U.S. patent application number 16/102640 was filed with the patent office on 2019-07-25 for bank of stem cells for producing cells for transplantation having hla antigens matching those of transplant recipients and metho.
The applicant listed for this patent is Advanced Cell Technology, Inc.. Invention is credited to Michael D. West.
Application Number | 20190225937 16/102640 |
Document ID | / |
Family ID | 32233212 |
Filed Date | 2019-07-25 |
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United States Patent
Application |
20190225937 |
Kind Code |
A1 |
West; Michael D. |
July 25, 2019 |
BANK OF STEM CELLS FOR PRODUCING CELLS FOR TRANSPLANTATION HAVING
HLA ANTIGENS MATCHING THOSE OF TRANSPLANT RECIPIENTS AND METHODS
FOR MAKING AND USING SUCH A STEM CELL BANK
Abstract
Methods for producing stem cell banks, preferably human, which
optionally may be transgenic, e.g., comprised of homozygous MHC
allele cell lines are provided. These cells are produced preferably
from parthenogenic, IVF, or same-species or cross-species nuclear
transfer embryos or by de-differentiation of somatic cells by
cytoplasm transfer. Methods for using these stem cell banks for
producing stem and differentiated cells for therapy, especially
acute therapies, and for screening for drugs for disease treatment
are also provided.
Inventors: |
West; Michael D.; (Mill
Valley, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Advanced Cell Technology, Inc. |
Worcester |
MA |
US |
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Family ID: |
32233212 |
Appl. No.: |
16/102640 |
Filed: |
August 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13721618 |
Dec 20, 2012 |
10047340 |
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16102640 |
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10445195 |
May 27, 2003 |
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13721618 |
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60382616 |
May 24, 2002 |
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60448585 |
Feb 21, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0606 20130101;
A01K 2217/05 20130101; A61K 35/12 20130101; C12N 15/85 20130101;
C12N 2510/00 20130101; C12N 2517/10 20130101; A01K 2267/0393
20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; C12N 15/85 20060101 C12N015/85 |
Claims
1-62. (canceled)
63. A method for treatment, preferably acute treatment, comprising
transplanting cells or tissue that are homozygous for at least HLA
one allele in a person in need of such a transplant, comprising: a.
identifying the MHC alleles of a person in need of a transplant
(the recipient); b. obtaining from a stem cell bank comprising a
plurality of stem cells homozygous for at least one MHC allele of
the transplant recipient; c. generating cells or tissue suitable
for transplant from said stem cells; and d. transplanting said
cells or tissue suitable for transplant into said recipient.
64. The method of claim 63 wherein said stem cell bank comprises at
least 10 different human stem cell lines, wherein each of said stem
cell lines are homozygous for a different combination of HLA
alleles relative to the other stem cell lines.
65. The method of claim 63 wherein said stem cell bank comprises at
least 15 different human stem cell lines wherein each of said stem
cell lines are homozygous for a different combination of HLA
alleles relative to the other human stem cell lines in the cell
bank.
66. The method of claim 63 wherein said stem cell bank comprises at
least about 100 to 1000 stem cell lines each homozygous for a
different combination of HLA alleles relative to the other human
stem cell lines in the cell bank.
67. The method of claim 63 wherein one or more of said human stem
cell lines are ES or inner cell mass-derived stem cells.
68. The method of claim 63 wherein one or more of said human stem
cell lines is derived from a parthenogenetic human embryo.
69. The method of claim 63 wherein one or more of said human stem
cell lines are produced by haploidization comprising the steps of
a) inserting or fusing a somatic donor cell or nucleus thereof into
or with an oocyte which is treated to remove or destroy its
endogenous genomic DNA before, during or after insertion or fusion;
b) activation of the reconstructed embryo to expel haploid genome
into a pseudopolar body; c) screening of the pseudopolar body for
the genetype of the remaining pronuclear; d) combination of the two
pronuclei to generate a reconstructured diploid embryo by
pronuclear transfer or alternatively producing a diploid embryo by
transferal of a pronucleus to an activated haploid oocyte
comprising desired haploid genome; e) optionally injecting human
morula stage embryo lysates into the reconstructed embryos; and f)
isolating human stem cell lines from said reconstructed diploid
embryo.
70. The method of claim 63 wherein one or more of said human stem
cell lines is produced by the insertion of first and second polar
bodies into a recipient cell.
71. The method of claim 63 wherein at least one of said stem cell
lines is produced by de-differentiation of a somatic cell by
cytoplasmic transfer.
72. The method of claim 63 wherein said human stem cell bank
comprises cells which are homozygous for one of the following HLA
serotypes: HLA-A1, HLA-A3, HLA-A11, HLA-A15, HLA-A22, HLA-A27,
HLA-A28, HLA-A29, HLA-A32, HLA-B5, HLA-B7, HLA-B8, HLA-B12,
HLA-B17, HLA-B18, HLA-B35 and HLA-B40.
73. The method of claim 63 wherein said human stem cell bank
comprises stem cells which are homozygous for at least one of the
following HLA-A, --B or -DR haplotypes: 1, 7, 2; 1, 8, 3; 2, 14, 1;
2, 35, 4; 2, 35, 8; 2, 44, 4; 3, 7, 2; 3, 7, 4; 3, 7, 8; 3, 35, 1;
31, 51, 4; and 32, 14, 7.
74. The method of claim 72 wherein said cell lines are
O-negative.
75. The method of claim 73 wherein said cell lines are
O-negative.
76. The method of claim 63, wherein step b comprises obtaining stem
cells selected from the group consisting of totipotent, nearly
totipotent, and pluripotent stem cells.
77. The method of claim 63, wherein step b comprises obtaining
embryonic stem cells.
78. The method of claim 63, wherein step b comprises obtaining stem
cells that can differentiate into hematopoietic stem cells.
79. The method of claim 63, wherein step b comprises obtaining
hematopoietic stem cells from the stem cell bank.
80. The method of claim 63, wherein step b comprises obtaining stem
cells homozygous for an MHC allele selected from HLA-A, HLA-B,
HLA-C, HLA-DR, HLA-DQ, and HLA-DP.
81. The method of claim 63, wherein step b comprises obtaining stem
cells homozygous for the MHC alleles encoding HLA-A, HLA-B, and
HLA-DR.
82. The method of claim 63, wherein step b comprises obtaining stem
cells derived from embryos produced by in vitro fertilization or
intracytoplasmic sperm injection.
83. The method of claim 63, wherein step b comprises obtaining
diploid stem cells derived from embryos produced by
parthenogenesis.
84. The method of claim 83, comprising obtaining diploid stem cells
in which all of the MHC alleles are homozygous.
85. The method of claim 63, wherein step b comprises obtaining stem
cells derived from embryos produced by cloning by nuclear
transfer.
86. The method of claim 85, comprising obtaining rejuvenated stem
cells.
87. The method of claim 86, comprising obtaining rejuvenated stem
cells having telomeres that are on average at least as long as the
telomeres of age-matched control cells of the same type that are
not generated by nuclear transfer techniques.
88. The method of claim 86, comprising obtaining rejuvenated stem
cells for which the proliferative life-span is at least as long as
the proliferative life-span of age-matched control cells of the
same type that are not generated by nuclear transfer
techniques.
89. The method of claim 86, comprising obtaining rejuvenated stem
cells for which the proliferative life-span is longer than the
proliferative life-span of age-matched control cells of the same
type that are not generated by nuclear transfer techniques.
90. The method of claim 86, comprising obtaining rejuvenated stem
cells having EPC-1 activity that is greater than EPC-1 activity in
age-matched control cells of the same type that are not generated
by nuclear transfer techniques.
91. The method of claim 86, comprising obtaining rejuvenated stem
cells having telomerase activity that is greater than telomerase
activity in age-matched control cells of the same type that are not
generated by nuclear transfer techniques.
92. The method of claim 85, comprising obtaining stem cells
comprising non-human mitochondria.
93. The method of claim 63, wherein step b comprises obtaining stem
cells having DNA that is genetically modified relative to the DNA
of the human donor from which the stem cells are derived.
94. The method of claim 92, comprising obtaining genetically
altered stem cells, the DNA of which is modified by adding,
modifying, substituting, or deleting one or more DNA sequences.
95. The method of claim 93, comprising obtaining genetically
altered stem cells, the DNA of which is modified so as to obtain,
increase, decrease, inhibit, or otherwise modify, the expression of
a gene that is native to or introduced into said cells, relative to
expression of said gene in a control cell without the genetic
modification.
96. The method of claim 93, comprising obtaining genetically
altered stem cells, the DNA of which is modified by homologous
recombination.
97. The method of claim 93, comprising obtaining genetically
altered stem cells, the DNA of which is altered to prevent the
expression of a gene encoding an antigenic protein that elicits an
immune response contributing to rejection.
98. The method of claim 96, comprising genetically altered stem
cells, the DNA of which is modified so as to inhibit production of
at least one HLA antigen by cells of said cell line.
99. The method of claim 96, comprising genetically altered stem
cells, the DNA of which is modified so as to inhibit production of
one or more HLA antigens selected from HLA-A, HLA-B, HLA-C, HLA-DR,
HLA-DQ, and HLA-DP.
100. The method of claim 96, comprising genetically altered stem
cells, the DNA of which is modified so as to inhibit production of
.beta.2-microglobulin.
101. The method of claim 96, comprising genetically altered stem
cells, the DNA of which is altered by replacing a non-homozygous
MHC allele with one that is homozygous.
102-133. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/721,618 filed Dec. 20, 2012, now issued as
U.S. Pat. No. 10,047,340; which is a continuation application of
U.S. application Ser. No. 10/445,195 filed May 27, 2003, now
abandoned; which claims the benefit under 35 USC .sctn. 119(e) to
U.S. Application Ser. No. 60/448,585 filed Feb. 21, 2003 and to
U.S. Application Ser. No. 60/382,616 filed May 24, 2002, both now
expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The invention described herein relates to methods for
producing a collection of human and non-human stem cell cultures,
preferably human stem cell cultures, each of which contains
totipotent or pluripotent stem cells that have genes encoding the
same set of critical cell surface antigenic proteins, e.g.,
histocompatibility antigens (e.g., HLA antigens in the case of
human) as are present on the cells of members of a human
population. (By critical antigens is meant the set of antigens that
form the major histocompatibility complex and other antigens such
as blood group antigens that are involved in immuno-mediated
rejection when collogenic cells and tissues are transplanted into
donors that express a different set of histocompatibility and other
critical antigens). The methods disclosed herein include deriving
such human stem cell cultures from cells of early embryos produced
e.g., by in vitro fertilization, parthenogenesis, and by nuclear
transfer. Also, stem cells can be produced by transfer of cytoplasm
from embryonic cells, e.g., oocytes, early embryonic cells or ES
cells into somatic cells.
[0003] The invention described herein also relates to methods
wherein such human and non-human stem cell cultures are induced to
differentiate ex or in vivo into cell types that are useful for
therapeutic cell transplantation; and to methods by which the
differentiated cells are isolated from other cell types. The
invention also relates to methods in which stem cell-derived
differentiated cells having a selected set of critical cell surface
antigens are therapeutically transplanted or engrafted to a
recipient, e.g., a human patient in need of a cell transplant
having cells that express the same critical cell surface antigens.
The invention further relates to a collection or "bank" of cultures
of different types of stem cells, each culture having a different
set of genes encoding cell surface antigenic proteins present in a
human population; to compositions comprising the individual stem
cell cultures that make up such a stem cell bank; and to
compositions comprising differentiated cells derived from such stem
cells.
[0004] Preferably, stem cell banks produced according to the
invention will comprise stem cell lines which are homozygous for
MHC alleles which occur very frequently in the human population.
Typically, a stem cell bank according to the invention will
comprise at least 15 stem cell lines and more preferably at least
100 to 1000 stem cell lines. Thereby, the stem cell bank will
provide maximal therapeutic and diagnostic efficacy as it will
contain cells that are histocompatible for a wide range of
potential transplant recipients.
Background Information
A. Histocompatibility and Transplant Rejection
[0005] Histocompatibility is a largely unsolved problem in
transplant medicine. Rejection of transplanted tissue is the result
of an adaptive immune response to alloantigens on the grafted
tissue by the transplant recipient. The alloantigens are "non-self
proteins, i.e., antigenic proteins that vary among individuals in
the population and are identified as foreign by the immune system
of a transplant recipient. The antigens on the surfaces of
transplanted tissue that most strongly evoke rejection are the
blood group (ABO) antigens and the major histocompatibity complex
(MHC) proteins and in the case of humans, the human leukocyte
antigen (HLA) proteins.
