U.S. patent application number 11/025893 was filed with the patent office on 2005-12-01 for method of differentiation of morula or inner cell mass cells and method of making lineage-defective embryonic stem cells.
This patent application is currently assigned to Advanced Cell Technology, Inc.. Invention is credited to Cibelli, Jose, Lanza, Robert, West, Michael D..
Application Number | 20050265976 11/025893 |
Document ID | / |
Family ID | 22573023 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050265976 |
Kind Code |
A1 |
Cibelli, Jose ; et
al. |
December 1, 2005 |
Method of differentiation of morula or inner cell mass cells and
method of making lineage-defective embryonic stem cells
Abstract
An improved method of producing differentiated progenitor cells
comprising obtaining inner cell mass cells from a blastocyst and
inducing differentiation of the inner cell mass cells to produce
differentiated progenitor cells. The differentiated progenitor
cells may be transfected such that there is an addition, deletion
or alteration of a desired gene. The differentiated progenitor
cells are useful in cell therapy and as a I source of cells for the
production of tissues and organs for transplantation. Also provided
is a method of producing a lineage-defective human embryonic stem
cell.
Inventors: |
Cibelli, Jose; (Holden,
MA) ; West, Michael D.; (Worcester, MA) ;
Lanza, Robert; (Worcester, MA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Advanced Cell Technology,
Inc.
Worcester
MA
|
Family ID: |
22573023 |
Appl. No.: |
11/025893 |
Filed: |
December 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11025893 |
Dec 29, 2004 |
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10625653 |
Jul 24, 2003 |
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10625653 |
Jul 24, 2003 |
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09689743 |
Oct 13, 2000 |
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60159550 |
Oct 15, 1999 |
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Current U.S.
Class: |
424/93.7 ;
435/368; 435/372 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
9/02 20180101; A61P 19/04 20180101; C12N 5/0606 20130101; A61P
25/14 20180101; A61P 35/00 20180101; A61P 3/10 20180101; A61P 1/16
20180101; A61P 37/06 20180101; A61P 31/18 20180101; A61P 25/28
20180101; C12N 5/16 20130101; A61P 17/02 20180101; A61P 11/00
20180101; A61P 25/00 20180101; C12N 2510/00 20130101; A61K 35/12
20130101; A61P 25/16 20180101; A61P 21/04 20180101; A61P 13/02
20180101 |
Class at
Publication: |
424/093.7 ;
435/368; 435/372 |
International
Class: |
A61K 048/00; C12N
005/08 |
Claims
1-27. (canceled)
28. A method of therapy comprising administering to a patient in
need of cell transplantation therapy, an isogenic differentiated
cell, wherein said cell transplantation therapy is selected from
the group consisting of Parkinson's disease, Huntington's disease,
Alzheimer's disease, ALS, spinal cord defects or injuries, multiple
sclerosis, muscular dystrophy, cystic fibrosis, liver disease,
diabetes, heart disease, cartilage defects or injuries, burns, foot
ulcers, vascular disease, urinary tract disease, AIDS and
cancer.
29. A method of producing a lineage-defective embryonic stem cell,
comprising: i) genetically modifying a somatic cell such that said
somatic cell is incapable of differentiating into a predetermined
cell lineage; ii) generating a nuclear transfer unit using the
genetically modified somatic cell or cell nucleus as the nuclear
donor; iii) activating the resultant nuclear transfer unit; iv)
culturing said activated nuclear transfer unit until greater than
the 2-cell developmental stage; and v) culturing cells obtained
from said cultured nuclear transfer unit under conditions suitable
for the formation of a lineage-defective embryonic stem cell, said
stem cell being unable to differentiate into at least one of the
embryonic germ layers.
30. The method according to claim 29, wherein generating said
nuclear transfer unit comprises inserting the genetically modified
human somatic cell or cell nucleus into an enucleated mammalian
oocyte under conditions suitable for formation of a nuclear
transfer unit.
