, hormones, and
cell-regulating factors such as LIF, GM-CSF, SCF, IL-3, thyroid
hormone (T3), stem cell factor (SCF), fibroblast growth factor
(FGF-2), -platelet derived growth factor (PDGF), ciliary
neurotrophic factor. While stimulating cytokines such as GM-CSF,
SCF, and IL-3 have been shown to promote differentiation (see Keil
F, et al., Ann. Hematol. 79(5):243-8, 2000 (incorporated by
reference herein)), inhibitory factors such as LIF has been shown
to maintain mouse embryonic stem (ES) cells in the undifferentiated
pluripotent state (Zandstra P W, et al., Blood 96(4):1215-22, 2000
incorporated by reference herein). While SCF has been shown to
stimulate the differentiation of chicken osteoclasts from their
putative progenitors (van't Hof R J, et al., FASEB J. March 1997;
11(4):287-93, 1997 incorporated by reference herein), FGF-2 has
been shown to play a role both in initiating lactotrope
differentiation and maintaining Prolactin expression in
immortalized GHFT cells, thereby suggesting a mechanism for
controlling differentiation of stem cells into different anterior
pituitary cells (Lopez-Fernandez J, et al., J. Biol. Chem.
275(28):21653-60, 2000 incorporated by reference herein).
Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports
neuronal differentiation while ciliary neurotrophic factor and
thyroid hormone T3 generate clones of astrocytes and
oligodendrocytes (Johe KK, et al., Genes Dev. 10(24):3129-40, 1996
incorporated by reference herein).
[0154] HS cells can be induced to differentiate by transplantation
in vivo, preferably in situ, where the cells undergo histologic and
functional differentiation and form appropriate connections with
host cells. Endogenous regulation factors located in the transplant
site can direct the differentiation of the stem cell into a
particular type of differentiated cell or tissue. Alternatively,
groups of divergent differentiated cells and/or tissues result from
stem cells transplanted to the hypodermis, the peritoneum, and the
renal capsule. See Hogan, supra, pp. 183 to 184, for a detailed
description of the kidney capsule implantation procedure.
[0155] Differentiation of pluripotent HS cells into various
endodermal cell types has great therapeutic implications, including
use for transplantation purposes, or to enhance the HS cells can be
uptake and processing of nutrients or to direct pattern formation,
induced to differentiate into endodermal progenitor cells by
treatment with high doses of RA or by members of the transforming
growth factor .beta. superfamily including bone morphogenetic
protein (BMP)-2 (Pera and Herzfeld, Reprod. Fert. Dev. 80:551-555
1998). Some HS cell lines can also be induced to differentiate in
distinct culture systems. For example, non-neural differentiation
can be induced by hexamethylene bisacetamide (HMBA) (Andrews, APMIS
106: 158-168 1998). BMP-2 can be used to specifically trigger
differentiation into parietal, or visceral endoderm (Rogers et at.,
Mol. Bio. Cell. 3:189-196 1992). BMPs are molecules that can induce
cartilage and bone growth in vivo, but BMP messages are also
expressed in many non-bony tissues, including developing heart,
hair follicles and central nervous system, indicating a pivotal
role in cell commitment and differentiation.
[0156] F. Prophylactic and Therapeutic Utilities
[0157] One embodiment of the present invention is a method of
growing cells and tissues that may be transplanted into an affected
person for the treatment of diseases including but not limited to
hereditary, or genetic diseases, neurological or neurodegenerative
diseases, traumatic injuries and cancers. Another cosmetic
application of the present invention is skin grafts for hair
replacement and/or other such applications.
[0158] HS cells can be induced to differentiate into various types
of tissues originating from all three germ layers (endoderm,
mesoderm, and ectoderm) including but not limited to skin, hair,
nervous tissue, pancreatic islet cells, bone, bone marrow,
pituitary gland, liver, bladder, and other tissues having
diagnostic or therapeutic utility in animals, including humans as
described in U.S. patent application Ser. No. 09/997,240, and U.S.
Provisional Application Ser. No. 60/253,943.
[0159] The present invention provides a method of treating a
disorder or disease state by generating, in situ or in vitro,
suitable replacement cells, groups of cells, tissues or organs from
isolated HS cells with pre-selected immunotype and/or genotype.
Methods of directing differentiation of cells in situ or in vitro
are described in the preceding section, and in U.S. patent
application Ser. No. 09/997,240, and U.S. Provisional Application
Ser. No. 60/253,943.
[0160] Illustrative disorders and disease states include but are
not limited to traumatic injury (e.g., post-trauma repair and
reconstruction, for limb replacement, spinal cord injury, burns,
and the like) and birth defects; pathological and malignant
conditions of the cells, tissues, and organs (e.g., cancer); and
degenerative and congenital diseases of the cells and tissues of
the muscles (e.g., cystic fibrosis, muscular dystrophy, cardiac
conditions), nerves (e.g., Alzheimer's, Parkinson's, and multiple
sclerosis), epithelium (e.g., blindness and myopathy,
atherosclerosis and other stenotic vascular conditions, enzyme
deficiencies such as Crohn's disease, and hormone deficiencies such
as diabetes), and connective tissues (e.g., immune conditions and
anemia). HS-derived cells and tissues may be grafted or
transplanted to a subject in need, preferably using the subject's
own donor material.
[0161] Currently, gene therapy for the treatment of hereditary
diseases has not had much success. There are many reasons for this,
the most significant being the inability to place normal DNA into a
patient's cells such that it is stably integrated and expressed.
Additionally, gene-replacement therapy may be too targeted, for
example, gene-corrected T-cell therapy, where T-cells with normal
DNA have been transplanted into patients to treat severe combined
immunodeficiency disease (SCID), including the ADA deficiency type.
The problem faced here is that the corrected T-cells are primed for
only one type of antigen, and a whole range of immune response
cannot be derived from them. Yet another problem faced by current
application of gene therapy, is that the product or the exact
function of the mutant gene may not be known or the genetic disease
may involve an entire cascade of factors untreatable by
gene-replacement.
[0162] The present invention provides a means to bypass these
concerns. HS cells, for example in the context of SCID may be
transplanted into the bone marrow of the patient such that local
endogenous factors derive an entire range of T- and B-cells. Issues
of HLA compatibility, or graft vs. host disease would not be an
issue here as the cells would be immunologically compatible.
Similarly, HS cells may be useful for the treatment of genetic
diseases that are caused by mutations that result in a missing gene
product or the absence of an enzyme. For example, currently the
treatment of Phenylketonuria is elimination of phenylalanine from
the diet of the patient, as the patient does not produce the enzyme
required to convert it to tyrosine. Differentiated HS cells may be
transplanted into these patients to produce the required enzyme.
Similarly, HS cells may find transplant applications in diseases
like Haemophilia A, where differentiated HS cells may produce a
missing gene product, namely Clotting Factor VIII.