[0006] The blood group antigens were first described by Landsteiner
in 1900; they are branched oligosaccharides that are attached to
proteins and lipids on the surfaces of red blood cells, endothelial
cells, and other cells, and are also present in secretions such as
saliva. Compatibility of the blood group antigens of the ABO system
of a vascularized organ or tissue transplant with those of the
transplant recipient is generally required; but blood group
compatibility may be unnecessary for many types of cell
transplants.
[0007] The HLA proteins are encoded by clusters of genes that form
a region located on chromosome 6 known as the Major
Histocompatibility Complex, or MHC, in recognition of the important
role of the proteins encoded by the MHC loci in graft rejection.
Accordingly, the HLA proteins are also referred to as MHC proteins.
The MHC genes and proteins will be used interchangeably in this
application as the application encompasses human and non-human
animal applications. The HLA or MHC proteins normally play a role
in defending the body against foreign pathogens such as viruses,
bacteria, and toxins. They are cell surface glycoproteins that bind
peptides at intracellular locations and deliver them to the cell
surface, where the combined ligand is recognized by a T cell. Class
I MHC proteins are found on virtually all of the nucleated cells of
the body. The class I MHC proteins bind peptides present in the
cytosol and form peptide-MHC protein complexes that are presented
at the cell surface, where they are recognized by cytotoxic CD8+ T
cells. Class II MHC proteins are usually found only on
antigen-presenting cells such as B lymphocytes, macrophages, and
dendritic cells. The class II MHC proteins bind peptides present in
a cell's vesicular system and form peptide-MHC protein complexes
that are presented at the cell surface, where they are recognized
by CD4+ T cells. CD4+ T cells activated by class II MHC proteins
undergo clonal expansion with production of regulatory cytokines
that signal helper and cytotoxic T cells. Unfortunately for those
in need of transplants, the frequency of T cells in the body that
are specific for non-self MHC molecules is relatively high, with
the result that differences at MHC loci are the most potent
critical elicitors of rejection of initial grafts. Rejection of
most transplanted tissues is triggered predominantly by the
recognition of class I MHC proteins as non-self proteins. T cell
recognition of foreign antigens on the transplanted tissue sets in
motion a chain of signaling and regulatory events that causes the
activation and recruitment of additional T cells and other
cytotoxic cells, and culminates in the destruction of the
transplanted tissue. (Charles A. Janeway et al., Immunobiology.
Garland Publishing, New York, N.Y., 2001, p. 524).
B. The Genes Encoding MHC Proteins
[0008] The MHC genes are polygenic--each individual possesses
multiple, different MHC class I and MHC class II genes. The MHC
genes are also polymorphic--many variants of each gene are present
in the human and non-human population. In fact, the MHC genes are
the most polymorphic genes known. Each MHC Class I receptor
consists of a variable a chain and a relatively conserved
.beta.2-microglobulin chain. Three different, highly polymorphic
class I a chain genes have been identified. These are called HLA-A,
HLA-B, and HLA-C. Variations in the a chain chains account for all
of the different class I MHC genes in the population. MHC Class II
receptors are also made up of two polypeptide chains, an a chain
and a .beta. chain, both of which are polymorphic. In humans, there
are three pairs of MHC class II a and .beta. chain genes, called
HLA-DR, HLA-DP, and HLA-DQ. Frequently, the HLA-DR cluster contains
an extra gene encoding a .beta. chain that can combine with the DR
a chain; thus, an individual's three MHC Class II genes can give
rise to four different MHC Class II molecules.
[0009] In humans, the genes encoding the MHC class I a chains and
the MHC class II a and .beta. chain are clustered on the short arm
of chromosome 6 in a region that extends over from 4 to 7 million
base pairs that is called the major histocompatibility complex.
Every person usually inherits a copy of each HLA gene from each
parent. If an individual's two alleles for a particular MHC locus
encode structurally different proteins, the individual is
heterozygous for that MHC allele. If an individual has two MHC
alleles that encode the same MHC molecule, the individual is
homozygous for that MHC allele. Because there are so many different
variants of the MHC alleles in the population, most people have
heterozygous MHC alleles. The numbers of different alleles found
for each type of MHC class I a chain and MHC class II .alpha. and
.beta. chains as of January 2003 are shown in Table 1.
TABLE-US-00001 TABLE 1 The numbers of different alleles for the
polymorphic MHC class I and class II chains identified as of
January, 2003. MHC Chain No. of Alleles HLA-A 266 HLA-B 511 HLA-C 6
HLA-DRA 3 HLA-DRB 403 HLA-DQA1 23 HLA-DQB1 53 HLA-DPA1 20 HLA-DPB1
101
[0010] The data in Table 1 is from the Internet web site of the
Informatics Group of the Anthony Nolan Trust, The Royal Free
Hospital, Hampstead, London, England. Lists of identified HLA Class
I and Class II alleles are also available at the same web site.
C. Matching MHC Types to Inhibit Rejection of Transplants
[0011] Since the recognition that-non-self-M1-G-molecules are a
major determinant of graft rejection, much effort has been put into
developing assays to identify the MHC types present on the cells of
tissue to be transplanted, and on the cells of transplant
recipients, in order to match the types of MHC molecules present in
the transplant tissue with those of the recipient. Tissue typing,
the detection of MHC antigens, is performed by various means; for
example, (i) by serology, using antibodies specific for particular
MHC molecules to detect the presence of the targeted MHC molecules
on donor or recipient cells, e.g., by the lymphocytotoxicity test;
(ii) by detection of antibodies of a transplant recipient that bind
specifically to a MHC protein of transplant tissue; and (iii) by
direct analysis of the nucleotide sequence of the DNA of the MHC
alleles. Most tissue typing for organ banking purposes is done by
determining the blood type (ABO typing) and by typing the patient's
and donor cells using serological methods; however, the use of
rapid and reliable DNA-specific methods is increasing. Such methods
can employ sequence-specific oligonucleotide primers and
amplification by the polymerase chain reaction (PCR), and can be
augmented by combining fluorescent detection methods with the use
of a DNA chip to which are bound sequence specific oligonucleotides
designed to detect unique sequences present in the different MHC
alleles.
[0012] At present, tissue typing to match the HLA antigens of a
transplant with those of a recipient is usually limited to the
Class I HLA-A and -B antigens, and the Class II HLA-DR antigens.
Most transplant donors are unrelated to the transplant recipient,
and finding a tissue type to match that of the recipient usually
involves matching the blood type and as many as possible of the 6
HLA alleles--two for each HLA-A, --B, and -DR locus. Transplant
centers do not usually consider potential incompatibilities at
other FILA loci, such as HLA-C and HLA-DPB1, although mismatches at
these loci can also contribute to rejection. Considering only the
combinations of maternal and paternal alleles of the HLA-A. HLA-B,
and HLA-DR loci identified to date, there is a complexity of
billions of possible tissue types. The task of matching HLA types
of organs for transplant is simplified in that HLA-A and HLA-B are
usually identified serologically. The number of HLA antigens
identified serologically is considerably less than the number of
different MLA antigens based on DNA sequencing. The World Health
Organization (WHO) has recognized 28 distinct antigens in the HLA-A
locus and 59 in the HLA-B locus, based on serological typing.
Matching organs is also simplified to some extent by the fact that
some alleles are much more common than others. Some of the more
common HLA-A and HLA-B alleles are shown in Table 2:
TABLE-US-00002 TABLE 2 Frequency of Common HLA-A and HLA-B Alleles
in the Population HLA-A (Frequency (%)) HLA-B (Frequency (%)) HLA-A
1 (25.1) HLAB5 (15.2) HLA-A2 (44.8) HLA-B 7 (18.2) HLA-A3 (22.6)
HLA-B 8 (16.7) HLA-A24 (18.2) HLA-B 12 (32.5) HLA-A 11 (11.8)
HLAB14 (8.8) HLA-A28 (9.8) HLA-B 18 (11.3) HLA-A29 (10.3) HLA-B35
(15.2) HLA-A3 2 (9.8) HLA-B40 (13.7) HLA-B 15 (12.3) (from Snell G
D et al, Histocompatibility, New York, Academic Press, 1976)
[0013] The frequencies with which the various alleles appear in a
population is not random; it depends on the racial makeup of the
population. Dr. Motomi Mori has determined the frequencies with
which thousand of different haplotypes of HLA-A, --B, and -DR loci
appear in Caucasian, African-American, Asian-American, and Native
American populations. Each haplotype is a particular combination of
HLA-A, HLA-B, and HLA-DR loci that is present on a single copy of
chromosome no. 6. The frequencies of several relatively common
HLA-A, --B, and -DR haplotypes are shown in Table 3 to illustrate
the wide variation in HLA haplotype frequencies in some of the
racial groups that make up the North American population. In
interpreting haplotype frequency data such as that shown in Table
3, one must bear in mind that cells of patients and organs are
diploid and have an HLA type that is the product of the HLA
haplotypes of the chromosomes inherited from both parents.
TABLE-US-00003 TABLE 3 Examples of HLA-A, -B, -DR haplotype
frequencies HLA-A, -B, and -DR haplotype frequencies (expressed in
percent) and their respective rankings within each racial group:
Caucasian (CAU), African-American (AFR), Asian-American (ASI) and
Native American (NAT). Haplotype Frequency (%) Ranking A B DR CAU
afr ASI LAT NAT CAU AFR ASI LAT NAT 1 7 2 0.5349 0.2094 0.0798
0.1888 0.2812 21 58 262 91 62 1 8 3 5.1812 1.2491 0.3195 1.6733
4.7439 1 2 54 3 1 2 14 1 0.1563 0.0444 0.0076 0.3794 0.0624 107 539
1451 39 312 2 35 4 0.1457 0.0737 0.3293 1.2858 0.6342 115 302 49 4
12 2 35 8 0.0823 0.0931 0.1756 1.7641 0.3289 241 226 122 1 46 2 44
4 2.1507 0.6506 0.1276 0.6906 2.0004 3 4 170 12 3 3 7 2 2.6285
0.7596 0.1891 1.1986 2.7083 2 3 113 5 2 3 7 4 0.4411 0.1534 0.0498
0.1795 0.4448 30 104 408 98 29 3 7 8 0.0848 0.0367 0.0000 0.0622
0.0537 230 653 14053 310 366 3 35 1 1.0224 0.2741 0.1372 0.3552
0.8125 7 29 156 44 8 31 51 4 0.0915 0.0342 0.1646 0.2597 0.5691 209
699 135 64 16 32 14 7 0.2617 0.0513 0.0046 0.1324 0.1775 57 479
1858 140 104
[0014] The data in Table 3 was produced for The National Marrow
Donor Program Donor Registry, and is available at the Internet web
site of Motomi Mori, Ph.D., Huntsman Cancer Institute, Salt Lake
City, Utah.
D. Rejection Triggered by Minor Histocompatibility Antigens
[0015] Matching the MHC molecules of a transplant to those of the
recipient significantly improves the success rate of clinical
transplantation; however, it does not prevent rejection, even when
the transplant is between HLA-identical siblings. This is because
rejection is also triggered by differences between the minor
histocompatibility antigens. These polymorphic antigens are
actually "non-self peptides bound to MHC molecules on the cells of
the transplant tissue. The rejection response evoked by a single
minor histocompatibility antigen is much weaker than that evoked by
differences in MHC antigens, because the frequency of the
responding T cells is much lower (Janeway et al., supra, page 525).
Nonetheless, differences between minor histocompatibility antigens
will often cause the immune system of a transplant recipient to
eventually reject a transplant, even where there is a match between
the MHC antigens, unless immunosuppressive drugs are used.
E. Inadequate Supply of Cells, Tissues, and Organs for
Transplant
[0016] The number of people in need of cell, tissue, and organ
transplants is far greater than the available supply of cells,
tissues, and organs suitable for transplantation. Under these
circumstances, it is not surprising that obtaining a good match
between the MHC proteins of a recipient and those of the transplant
is frequently impossible, and many transplant recipients must wait
for an MHC-matched transplant to become available, or accept a
transplant that is not MHC-matched. If the latter is necessary, the
transplant recipient must rely on heavier doses of
immunosuppressive drugs and face a greater risk of rejection than
would be the case if MHC matching had been possible. There is
presently a great need for new sources of cells, tissues, and
organs suitable for transplantation that are histocompatible with
the patients in need of such transplants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows cynomolgus monkey blastocysts derived from
parthenogenetic embryos.
[0018] FIG. 2 shows an ES-like cell line (Cyno 1) derived from a
cynomolgus parthenogenetic blastocyst.
[0019] FIG. 3 shows the Cyno 1 cell line before and after
immunosurgery.
[0020] FIG. 4 shows the Cyno 1 cell line before and after
immunosurgery.
[0021] FIG. 5 shows the Cyno 1 cell line 5 days after plating.