31. The method according to claim 29, wherein said
lineage-defective human embryonic stem cell is incapable of
differentiating into mesoderm.
32. The method according to claim 29, wherein said
lineage-defective human embryonic stem cell is incapable of
differentiating into endoderm.
33. The method according to claim 29, wherein said
lineage-defective human embryonic stem cell is incapable of
differentiating into ectoderm.
34. A lineage-defective human embryonic stem cell produced
according to the method of claim 29.
35. The method according to claim 29, wherein said
lineage-defective embryonic stem cell is human.
36. The method of claim 29 wherein the somatic cell comprises a
genetic construct comprising an inducible promoter operably linked
to a gene the expression of which blocks the growth of
undifferentiated cells.
37. The method of claim 29 wherein the somatic cell comprises a
genetic construct that comprises a promoter that is germ-like
specific that regulates the expression of a cell cycle blocker or
an apoptosis gene.
38. The method of claim 29 wherein the somatic cell comprises a
genetic construct that comprises an inducible promoter operably
linked to a gene that induces the differentiation of
undifferentiated cells.
39. The method of claim 36, wherein the somatic cell is human.
40. The method of claim 37, wherein the somatic cell is human.
41. The method of claim 38, wherein the somatic cell is human.
42. An isolated embryonic cell that comprises an inducible promoter
operably linked to a gene the expression of which prevents the
growth of undifferentiated cells.
43. An isolated embryonic cell that comprises a promoter operably
linked to a gene which regulates cell oocytes or apoptosis.
44. The embryonic cell of claim 42 which is human.
45. The embryonic cell of claim 43 which is human.
46. The embryonic cell of claim 42 which is derived from a nuclear
transfer embryo.
47. The embryonic cell of claim 43 which is from a nuclear transfer
embryo.
48. The embryonic cell of claim 42 which is an inner cell mass cell
or a morula cell.
49. The embryonic cell of claim 43 which is an inner cell mass or
morula cell.
50. The embryonic cell of claim 44 which is an inner cell mass or
morula cell.
51. (canceled)
52. The isolated embryonic cell of claim 50 which is human.
53. A method of producing differentiated cells comprising culturing
an embryonic cell according to claim 42 under conditions that
promote differentiation and which prevent the growth of embryonic
stem cells.
54. The method of claim 50 which is used to produce human
differentiated cells.
55. Differentiated cells produced according to claim 53.
56. The differentiated cells of claim 55 which are human.
57. The differentiated cells of claim 56 which are transgenic.
58. The method of claim 28, wherein the differentiated cell is a
neural cell.
59. The method of claim 28, wherein the differentiate cell is a
human differentiated progenitor cell.
60. The method of claim 59, wherein the human differentiated
progenitor cell is obtained by: i) obtaining morula-derived cells
or inner cell mass cells from a blastocyst; ii) inducing
differentiation of the morula-derived cells or inner cell mass
cells to produce differentiated progenitor cells; and iii)
isolating said differentiated progenitor cells.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method of differentiation
of the cells of a morula or the inner cells of a blastocyst. These
cells can be used for cell therapy and for the generation of cells
and organs for isogenic, allogeneic and/or xenogeneic
transplantation. The present invention also relates to the
production of lineage-defective embryonic stem cells which will not
differentiate into specific differentiated lineages, such as
mesoderm, endoderm or ectoderm.
[0003] 2. Description of the Related Art
[0004] Methods for deriving embryonic stem (ES) cell lines in vitro
from early preimplantation mouse embryos are well known. (See,
e.g., Evans et al., Nature, 29:154-156 (1981); Martin, Proc. Natl.
Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be passaged in
an undifferentiated state, provided that a feeder layer of
fibroblast cells (Evans et al., Id.) or a differentiation
inhibiting source (Smith et al., Dev. Biol., 121:1-9 (1987)) is
present.