[0163] Another embodiment of the invention is a method of inducing
HS cells to differentiate by transplantation in vivo, preferably in
situ, thereby generating a stemplasm, wherein the cells undergo
histologic and functional differentiation and form appropriate
connections with host cells. Endogenous regulation factors located
in the transplant site can direct the differentiation of the stem
cell into a particular type of differentiated cell or tissue.
Alternatively, groups of divergent differentiated cells and/or
tissues result from stem cells transplanted to the hypodermis, the
peritoneum, and the renal capsule. These and other methods are
described in U.S. patent application Ser. No. 09/997,240, and U.S.
Provisional Application Ser. No. 60/253,943.
[0164] The pluripotent isolated HS cells of the present invention
can also be differentiated into selected tissues by in vivo
differentiation in immuno-compromised animal followed by isolation
of the said tissues for a variety of therapeutic uses. And the HS
cells can also be cultured and differentiated in vitro for purposes
of study, or diagnostics.
[0165] G. Depository of HLA typed HS cells
[0166] HS cells are homozygous and carry only one HLA haplotype.
Thus, a donor HS cell has only one HLA haplotype to be matched with
the recipient instead of two, as in heterozygous stem cells. As
discussed above, there are 22 different HLA A antigens, 42
different B antigens, 9 different Cw antigens and 18 DR antigens.
With such a large number of polymorphic HLA haplotypes, a complete
match of two haplotypes between a recipient and a donor is quite
difficult. In a best case scenario, such as when there is linkage
disequilibrium and the presence of specific ethnic HLA haplotypes,
the chance of finding a two haplotype match ranges from one in
1,000 to one in several million. However, in the same scenario, a
one haplotype match could be achieved in less than 1 in 200 persons
for the common haplotypes (see Martin et al., supra., showing total
disequilibrium of common haplotype of around 172 for the Caucasian
population in the United States). Thus, HS cells can be taken from
random donors of multiple ethnicities to provide a depository or
bank of HS cell populations that immunohistologically match at
least one haplotype carried by individuals in the population at
large. The depository needs only contain about 200 different HS
cell lines, each having a distinct HLA haplotype, to service most
of the general population (i.e., provide immunohistocompatible
cells suitable for transplant).
[0167] In a preferred embodiment, the depository contents are
catalogued, for example as a searchable computer database, to
facilitate remote, rapid screening by practitioners around the
world. The present invention provides a method for producing a
population of genotyped homozygous stem (HS) cells from multiple
donor. Ideally, the donors will be selected for having a high
probability of having widely divergent HLA haplotypes. The donors
will then be assayed for the HLA haplotype and genotype. Then HS
are derived from said donors by mitotically activating donor
homozygous post-meiosis I diploid germ cells to develop multiple
blastocyst-like masses, each of which contains an inner cell mass
(ICM) that is homozygous for a particular allele, isolating HS
cells from the selected ICM, and culturing said HS cells to create
each of the HS cell lines, assaying the genotype of each said HS
cell line, and cataloging each HLA haplotype and genotype
associated with each HS cell line generated.
[0168] The following examples further illustrate experiments which
have demonstrated reduction to practice and utility of selected
preferred embodiments of the present invention, although they are
in no way a limitation of the teaching or disclosure of the present
invention as set forth herein.
EXAMPLES
Example 1
Homozygous Stem Cell Formation, And Their Differentiation Into
Progenitor Cells and Various Tissues Of The Three Embryonic Germ
Layers Within Stemplasms
Example 1(a)
Derivation of HS Cells from Mouse Post-Meiosis I Oocytes By
Activation Followed by Prevention of the Extrusion of the Secondary
Polar Body
[0169] Three eight-week old female C57/BDA2 F1 mice (Charles River
Laboratories, Wilmington, Mass.), were superovulated by
sub-peritoneal injections of 5 IU/100 ul of pregnant mare's serum
gonadotropin (PMS; PCCA, Houston, Tex. (29-1000-1BX)), and 5 IU/100
.mu.I of human chorionic gonadotropin (HCG; Sigma, St. Louis, Mo.,
(C8554)) with 48 hours apart between the injections. Seventy-three
oocytes were harvested about 17 hours after the HCG injection, and
the cumulus was removed by incubating the freshly obtained oocytes
in a drop (.about.300 .mu.l) of hyaluronidase (Sigma, H4272)
diluted in M2 media (M7167, Sigma) at final concentration of 0.3
mg/ml, followed by 3 washes with HEPES buffered M2 media before
further handling. Oocytes were then activated by treatment with 5
.mu.M calcium ionophore (Sigma,C7522) solution at room temperature
for five minutes followed by 2 washes with HEPES buffered M2 media.
The oocytes were further subjected to incubation in 5 mM
6-dimethylaminopurine (6-DMAP) (Sigma, D2629) in M16
bicarbonate-buffered culture media (Sigma, M7292) at 5% CO.sub.2
and 37.degree. C. for 3 hours. After the incubation, oocytes were
washed three times with M16 media and incubated in a drop of M16
media under mineral oil until blastocyst-like masses developed (4
to 5 days). The blastocyst-like masses then naturally hatch out of
the surrounding zona shell, and the blastocyst-like masses were
then transferred to a mitomycin-C treated murine embryonic feeder
cell layer and cultured for at least 15 days in ES medium (DMEM
(Gibco, Life Technologies, Rockville, Md., 11995-065) with 20%
fetal bovine serum (Gibco, 16141-079) and 1,400 U/ml of LIE
(Chemicon, ESG1106) for stem cell line formation.
[0170] Alternatively, immunosurgery was performed to remove
trophoblast cells from blastocyst-like masses before culturing on
the feeder by the following procedure. Hatched blastocyst-like
masses were first incubated with anti-mouse Thy-1 rabbit serum
(1:10 dilution in stem cell medium, Accurate Chemical, Westbury,
N.Y., ACL2001) and anti-human lymphocytes rabbit serum (1:10
dilution in stem cell medium, Accurate Chemical, CL8010) for one
hour at 37.degree. C. After washes three times with M2 medium the
blastocyst-like masses were then incubated with guinea pig
complement (1:10 dilution in stem cell medium, Accurate Chemical,
ACL4051) for 30 minutes at 37.degree. C . The complement-treated
cell masses were then washed 3 times in the M2 medium and
transferred to a mitomycin-C treated murine embryonic feeder cell
layer for further culturing in stem cell medium consisted of 80%
Dulbecco's modified Eagle's medium (no pyruvate, high glucose
formulation; Gibco-BRL) supplemented with 20% fetal bovine serum
(Gibco-BRL), 1 mM glutamine, 0.1 mM --mercaptoethanol (Sigma), 1%
nonessential amino acid stock (Gibco-BRL) and 1,400 U/ml LIF
(Chemicon, ESG1106).
[0171] After 15 days, inner cell mass-derived outgrowths formed and
were dissociated into clumps by mechanical dissociation with a
micropipette and replated on murine fibroblasts feeder in fresh
stem cell medium. Individual colonies with a uniform
undifferentiated morphology were individually selected by
micropipette, mechanically dissociated into clumps, and replated.