[0022] FIG. 6 shows the Cyno 1 cell line growing on top of a feeder
layer.
[0023] FIG. 7 shows the results of an RT-PCR showing that the
Cyno-1 cell line is homozygous for the Snrpn gene (contains
paternal allele).
[0024] FIG. 8 shows metaphase II oocytes at retrieval.
[0025] FIG. 9 shows 4 and 6 cell embryo 48 hours after
parthenogenetic activation.
[0026] FIG. 10 shows blastocoele cavities in human parthenogenetic
activation 48 hours after activation.
[0027] FIG. 11 shows human parthenogenetic embryo having an inner
cell mass.
[0028] FIG. 12 shows human ES-like cells derived from cultured ICM
cells.
DETAILED DESCRIPTION OF THE INVENTION
A. A Bank of Stem Cell Lines Homozygous for MHC Loci
[0029] It is an object of the present invention to prepare a bank
of totipotent, nearly totipotent, and/or pluripotent stem cell
lines that are homozygous for one or more critical antigen genes,
i.e., genes which encode histocompatibility antigens, e.g., in the
case of human stem cells and "stem-like" cells, MHC alleles that
are present in the human population. Preferably, this work will be
homozygous for MHC alleles that are representative of at least most
prevalent in the particular species, preferably human. Many of
these lines will also have an ABO blood group type 0-negative to
make them broadly compatible across the different blood types. Stem
cell lines of the present invention can be induced to differentiate
into cell types suitable for therapeutic transplant. Because the
cells of the present invention have homozygous MHC alleles, the
chance of obtaining cells for transplant that have MHC alleles that
match those of a patient in need of a transplant is significantly
enhanced. Instead of having to find a six of six match between two
sets of HLA-A, HLA-B, and HLA-DR antigens, a high level of
histocompatibility is provided by the cells for transplant of the
present invention when either of the two HLA-A, HLA-B, and HLA-DR
antigens of the prospective transplant recipient matches one of the
corresponding homozygous HLA antigens of the cells for transplant.
For example, a stem cell bank able to provide cells having an
HLA-A/HLA-B match to a patient having any of the eight HLA-A and
nine HLA-B antigens listed in Table 2 would require only 72 stem
cell lines with homozygous HLA-A and HLA-B antigens; whereas a bank
of stem cells with heterozygous HLA-A and HLA-B antigens would need
to have 4032 different stem cell lines. To provide a library of
heterozygous stem cell lines that match the WHO list of serological
types would require obtaining stem cells having every combination
of 28 different pairs of HLA-A antigens and 59 different pairs of
HLA-B, to account for both the maternal and paternal alleles for
each loci. The complexity of such a stem cell bank, i.e., the
number of different cell lines required, would be 2,587,032. In
contrast, a bank of stem cells homozygous for the same HLA-A and
HLA-B antigens would only need to have a complexity of 1,652 stem
cell lines to guarantee a match to a patient with HLA-A and -B
antigens on the WHO list of serological types. The actual number
required to meet the needs of a majority of patients will actually
be less than this due to the nonrandom distribution of alleles in
various populations around the world. Patients in need of bone
marrow stem cell grafts who are homozygous in particular alleles
are particularly sensitive to graft versus host disease when
heterozygous bone marrow grafts are used. Stem cell grafts using
stem cells having homozygous alleles made according to the methods
of the present invention would alleviate this common complication
of transplants.
[0030] This present invention provides novel means for making
libraries of totipotent and/or pluripotent stem cells that can
serve as sources of cells for therapeutic transplant that are
highly histocompatible with human or nonhuman patients in need of
cell transplants. Additionally, those cell lines are useful in
creating animal models for specific diseases that may be used to
evaluate potential treatments and drug antidotes. In one
embodiment, the invention comprises preparing a bank of stem cell
lines that are homozygous for one or more critical antigen alleles,
in the case of human stem cells. MHC alleles that are present in
all or most of the world's populations, including the populations
of North America, Central and South America, Europe, Africa, and
Asia, and the Pacific islands. It is an object of the present
invention to provide a stem cell bank comprising stem cells
generated from vertebrate somatic cells, preferably mammalian
somatic cells, and more preferably human research cells that are
homozygous for one or more critical antigen alleles, e.g., MHC
alleles using nuclear transfer or parthenogenic produced embryos. A
preferred object of the present invention is to provide a stem cell
bank comprising diploid vertebrate, preferably mammalian and more
preferably human stem cells generated by parthenogenesis that are
homozygous for MHC alleles. Another object of the present invention
is to provide a stem cell bank comprising diploid vertebrate,
preferably mammalian and more preferably human stem cells generated
by union of sperm and egg in vitro that are homozygous for one or
more MHC alleles. S&M further, an object of the invention is to
preview a bank of homozygous IES cell lines by introducing
cytoplasm from embryonic cells into growth cells that are
homozygous for specific MITC allele or are rendered homozygous by
genetic manipulation. (The embryonic cytoplasm contains
constituents that de-differentiate the differentiated growth cell
into stem cell lineages.
[0031] The stem cell bank of the present invention comprises lines
of totipotent, nearly totipotent, and/or pluripotent stem cells
that are homozygous for at least one histocompatibility antigen
collection. In the case of human stem cells this will be an MHC
allele selected from the group consisting of HLA-A, HLA-B, HLA-C,
HLA-DR, HLA-DQ, and HLA-DP. In a useful embodiment, the stem cell
bank comprises totipotent, nearly totipotent, and/or pluripotent
stem cells stem cells that are homozygous for the significant
histocompatibility antigen alleles, e.g., the HLA-A, HLA-B, and
HLA-DR alleles. In another embodiment, the stem cell bank comprises
stem cells that are homozygous for all of the histocompatibility
antigen alleles, e.g., MHC alleles.
[0032] The stem cell bank of the present invention comprises
totipotent and/or nearly totipotent stem cells such as embryonic
stem (ES) cells, that can differentiate in vivo or ex vivo into a
wide variety of different cell types having one or more homozygous
MHC alleles. The stem cell bank of the present invention can also
comprise partially differentiated, pluripotent stem cells such as
neuronal stem cells and/or hematopoietic stem cells, that
differentiate in vivo or ex vivo into a more limited number of
differentiated cell types having one or more homozygous MHC
alleles. These stem cells optionally may be transgenic, e.g., they
may express antigens that encode therapeutic or diagnostic proteins
and polypeptides. For example, the stem cells may be genetically
engineered to express proteins that inhibit immune rejection
responses such as CD4O-L (CD154 or gp139) or in the case of porcine
stem cells May be genetically engineered to knock-out a
glycosylated antigen that is known to trigger immune rejection
responses.
[0033] An object of the present invention is to provide a stem cell
bank comprising stem cells having homozygous histocompatibility
alleles, i.e., MHC alleles that are available "off the shelf" for
providing histocompatible cells suitable for transplant to patients
in need of such a transplant. Desirably, this stem bank will
include stem cell lines that are representative of the different
histocompatibility antigens expressed in the particular species,
e.g., human. In a useful embodiment, the stem cell bank comprises
stem cells that are isolated and maintained without feeder cells or
serum of non-human animals, to minimize concerns of contamination
by pathogens. In another useful embodiment, the stem cell bank
comprises stem cells that are genetically modified relative to the
cells of the donor, e.g., human donor from which they are derived.
In another embodiment, the stem cell bank comprises stem cells
generated by nuclear transfer techniques that are rejuvenated, or
"hyper-youthful," relative to the cells of the donor, e.g.,
non-human mammal or human donor from which they are derived, and
also relative to age-matched control cells of the same type and
species that are not generated by nuclear transfer techniques. Such
rejuvenated or "hyper-youthful" cells have extended telomeres,
increased proliferative life-span, and metabolism that is more
characteristic of youthful cells, e.g., increased EPC-1 and
telomerase activities, relative to the human donor cells from which
they are derived, and also relative to age-matched control cells of
the same type that are not generated by nuclear transfer
techniques.
[0034] Another object of the present invention is to provide a stem
cell bank comprising stem cells having homozygous recessive alleles
responsible for genetically inherited diseases. Recessive
disease-causing genes are endemic in the population, and such stem
cells can be generated by parthenogenesis using oocytes collected
from female carriers of the recessive disease-causing alleles.
There is great need for totipotent, nearly totipotent, and/or
pluripotent stem cells that having homozygous recessive
disease-causing alleles that can be induced to differentiate into
cells useful for basic research directed to studying the disease
phenotype, both ex vivo and in vivo (e.g., in immunodeficient
laboratory animals), and for screening to discover drugs and other
therapies that treat or cure the disease.
B. Terms Used in Describing the Invention
[0035] As used herein, a "stem cell" is a cell that has the ability
to proliferate in culture, producing some daughter cells that
remain relatively undifferentiated, and other daughter cells that
give rise to cells of one or more specialized cell types; and
"differentiation" refers to a progressive, transforming process
whereby a cell acquires the biochemical and morphological
properties necessary to perform its specialized functions. Stem
cells therefore reside immediately antecedent to the branch points
of the developmental tree.
[0036] As used herein, a "totipotent" cell is a stem cell with the
"total power to differentiate into any cell type in the body,
including the germ line following exposure to stimuli like that
normally occurring in development. Examples of totipotent cells
include an embryonic stem (ES) cell, an embryonic germ (EG) cell,
an inner cell mass (ICM)-derived cell, or a cultured cell from the
epiblast of a late-stage blastocyst.
[0037] As used herein, a "nearly totipotent cell" is a stem cell
with the power to differentiate into most or nearly all of the cell
types in the body following exposure to stimuli like that normally
occurring in development.
[0038] As used herein, a "pluripotent cell" is a stem cell that is
capable of differentiating into multiple somatic cell types, but
not into most or all cell types. This would include by way of
example, but not limited to, mesenchymal stem cells that can
differentiate into bone, cartilage and muscle; hematopoietic stem
cells that can differentiate into blood, endothelium, and
myocardium; neuronal stem cells that can differentiate into neurons
and glia; and so on.
[0039] As used herein, an "embryonic stem cell line is a cell line"
with the characteristics of the murine ES cells isolated from
morulae or blastocyst inner cell masses, as reported by Martin
(Proc. Natl. Acad. Sci. USA (1981) 78:7634-7638); and by Evans et
al. (Nature (1981) 292: 154-156). ES cells have high
nuclear-to-cytoplasm ratio, prominent nucleoli, are capable of
proliferating indefinitely and can be differentiate into most or
all of the specialized cell types of an organism, such as the three
embryonic germ layers, all somatic cell lineages, and the germ
line. ES cells that can differentiate into all of the specialized
cell types of an organism are totipotent. In some cases, ES cells
are obtained that can differentiate into almost all of the
specialized cell types of an organism; but not into one or a small
number of specific cell types. For example, Thomson et al. describe
isolating a primate ES cell that, when transferred into another
blastocyst, does not contribute to the germ line (Proc. Natl. Acad.
Sci. USA. (1995) 92:7844-7848). Such ES cells are an example of
stem cells that are nearly totipotent.
[0040] As used herein, "inner cell mass-derived cells" (ICM-derived
cells) are cells directly derived from isolated ICMs or morulae
without passaging them to establish a continuous ES or ES-like cell
line. Methods for making and using ICM-derived cells are described
in co-owned U.S. Pat. No. 6,235,970, the contents of which are
incorporated herein in their entirety.
[0041] As used herein, "enucleation" refers to removal of the
genomic DNA from an cell, e.g., from a recipient oocyte.
Enucleation therefore includes removal of genomic DNA that is not
surrounded by a nuclear membrane, e.g., removal of chromosomes
aligned to form a metaphase plate. As discussed below, the
recipient cell can be enucleated by any of the known means either
before, concomitant with, or after nuclear transfer.
[0042] As used herein, "ex vivo" cell culture refers to culturing
cells outside of the body. Ex vivo cell culture includes cell
culture in vitro, e.g., in suspension, or in single- or multi-well
plates. Ex vivo culture also includes co-culturing cells with two
or more different cell types, and culturing in or on 2- or
3-dimensional supports or matrices, including methods for culturing
cells alone or with other cell types to form artificial
tissues.
[0043] As used herein, "parthenogenetic embryos" refers to an
embryo that only contains male or female chromosomal DNA that is
derived from male or female gametes. For example, parthenogenetic
embryos can be derived by activation of unfertilized female
gametes, e.g., unfertilized human, murine, cynomolgus or rabbit
oocytes.