[0005] ES cells have been previously reported to possess numerous
applications. For example, it has been reported that ES cells can
be used as an in vitro model for differentiation, especially for
the study of genes which are involved in the regulation of early
development. Mouse ES cells can give rise to germline chimeras when
introduced into preimplantation mouse embryos, thus demonstrating
their pluripotency (Bradley et al., Nature, 309:255-256
(1984)).
[0006] In view of their ability to transfer their genome to the
next generation, ES cells have potential utility for germline
manipulation of livestock animals by using ES cells with or without
a desired genetic modification. Moreover, in the case of livestock
animals, e.g., ungulates, nuclei from like preimplantation
livestock embryos support the development of enucleated oocytes to
term (Smith et al., Biol. Reprod., 40:1027-1035 (1989); and Keefer
et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to
nuclei from mouse embryos which beyond the eight-cell stage after
transfer reportedly do not support the development of enucleated
oocytes (Cheong et al, Biol. Reprod., 48:958 (1993)). Therefore, ES
cells from livestock animals are highly desirable because they may
provide a potential source of totipotent donor nuclei, genetically
manipulated or otherwise, for nuclear transfer procedures.
[0007] Some research groups have reported the isolation of
purportedly pluripotent embryonic cell lines. For example,
Notarianni et al., J Reprod. Fert. Suppl., 43:255-260 (1991),
reports the establishment of purportedly stable, pluripotent cell
lines from pig and sheep blastocysts which exhibit some
morphological and growth characteristics similar to that of cells
in primary cultures of inner cell masses isolated immunosurgically
from sheep blastocysts. Also, Notarianni et al., J. Reprod. Fert.
Suppl., 41:51-56 (1990) discloses maintenance and differentiation
in culture of putative pluripotential embryonic cell lines from pig
blastocysts. Gerfen et al., Anim. Biotech, 6(1):1-14 (1995),
discloses the isolation of embryonic cell lines from porcine
blastocysts. These cells are stably maintained in mouse embryonic
fibroblast feeder layers without the use of conditioned medium, and
reportedly differentiate into several different cell types during
culture.
[0008] Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141
(1992) reports cultured, bovine embryonic stem cell-like cell lines
which survived three passages, but were lost after the fourth
passage. Handyside et al., Roux's Arch. Dev. Biol., 196:185-190
(1987) discloses culturing of immunosurgically isolated inner cell
masses of sheep embryos under conditions which allow for the
isolation of mouse ES cell lines derived from mouse inner cell
masses. Handyside et al. reports that under such conditions, the
sheep inner cell masses attach, spread, and develop areas of both
ES cell-like and endoderm-like cells, but that after prolonged
culture only endoderm-like cells are evident.
[0009] Recently, Chemy et al., Theriogenology, 41:175 (1994)
reported purportedly pluripotent bovine primordial germ
cell-derived cell lines maintained in long-term culture. These
cells, after approximately seven days in culture, produced ES-like
colonies which stained positive for alkaline phosphatase (AP),
exhibited the ability to form embryoid bodies, and spontaneously
differentiated into at least two different cell types. These cells
also reportedly expressed mRNA for the transcription factors OCT4,
OCT6 and HES1, a pattern of homeobox genes which is believed to be
expressed by ES cells exclusively.
[0010] Also recently, Campbell et al., Nature, 380:64-68 (1996)
reported the production of live lambs following nuclear transfer of
cultured embryonic disc (ED) cells from day nine ovine embryos
cultured under conditions which promote the isolation of ES cell
lines in the mouse. The authors concluded that ED cells from day
nine ovine embryos are totipotent by nuclear transfer and that
totipotency is maintained in culture.