Once established and expanded, cultures were passaged by 5 minutes
exposure to Trypsin/EDTA solution (0.05%/0.5%, Gibco-BRL) followed
by firther culturing on feeder cells and fresh stem cell
medium.
[0172] Murine embryonic fibroblasts feeder cells were purchased
from Stemcell, Inc. (00308), and passaged 2-3 times before use. To
mitotically inhibit the feeder cells, one 60-mm dish of
confluent-expanded feeder cells was treated with 5 ml of 10
.mu.g/ml mitomycin-C (Sigma, M4287) in DMEM/10% FBS medium at
37.degree. C. for three hours. Treated feeder cells were then
washed with 5 ml DMEM/10% FBS three times, and collected by
trypsinization at 37.degree. C. for 5 minutes, followed by
neutralization with 5 ml DMEM/10% FBS medium and centrifugation at
1000 rpm for 5 minutes. The mitomycin-treated cell pellet obtained
was then resuspended in 15 ml DMEM/10% FBS medium, plated on three
60-mm dishes (5 ml of cell suspension/dish), and incubated at
37.degree. C. overnight before use.
Example 1(b)
Derivation of HS Cells Homozygous For a Specific Genotype and an
Immunotype From Female C57/DBA2 Hybrid Mice
[0173] Five female C57DBA2 hybrid mice heterozygous for most
genetic loci and H-2 (mouse MHC) were superovulated as described
example 1(a) for HS cells population. Eleven cell lines were
derived and were propagated in culture and then cryopreserved.
Cells from each line at around passage 5 were immunotyped for
I-E-beta in H-2 loci (sequences of PCR primers are: Eb1:CAG AAC CTG
AGT CCT GGG CG; Eb2: AGC AGA CCA GGA GGT TGT GG) and genotyped for
D2mit42 using microsatellite markers (Research Genetics).
[0174] Methods for genomic DNA extraction, PCR amplification, and
genetic analysis (using D2mit42 microsatellite marker) of mouse HS
cells were as describe in example 1(f). Immunotyping of H-2
(I-E-beta) was performed by separating the PCR products of the H-2
alleles using single-strand conformational polymorphism (SSCP)
analysis on a MDE gel (FMC Bioproducts). Results of genotyping and
immunotyping of two representative HS cell lines derived from
C57/DBA2 hybrid mice are shown in FIGS. 9A-F, in which a C57/DBA2
mouse (FIG. 9A), and her superovulated oocytes-derived
blastocyst-like masses (FIG. 9B) and two representative colony
(FIGS. 9C and 9D) from two different HS cell lines are shown. The
genotype (FIG. 9E) and immunotype (FIG. 9F) of the donor mouse
(Lane 1), HS cell line-1 (Lane 2), and HS-cell line-2 (Lane 3) are
also demonstrated.
Example 1(c)
Development Of Blastocyst-Like Cell Masses From Human Diploid
Oocytes By Activation Followed By The Prevention Of The Extrusion
Of The Secondary Polar Body
[0175] Female ovum donors underwent down-regulation with leurpolide
acetate (Lupron: TAP Pharmaceuticals, Deerfield, Ill.) and then
began COH (Controlled Ovarian hyper-stimulation) by receiving
follicle stimulating hormone (FSH) (Serono, Gona-F) treatment at a
dosage of 300 IU/day to induce an appropriate multifollicular
response. When ultrasonographic criteria for follicular maturity
were met, a single 10,000 IU dose of human chorionic gonadotropin
(hCG) (Serono, Profasi) was administered, and transvaginal
follicular aspiration was performed approximately 36 hours after
hCG administration. Cumulus from retrieved oocytes were removed by
exposing them to 80 IU/ml hyaluronidase for approximately 30
seconds followed by HEPES-buffered human tubal fluid supplemented
with 10% humans serum albumin (HEPES-HTF-HSA) (InVitroCare, Inc.,
San Diego, Calif., 2002 and 2101).
[0176] To accomplish mitotic activation, the cumulus free mature
M-II oocytes were treated with 5 .mu.M calcium ionophore (A23187,
Sigma) in HEPES-HTF-HSA for 5 minutes at 33.degree. C. followed by
3 washes with HEPES-HTF-HSA and incubation in 1 to 5 mM
6-dimethylaminopurine (6-DMAP, Sigma) in IVC-TWO (InVitroCare,
2008) for 3 to 5 hours at 37.degree. C. The activated oocytes were
incubated in IVC-ONE medium (InVitroCare, 2006) for 3 days, and
further incubated in IVC-THREE (InVitroCare) for 2 days for cell
division and blastocyst-like mass formation.
[0177] Alternatively, oocytes were co-cultured with STO mouse
feeder cells. On day 6 assisted hatching was performed under a
micromanipulator under which a blastocyst-like mass was clamped
with the holding pipette (syringe suction system) so that the
micro-needle filled with acidified tyrodes solution (Medi-Cult,
1060-0002) at the 3 o'clock position is exposed to the empty
perivitelline space to expel the acidified tyrodes solution gently
over a small (30 microns) area by holding the needle tip very close
to the zona. Expulsion of the acidified tyrodes solution was ceased
immediately when the inside of the zona is pierced or softened. The
blastocyst-like cell mass would then release from the weakened
zona. After blastocyst-like mass detached from zona, immunosurgery
was performed to remove trophoblast cells by incubating the mass
with anti-human Thy-1 (1:10 dilution in IVC-THREE, Accurate
Chemical, Westbury, N.Y., CBL415-CD90) and anti-human lymphocytes
(1:10 dilution in IVC-THREE, Accurate Chemical, CL8010) for one
hour at 37.degree. C. After washes three times with IVC-THREE
medium the blastocystlike mass was then incubated with LOW-TOX
guinea pig complement (1:10 dilution in IVC-THREE, Cedarlane,
CL4051) for 30 minutes at 37.degree. C. The complement-treated cell
masses were then washed 3 times in the IVC-THREE medium and
transferred to a mitomycin-C treated STO (ATCC) feeder cell layer
and cultured in stem cell medium consisted of 80% Dulbecco's
modified Eagle's medium (no pyruvate, high glucose formulation;
Gibco-BRL) supplemented with 20% fetal bovine serum (Gibco-BRL), 1
mM glutamine, 0.1 mM -mercaptoethanol (Sigma), 1% nonessential
amino acid stock (Gibco-BRL) and 1,400 U/ml LIF (Chemicon,
ESG1106). See FIG. 10A.
Example 1(d)
Development Of Blastocyst-Like Cell Masses From Human Post Meiosis
I Diploid Oocytes By Activation Followed By Allowing The Extrusion
Of The Secondary Polar Body And Genomic Self-Replication
[0178] Procedures for superovulation, oocyte retrieval, and the
subsequent removal of cumulus were as described in Example 1(b). To
accomplish mitotic activation, the cumulus free mature M-II oocytes
were subjected to sham intracytoplasmic sperm injection (ICSI) to
mimic activation introduced by sperm followed by incubation with 25
.mu.M calcium iononphore (A23187, Sigma) for 5 minutes at
33.degree. C. Oocytes activated in this manner extrude the
secondary polar body and become haploid. Such haploid oocytes were
incubated in IVC-ONE medium (InVitroCare, Inc.) for 3 days, and
further incubated in IVC-THREE (InVitroCare) for 2 days for cell
division and blastocyst-like cell masses formation. The subsequent
manipulations of the blastocyst-like mass were as described in
Example 1(b). Haploid oocytes resulting from activation are able to
self-replicate their genome without cytokinesis and give rise to
diploid cells (Taylor, A. S., et al., Hum. Reprod. 9(12):2389-97
(1994); Kaufman, M. H. et al., J. Embryol. Exp. Morphol. 73:249-61
(1983). See FIGS. 10B-D.