[0044] As used herein, "nuclear transfer embryo" refers to an
embryo that is produced by the fusion or transplantation of a donor
cell or DNA from a donor cell into a suitable recipient cell,
typically an oocyte of the same or different species that is
treated before, concomitant or after transplant or fusion to remove
or inactivate its endogenous nuclear DNA. The donor cell used for
nuclear transfer include embryonic and differentiated cells, e.g.,
somatic and germ cells. The donor cell may be in a proliferative
cell cycle (G.sub.1, G.sub.2, S or M) or non-proliferating (G.sub.0
or quiescent). Preferably, the donor cell or DNA from the donor
cell is derived from a proliferating mammalian cell culture, e.g.,
a fibroblast cell culture. The donor cell optionally may be
transgenic, i.e., it may comprise one or more genetic addition,
substitution or deletion modifications.
[0045] As used herein, the term "gene" refers to the nucleotide
sequences at a genetic locus that encode and regulate expression of
a functional mRNA molecule or a polypeptide; i.e., as used herein,
a gene includes the nucleotide sequences that make up the coding
sequence (exons and introns), the promoter, enhancers, and other
DNA elements that regulate transcription, including as elements
conferring cell type-specific and differentiation stage-specific
expression, hormone responsive elements, repressor elements, etc.,
and nucleotide sequences that encode signals that regulate splicing
and translation of the mRNA, such as a cleavage signal, a
polyadenylation signal, or an internal ribosome entry site
(IRES).
C. Providing Histocompatible Transplants to Animal or Human
Recipients
[0046] Another object of the invention is to provide a method by
which a human or non-human animal, e.g., a person in need of a cell
or tissue transplant can be provided with cells or tissue suitable
for transplantation that have homozygous histocompatibility antigen
alleles, e.g., in the case of human recipients MHC alleles that
match the MHC alleles of the person needing the transplant. The
invention provides a method in which the MHC alleles of a person in
need of a transplant (the recipient) are identified, and a line of
stem cells homozygous for at least one MHC allele present in the
recipients cells is obtained from a stem cell bank produced
according to the disclosed methods. That line of stem cells is then
used to generate cells or tissue suitable for transplant that are
homozygous for at least one MHC allele present in .the recipients
cells. The method of the present invention further comprises
grafting the cells or tissue so obtained to the body of the person
in need of such a transplant. In a useful embodiment of the
invention, three, four, five, six or more of the MHC alleles of the
line of stem cells used to generate cells or tissue for transplant
are homozygous and match MHC alleles of the transplant
recipient.
[0047] In a useful embodiment, the line of stem cells used to
generate cells or tissue suitable for transplant is a line of
totipotent or nearly totipotent embryonic stem cells. In another
useful embodiment, the line of stem cells used to generate cells or
tissue suitable for transplant is a line of hematopoietic stem
cells. The lines of stem cells that can be used to generate cells
or tissue suitable for transplant are available "off the shelf" in
the stem cell bank of the present invention. In a useful
embodiment, the stem cell bank of the present invention comprises
lines of totipotent, nearly totipotent, and/or pluripotent stem
cells that are lines of rejuvenated, "hyper-youthful cells"
generated by nuclear transfer techniques. In another useful
embodiment, the stem cell bank of the present invention comprises
one or more lines of totipotent, nearly totipotent, and/or
pluripotent stem cell having DNA that is genetically modified
relative to the DNA of the human donor from which they are derived.
For example, the invention comprises altering genomic DNA of the
cells to replace a non-homozygous MHC allele with one that is
homozygous, or to inhibit the effective presentation of a class I
or class II HLA antigen on the cell surface, e.g., by preventing
expression of .beta.2-microglobulin, or by preventing expression of
one or more MHC alleles. Also, the invention encompasses
introducing one or more genetic modifications that result in
lineage-defective stem cells, i.e., stem cells which cannot
differentiate into specific cell lineages.
D. Methods for Making Stem Cell Lines with Homozygous MHC
Alleles
[0048] Totipotent, nearly totipotent, and/or pluripotent stem cell
lines that make up the stem cell banks of the present invention can
be derived from blastocyst embryos made up of cells that are
homozygous for some or all of the histocompatibility antigen
alleles, e.g., MHC alleles. Blastocyst embryos useful for the
present invention can be made by several different methods. In
preferred embodiments of the invention, human embryos are produced
by fertilization, parthenogenesis, or by same or cross-species
somatic cell nuclear transfer. In the case of human embryos, for
ethical reasons, they are never allowed to develop beyond the stage
of pre-implantation blastocysts of about 9-10 days before the inner
cell mass cells are isolated and are cultured to produce embryonic
stem (ES) cells. The cloning methods of the present invention which
utilize human embryos are restricted to human therapeutic cloning
techniques. The present invention does not include any methods that
permit development of human embryos beyond the pre-implantation
stage of about 9-10 days, nor does it include or contemplate
reproductive cloning in any form.
Stem Cells from Embryos Produced by Union of Sperm and Egg
[0049] In one embodiment of the invention, human or non-human stem
cells are derived from embryos produced in vitro by uniting sperm
and eggs by known means; for example, by in vitro fertilization
(IVF) or by intracytoplasmic sperm injection (ICSI). To produce
cells having homozygous MHC alleles, sperm and eggs can be obtained
from individuals that are closely related; e.g., brother and sister
or one determined to have similar MHC alleles. As in HLA typing for
a transplant between siblings, there is about a 25% chance that an
embryo produced with sibling's gametes will have matching HLA loci.
The embryos produced by uniting sperm and eggs of related
individuals are cultured in vitro to produce early embryo including
blastocysts from which ES cells or inner cell masses are derived.
HLA types of the resulting pluripotent cell lines are determined by
known means; e.g., by PCR, or by culturing a sample of the cells
under conditions that induce differentiation, and performing
serological testing of the cells using antibodies against specific
HLA antigens. Pluripotent cell lines having one or more homozygous
MHC alleles are then selected for inclusion in the stem cell bank.
Embryos produced by union of sperm and egg have normal genetic
imprinting, i.e., they have the epigenetic contributions of both
male and female parents, so they develop to form blastocysts from
which pluripotent cells can be derived with high efficiency.
[0050] In the case where sperm and egg donors are not closely
related sperm can be banked from individuals with characterized MHC
loci and used for IVF or ICSI fertilization of oocytes that also
have characterized MHC loci to produce embryos and stem cells with
a high likelihood of generating homozygosity in the MHC loci.
[0051] Persons skilled in the art would recognize that the human
embryos produced by uniting sperm and eggs of closely related
individuals according to the present invention may be viable and
could be implanted into human females to make pregnancies and
develop to live births of humans having homozygous HLA alleles.
This would be highly unethical, in view of the known risks to the
health of the child that result from close inbreeding. As stated
above, the present invention expressly does not comprise allowing
the embryos to develop beyond blastocysts of about 9-14 days.
Stem Cells Produced by Parthenogenesis
[0052] In another embodiment of the invention, totipotent and
pluripotent human stem cells are derived from embryos produced by
parthenogenesis. The stem cells obtained by this method are
diploid, because extrusion of the second polar body following
parthenogenetic activation is inhibited. Methods for producing a
diploid human embryo by parthenogenesis, for culturing the embryo
in vitro to form a blastocyst, and for culturing cells of the
blastocyst to obtain stem cells, are described in co-owned and
co-pending PCT Application PCT/US02/37899 (Methods for Making and
Using Reprogrammed Human Somatic Cell Nuclei and Autologous and
Isogenic Stem Cells) filed Nov. 26, 2002, the disclosure of which
is incorporated herein by reference in its entirety. Similar
methods for producing diploid embryos by parthenogenesis using
oocytes of rhesus monkeys and cynomolgus monkeys have been
described by Mitalipov et al. (2001, Biology of Reproduction,
65:253-259) and Cibelli et al. (2002, Science, 295:81),
respectively, the contents of both of which are incorporated herein
by reference in their entirety.
[0053] In general, production of a diploid human embryo by
parthenogenesis comprises [0054] a. obtaining oocytes from human
donors induced to superovulate by treatment with gonadotropins
followed by hCG injection; [0055] b. activating the oocytes at
about 38-45 hours after hCG stimulation; [0056] c. exposing the
activated oocytes to chemical treatment that inhibits extrusion of
the second polar body; and [0057] d. culturing the embryo in vitro
under conditions resulting in formation of a blastocyst.
[0058] Oocyte activation is normally mediated by oscillations of
intracellular Ca+2 ion triggered by the sperm cell. Parthenogenetic
activation of the oocytes can be achieved by any of the known means
for inducing oocyte activation. Such methods generally involve
exposing the oocyte to ethanol, electroporation, calcium ionophore,
ionomycin, inositol 1,4,5-triphosphate to increase the
intracellular Ca.sup.+2 ion concentration in the oocyte, in
combination with a treatment that temporarily inhibits protein
synthesis or protein phosphorylation. For example, Mitalipov et al.
(supra, p. 254) describe two such methods that result in production
of diploid parthenogenetic blastocysts from oocytes of rhesus
monkeys. In one method, the oocytes are incubated briefly in medium
containing ionomycin and calcium, followed by incubation for
several hours in medium containing 6-aminomethylpurine (DMAP), an
inhibitor of protein phosphorylation. In the other method, the
oocytes are electroporated three times in medium containing
calcium, and between each electroporation, the oocytes are
incubated for about 30 minutes in medium containing cycloheximide,
an inhibitor of protein synthesis, and cytochalasin B, an inhibitor
of microfilament synthesis.
[0059] Using a similar method Cibelli et al. (supra)
parthenogenetically activated oocytes of a cynomolgus monkey;
cultured the activated oocytes in vitro to produce a diploid
blastocysts; and isolated a line of diploid ES cells from cells of
the inner cell mass of a parthenogenesis-derived embryo; and showed
that the ES cells are capable of differentiating into cell types of
all three embryonic germ layers. This is also described in U.S.
Ser. No. 09/697,297 by Cibelli et al, which is incorporated by
reference in its entirety here.
[0060] Oocytes are obtained from women having MHC alleles of the
type needed for the stem cell bank. The oocytes are
parthenogenetically activated and are cultured to form blastocysts.
Using known methods, the inner cell mass cells of the blastocysts
are cultured in vitro to generate diploid embryonic stem cells.
Because extrusion of the second polar body after meiosis II was
prevented, the homologous chromosomes of such ES cells are actually
the sister chromatids that were joined together as a dyad during
meiosis I. Since the sister chromatids were formed by replication
of a single set of chromosomes at the outset of meiosis, they will
have identical DNA sequences, except for those regions that were
exchanged with the homologous dyad during the recombination stage
of meiosis. The HLA genes of the MHC are tightly linked, and
recombination in this region is rare occurring with a frequency of
about 1%. The two sets of homozygous HLA alleles in the
parthenogenetically-derived stem cell lines obtained with oocytes
from a given donor reflect the HLA haplotypes of the maternal and
paternal copies of chromosome 6 that the donor inherited from her
parents. Known screening methods can be performed to identify the
cell lines that have non-homozygous HLA antigens due to genetic
recombination, and to identify the homozygous HLA alleles of each
stem cell line.
Stem Cells Produced by Haploidization
[0061] In another embodiment of the invention, totipotent and
pluripotent human stem cells are derived from embryos produced by
union of two haploids that are homozygous for one or more MHC
alleles.
[0062] Methods for producing embryos by fusion of two haploid
genomes are described in U.S. Ser. No. 10/344,724, filed on Feb.
14, 2003 entitled, "Use of Haploid Genomes for Genetic Diagnosis,
Modification and Multiplication," which is incorporated by
reference in its entirety herein.
[0063] A bank of stem cell lines according to the present invention
can be .obtained by screening the population- and -identifying
individuals having cells which express desired MHC antigens, and
obtaining donations of the somatic cells from these individuals.
However, individuals that are homozygous for MHC antigens are rare,
because they are only found in inbred population. Thus, the useful
embodiment of the invention is utilization of heterozygous donor
cells to create homozygous stem cells.
[0064] In this method, somatic cells are introduced into enucleated
human oocytes, and the newly constructed oocytes are activated to
induce haploidization (Tesarik et al., 2001 R B Online. 2:160-164),
Lachem-Kaplan et al, 2001 R B Online 3: 205-211. When a protocol
for primate oocyte activation are used, approximately 90% of eggs
yield pseudo-polar body (Shoukhrat et al, 2001 Biol Reprod
65:253-259). These pseudo-polar bodies a re used for genotyping
using well established techniques. Other haploid embryos also can
be constructed by transferring cells from other donors using the
same protocol. Or the donor oocytes can be screened for the
presence of desired MHC allele after activation to generate haploid
oocytes. Screening of the first polar bodies will reveal the
genotype of the oocytes as in above the reconstructed eggs. The
activation can be done chemically and/or by injecting sperm factors
(see U.S. Application No. 60/191,089 of Rafael Fissore filed Mar.