[0011] Van Stekelenburg-Hamers et al., Mol. Reprod. Dev.,
40:444-454 (1995), reported the isolation and characterization of
purportedly permanent cell lines from inner cell mass cells of
bovine blastocysts. The authors isolated and cultured inner cell
masses from 8 or 9 day bovine blastocysts under different
conditions to determine which feeder cells and culture media are
most efficient in supporting the attachment and outgrowth of bovine
inner cell mass cells. They concluded that the attachment and
outgrowth of cultured inner cell mass cells is enhanced by the use
of STO (mouse fibroblast) feeder cells (instead of bovine uterus
epithelial cells) and by the use of charcoal-stripped serum (rather
than normal serum) to supplement the culture medium. Van
Stekelenburg et al reported, however, that their cell lines
resembled epithelial cells more than pluripotent inner cell mass
cells.
[0012] Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et
al, WO 90/03432, published Apr. 5, 1990, and Wheeler et al, WO
94/26889, published Nov. 24, 1994, report the isolation, selection
and propagation of animal stem cells which purportedly may be used
to obtain transgenic animals. Evans et al. also reported the
derivation of purportedly pluripotent embryonic stem cells from
porcine and bovine species which assertedly are useful for the
production of transgenic animals. Further, Wheeler et al, WO
94/26884, published Nov. 24, 1994, disclosed embryonic stem cells
which are assertedly useful for the manufacture of chimeric and
transgenic ungulates.
[0013] Thus, based on the foregoing, it is evident that many groups
have attempted to produce ES cell lines, e.g., because of their
potential application in the production of cloned or transgenic
embryos and in nuclear transplantation.
[0014] The use of ungulate inner cell mass (ICM) cells for nuclear
transplantation has also been reported. For example, Collas et al.,
Mol. Reprod. Dev., 38:264-267 (1994) discloses nuclear
transplantation of bovine ICMs by microinjection of the lysed donor
cells into enucleated mature oocytes. Collas et al. disclosed
culturing of embryos in vitro for seven days to produce fifteen
blastocysts which, upon transferral into bovine recipients,
resulted in four pregnancies and two births. Also, Keefer et al.,
Biol. Reprod., 50:935-939 (1994), disclosed the use of bovine ICM
cells as donor nuclei in nuclear transfer procedures, to produce
blastocysts which, upon transplantation into bovine recipients,
resulted in several live offspring. Further, Sims et al., Proc.
Natl. Acad. Sci., USA, 90:6143-6147 (1993), disclosed the
production of calves by transfer of nuclei from short-term in vitro
cultured bovine ICM cells into enucleated mature oocytes.
[0015] The production of live lambs following nuclear transfer of
cultured embryonic disc cells has also been reported (Campbell et
al., Nature, 380:64-68 (1996)). Still further, the use of bovine
pluripotent embryonic cells in nuclear transfer and the production
of chimeric fetuses has been reported (Stice et al., Biol. Reprod.,
54:100-110 (1996); Collas et al, Mol. Reprod. Dev., 38:264-267
(1994)). Collas et al. demonstrated that granulosa cells (adult
cells) could be used in a bovine cloning procedure to produce
embryos. However, there was no demonstration of development past
early embryonic stages (blastocyst stage). Also, granulosa cells
are not easily cultured and are only obtainable from females.
Collas et al. did not attempt to propagate the granulosa cells in
culture or try to genetically modify those cells.
[0016] Thomson, U.S. Pat. No. 5,843,780, issued Dec. 1, 1998,
reports the purification of primate embryonic stem cells. These
cells are reported to be negative for the cell surface marker
SSEA-1, positive for the cell surface markers SSEA-3, SSEA4,
TRA-1-60, TRA-1-81 and alkaline phosphatase, and to differentiate
into all tissues derived from all three embryonic germ layers
(endoderm, mesoderm and ectoderm). Pluripotent embryonic stem cell
lines derived from human blastocysts are described by Thomson et
al, Science, 282:1145-1147 (1998).