Example 1(e)
Mouse HS Cell Growth, Differentiation Of Such HS Cells Under Mouse
Kidney Capsule, And Embryoid Body Formation Of Such Cells
[0179] HS cells obtained from blastocyst-like masses as described
in Example 1(a) were seeded on mouse feeder cells in 0.1% gelatin
coated dishes (10 cm) with stem cell medium as described in Example
1(a) to colony formation.
[0180] One colony of HS cells was dissected into several pieces and
implanted in one of the two kidney capsules of 26 hybrid mice to
induce stemplasm formation. Stemplasms were then harvested by
sacrificing the mice in the post-implantation week 1, 3, 6, 9.5,
10.5, 11, 12, and 14. Half of each stemplasm was fixed in formalin
for morphological studies, and the other half was frozen in
-80.degree. C. for molecular characterization. Stemplasm started to
be formed to a visible size around week three. By staggering the
harvesting of stemplasms, various tissue types that developed
within the stemplasms were studied. All tissue types identified
herein were produced within said stemplasms. Stemplasm genotype was
verified by PCR-based allelic analysis described in section C.2.,
supra.
[0181] To create embryoid bodies (EB), HS cells or a 60 mm dish
were first washed with PBS twice. 1 ml of TrypsinIEDTA solution was
then added, and cells were held at a temperature of 37.degree. C.
for five minutes. 5 ml of ES medium was then added, and cells were
lifted by a cell scraper and spun down at 1000 rpm for five
minutes. The cell pellet thus obtained was then resuspended in 5 ml
stem cell medium without LIF, and the cell number was counted.
Cells were then seeded onto a suspension dish with lid and vent
(Nalge Nunc International, 171099,35.times.10 mm) at
2.times.10.sup.6/10 cm dish. Cells were fed in stem cell medium for
4 days, where medium was changed every two days by transferring
cells into 15 ml tubes, waiting about five minutes until the cells
settle to the bottom of the tube, then replacing medium. Cells were
then transferred back to the original dish and were allowed to
aggregate into embryoid bodies for further differentiation.
Example 1(f)
Differentiation Of Human HS Cells Within Teratomas, And The Genetic
Homozygosity Of Such Differentiated Tissue
[0182] Thirty-one teratomas were retrieved from the files of the
Armed Forces Institute of Pathology, Washington, D.C., and
Department of Pathology, New York University. New York, N.Y. (Dr.
J. Liu). A variety of different kinds of exclusively differentiated
tissue were found in twenty ovarian tumors from female patients.
Differentiated tissue was found to be diploid as confirmed by FISH
analysis carried out in representative cases using methods known in
the art, and alpha-satellite probes to chromosomes 3 and 8. Between
3 and 12 histological areas of undifferentiated and differentiated
tissue found in seven ovarian tumors from female patients and four
testicular tumors from male patients were identified and
selectively microdissected from each case for genetic analysis. In
each case, differentiated tissue was found to be genetically
homozygous, and undifferentiated tissue was found to be genetically
heterozygous.
[0183] Microdissection. Unstained 6-micron sections on glass slides
were deparaffinized with xylene, rinsed in ethanol from 100% to
80%, briefly stained with hematoxylin and eosin, and rinsed in 10%
glycerol in TE buffer. Tissue microdissection was performed under
direct light microscopic visualization. From each case, between 6
and 12 areas of different tissue differentiation were separately
micro dissected for genetic analysis. In addition, several areas of
normal, non-neoplastic tissue were procured.
[0184] DNA Extraction. Procured cells were immediately resuspended
in 25 .mu.l buffer containing Tris-HCI, pH 8.0; 1.0 mM
ethylenediamine tetraacetic acid, pH 8.0; 1% Tween 20; and 0.5
mg/ml proteinase K, and were incubated at 37.degree. C. overnight.
The mixture was boiled for 5 minutes to inactivate the proteinase K
and 1.5 .mu.l of this solution was used for PCR amplification of
the DNA.
[0185] Genetic Analysis. In order to reliably identify homozygosity
in the limited amounts of DNA that were available after
microdissection, multiple different microdissected tissue samples
were analyzed with up to 14 distinct highly polymorphic
microsatellite markers including DIS1646 and D1S243 (1p), D3S2452
(3p), D5S346 (5q), D7S1822 (7q), Ank-1 (8p), D9S171 (9p), D9S303
(9q), Int-2 and PYGM (11q), IFNA (9p), D17S250 (17q), CYP2D (22q),
and AR (Xq). Each PCR sample contained 1.5 .mu.l of template DNA as
described above, 10 pmol of each primer, 20 nmol each of dATP,
dCTP, DGTP, and DTTP, 15 mM MgCl.sub.2, 0.1U Taq DNA polymerase,
0.05 ml [.sup.32P]dCTP (6000 Ci/mmol), and 1 .mu.l of 10.times.
buffer in a total volume of 10 .mu.l. PCR was performed with 35
cycles: denaturing at 95.degree. C. for 1 min, annealing for 1 min
(annealing temperature between 550 and 60.degree. C. depending
on-the marker) and extending at 72.degree. C. for 60 sec. The final
extension was continued for 10 minutes. Labeled amplified DNA was
mixed with an equal volume of formamide loading dye (95% formamide,
20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol).
[0186] Samples can be denatured for 5 min at 95%, loaded onto a gel
consisting of 6% acrylamide (acrylamide:bisacrylamide 49:1), and
electrophoresed at 1800 V for 90 minutes. After electrophoresis,
the gels can be transferred to 3 mm Whatman paper and dried.
Autoradiography can be performed with Kodak X-OMAT film (Eastman
Kodak, Rochester, N.Y.).
[0187] Results. Differentiated teratomous tissue showing consistent
homozygosity of the same allele included microdissected samples of
squamous epithelium, glia, and cartilage (analyzed with markers
Ank1 (top) and D1S1646 (bottom)). Normal ovarian tissue was
included as control.
[0188] In a subset of teratomas, differentiated teratomous tissue
found to have discordant homozygous alleles (analyzed with markers
Int-2, D9S303, D1S1646, D3S2452, and Ank1) included samples of
epidermis, sebaceous gland respiratory epithelium, and glia. Normal
ovarian tissue was included as a control. In such tumors, it is
believed that allelic heterozygosity results from the initiation of
tumorigenesis before meiosis I in germ cells. After teratogenic
tumor cell initiation, random, independent events then lead to
progenitor cells with a postmeiotic genotype.