22, 2000 incorporated by reference in its entirety herein) easily
unless 2nd polar body extrusion is blocked systematically
(incorporated by reference in its entirety herein). The remaining
pronuclei are transferred to construct diploid embryos by
pronuclear transfer techniques. These techniques have been well
established and used widely in developmental biology fields for
more than a decade. To avoid possible imprinting disturbance,
morula stage human embryo lysates are injected into the newly
constructed eggs. These embryo lysates are known to have ability to
modify imprinting status of murine androgenone so effectively to
make live born animals, otherwise develop very poorly in vitro and
died out after implantation (Hagemman and First, 1992 Development
114:997-1001).
[0065] More particularly, the invention includes methods for
generating stem cells by haploidization comprising the steps of:
[0066] a. Inserting a somatic donor cell, or the nucleus of such a
cell, into an oocyte that is free of oocyte genomic DNA. [0067] b.
Activation of the reconstructed embryos to expel haploidal genome
into a pseudopolar body. [0068] c. Screening of the pseudopolar
body for the genotyping of remaining pronucleus. [0069] d. Union of
the two pronuclei to generate diploid embryos by pronuclei
transfer. Or alternatively, transferring a pronucleus to an
activated haploid oocyte which has desired haploid genome [0070] e.
Injection of human morular stage embryo lysates to the
reconstructed embryos. [0071] f. Culturing embryo and generating
stem cells/or differentiated cells or tissue needed for transplant
from cells of said embryos.
[0072] In addition, haploid genomes can be derived by other means
known in the art, including the use of the first and second polar
bodies. While occasionally, such DNA is fragmented, intact genomes
can be obtained as evidenced by the production of live mice from
polar body DNA (Wakayama, T., and Yanagimachi, R. Biol. Reprod.
1998. 59(1) 100-4) and these haploid or diploid genomes can be used
as described above.
Stem Cells Produced by Cytoplasm Transfer
[0073] Totipotent and pluripotent stem cells homozygous for
histocompatibility antigens, e.g., MHC antigens can also be
produced by transferring cytoplasm from an oocyte or an ES cell
into a somatic cell that is homozygous for MHC antigens, so that
the chromatin of the somatic cell is reprogrammed and the somatic
cell de-differentiates to generate a pluripotent or totipotent stem
cell. Methods for converting differentiated cells into
de-differentiated, pluripotent, stem or stem-like cells that can be
induced to re-differentiate into a cell type other than that of the
initial differentiated cells, are described in co-owned and
co-pending U.S. application Ser. No. 09/736,268, filed Dec. 15,
2000, and U.S. application Ser. No. 10/112,939 filed Apr. 2, 2002,
both by Karen B. Chapman, the disclosures of both of which are
incorporated herein by reference in their entirety.
Stem Cells from Embryos Produced by Nuclear Transfer
[0074] In another embodiment of the invention, totipotent, nearly
totipotent, and/or pluripotent human stem cells that are homozygous
for one or more MHC alleles are derived from embryos produced in
vitro by somatic cell nuclear transfer techniques. The totipotent
and/or pluripotent stem cells generated by this embodiment of the
invention will have the genomic DNA of the somatic donor cell used
for nuclear transfer. When the somatic donor cell is homozygous for
an MHC allele, the stem cells generated by nuclear transfer cloning
will also be homozygous for the MHC allele.
[0075] A bank of stem cell lines according to the present invention
can be obtained by screening a species, preferably human population
and identifying individuals that are homozygous for clinical MHC
antigens, and obtaining donations of somatic cells from these
individuals. Individuals having homozygous MHC alleles are often
found in inbred populations. Alternatively, somatic cells,
preferably human, homozygous for MHC loci that are useful for the
present invention can be produced by obtaining somatic cells that
are heterozygous for an MHC allele, and genetically altering the
DNA of the cells using known methods so that they are homozygous
for one or more MHC loci. This can be done, for example, by using
well-known homologous recombination techniques to replace a
non-homozygous MHC allele with one that is homozygous.
[0076] In a useful embodiment of the invention, donors of somatic
cells to be used in nuclear transfer according to the present
invention may be selected to provide cells that are relatively
resistant to blood cell cancers, for use in reconstituting the
blood of blood cancer patients. Such blood cells can be chosen
based on their natural killer (NK) cell phenotype. The somatic cell
donors who having resistance to blood cell cancers can be selected
to have homozygous MHC alleles, or the donated cells can be
genetically altered to have one or more homozygous MHC alleles as
discussed above.
[0077] The donated cells are cloned by nuclear transfer techniques
that result in production of blastocyst embryos from which are
obtained totipotent and/or pluripotent stem cells that are
homozygous for one or more MHC loci. For each cell line to be
produced, a somatic donor cell that is homozygous for a MHC allele,
or the nucleus or set of chromosomes of such a cell, is inserted
into a human oocyte that is coordinately enucleated to produce a
nuclear transfer unit that develops as an embryo. The embryo is
cultured ex vivo to the blastocyst stage, and totipotent and/or
pluripotent stem cells are derived from inner cell mass (ICM) cells
of the embryo that have the genomic DNA of the donor cell. In a
useful embodiment, the stem cell bank comprises totipotent, nearly
totipotent ES cells homozygous for MHC antigens. Totipotent and
pluripotent stem cells homozygous for various combinations of MHC
antigens are assembled and maintained as a bank of cells available
for therapeutic transplantation.
[0078] Methods for transferring the nuclear DNA of a somatic cell
of a patient into an oocyte to effect the reprogramming of the
chromatin and produce an NT unit from which are generated
pluripotent stem cells and totipotent ES cells are described, for
example, in co-owned and co-pending U.S. application Ser. No.
09/655,815 filed Sep. 6, 2000; and U.S. application Ser. No.
09/797,684 filed Mar. 5, 2001; and also in PCT Application No.
PCT/US02/37899 (Methods for Making and Using Reprogrammed Human
Somatic Cell Nuclei and Autologous and Isogenic Stem Cells) filed
Nov. 26, 2002, the disclosures of all three of which are
incorporated herein by reference in their entirety. Similar methods
are described in co-owned and co-pending U.S. application Ser. No.
09/527,026 filed Mar. 16, 2000, Ser. No. 09/520,879 filed Apr. 5,
2000, and Ser. No. 09/656,173 filed Sep. 6, 2000, the disclosures
of which are incorporated herein by reference in their entirety. In
general, methods for cloning by somatic cell nuclear transfer to
produce stem cells for generating cells or tissue useful for
transplantation comprise the steps of: [0079] a. inserting a
somatic donor cell, or the nucleus of such a cell, into an oocyte
and removing the oocyte genomic DNA (enucleation) under conditions
that produce an activated nuclear transfer unit that develops as an
embryo; and [0080] b. generating stem cells and/or differentiated
cells or tissue needed for transplant from cells of said
embryo.
[0081] Such a method can be used to generate lines of totipotent or
nearly totipotent ES cells that can be cultured under conditions in
which they differentiate into specific, recognized cell types. Such
ES cells have the capacity to differentiate into every cell type of
the body, including the germ cells. The stem cells produced by
somatic cell nuclear transfer have the patients genomic DNA, so the
differentiated cells and tissues generated from such stem cells are
nearly completely autologous--all of the cells' proteins, are
encoded by the patients own DNA except for those proteins encoded
by the cells' mitochondria, which derive from the oocyte.
Accordingly, differentiated cells and tissues generated from stem
cells produced by nuclear transfer methods can be transplanted to
the person who provided the nuclear donor cell without triggering
the severe rejection response that results when foreign cells or
tissue are transplanted.
[0082] As described in the above-identified co-pending
applications, the somatic donor cell used for nuclear transfer to
produce human stem cells homozygous for a MHC allele according to
the present invention can be of any. somatic cell type in the body.
For example, the somatic donor cell can be a cell selected from the
group consisting of fibroblasts. B cells, T cells, dendritic cells,
keratinocytes, adipose cells, epithelial cells, epidermal cells,
chondrocytes, cumulus cells, neural cells, glial cells, astrocytes,
cardiac cells, esophageal cells, muscle cells, melanocytes,
hematopoietic cells, macrophages, monocytes, and mononuclear cells.
The somatic donor cell can be obtained from any organ or tissue in
the body; for example, it can be a cell from an organ selected from
the group consisting of liver, stomach, intestines, lung, stomach,
intestines, lung, pancreas, cornea, skin, gallbladder, ovary,
testes, kidneys, heart, bladder, and urethra.
[0083] Methods for generating rejuvenated, "hyper-youthful" stem
cells and differentiated somatic cells having the genomic DNA of a
human somatic donor cell are described in co-owned and co-pending
U.S. application Ser. No. 09/527,026 filed Mar. 16, 2000, Ser. No.
09/520,879 filed Apr. 5, 2000, and Ser. No. 09/656,173 filed Sep.
6, 2000, the disclosures of which have been incorporated herein by
reference in their entirety. For example, rejuvenated,
"hyper-youthful" stem cells having the genomic DNA of a human
somatic cell donor can be produced by a method comprising: [0084]
a. isolating normal, somatic cells from a human donor, and
passaging or otherwise inducing the cells into a state of
checkpoint-arrest, senescence, or near-senescence, [0085] b.
transferring a checkpoint-arrested, senescent, or near-senescent
donor cell, the nucleus of said cell, or chromosomes of said cell,
into a recipient oocyte, and coordinately removing the oocyte
genomic DNA from the oocyte, to generate an embryo; and [0086] c.
generating rejuvenated stem cells from said embryo having the
genomic DNA of the donor cell.
[0087] As described in the above-identified co-pending
applications, the pluripotent and totipotent stem cells homozygous
for a MHC allele of the present invention that are produced by
nuclear transfer using a checkpoint-arrested, senescent, or
near-senescent donor cell are rejuvenated cells that are
distinguished from other cells in having telomeres that are longer
than the corresponding telomeres of the checkpoint-arrested,
senescent, or near-senescent donor cell. Moreover, the telomeres of
such rejuvenated cells are on average at least as long as the
telomeres of age-matched control cells of the same type and species
that are not generated by nuclear transfer techniques. In addition,
the nucleotide sequences of the tandem (TTAGGG).sub.n repeats that
comprise the telomeres of such rejuvenated cells are more uniform
and regular; i.e., have significantly fewer non-telomeric
nucleotide sequences, than are present in the telomeres of
age-matched control cells of the same type and species that are not
generated by nuclear transfer. Such rejuvenated cells are
"hyper-youthful", in that the proliferative life-span of the
rejuvenated cells is at least as long as, and is typically longer
than, the proliferative life-span of age-matched control cells of
the same type and species that are not generated by nuclear
transfer techniques. Such rejuvenated cells also have patterns of
gene expression that are characteristic of youthful cells; for
example, activities of EPC-1 and telomerase in such rejuvenated
cells are typically greater than EPC-1 and telomerase activities in
age-matched control cells of the same type and species that are not
generated by nuclear transfer techniques.
[0088] As described in the above-identified co-pending
applications, rejuvenated totipotent and/or pluripotent stem cells
can be generated from an embryo produced by nuclear transfer by
methods comprising obtaining a blastocyst, an embryonic disc cell,
inner cell mass cell, or a teratoma cell using said embryo, and
generating the pluripotent and/or totipotent stem cells from said
blastocyst, inner cell mass cell, embryonic disc cell, or teratoma
cell.
[0089] As described in co-owned and co-pending U.S. application
Ser. No. 09/685,061 filed Oct. 6, 2000, Ser. No. 09/809,018 filed
Mar. 16, 2001, and Ser. No. 09/874,040 filed Jun. 6, 2001, the
recipient oocyte may be derived from a non-human mammal. For
example, the oocyte may be from a mammal selected from the group
consisting of sheep, bovines, bovines, pigs, horses, rabbits,
guinea pigs, mice, hamsters, rats, and non-human primates. In a
preferred embodiment, the oocyte is from a bovine mammal, or a
rabbit. A stem cell line having the genome of a human cell that is
derived using a nonhuman oocyte is referred to herein as a "human"
stem cell line, even though the mitochondria of such cells are of a
non-human type.
Genetically Modified Stem Cells
[0090] The methods of the present invention include producing
totipotent and/or pluripotent stem cells homozygous for MHC
antigens that are genetically modified relative to the cells of the
human donor from which they were originally obtained. The stem
cells can be genetically modified in any manner that enhances or
improves the overall efficiency by which cells for transplant are
produced and the therapeutic efficacy of the cell transplantation.