[0017] In addition, Stice et al, U.S. Pat. No. 5,905,042, issued
May 18, 1999, describes cultured inner cell mass cells, and cell
lines, derived from ungulates. These cultured inner cell mass cells
possess similar morphology and express cell markers identically or
substantially similarly to inner cell masses of undifferentiated
developing embryos for prolonged culturing periods.
[0018] A potential application of embryonic stem cells is to use
those cells as a source to produce differentiated cells for cell
therapy and for the generation of tissues and organs for
transplantation. However, stable embryonic stem cell lines and
reliable methods for expansion of those cells into differentiated
cells/tissues/organs are not yet available. Therefore,
notwithstanding what has previously been reported in the
literature, there exists a need for improved sources of cells for
cell therapy and for the generation of tissues and organs for
transplantation.
OBJECTS AND SUMMARY OF THE INVENTION
[0019] It is an object of the invention to provide novel and
improved methods for producing mammalian cells which can be used as
sources of cells for cell therapy and for the generation of tissues
and organs for transplantation.
[0020] It is a more specific object of the invention to provide a
novel method for inducing inner cell mass cells to differentiate
into progenitor cells which can be used for cell therapy or for the
generation of tissues and organs for transplantation.
[0021] It is an object of the invention to provide an improved
method for producing genetically engineered mammalian progenitor
cells which can be used as sources of cells for cell therapy and
for the generation of tissues and organs for transplantation.
[0022] It is a more specific object of the invention to provide a
method for inducing genetically engineered inner cell mass cells to
differentiate into progenitor cells which can be used for cell
therapy or for the generation of tissues and organs for
transplantation, wherein a desired gene is inserted, removed or
modified in the genetically engineered inner cell mass cells.
[0023] It is an object of the invention to provide a method by
which progenitor cells derived from an inner cell mass are
genetically engineered, and the genetically engineered cells are
used as a source of cells for cell therapy and for the generation
of tissues and organs for transplantation.
[0024] It is another object of the invention to provide a novel
method for producing differentiated progenitor cells which involves
using a differentiated cell as a nuclear donor for forming a
nuclear transfer (NT) unit, producing a morula or blastocyst from
the nuclear transfer unit, and inducing cells of the morula or
inner cell mass cells from the blastocyst to differentiate into
progenitor cells which may be used as a source of cells for cell
therapy and for the generation of tissues and organs for
transplantation.
[0025] It is another object of the invention to provide
differentiated progenitor cells produced by using a differentiated
cell as a nuclear donor to form a nuclear transfer unit, producing
a morula or blastocyst from the nuclear transfer unit, and inducing
cells of the morula or inner cell mass cells from the blastocyst to
differentiate into progenitor cells.
[0026] It is another object of the invention to provide human
differentiated progenitor cells produced by using a differentiated
human cell as a nuclear donor to form a nuclear transfer unit,
producing a morula or blastocyst from the nuclear transfer unit,
and inducing cells of the morula or inner cell mass cells from the
blastocyst to differentiate into progenitor cells.
[0027] Another object of the invention is to culture cells of a
morula or inner cell mass cells such that the growth of embryonic
stem cells is prevented while the growth of differentiated
progenitor cells is promoted.
[0028] It is another object of the invention to use such
differentiated progenitor cells for therapy or diagnosis.
[0029] It is a specific object of the invention to use such
differentiated progenitor cells, including human and ungulate
differentiated progenitor cells, for treatment or diagnosis of any
disease wherein cell, tissue or organ transplantation is
therapeutically or diagnostically beneficial. The differentiated
progenitor cells may be used within the same species or across
species.
[0030] It is another object of the invention to use cells derived
from NT embryos, including human and ungulate cells, for treatment
or diagnosis of any disease wherein cell, tissue or organ
transplantation is therapeutically or diagnostically beneficial.
The tissues may be used within the same species or across
species.
[0031] It is another specific object of the invention to use the
differentiated cells produced according to the invention in vitro,
e.g. for study of cell differentiation and for assay purposes, e.g.
for drug studies.