[0189] A series of ovarian teratomas and testicular germ cell
tumors containing both differentiated and undifferentiated tissue
were also analyzed. In each tumor, both undifferentiated and
differentiated tissue elements were procured. Homozygous and
heterozygous components were detected using markers D3S2452,
D3S303, CYP2D, and D17S250. Normal ovarian and testicular tissues
were included as controls. Heterozygous alleles were detected in
undifferentiated tissue elements including immature squamous
epithelium, neural tissue (sometimes from separate areas of neural
tissue within the same tumor), cartilage, glandular structures, and
mesenchyme. Differentiated tissue elements isolated from the same
tumors by microdissection were found to be homozygous for the same
markers. Mature elements tested included: sebaceous gland tissue,
hair follicle, and mature squamous epithelium (sometimes from
separate areas of squamous epithelium within the same tumor). In
some tumors, differentiated elements showed opposite homozygous
alleles, indicating recombination or suggesting that various
elements arose separately from distinct postmeiotic cells.
Example 1(g)
Derivation Of Progenitor Cells From Human HS Cells Primary
Differentiation
[0190] HS cells grown on 60 mm dish (Falcon, #353802) with primary
embryonic fibroblast layer and/or 0.1% gelatin coated dishes are
trypsinized with 1.5 ml Trypsin/EDTA (Invitrogen, #25300-050) and
transferred to 1.5 ml ES-LT medium in a 15 ml conical tube. Cells
are then spun down at 1200 rpm, and the supernatant is removed. The
cell pellet is resuspended into single cell suspension in 2 ml
ES-LIF medium, and cultured as suspension cells in suspension
culture-35*10 mm-dishes (NalgeNunc, #171099) at a density of
1-3.times.10.sup.6 cells to allow stem cells to form rounded
spherical clusters, known as embryoid bodies (EBs) for 4-6 days.
Forming EBs are washed every two days by transferring the EBs to 15
ml conical tubes, and then allowed to settle to the bottom. The
supernatant is removed and new ES-LIF is added. EBs are then
transferred back into suspension culture dish. HS cells grown as
embryoid bodies are comprised of all the germ cell layers,
ectodermal, endodermal, and mesodermal.
[0191] Ectodermal Progenitors: After 4-6 days, EBs are trypsinized
in 1 ml of Trypsin/EDTA, washed in 4 ml ES-LIF medium, and
resuspended into single cell suspension in DMEM/Knockout medium
(Invitrogen, #10829-018) supplemented with 10% Serum Replacement
(Invitrogen, #10828), and G5 (Invitrogen, #17503), N2 (Invitrogen,
#17502-048) or beta NGF (100 ng/ml) (R&D Systems, #256-GF).
These cells are cultured at 3-5.times.10.sup.5/3 ml in
fibronectin-coated 35 mm dishes (50 ug/ml)(Sigma, #F-0895) for 10
days, with media changes every two-three days.
[0192] Alternatively, the EBs are cultured in 0.1% gelatin-coated
dish in ES-LIF medium for 1-2 days, and then the medium is changed
to serum-free medium supplemented with Insulin (5 ug/ml), Selenium
chloride (0.015 nM), Transferrin (50 ug/ml), and fibronectin (5
ug/ml)(Sigma) for 6 days. The cells are trypsinized, and single
cell suspensions are cultured in N2 medium (serum free-DMEM/F12
supplemented with N2 (Invitrogen, # 17502-048), B27 (Invitrogen,
#1750444), and bFGF (10 ng/mL) (Invitrogen, #13256-029)). Cells are
then counted and seeded at a density of 2-5.times.10.sup.4
cells/well/400 uL N2 medium in 24-well plates pre-coated with
poly-L-ornithine (15 ug/ml)(Sigma, #P36550), and expanded for six
days.
[0193] These progenitors are further differentiated into different
neuronal cell types by adding G5, RA, FGF, NGF, GNDF, or BNDF. They
are also maintained in their presence conditioned media for cell
expansion.
[0194] Mesodermal Progenitors: For mesodermal progenitors, the
single cell suspension in DMEM/Knockout medium supplemented with
10% Serum Replacement and beta-NGF as described above are cultured
for 10 days with media change every two/three days. After this
period, the cells are further cultured in Activin A supplemented
(20 ng/ml) (Sigma, #A4941) conditioned medium for another 10 days
for heart progenitor cells. Alternatively, for kidney and Mullerian
duct progenitor cells the cells are further cultured in Activin A
supplemented (20 ng/ml) (Sigma, #A4941) conditioned medium for 4-6
days after which 2 ng/ml of TGF-beta (R&D Systems, #) is added
to the medium, and the cells are cultured for another 4-6 days.
[0195] Endodermal Progenitors: For endodermal progenitors, the
single cell suspension in DMEM/Knockout medium supplemented with
10% Serum Replacement, along with G5 or beta-NGF on laminin-coated
(10 ug/ml)(Sigma, #L2020), or Collagen I-coated (10 ug/ml)(Sigma,
#C-7661) is cultured for 10 days. HGF (20ng/ml) and/or TGF-alpha (2
ng/ml) are added to the medium to replace G5 or beta-NGF, and the
cells are cultured for another 6-8 days.
[0196] Alternatively, EBs are plated onto Collagen I-coated dishes
and cultured in ES-LIF medium for 4 days. FGF (20 ng/ml) is added
and the cells are cultured for another 3 days. After this period,
HGF (20 ng/ml) and/or TGF-alpha (2 ng/ml) are added and cultured
for another 6 days.
[0197] EBs are also transferred to laminin-coated adherent dishes
(10 ng/ml) (Sigma, #L2020) or 0.1% gelatin coated 35*10 mm adherent
dish, and cultured 1-2 days in ES-LIF medium. The medium is removed
and serum-free DMEM/F12 (Invitrogen, #11330-0321) medium
supplemented with Insulin (5 ug/ml)(Invitrogen, # I1882), Selenium
chloride (0.015 nM) (Sigma, #S5261), Transferrin (50 ug/ml) (Sigma,
#T-2036), and Fibronectin (5 ug/ml) (Sigma). This medium is
designated as ITSFn medium. Cells are fed for 6 days in ITSFn
medium, where medium is changed every two days.
Example 1(h)
Development And Isolation of Homozygous Progenitor Cells From
Transplanted HS Cells
[0198] To obtain homozygous progenitor cells, pluripotent HS cells
derived from methods disclosed in the foregoing in the foregoing
description and examples are transplanted into immuno-compromised
mice under kidney capsules and are allowed to grow in vivo for 4 to
6 weeks. The cell mass obtained is then minced into single cells
and cultured on feeder cells for further propagation and
development into cell lines.
[0199] To assess the lineage commitment (the types of progenitor
cells ), gene expression assays, such as RT-PCR, northern blot,
immunohistochemistry, and so forth, are performed for known
lineage-specific markers, for example, NF-H, keratin, D-beta-H for
the ectoderm, enolase, CMP, rennin, kallikerein, WTI, delta-globin,
beta-globin for the mesoderm, and albumin, alpha-1-AT, amylase,
PDX-1, insulin, alpha-FP for the endoderm progenitor lineages.