Methods that use recombinant DNA techniques to introduce
modifications at selected sites in the genomic DNA of cultured
cells are well known. Such methods can include (1) inserting a DNA
sequence from another organism (human or non-human) into target
nuclear DNA, (2) deleting one or more DNA sequences from target
nuclear DNA, and (3) introducing one or more base mutations (e.g.,
site-directed mutations) into target nuclear DNA. Such methods are
described, for example, in Molecular Cloning, a Laboratory Manual,
2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor
Laboratory Press; U.S. Pat. No. 5,633,067, "Method of Producing a
Transgenic Bovine or Transgenic Bovine Embryo," DeBoer et al.,
issued May 27, 1997; U.S. Pat. No. 5,612,205, "Homologous
Recombination in Mammalian Cells," Kay et al., issued Mar. 18,
1997; and PCT publication WO 93/22432, "Method for Identifying
Transgenic Pre-Implantation Embryos," all of which are incorporated
by reference herein in their entirety. Such methods include
techniques for transfecting cells with foreign DNA fragments and
the proper design of the foreign DNA fragments such that they
effect insertion, deletion, and/or mutation of the target DNA
genome. For example, known methods for genetically altering cells
that use homologous recombination can be used to insert, delete, or
rearrange DNA sequences in the genome of a cell of the present
invention. A genetic system that uses homologous recombination to
modify targeted DNA sequences in a mammalian cell to "knock-out" a
cell's ability to express a selected gene is disclosed by Capecchi
et al. in U.S. Pat. Nos. 5,631,153 and 5,464,764, the contents of
which are incorporated herein in their entirety. Such known methods
can be used to insert into the genomic DNA of a cell an additional
(exogenous) DNA sequence comprising an expression construct
containing a gene that is to be expressed in the modified cell. The
gene to be expressed can be operably linked to any of a wide
variety of different types of transcriptional regulatory sequences
that regulate expression of the gene in the modified cell. For
example, the gene can be under control of a promoter that is
constitutively active in many different cell types, or one that is
developmentally regulated and is only active in one or a few
specific cell types. Alternatively, the gene can be operably linked
to an inducible promoter that can be activated by exposure of the
cell to a physical (e.g., cold, heat, light, radiation) or chemical
signal. Many such inducible promoters and methods for using them
effectively are well known. Examples of the characteristics and use
of such promoters, and of other well-known transcriptional
regulatory elements such as enhancers, insulators, and repressors,
are described, for example, in Transgenic Animals, Generation and
Use, 1997, edited by L. M. Houdebine, Hardwood Academic Publishers,
Australia, the contents of which are incorporated herein by
reference.
[0091] Stem cells homozygous for MI-IC antigens that have multiple
genetic alterations can be produced using known methods. For
example, one can produce cells that are modified at multiple loci,
or cells that are modified at a single locus by complex genetic
alterations requiring multiple manipulations. To produce stem cells
having multiple genetic alterations, it is useful to perform the
genetic manipulations on somatic cells cultured in vitro, and then
to clone the genetically altered cells by somatic cell nuclear
transfer and generate ES cells having multiple genetic alterations
from the resulting blastocysts. Methods for generating genetically
modified cells using nuclear transfer cloning techniques are
described, for example, in co-owned and co-pending U.S. application
Ser. No. 09/527,026 filed Mar. 16, 2000, Ser. No. 09/520,879 filed
Apr. 5, 2000, and Ser. No. 09/656,173 filed Sep. 6, 2000, the
disclosures of which have been incorporated herein by reference in
their entirety.
[0092] Alternatively, the totipotent and/or pluripotent stem cells
having homozygous MHC alleles that are produced by any of the
methods described above can be genetically modified directly using
known methods. For example, Zwaka et al. have described a method
for genetically modifying human ES cells in vitro by homologous
recombination (Nature Biotechnology, Vol. 21, No. 3, March,
2003).
[0093] In generating stem cells by nuclear transfer, it is useful
to genetically modify the nuclear donor cell to enhance the
efficiency of embryonic development and the generation of ES cells.
The gene products of the Ped type, which are members of the Class I
MHC family and include the Q7 and Q9 genes, are reported to enhance
the rate of embryonic development. Modification of the DNA of
nuclear donor cells by insertion of DNA expression constructs that
provide for the expression of these genes, or their human
counterparts, will give rise to nuclear transfer embryos that grow
more quickly. It appears that these genes are only expressed early
in blastocyst development and so are not expected to be disruptive
of later development.
[0094] The efficiency of embryonic development can also be enhanced
by genetically modifying the nuclear donor cell to have increased
resistance to apoptosis. Genes that induce apoptosis are reportedly
expressed in preimplantation stage embryos (Adams et al, Science,
281(5381):1322-1326 (1998). Such genes include Bad, Bok, BH3, Bik,
Hrk, BNIP3, BimL, Bad, Bid, and EGL-1. By contrast, genes that
reportedly protect cells from programmed cell death include BcL-XL,
Bcl-w, Mcl-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K,
and CED-9. Nuclear donor cells can be constructed in which genes
that induce apoptosis are "knocked out" or in which the expression
of genes that protect the cells from apoptosis is enhanced or
turned on during embryonic development. Expression constructs that
direct synthesis of antisense RNAs or ribozymes that specifically
inhibit expression of genes that induce apoptosis during early
embryonic development can also be inserted into the DNA of nuclear
donor cells to enhance development of nuclear transfer-derived
embryos. Apoptosis genes that may be expressed in the antisense
orientation include BAX, Apaf-1, and capsases. Many DNAs that
promote or inhibit apoptosis have been reported and are the subject
of numerous patents. The construction and selection of genes that
affect apoptosis, and of cell lines that express such genes, is
disclosed in U.S. Pat. No. 5,646,008, the contents of which are
incorporated herein by reference.
[0095] Stem cells can be produced that are genetically modified
grow more efficiently in tissue culture than unmodified cells;
e.g., by increasing the number of growth factor receptors on the
cells' surface. Use of stem cells having such modifications reduces
the time required to generate an amount of cells for transplant
that is sufficient to have therapeutic effect.
[0096] The histocompatibility of a line of cells to be used for
transplant with a patient in need of such as transplant may be
increased by altering the genomic DNA of the cells to replace a
non-homozygous MHC allele with one that is homozygous and matches
an HLA allele of the patient. Alternatively, the genomic DNA of the
cells can be modified to inhibit the effective presentation of a
class 1 or class II HLA antigen on the cell's surface; for example,
by introducing a genetic alteration that prevents expression of
.beta.2-microglobulin, which is an essential component of class I
HLA antigens; by introducing genetic alterations in the promoter
regions of the class I and/or or class II MHC genes; or simply by
deleting a portion of the DNA of one or more of the class I and/or
or class II MHC genes sufficient to prevent expression of the
gene(s).
[0097] Stem cells of the invention can be genetically modified
(e.g., by homologous recombination) to have a heterozygous
knock-out of the Id1 gene, and a homozygous knockout of the Id3
gene. As described in co-owned and co-pending PCT Application No.
PCT/USU3/01827 (Stem Cell-Derived Endothelial Cells Modified to
Disrupt Tumor Angiogenesis), filed Jan. 22, 2003, these stem cells
can be induced to differentiate into Id1+/-, M3-/- endothelial cell
precursor cells that are useful for the treatment of cancer because
they give rise to endothelial cells that disrupt and inhibit tumor
angiogenesis.
[0098] Stem cells of the invention can also be genetically modified
to provide a therapeutic gene product that the patient requires,
e.g., due to an inborn error of metabolism. Many genetic diseases
are known to result from an inability of a patient's cells to
produce a specific gene product. The present invention making
genetically altered stem cells that can be used to produce cells
with homozygous MHC alleles for transplantation that are
genetically modified to synthesize enhanced amounts of a gene
product required by the transplant recipient. For example,
hematopoietic stem cells that are genetically altered to produce
and secrete adenosine deaminase can be prepared for transplant to a
patient suffering from adenosine deaminase deficiency. The methods
of the present invention permit production of such cells without
the use of recombinant retrovirus, which can insert at a site in
the genomic DNA that disrupts normal growth control and causes
neoplastic transformation.
[0099] Stem cells of the invention can also be genetically modified
by introduction of a gene that causes the cell to die. The gene can
be put under control of in inducible promoter. If for any reason
the transplanted cells react in a in a way that can harm the
recipient, expression of the suicide genes can be induced to kill
the transplanted cells. Use of inducible suicide genes in this
manner is known in the art. Suitable suicide genes include genes
encoding HSV thymidine kinase and cytodine deaminase, with which
cell death is induced by gancyclovir and 5-fluorocytosine,
respectively.
[0100] The cells may be modified to knockout one or more
histocompatibility antigen alletes, e.g., MHC alleles such that
only one set remains. This leads to an underexpression of the MHC
genes, but a phenotype effective in reducing the complexity of the
MHC serotype and effective in producing cells capable of otherwise
functioning and useful in the treatment of disease. Alternatively,
homozygosity can be engineered into the cell lines by the targeted
introduction of the appropriate alleles to the nonhomologous set,
to result in homozygosity. In addition, the chromosome carrying the
MHC genes can be removed from cells by laser ablation and a
chromosome carrying the identical chromosome as remains in the cell
can be added by microsome-mediated chromosome transfer, or by other
techniques known in the art.
[0101] The present invention is by no means limited to the
foregoing examples of genetic alterations. Persons skilled in the
art will be able to identify numerous other ways by which stem
cells produced according to the present invention can be
genetically modified to enhance their utility.
Preparing Totipotent and/or Pluripotent Stem Cells
[0102] Stem cells are present in the earliest stages of embryo
formation. Embryonic stem cells (ES cells) are undifferentiated
stem cells that are derived from the inner cell mass (ICM) of a
blastocyst embryo. Totipotent and/or nearly totipotent ES cell
lines can be derived from human blastocysts using known methods
comprising removing cells of the inner cell mass of an early
blastocyst by microsurgery or immuno-surgery and culturing the
cells in vitro (e.g., see U.S. Pat. No. 6,235,970, the contents of
which are incorporated herein by reference in their entirety). For
example, such methods are described in co-owned and co-pending PC7
application, PCT/US02/37899 (Methods for Making and Using
Reprogrammed Human Somatic Cell Nuclei and Autologous and Isogenic
Stem Cells) filed Nov. 26, 2002, using blastocysts produced both by
nuclear transfer and by parthenogenesis, the disclosure of which
are incorporated herein by reference in its entirety. Thomson et
al. also describes methods by which ES cell lines can be derived
from primate/human blastocysts (Science, 1988, 282:1145-1147; and
Proc. Natl. Acad. Sci., USA, 1995, 92:7544-7848), which are
incorporated by reference herein in their entirety. A detailed
.method for preparing human ES cells is also described in Thomson's
U.S. Pat. No. 6,200,806, "Primate Embryonic Cells," issued Mar. 13,
2001, the disclosure of which is incorporated herein by reference
in its entirety. As described therein, a human ES cell line can be
derived from cells of a blastocyst by a method comprising: [0103]
a. isolating a human blastocyst; [0104] b. isolating cells from the
inner cell mass of the blastocyst; [0105] c. plating the inner cell
mass cells on embryonic fibroblasts so that inner-cell mass-derived
cell masses are formed; [0106] d. dissociating the mass into
dissociated cells; [0107] e. replating the dissociated cells on
embryonic feeder cells; [0108] f. selecting colonies with compact
morphologies and cells with high nucleus to cytoplasm ratios and
prominent nucleoli; and [0109] g. culturing the selected cells to
generate a pluripotent human embryonic stem cell line.
[0110] Methods for growing human ES cells and maintaining them in
an undifferentiated state without culturing them on a layer of
feeder cells have also been described (Xu et al., Nature
Biotechnology, 2001, 19:971-4, the contents of which are
incorporated herein by reference in their entirety). Feeder-free
culture of stem cells can reduce the risk of contamination of the
cells by pathogens that may reside in the feeder cells.
Generating Differentiated Cells
[0111] Stem cells are widely regarded as an abundant source of
pluripotent cellular material that can be directed to differentiate
into cells and tissues that are suitable for transplantation into
patients in need of such cell and tissue transplants. ES cells
appear to have unlimited proliferative potential and are capable of
differentiating into all of the specialized cell types of a mammal,
including the three embryonic germ layers (endoderm, mesoderm, and
ectoderm), and all somatic cell lineages and the germ line. Using
known methods, totipotent or nearly totipotent ES cells can be
cultured under conditions in which they differentiate into
pluripotent or multipotent stem cells such as hematopoietic or
neuronal stem cells. Alternatively, totipotent ES cells can be
cultured under conditions in which they differentiate into a
terminally differentiated cell type such as a cardiac muscle cell.