[0032] It is another object of the invention to provide improved
methods of transplantation therapy, comprising the usage of
isogenic or syngenic cells, tissues or organs produced from the
differentiated cells produced according to the invention. Such
therapies include by way of example treatment of diseases and
injuries including Parkinson's, Huntington's, Alzheimer's, ALS,
spinal cord injuries, multiple sclerosis, muscular dystrophy,
diabetes, liver diseases, heart disease, cartilage replacement,
burns, vascular diseases, urinary tract diseases, as well as for
the treatment of immune defects, bone marrow transplantation,
cancer, among other diseases.
[0033] It is another object of the invention to use the transgenic
or genetically engineered differentiated cells produced according
to the invention for gene therapy, in particular for the treatment
and/or prevention of the diseases and injuries identified,
supra.
[0034] It is another object of the invention to use the
differentiated cells produced according to the invention or
transgenic or genetically engineered differentiated cells produced
according to the invention as nuclear donors for nuclear
transplantation.
[0035] Thus, in one aspect, the present invention provides a method
for producing differentiated progenitor cells, comprising:
[0036] (I) obtaining cells of a morula or inner cell mass cells
from a blastocyst; and
[0037] (ii) inducing differentiation of cells of the morula or
inner cell mass cells to produce differentiated progenitor
cells.
[0038] The differentiated progenitor cells can used to derive
cells, tissues and/or organs which are advantageously used in the
area of cell, tissue and/or organ transplantation.
[0039] In another aspect the present invention provides a method of
producing a genetically altered differentiated progenitor cell, by
which a desired gene is inserted, removed or modified in the cell
used to generate a nuclear transfer unit for use to produce morula
for obtaining morula cells or a blastocyst for obtaining inner cell
mass cells.
[0040] In yet another aspect, the present invention provides a
method of producing a lineage-defective human embryonic stem cell,
comprising:
[0041] I) genetically modifying a human somatic cell such that said
somatic cell is incapable of differentiating into a predetermined
cell lineage;
[0042] ii) generating a nuclear transfer unit using the genetically
modified human somatic cell or cell nucleus as the nuclear
donor;
[0043] iii) activating the resultant nuclear transfer unit;
[0044] iv) culturing said activated nuclear transfer unit until
greater than the 2-cell developmental stage; and
[0045] v) culturing cells obtained from said cultured nuclear
transfer unit under conditions suitable for the formation of a
lineage-defective human embryonic stem cell, said stem cell being
unable to differentiate into specific differentiated lineages, such
as at least one of the embryonic germ layers.
[0046] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] The present invention provides improved procedures for
making differentiated progenitor cells for use, for example, in
cell therapy or as a source of cells to provide tissues and organs
for transplantation. More particularly, morula-derived cells or
inner cell mass cells derived from blastocysts are induced to
differentiate to differentiated progenitor cells and those
differentiated cells are used in cell therapy or as a source of
cells to provide tissues and organs for transplantation.
[0048] In the past, long-term culture of inner cell mass cells was
used to produce embryonic stem cells. Subsequently, the embryonic
stem cells were cultured and genetically modified, and induced to
differentiate in order to produce cells to make transgenic animals
or cells for therapy. By the present invention, the production of
embryonic stem cells is bypassed, i.e., morula-derived cells or
inner cell mass cells are induced to differentiate directly into
differentiated progenitor cells which are then used for cell
therapy and for the generation of tissues and organs for
transplantation. If desired, genetic modifications can be
introduced, for example, into somatic cells prior to nuclear
transfer to produce a morula or blastocyst. Thus, the
differentiated progenitor cells of the present invention are not
pluripotent and are, in essence, tissue-specific stem cells. The
differentiated progenitor cells may give rise to cells from all
three embryonic germ layers, i.e., endoderm, mesoderm and ectoderm.