Example 2
Selection of HS Cells Having a Target Immunotype from Populations
of HS cells Derived from Material Donated by a Relative (e.g,
Parent of the Intended Recipient)
[0200] Oocytes are obtained from the recipient's mother by
super-ovulation using methods described in the foregoing examples.
Once HS cell populations have developed, following methods
described in the foregoing examples, a sample of each HS population
can be subjected to in vitro differentiation for the optimal
expression of HLA molecule (e.g., hemapoietic lineage), the
HS-derived sample cells from each population are then tested for
HLA-A, -B, and -C specificities using the microlymphotoxicity
assay. The test is performed in a 60- or 72 well
microlymphotoxicity plate. A panel of antisera, obtained from a
commercial source, are selected and prepoured onto the plates and
cooled in a -40.degree. or -70.degree. C. freezer. 0.5-1 .mu.l of
HS cell suspension (prepared by suspending HS cells in RPMI, or a
desired diluent for typing, to 1.5.times.10.sup.6 HS-derived sample
cells per milliliter using standard techniques) is dispensed into
each well of the plate and incubated at 20-22.degree. C. for thirty
minutes. 2-5 .mu.l of complement is then added to each well of the
plate and incubated at 20-22.degree. C. for sixty minutes.
Complement is available commercially as pooled rabbit serum in a
freeze-dried form. 1 .mu.l of 5% Eosin solution followed by 1 .mu.l
of formaldehyde is then added to each well. Using an appropriate
microscope, cell death in each well is established as determined by
the amount of dye fixed onto the cells. A standard scoring system
is used, from 0 to 8, where 8 is 80-100% representing a strong
positive, and 2 is 20-40% representing a doubtful positive.
[0201] The sample cells are then tested for HLA-DR, -DQ and -DB
using PCR-RFLP analysis. DNA is extracted from HS cells using
standard procedures known in the art. Extracted DNA, 200-300 ng, is
then amplified by PCR with 2.5 U of the Taq polymerise. PCR primers
used for amplification of HLA class II are listed in FIG. 11. After
amplification, 4-7 .mu.l of a reaction mixture are added to a
solution containing a restriction enzyme chosen from the list in
FIG. 5, and an appropriate restriction buffer, and incubated for 3
hrs. Twenty-nine different enzymes are used for full class II
typing. Samples of the amplified DNAs cleaved by restriction
enzymes are then subjected to electrophoresis using a 12%
polyacrylamide horizontal gel in a mini-apparatus (e.g. Mupid-2
obtained from E-Y laboratories Inc., San Mateo, Calif.). RFLP
fragments are detected by staining with ethidium bromide.
[0202] The immunotype of the recipient is determined using the
serological and molecular methodology described above. 20 ml of
venous blood is obtained from the recipient (target) into sodium
citrate anticoagulant (1 ml of 3.3% sodium citrate per 10 ml of
blood.) The citrated blood is then diluted with an equal volume of
heparinized HBSS (1 ml of 1000 U/ml heparin to 100 ml of HBSS). A
10 ml volume of diluted blood is layered onto 4ml of
Ficoll-isopaque in 17-120 mm screw top centrifuge tubes using a
pipette. The tubes are then centrifuged at 700.times. g for fifteen
to twenty minutes. Red blood cell and polymorphonuclear cells form
a pellet at the end of the tube, and lymphocytes form a discrete
layer at the interface of Ficoll-isopaque. The lymphocyte layer is
aspirated into another tube, topped off with HBSS, and centriftiged
again at 250.times. g for five minutes. The lymphocyte pellet is
then resuspended, washed with HBSS, and centrifuged again at
120.times. g for five minutes. This step separates most of the
platelets, suspended in the supernatant, while pelleting the
lymphocytes at the bottom of the tube. The HBSS is discarded, and
the lymphocyte pellet is resuspended in HBSS, and spun for the last
time at 250.times. g for five minutes. The HBSS is then discarded,
and the lymphocyte pellet is resuspended in RPMI or a desired
diluent for typing. HLA-A, B and C typing procedure described above
with the sample stem is repeated to determine the target
HLA-haplotype.
[0203] Another 20 ml or less of venous blood is obtained from the
recipient for molecular typing of HLA class II. Genomic DNA
extracted from the blood cell of the recipient using techniques
known in the art is amplified using PCR. An RFLP analysis as
described above is then performed.
[0204] The recipient's HLA haplotype may be homozygous or
heterozygous, and in either situation one HLA haplotype (e.g.,
maternal II) will match with one of the HS cell populations derived
from the mother's oocytes. Once a HS population is determined to
have the target immunotype, it may then be differentiated in vivo,
or in vitro using the techniques described in U.S. patent
application Ser. No. 09/997,240, and U.S. Provisional Application
Ser. No. 60/253,943, fully incorporated by reference herein, and
transplanted into the recipient.
Example 3
Selection of HS Cells Having Target Genotype From Populations of HS
Cells Derived from Material Extracted from Intended Recipient (Self
Donor)
[0205] In those instances where the intended recipient of the
transplant suffers from a genetic disorder linked to the expression
of a mutant gene sequence, it becomes necessary to select the
population of HS cells that are homozygous for non-mutant haplotype
(e.g., the "target" genotype). An exemplary protocol for the
selection for genotype is as follows:
[0206] In this example, the intended recipient is a women afflicted
with sickle cell anemia. A number of human disease states have been
attributed to genetic mutations effecting one or more of the genes
encoding hemoglobin polypeptide chains, including sickle cell
anemia, which results from a point mutation in the hemoglobin
13-chain. The genetic sequence of the mutant gene associated with
sickle cell anemia has been disclosed and extensively studied. See,
for example, Saiki et al., 1985, Science, 230, 1350-1354,
incorporated by reference herein.
[0207] A series of populations of HS cells are created from oocytes
harvested from the intended recipient according to the procedures
described above in Example 1. The populations are then assayed for
genotype. Only those populations that homogeneously carry the
target genotype (e.g., the normal or wild type gene rather than the
mutant gene) are selected for further development.
[0208] In this example, the mutant gene associated with sickle cell
anemia is detected by allele-specific hybridization, or "ASH." This
technology is based on the stable annealing of a short,
single-stranded oligonucleotide probe to a single-stranded target
nucleic acid only when base pairing is completely complementary.
The hybridization can then be detected from a radioactive or
non-radioactive label on the probe (methods of labeling probes and
other nucleic acids are set forth in detail below). ASH markers are
used as dominant markers where the presence or absence of only one
allele (or haplotype) is determined from hybridization or lack of
hybridization by only one probe. The alternative allele may be
inferred from the lack of hybridization.
[0209] ASH markers have been developed to diagnose susceptibility
to human diseases caused by point mutations in DNA sequence. An ASH
marker useful in detecting the .beta..sup.s-globin allele
associated with sickle-cell anemia is described by Conner et al.,
Proc. Natl. Acad. Sci. USA 80:278-282, 1983 and incorporated by
reference herein.