Totipotent and/or pluripotent stem cells homozygous for MHC alleles
produced by the methods of the present invention can be cultured
using methods and conditions known in the art to generate cell
lineages that differentiate into many, if not all, of the cell
types of the body, for transplant into human patients in need of
such transplants. Such stem cells having one or more homozygous MHC
alleles can differentiate into cells selected from the group
consisting of immune cells, neurons, skeletal myoblasts, smooth
muscle cells, cardiac muscle cells, skin cells, pancreatic islet
cells, hematopoietic cells, kidney cells, and hepatocytes. For
example, methods have been described by which totipotent or nearly
totipotent ES cells are induced to differentiate in vitro into
cardiomyocytes (Paquin et al., Proc. Nat. Acad. Sci. (2002)
99:95509555), hematopoietic cells (Weiss et al., Hematol. Oncol.
Clin. N. Amer. (1997) 11(6): 1185-98; also U.S. Pat. No.
6,280,718), insulin-secreting beta cells (Assady et al., Diabetes
(2001) 50(8):1691-1697), and neural progenitors capable of
differentiating into astrocytes, oligodendrocytes, and mature
neurons (Reubinoff et al., Nature Biotechnology (2001)
19:1134-1140; also U.S. Pat. No. 5,851,832).
[0112] Novel screening methods that make use of gene trapped cell
lines and provide means for efficiently identifying combinations of
biological, biochemical, and physical agents or conditions that
induce stem cells to differentiate into cell types useful for
transplant therapy, and for preparing and isolating specific
differentiated cell types, are described in co-owned and co-pending
U.S. application Ser. No. 10/227,282, filed Aug. 26, 2002, and in
U.S. Provisional Application No. 60/418,333 ("Methods Using Gene
Trapped Stem Cells for Marking Pathways of Stem Cell
Differentiation And Making and Isolating Differentiated Cells"),
filed Oct. 16, 2002, the contents of both of which are also
incorporated herein by reference in their entirety.
[0113] In a useful embodiment of the present invention, a stem cell
bank is produced that comprises hematopoietic stem cells homozygous
for MHC antigens. A method for inducing the differentiation of
pluripotent human embryonic stem cells into hematopoietic cells
useful for transplant according to the present invention is
described in U.S. Pat. No. 6,280,718, "Hematopoietic
Differentiation of Human Pluripotent Embryonic Stem Cells," issued
to Kaufman et al. on Aug. 28, 2001, the disclosure of which is
incorporated herein by reference in its entirety. The method
disclosed in the patent of Kaufman et al. comprises exposing a
culture of pluripotent human embryonic stem cells to mammalian
hematopoietic stromal cells to induce differentiation of at least
some of the stem cells to form hematopoietic cells that form
hematopoietic cell colony forming units when placed in
methylcellulose culture.
[0114] Those skilled in the art will appreciate that, using
currently available methodologies, the totipotent and pluripotent
stem cells of the present invention can also be used to generate
tissues formed of two or more different cell types homozygous for a
MHC allele, for transplant to a person in need of such a tissue
transplant.
[0115] The pluripotent and totipotent stem cells homozygous for MHC
antigens that are generated according to the present invention, and
the lines of differentiated cells obtained from these stem cells,
are produced and isolated under Good Manufacturing Practices (GMP)
conditions.
Providing Histocompatible Transplants to People Needing them
[0116] The methods for generating stem cells and differentiated
cells having homozygous MHC alleles described above provide
effective solutions to many of the problems associated with
obtaining cells for transplant that are histocompatible with a
transplant recipient. However, de novo production of
histocompatible cells and tissue for transplantation by in vitro
fertilization, parthenogenesis, or nuclear-transfer-based methods
for each patient in need of transplant is time-consuming. The time
required to prepare "customized" cells or tissue for
transplantation having the same HLA antigens as the transplant
recipient can be problematic when the health of the would-be
recipient is rapidly deteriorating for want of a transplant.
Therefore, one or more of the above-described methods for
generating stem cells and differentiated cells having homozygous
MHC alleles are used to produce a stem cell bank comprising many
different lines of stem cells, each having a different combination
of homozygous MHC alleles present in the population. When a patient
is found to be in need of a particular type of cell transplant, a
line of stem cells from the stem cell bank having homozygous MHC
alleles matching those of the patient can be taken "off the shelf"
and cultured under conditions causing them to differentiate into
the type(s) of cells needed. The differentiated cells are then
isolated using known methods, and are provided to the patients
physician for transplant.
[0117] Accordingly, the present invention includes the process of
identifying the type of cells needed for transplant, and the blood
type and HLA antigens of the transplant recipient, selecting stem
cells from the stem cell bank that differentiate into the cell type
needed and have homozygous HLA antigens that match those of the
transplant recipient; culturing the stem cells under conditions in
which they differentiate into the cell type needed; isolating the
differentiated cells needed for transplant; and providing these to
the patient's physician for transplant into the patient.
[0118] The differentiated cells for transplant produced by these
methods are homozygous for at least one HLA antigen present on
cells of the transplant recipient. Histocompatibility of the cells
for transplant and the recipient increases as a function of the
number of homozygous HLA antigens of the cells for transplant that
match HLA antigens of the recipient. The greater the number of
homozygous HLA antigens of the cells for transplant that match HLA
antigens of the recipient, the longer the graft is expected to
survive without being rejected. The cells for transplant provided
by the invention will therefore have one, two, three, four, five,
six, or more homozygous HLA antigens that match HLA antigens of the
recipient. For example, cells for transplant produced by the
present invention can have homozygous HLA-A, HLA-B, and HLA-DR
antigens that match HLA antigens of the recipient. Alternatively,
the cells for transplant produced by the present invention can have
homozygous HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP antigens
that match HLA antigens of the recipient. The ability to select
stem cells "off the shelf to produce cells for transplant having a
relatively high number of homozygous HLA antigens that match those
of a prospective transplant recipient depends on the size and
complexity of the stem cells bank. A stem cell bank containing from
100,000 to 200,000 different stem cell lines, each having a
different combination of homozygous HLA-A, HLA-B, and HLA-DR
antigens, is required in order to be able to provide cells with
homozygous HLA-A. HLA-B, and HLA-DR antigens that match the
corresponding HLA antigens of a large percentage of people in a
diverse population such as that of North America. Use of cells from
individuals with blood type 0 can avoid rejection based on ABO
blood type; but there would have to be two versions of each cell
type in the stem cell bank in order to provide matches to the Rh(+)
and Rh(-) blood types. Accordingly, a stem cell bank containing
several hundred thousand stem cell lines can be expected to provide
"off the shelf stem cells that can be used to generate
differentiated cells needed for transplant that have homozygous
HLA-A, HLA-B, and HLA-DR antigens matching those of a person in
need of such a transplant.
[0119] The stem cell bank of the present invention contains lines
of totipotent, nearly totipotent, and/or pluripotent human stem
cells, each having a specific combination of one or more homozygous
HLA antigens. The lines of stem cells that can be used to generate
cells or tissue suitable for transplant can be lines of totipotent
or nearly totipotent human ES cells. The stem cell lines can also
be pluripotent, partially differentiated stem cells such as
myoblasts, hematopoietic stem cells, neuronal precursor cells, and
endothelial cell precursor cells.
Therapeutic Cell Transplantation
[0120] Using the methods of the present invention, a line of
totipotent or pluripotent stem cells can be selected from a bank of
such stem cells that are homozygous for one or more
histocompatibility antigen alleles, in the case of human stem
cells. MHC alleles that match an MHC allele of a patient in need of
transplant. For example, the stem cells can have homozygous HLA-A,
HLA-B, and HLA-DR alleles that match HLA-A, HLA-B, and HLA-DR
alleles of the patient. The stem cells are cultured ex vivo under
conditions in which they are induced to differentiate into
partially or fully differentiated cell types that are suitable for
transplant and have homozygous MHC alleles that match MHC alleles
of the patient in need of the transplant.
[0121] The partially or fully differentiated cells needed for
transplant are isolated from other cell types, e.g., using
antibody-based separation methods such as cell sorting or
immunomagnetic beads, and antibodies that are specific for one or
more differentiation antigens on the surface of the cell type
needed for transplant, as described in U.S. Provisional Application
No. 60/418,333, filed Oct. 16, 2002, the disclosure of which is
incorporated herein by reference in its entirety. The isolated
partially or fully differentiated cells are then administered by
transplantation to the patient using known methods. Methods for
transplantation of epidermal cells, hematopoietic stem cells. Islet
of Langerhans cells, chondrocytes, hepatocytes, myoblasts, neural
cells, and endothelial cells are reviewed by Inverardi et al.
(Transplantation Biology, Cellular and Molecular Aspects, Chapter
56, 1996, ed. by Tilney et al., Lippincott-Raven, Philadelphia, Pa.
The method to be used to transplant or engraft cells to a patient
is recognized as depending on the type of cells to be transplanted,
and on the pathology of the patient.
Cells Homozygous for Recessive Disease-Causing Genes
[0122] Recessive alleles responsible for genetically inherited
diseases are endemic in the population. If cells of people carrying
a recessive disease-causing gene are used to produce stem cells
having homozygous HLA alleles from embryos generated by
parthenogenesis, or with sperm and eggs of closely related
individuals, there is a relatively high likelihood that some of the
stem cell lines obtained also be homozygous for the recessive
disease-causing gene. The stem cell lines produced by the methods
of the present invention can therefore be screened to identify
those which are homozygous for a recessive disease-causing gene.
Such screening can be carried out using known methods. For example,
DNA sequences of the cells can be amplified by the polymerase chain
reaction (PCR) and analyzed by DNA sequencing, restriction enzyme
cleavage, or by hybridization to an array of oligomers, e.g., on a
microchip. Examples of recessive-disease causing genes to be
screened for include, but are not limited to, of recessive genes
causing the following conditions:
[0123] Adenosine deaminase deficiency
[0124] Albinism
[0125] Adenylosuccinate lyase deficiency
[0126] Alpha-1 antitrypsin deficiency
[0127] Cystic Fibrosis
[0128] Friedreich's ataxia
[0129] Gaucher's disease
[0130] Hypercholesterolemia
[0131] Alzheimer's Disease
[0132] Autoimmune polyendocrinopathy candidiasis-ectodermal
dystrophy
[0133] AID--deficiency of activation-induced cytidine deaminase
[0134] Ataxia-telangiectasia
[0135] CD3-epsilon deficiency (causes SCID)
[0136] CD3-gamma deficiency (causes SCID)
[0137] chronic granulomatous disease--deficiency of
p47.sup.phox
[0138] Phenylketonuria--Phenylalanine hydroxylase (PAH)
deficiency
[0139] Tetrahydrobiopterin deficiencies:
[0140] GTP cyclohydrolase I (GTPCH) deficiency
[0141] 6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency
[0142] Dihydropteridine reductase (DHPR) deficiency
[0143] Pterin-4a-carbinolamine dehydratase (PCD) deficiency
[0144] Janus Kinase 3 (JAK3) deficiency (causes SCID)
[0145] Hereditary fructose intolerance
[0146] Porphyria (one of the six forms is caused by a recessive
gene)
[0147] Sickle Cell Anemia
[0148] Tay Sachs syndrome
[0149] Thalassemia
[0150] Wilson's disease
[0151] Xeroderma pigmentosum
[0152] Zeta-chain-associated protein kinase deficiency (causes
SCID)
[0153] The totipotent and/or pluripotent stem cell lines having a
homozygous recessive disease-causing gene that are produced by the
methods of the present invention are highly useful. They can be
cultured under conditions in which they differentiate into cell
types related to manifestation of the disease phenotype. Such cells
having a homozygous recessive disease-causing gene are useful for
basic research directed to studying the disease phenotype ex vivo.
They can also be implanted into experimental animals (e.g.,
immunodeficient animals), for study of their metabolic activities
in vivo. Persons skilled in the art would recognize that studies in
which such cells are genetically modified can be useful for gaining
understanding of the disease phenotype. Such cells having a
homozygous recessive disease-causing gene can also be used in drug
discovery; e.g., in screening for drugs or other therapies that
will treat or cure the disease caused by the recessive gene.
[0154] In order to further illustrate the invention and its
preferred embodiments, the following examples are provided. These
examples are intended to be exemplary and in no way limitative of
the scope of the present invention.
Example 1
Production of Parthogenic Primate Primordial Stem Cells
(PPSC's)
Materials and Methods
[0155] 1. Cynomolgus Monkey (Macaca fascicularis) were
superovulated using a single injection of 1000 IU of pregnant
mare's serum gonadrophin (PMSG) and 500 IU of human chorionic
gonadoprophin (hCG) four days later.
[0156] 2. Ovaries were retrieved by laparotomy and oocytes
dissected from the follicles and placed in maturation media 36 to
48 hrs after (hCG). Maturation media consisted of medium-199 (Gibco
BRL) with Earle's balanced salt solution supplemented with 20%
fetal bovine serum, 10|U/ml of PMSG, 10 Mimi of hCG, 0.05 mg/ml of
penicillin G and 0.075 mg/ml of steptomycin sulfate (Hong,
1999).