For example, the differentiated progenitor cells may differentiate
into bone, cartilage, smooth muscle, striated muscle and
hematopoietic cells (mesoderm); liver, primitive gut and
respiratory epithelium (endoderm); or neurons, glial cells, hair
follicles and tooth buds (ectoderm).
[0049] Furthermore, it is not necessary for the differentiated
progenitor cells of the present invention to be immortal, or that
the progenitor cells express cell surface markers found on
embryonic stem cells, such as the cell surface markers
characteristic of primate embryonic stem cells: positive for
SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase activity,
and negative for SSEA-1. Moreover, the differentiated progenitor
cells of the present invention are distinct from embryoid bodies,
i.e., embryoid bodies are derived from embryonic stem cells whereas
the differentiated stems cells of the present invention are derived
from morula-derived cells or from inner cell mass cells.
[0050] Preferably, the differentiated progenitor cells of the
present invention are produced by culturing morula-derived cells or
inner cell mass cells in the absence in the culture of
undifferentiated embryonic stem cells. Growth of undifferentiated
embryonic stem cells can be prevented, for example, by culturing
morula-derived cells or inner cell mass cells in the presence of
differentiation-inducing agents or by introducing genetic
modifications into the cells such that the growth of embryonic stem
cells is prevented.
[0051] Any blastocyst may be used as the source of the inner cell
mass, including in vitro-fertilization produced blastocysts and
blastocysts derived from nuclear transfer units. For methods of
producing blastocysts via nuclear transfer units, see U.S. Pat. No.
5,945,577 to Stice et al, the contents of which are hereby
incorporated by reference.
[0052] Blastocysts may be from any mammalian species, including
humans. When blastocysts are derived from nuclear transfer units,
the nuclear transfer unit may be the result "same species"
transfer, e.g., transfer of a nucleus from a human differentiated
cell into a human enucleated oocyte, or "cross species transfer,
e.g., transfer of a nucleus from a human differentiated cell into a
bovine enucleated oocyte. For production of nuclear transfer units
by cross species transfer, see, for example, WO 98/07841, the
contents of which are hereby incorporated by reference.
[0053] Nuclei from either differentiated or embryonic cells may be
used to produce nuclear transfer units. Differentiated mammalian
cells are those cells which are past the early embryonic stage.
More particularly, differentiated cells are those from at least
past the embryonic disc stage (day 10 of bovine embryogenesis).
[0054] Methods for isolating inner cell mass cells from blastocysts
are known to those of skill in the art. See, for example, U.S. Pat.
No. 5,905,042 to Stice et al, and U.S. Pat. No. 5,843,780 to
Thomson, the contents both of which are hereby incorporated by
reference. Inner cell mass cells from early blastocyte development
can be used, or partially differentiated inner cell mass cells from
later in blastocyst development can be used according to the
present invention.
[0055] Isolated inner cell mass cells are induced to differentiate
in the absence or presence of cytokines, growth factors,
extracellular matrix components, and other factors by any
appropriate method. For example, inner cell mass cells can be
induced to differentiate in a flat adhesive environment (liquid) or
in a 3D adhesive environment (e.g., 1% collagen gel). A
microgravity environment can also be used to induce inner cell mass
cell differentiation, Ingram et al, In Vitro Cell Dev Biol Anim,
33(6):459-466 (1997). Another method of inducing inner cell mass
cell differentiation is by generation of teratomas in
immunodeficient mice, Thomson et al, Science, 282(5391):1145-1147
(1998), or other animals. Differentiation may also be induced by
encapsulating the inner cell mass cells and allowing them to form
teratomas in an appropriate host. For example, human inner cell
mass cells may be encapsulated and place in the same patient from
which the inner cell mass cells were derived (isogenic) or a
different human (allogeneic). There are currently a number of
systems available that allow separation of cells from the immune
system of the body by a synthetic, selectively permeable membrane.