[0210] Cell samples are taken from each HS population and genomic
DNA is extracted using conventional methodology. Following the
procedures outlined by Conner et al., supra, the genomic DNA is
digested with restriction endonucleases. The resulting nucleic acid
fragments are amplified using PCR (see Mullis, K. B. et al.,
Methods Enzymol. 155:335-350 (1987) incorporated by reference
herein) to create amplicons. The amplicons are then transferred to
a membrane having a radio-labeled oligonucleotide probe specific
for sickle cell anemia bound thereto in a dot-blot format.
Hybridization dots are detected by autoradiography. Alternatively,
the amplicons can be labeled, and the probes membrane bound.
[0211] Once a sample is determined to have the target immunotype
and genotype, it may then be differentiated in vivo or in vitro,
using the techniques described in U.S. patent application Ser. No.
09/997,240, and U.S. Provisional Application Ser. No. 60/253,943
incorporated by reference herein, and transplanted into the
intended recipient.
[0212] For male recipients and female recipient who are not
suitable for self-donors due to medical conditions, HS cells having
both the target immunotype and genotypes can be derived from female
family member donors. Once a sample population is determined to
have the target immunotype and genotype, it may then be
differentiated in vivo or in vitro, using the techniques described
in U.S. patent application Ser. No. 09/997,240, and U.S.
Provisional Application Ser. No. 60/253,943 incorporated by
reference herein, and transplanted into the intended recipient.
Example 4
Selection of HS Cells Having a Target Immunotype and a Target
Genotype From Populations of HS Cells Derived from Material Donated
by A Non-Related Individual (Non-Self, Non-Relative Donor)
[0213] In certain instances, donor material cannot be obtained from
the intended recipient or a close relative. In such cases, it may
be necessary to screen the HS populations both for target
immunotype and for target genotype. An exemplary protocol for the
selection for target immunotype and target genotype is as
follows.
[0214] Populations of HS cells are derived from oocytes extracted
from the donor individual using the protocol cited above in Example
1. Once HS cell populations have developed, sample cells are typed
for HLA-A, -B, and -C specificities using the microlyphotoxicity
assay described in Example 2 above.
[0215] HS cells are further typed to HLA class II antigens using
the PCR-SSOP procedure. DNA is extracted from the cells using
standard procedures known in the art. Extracted DNA, 200-300 ng, is
then amplified by PCR with 2.5 U of the Taq polymerase. PCR primers
used for amplification of HLA-DRB/DQB/DPB, and cycling conditions
for all primer combinations for oligotyping are listed in FIG. 14.
2.5 .mu.l aliquots of the reaction mixture are then mixed with 50
.mu.l NH4-acetate 1M, and manually applied on a filter (Nytran,
Schleicher & Schuell) using a Minifold II stot blotter
(Schleicher & Schuell). The membranes are denatured by placing
them in 0.4N NaOH, for ten minutes, then neutralized for 10 minutes
in 1M NH.sub.4-acetate, and then dried at room temperature.
Positive and negative controls for hybridization are included on
each membrane.
[0216] Oligonucleotides for hybridization are then labeled with
[.sup.32P].gamma.-ATP and polynucleotide kinase, in a 10 .mu.l
reaction. The reaction contains 6 pmol oligo, 25 .mu.Ci ATP, 1
.mu.l kinase buffer (0.5 M tris-HCL, pH 7.6, 0.1 M MgCl.sub.2, 50
mM dithiothreitol (DTT), 1 mM spermidine, 1 mM EDTA), and 5UT4
polynucleotide kinase (Pharmacia). The reaction is incubated for
thirty minutes at 37.degree. C., and the labeled oligo is purified
on a DE52 column (Whatman, Cat. No. 4057-050). After loading the
column, the oligo is washed with 10 ml washing buffer (150 mM NaCl,
20 mM Tris-HCl, pH 7.8, 10 mM EDTA), and then eluted with 0.2-ml
fractions of elution buffer (1M NaCl, 20 mM Tris-HCL, pH 7.8, 1 mM
EDTA).
[0217] Each membrane is then hybridized with radioactive oligos.
First, a prehybridization step is performed for thirty minutes at
54.degree. C. with tetramethylammonium chloride hybridization
(TMAC) buffer (50 mM Tris-HCl, pH 8.0, 3M TMAC (Fluka), 2 mM EDTA,
pH 8.0, 5.times. Denhardt's, 0.1% Na-dodecyl sulphate, 100 .mu.g/ml
denatured salmon sperm DNA, filtered through Whatman 3MM paper).
The membranes are then hybridized for one to three hours in a TMAC
hybridization buffer containing 0.5.times.10.sup.6 cpm/ml of the
labeled oligo at 54.degree. C. The blots are washed twice for ten
minutes at room temperature in a TMAC washing buffer, and then
twice again for fifteen minutes at 59.degree. C. (19-mers) in the
same. The procedure is fully described in Hui et al., Handbook, at
119-123, incorporated by reference herein.
[0218] The target immunotype is determined using the serological
and molecular methodology described above in Example 2 above.
Target haplotype may be homozygous or heterozygous. In either
situation there is a high probability that at least one HLA
haplotype of the recipient will match the HLA of a sample HS
population derived from the populations of cells from unrelated
oocyte donors of the same ethnicity as the recipient (See
above).
[0219] In this invention, unrelated women afflicted with genetic
diseases do not need to be excluded since HS cells with the absence
of disease genotype can be derived. For example, the foreign donor
(e.g., the unrelated individual) is a women afflicted with
betathalassemia. Beta-thalassemia is a genetically determined
defect in hemoglobin synthesis. There is an inability to
manufacture sufficient quantities of globin chains. Alpha-and
beta-thalassemia conditions are blood-related disorders which
result from genetic mutations manifested phenotypically by
deficient synthesis of one type of globin chain, resulting in
excess synthesis of the other type of globin chain. See generally
Weatherall et al., The Thalassaemia Syndromes, 3rd ed., Oxford,
Blackwell Scientific, 1981 and U.S. Pat. No.5,750,345. The
.beta..sup.0-thalassemia allele associated with .beta.-thalassemia
is described by Pirastu et al., New England J. Med. 309:284-287,
1983 incorporated by reference herein. Thus, in the context of this
example, the "target" genotype will be the absence of the
13-thalassemia allele, the mutant haplotype associated with
pathology.
[0220] In assaying for genotype, only those populations that
homogeneously carry the target genotype (e.g., the normal or wild
type gene rather than the mutant gene) are selected for further
development. The target genotype, e.g., the absence of the gene
corresponding to beta-thalassemia, is detected and selected by a
procedure analogous to that described in Example 3 above with
respect to sickle cell anemia, using an ASH probe specific for
beta-thalassemia (see Weatherall et al. and Piratsu et al.,
supra.).
[0221] Once a sample population is determined to have the target
immunotype and genotype, it may then be differentiated in vivo or
in vitro, using the techniques described in U.S. patent application
Ser. No. 09/997,240, and U.S. Provisional Application Ser. No.