[0157] 3. Oocyte Activation
[0158] After 40 hrs in maturation, metaphase II eggs were placed in
10 micromoles of lonomycin followed incubation in 200 mM 6-DMAP
(dimethylaminopurine) for 3 to 4 hrs.
[0159] 4. Embryo Culture. Commercially available embryo culture
media `Cooks` was used (modified SOF). Embryos were cultured with a
co-culture of mitotically inactivated mouse embryonic fibroblasts
as feeder layer.
[0160] 5. Isolation of Inner Cell Mass [0161] a) Upon development
to blastocyst, embryos were placed in a buffered solution of 0.3%
pronase for 2 minutes to digest zone pellucida [0162] b) Blastocyts
were then rinsed in buffered solution and moved to solution of 01
culture media and polyclonal antibodies (antihuman whole serum) 1:3
dilution for 30 minutes. [0163] c) Embryos were rinsed 5 times in a
buffered solution. [0164] d) Embryos were moved into a solution of
G1 culture media and guinea pig complement 1:3 dilution for 30
minutes. [0165] e) Remaining of the embryos (dead trophoblast cells
and ICM) wee
[0166] rinsed 5 times in buffered solution the Inner Cell Mass
(ICM) was isolated and placed on top of a mouse embryonic
fibroblast feeder layer for isolation and growth of Primordial Stem
Cells (PSC's).
Results
[0167] We have obtained 450 eggs total, after maturation, 224 were
still at germinal vesicle stage (GV=no maturation), 79 were dead,
56 were at metaphase one (MI) and 91 at metaphase two (Mil).
[0168] We have parthenogenically activated all of them. As
expected, there was no cleavage on the GY group, 32% cleavage on
the MI and 57% on the MIL When put in culture, 7 embryos developed
to the blastocyst stage (See FIG. 1).
[0169] After attempting to establish ES-like culture cells, four
Inner cell masses attached nicely one differentiated immediately,
and out of the three remaining, one cell line was obtained. This
cell line is called Cyno 1 (FIG. 2). This cell line before and
after immunosurgery is shown in FIG. 3 and FIG. 4. FIG. 5 shows the
Cyno 1 Cell line five days after plating.
[0170] FIG. 6 shows the Cyno 1 cell line growing on top of a mouse
fibroblast feeder layer. These cells show typical morphology of
pluripotent-embryonic-cells such as small nuclear cytoplasmic
ration and the presence of cytoplasmic granules.
[0171] These cells were maintained in an undifferentiated state for
a period of months. This is evidenced by screening of such cells
after prolonged culturing for the expression of a cell marker
characteristic of undifferentiated cells, Alkaline Phosphatase. As
expected, cells were positive on passage 3 and on passage 5.
[0172] The fact that these cells maintain their pluripotency is
also shown by their spontaneous differentiation into many
differentiated cell types after being placed in tissue culture in
the absence of a feeder layer. In the days following, the cells
were observed to differentiate into cuboidal epithelium,
fibroblasts, beating myocardial cells and other cells. Two colonies
of beating myocardial cells were observed in one well of a 4-well
tissue culture plate.
[0173] To determine whether differentiated cells of various somatic
cell lineages were observed from the differentiating PPSC's, we
extracted mRNA from differentiated cell cultures, performed RT-PCR,
using human sequence primers specific for various differentiated
cell types. As shown in FIG. 6, transcripts of a predicted size for
the mesodermally-derived transcripts brachyury and skeletal muscle
myosin heavy polypeptide 2 were observed. The transcript sonic
hedgehog essential for endoderm development was observed. In
addition, the neuron-specific ectoderm marker enolase was observed
as well as keratin (not shown) as markers of ectodermally derived
cells. These PCR products were not observed in the mouse feeder
layer controls or in the absence of reverse transcriptase.
[0174] To establish that the imprinting status of parthogenetic
PPSC's different than that of di-parental PPSCs we looked at the
expression of several imprinted genes. Genes that are
mono-allelically expressed from the paternal allele, would not be
expected to be expressed in parthogenetic cells, as these cells are
derived exclusively from the maternal genome. The Snrpn gene is
mono-allelically expressed from the paternal allele in mouse
blastocyst inner cell mass [Szabo, P E and Mann, JR; Genes &
Development 9:3097-3108 (1995)]. We looked at the expression of
this gene in the parthogenetic Macaca facicularis PPSCs and found
that the express was undetectable by RT-PCR, whereas under
identical conditions, this gene is readily detected in fibroblast
cell cultures from the same species. The Snrpn gene is expected to
be expressed in diparental PPSCs, as these cells contain a paternal
allele. In FIG. 7, the expected size RT-PCR product for the Snrpn
gene is 260 bp.
Example 2
Stable Engraftment of Homozygous Fibroblasts in Histocompatible or
Non-Histocompatible Cynomolgus Recipients
[0175] Connective tissue fibroblasts are generated from the cyno-1
stem cell line described above which are labeled with green
fluorescent protein (GFP) gene. This cell line is homozygous as
evidenced by the portion of a single allete of 225 basepairs using
a primer set specific for DQBlu6011-17. These cells are propagated
in vitro until several million cells are obtained.
[0176] Thereafter, approximately a million labeled connective
fibroblasts are transplanted into histocompatible cynomolgus monkey
recipients, and non-histocompatible cynomolgus controls. Each
monkey is transplanted with a million labeled cells administered by
injection in the upper arm at for different sites, in four equal
parts.
[0177] The degree of engraftment of these engrafted labeled cells
is assessed at three different times, at four weeks, six months and
a year. Three of the four grafts are removed at three different
times and the number of GFP labeled cells is determined in the
histocompatible transplant recipients and controls. The number of
GFP cells is compared for both groups.
[0178] Also, a histological examination is effected to look for any
signs of lymphocyte infiltration and any signs of rejection.
Example 3
Production of Homozygous Stem Cell Lines from Rabbit
Parthenogenically Activated Oocytes
[0179] Rabbit ES cells were similarly obtained from parthenogenetic
embryos. Specifically, rabbit oocytes were obtained from
superovulating rabbits and were actuated using ionomycin and DMAP.
This resulted in blastocystes, the inner cell masses of which were
transferred to fibroblast factor layer. This in turn resulted in
the production of rabbit ES cell lines which stained positive for
characteristic embryonic antigens and which gave rise to various
differentiated cell types when removed from the front layer.
[0180] More specifically, true rabbit ES cell lines morphlogically
looked like ES cells and differentiated [into] into all three germ
cell lineages. Among the cell types that observed from this cell
line were myocordial, vascular endothalial, neuronal, and
hemotopoiath cell lineages.
Example 4
Protection of Homozygous Stem Cell Lines from Human
Parthenogenically Activated Oocytes
Production of Autologous Cells by Parthenogenetic Activation of
Oocytes
[0181] Oocytes from three volunteers were used for parthenogenetic
activation. The donors were induced to superovulate by 11 days of
low dose (75 IU bid) gonadotropin injections prior to hCG
injection. A total of 22 oocytes were obtained from the donors 34
hours after HCG stimulation, and were activated at 40-43 h after
hCG stimulation.
[0182] The oocytes ere activated on day 0, using the ionomycin/DMAP
activation protocol described above. Twelve hours after activation,
20 oocytes (90%) developed one pronucleus and cleaved to the
two-cell to four-cell stage on day 2. On day 5 of culture, evident
blastocoele cavities were observed in six of the parthenotes (30%
of the cleaved oocytes) though none of the embryos displayed a
clearly discernible inner cell mass. The results of parthenogenetic
activation of the human oocytes are summarized in Table 4.
TABLE-US-00004 TABLE 4 Parthenogenetic Activation of Human Oocytes
Embryos with No. of Pronucleus Cleaved blastocoele Cavity Donor
Oocytes (%).sup.a (%).sup.a (%).sup.b 1 5 4 (80) 4 (80) 2 14 13
(93) 13 (93) 4 (31) 6 5 3 3 (100) 2 (67) Total 22 20 (90) 20 (90) 6
(30) .sup.aAs a percentage of activated oocytes. .sup.bAs
percentage of cleaved oocytes.
[0183] FIG. 8 shows MI I oocytes at the time of retrieval. FIG. 9
shows four-to six-cell embryos 48 h after activation.
Distinguishable single-nucleated blastomeres (labeled "n" in FIG.
6) were consistently observed. FIG. 10 shows embryos with
blastocoele cavities (arrows) that were detected on day 6 and
maintained in culture until day 7. The scale bars for FIG. 6, FIG.
7 and FIG. 8=100.
[0184] In a study similar to the one described above, human oocytes
were activated using the ionomycin/DIVIAP activation protocol and
were cultured in vitro. One of the activated embryos developed a
pronucleus, cleaved, formed a blastocoele cavity, and then
developed into a blastocyst having an inner cell mass, shown in
FIG. 11. The inner cell mass was isolated and plated on mouse
feeder layers as described (Cibelli, J. B., et al. 2002.
Parthenogenetic stem calls in non-human primates. Science 295:
819). The cultured ICIM cells increased in number over the first
week, and cells indistinguishable from human embryonic stem cells
were observed. These grew in close association as a colony with a
distinct boundary, as shown in FIG. 12; they had a high
nuclear-to-cytoplasmic ratio, prominent nucleoli, and were observed
to differentiate in vitro into multiple differentiated cell
types.
Example 5
Production of Homozygous Stem Cell Lines from Mouse
Parthenogenically Activated Oocytes
[0185] Using substantially the same methods described in the
present application, another research group, Lin et al., Stem Cells
21:152-161 (2003) incorporated by reference in its entirety,
generated stem cell lines from unfertilized mouse metaphase II
oocytes. These oocytes were activated by 5 minute exposure to 5 mm
calcium ionophore (ionomycin) followed by a 3 hour exposure to
6-methyldiaminopurine (DMAP). Those stem cell lines were
characterized as stem cell lines based on their expression of
characteristic embryonic antigens (SSEAs, OCT-4, alkaline
phosphatase and telomerase) and their pluripotency (give rise to
ectodermal, endodermal and mesodermal cell types).
[0186] Specifically, activated, unfertilized oocytes from F1 hybrid
mice (H-2-B/DO were used to establish those stem cell lines
homozygous for H-2-B and H-2-D respectively. The stem cell lines
appeared karyotypically normal. When cultured in vitro in the
pressures of specific growth factors, these cell lines gave rise to
ectodermal, mesodermal, and endodermal cell types. Histological
examination of cultures revealed cells having the morphology of
neuronal cells and hemotopviette lineages (lymphocytes, monocytes
and erythrocytes).
[0187] Further, when these cell lines were implanted in the kidney
of syngenetic F1 mice they similarly resulted in teratomas that
comprised cells of all three germ layers. The teratomas when
histologically examined showed evidence of hair follicles, thyroid
glands, lung epithelium and connective tissue.
CONCLUSIONS
[0188] The results in the foregoing examples provides proof of
principle, namely that homozygous stem cell lines may be generated
from embryos, e.g., parthenogenically activated embryos, and used
to produce differentiated cell types for cell therapy. More
specifically, the present instruction provides methods for making
libraries or banks of stem cell lines that are homozygous for
specific MHC alleles. Thereby, a bank of cells is available which
can be used to produce differentiated cells which are
histocompatible for a wide range of transplant recipients. This is
feasible with a relatively few number of stem cell lines given that
certain HLA haplotyes are expressed with relatively high frequency
in the human population.
[0189] These differentiated cells should be well tolerated and be
stably engrafted given their antigenic expression relative to the
transplant recipient. Also, in the case of stem cell lines derived
from parthenogenically activated oocytes, these cells eliminate
certain ethical issues with therapeutic cloning, namely a viable
embryo (capable of giving rise to an offspring) is never obtained
or destroyed. These cells are useful for treating any condition
wherein cell or tissue transplantation is therapeutically
desirable, e.g., immune deficiencies, age-related deficiencies,
cancer, autoimmune disorder, organ deficiencies, disease, or
injury, burn, malignancy, cell proliferation disorders,
hemotopoietic disorders, e.g., blood malignancy such as
non-Hodgkins lymphoma, leukemia, inflammatory disorders, connective
tissue disorder, dermatological disorder, ischemia, stroke,
neurological disorders and the like. The present cell banks on
particularly well suited for treating acute disease, particularly
when there is not sufficient time to do therapeutic cloning. For
example, those cells are useful in obtaining differentiated cells
for treatment of conditions where the patient is near death, e.g.,
sepsis, stroke and other conditions where cell therapy is urgently
needed. Also, the invention provides means for having cells on hand
that express desired therapeutic polypeptides which are
histocompatible.
* * * * *