60/253,943 incorporated by reference herein, and transplanted into
the intended recipient.
Sequence CWU 1
1
86 1 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 1 ttcaaactta agctgccac 19 2 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 2 ctcatactta tcctgctgc 19
3 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 3 tctgcagtag ttgtccacc 19 4 20 DNA Artificial Sequence
Description of Artificial Sequence Probe 4 ctcttggtga tagaagtatc 20
5 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 5 ccagtactcc tcatcaggc 19 6 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 6 agcgcaggag ctcctcctg 19
7 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 7 cttatactta ccctgccac 19 8 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 8 gtgtccacca gggcccgcc 19
9 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 9 ctgtgcagat accgcaccc 19 10 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 10 ctcaaactta acctcctcc 19
11 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 11 ggcccgctcg tcttccagg 19 12 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 12 cggcccgctt gtcttccag 19
13 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 13 gttccagtgc tccgcagca 19 14 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 14 agcgcacgtt ctcctcctg 19
15 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 15 gaagcgcaag tcctcctct 19 16 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 16 gctctccaca gccccgtag 19
17 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 17 acactcaccc gtagagtac 19 18 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 18 atagaagtgt ctgtccagg 19
19 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 19 agcgcacgtt ctcctcctg 19 20 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 20 agcgcacgga ctcctcttg 19
21 18 DNA Artificial Sequence Description of Artificial Sequence
Probe 21 gcctagcgcc gagtactg 18 22 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 22 gttccagtgc tccgcagca 19
23 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 23 cttccaggaa gtccttctg 19 24 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 24 cttccaggat gtccttctg 19
25 18 DNA Artificial Sequence Description of Artificial Sequence
Probe 25 acatcctgga agacgagc 18 26 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 26 ggcccgctcg tcttccagg 19
27 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 27 cggcccgctt gtcttccag 19 28 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 28 cggcccgctt ctgctcgac 19
29 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 29 ccgcggcccg cctctgctc 19 30 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 30 cggcccgcct ctgctccag 19
31 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 31 ggcccgcctg tcttccagg 19 32 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 32 ccgcggcgcg cctgtcttc 19
33 18 DNA Artificial Sequence Description of Artificial Sequence
Probe 33 agcggaggcg ggccgagg 18 34 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 34 gtgtccacct cggcccgcc 19
35 19 DNA Artificial Sequence Description of Artificial Sequence
Probe 35 gtgtccacca gggcccgcc 19 36 18 DNA Artificial Sequence
Description of Artificial Sequence Probe 36 aactacgggg ttgtggag 18
37 18 DNA Artificial Sequence Description of Artificial Sequence
Probe 37 aactacgggg ttggtgag 18 38 19 DNA Artificial Sequence
Description of Artificial Sequence Probe 38 gctctccaca gccccgtag 19
39 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 39 tcaatcaaat catccccaga ag 22 40 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 40 gggattacag gcaggagcca
c 21 41 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 41 gaaatgtgag aataaaggag a 21 42 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 42 agtcacccta
ctgtgctatc 20 43 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 43 gcttcacccg atcagtagaa gac 23 44 21
DNA Artificial Sequence Description of Artificial Sequence Primer
44 ggagaagttg agtatttctg c 21 45 31 DNA Artificial Sequence
Description of Artificial Sequence Primer 45 gcactatcat taaatttgct
ttccacagta c 31 46 28 DNA Artificial Sequence Description of
Artificial Sequence Primer 46 gaaacatata ttaacagaga cagacaaa 28 47
20 DNA Artificial Sequence Description of Artificial Sequence
Primer 47 ggtaaaattc ctgactggcc 20 48 18 DNA Artificial Sequence
Description of Artificial Sequence Primer 48 gctttgatct cccccctc 18
49 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 49 tgcttatagg gagactaccg 20 50 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 50 ggcattcagg catgcctggc
20 51 21 DNA Artificial Sequence Description of Artificial Sequence
Primer 51 ccttctttgc agactgtcac c 21 52 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 52 gaaggagaat tgtaattccg
20 53 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 53 gataaagggg aactactaca 20 54 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 54 atcgaggtaa acagcagaaa
g 21 55 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 55 gcatggtgtc agagatagtc aggtc 25 56 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 56
accaaacttc aaattttcgg 20 57 31 DNA Artificial Sequence Description
of Artificial Sequence Primer 57 tgattcataa ggcaagaatc cagcatattg g
31 58 25 DNA Artificial Sequence Description of Artificial Sequence
Primer 58 catttctctt ccttatcact tcata 25 59 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 59 gacagctctt
cttaacctgc 20 60 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 60 ggacaatatt ttgctcctga gg 22 61 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
gaggtaatgt cacaggatgg g 21 62 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 62 ggggatgacg aattattcac
taact 25 63 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 63 gtgctgcagg tgtaaacttg taccag 26 64 28 DNA
Artificial Sequence Description of Artificial Sequence Primer 64
cacggatccg gtagcagcgg tagagttg 28 65 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 65 gcatgtgcta cttcaccaac
g 21 66 29 DNA Artificial Sequence Description of Artificial
Sequence Primer 66 cacctgcaga tcccgcggta cgccacctc 29 67 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 67
gcatgtgcta cttcaccaac g 21 68 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 68 cacctgcagt gcggagctcc
aactggta 28 69 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 69 ttcctgtggc agcctaagag g 21 70 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 70 gtttcttgga
gcaggttaaa c 21 71 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 71 gaagcaggat aagtttgagt g 21 72 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 72
ggttgctgga aagatgcatc t 21 73 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 73 agttcctgga aagactcttc
t 21 74 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 74 ggttgctgga aagacgcgtc c 21 75 22 DNA Artificial
Sequence Description of Artificial Sequence Primer 75 acgtttcttg
gagtactcta cg 22 76 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 76 ccgctgcact gtgaagctct 20 77 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 77
cccagcacgt ttcttggagc t 21 78 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 78 ccgctgcact gtgaagctct
20 79 19 DNA Artificial Sequence Description of Artificial Sequence
Primer 79 cttgcagcag gataagtat 19 80 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 80 ccgctgcact gtgaagctct
20 81 27 DNA Artificial Sequence Description of Artificial Sequence
Primer 81 ggaagcttga tccccctgag gtgaccg 27 82 27 DNA Artificial
Sequence Description of Artificial Sequence Primer 82 ggggatcccc
agtgcttgag gagcggc 27 83 28 DNA Artificial Sequence Description of
Artificial Sequence Primer 83 ggaagcttga ggcccaagag ccaatcca 28 84
26 DNA Artificial Sequence Description of Artificial Sequence
Primer 84 ggggatccgc cagaacgcag agactt 26 85 26 DNA Artificial
Sequence Description of Artificial Sequence Primer 85 gtgaagcttt
ccccgcagag aattac 26 86 27 DNA Artificial Sequence Description of
Artificial Sequence Primer 86 cacctgcagt cactcacctc ggcgctg 27
* * * * *
References