The membrane allows free exchange of nutrients, oxygen and
biotherapeutic substance between blood or plasma and the
encapsulated cells. These systems may also modulate the
bidirectional diffusion of antigens, cytokines and other
immunological moieties based on the chemical characteristics of the
membrane and matrix support, Lanza et al, Nat Biotechnol,
14(9):1107-1111 (1996). For systems involving implantation of inner
cell masses in animals or humans, individual as well as multiple
inner cell masses may be implanted in a single animal or human.
[0056] Preferably, a screening test is used to detect agents that
induce the differentiation of morula-derived cells or inner cell
mass cells into desired differentiated cell types. A library of
various combinations of differentiation agents is generated. The
library of differentiation agents includes, for example, growth
factors, cytokines, extracellular matrix components, hormones and
hormone antagonists, and neutralizing antibodies to the foregoing.
The library of differentiation agents is then tested for the
ability to induce differentiation of morula-derived cells or inner
cell mass cells. Any of the methods discussed above for inducing
differentiation of cells can be used. For example, the cells can be
cultured in tissue culture wells, with each well containing a
unique combination of differentiation factors. Nucleic acids or
cDNAs encoding such factors can also be plated out as naked DNA, or
constructs are prepared to carry such nucleic acids by transfection
or by viruses. Differentiated cells are identified by use of
differentiation-specific antibodies; morphology, PCR using
differentiation-specific primers, or any other applicable technique
for identifying specific types of differentiated cells.
[0057] Differentiation agents which can be used according to the
present invention include cytokines such as interferon-alpha A,
interferon-alpha A/D, interferon-.beta., interferon-gamma,
interferon-gamma-inducible protein-10, interleukin-1,
interleukin-2, interleukin-3, interleukin-4, interleukin-5,
interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10, interleukin-1, interleukin-12, interleukin-13,
interleukin-15, interleukin-17, keratinocyte growth factor, leptin,
leukemia inhibitory factor, macrophage colony-stimulating factor,
and macrophage inflammatory protein-1 alpha.
[0058] Differentiation agents according to the invention also
include growth factors such as 6Ckine (recombinant), activin A,
AlphaA-interferon, alpha-interferon, amphiregulin, angiogenin,
B-endothelial cell growth factor, beta cellulin, B-interferon,
brain derived neurotrophic factor, Cl0 (recombinant),
cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced
neutrophil chemoattractant-1, endothelial cell growth supplement,
eotaxin, epidermal growth factor, epithelial neutrophil activating
peptide-78, erythropoiten, estrogen receptor-alpha, estrogen
receptor-B, fibroblast growth factor (acidic/basic, heparin
stabilized, recombinant), FLT-3/FLK-2 ligand (FLT-3 ligand),
gamma-interferon, glial cell line-derived neurotrophic factor,
Gly-His-Lys, granulocyte colony-stimulating factor, granulocyte
macrophage colony-stimulating factor, GRO-alpha/MGSA, GRO-B,
GRO-gamma, HCC-1, heparin-binding epidermal growth factor like
growth factor, hepatocyte growth factor, heregulin-alpha (EGF
domain), insulin growth factor binding protein-1, insulin-like
growth factor binding protein-1/IGF-1 complex, insulin-like growth
factor, insulin-like growth factor II, 2.5S nerve growth factor
(NGF), 7S-NGF, macrophage inflammatory protein-1B, macrophage
inflammatory protein-2, macrophage inflammatory protein-3 alpha,
macrophage inflammatory protein-3B, monocyte chemotactic protein-1,
monocyte chemotactic protein-2, monocyte chemotactic protein-3,
neurotrophin-3, neurotrophin-4, NGF-B (human or rat recombinant),
oncostatin M (human or mouse recombinant), pituitary extract,
placenta growth factor, platelet-derived endothelial cell growth
factor, platelet-derived growth factor, pleiotrophin, rantes, stem
cell factor, stromal cell-derived factor 1B/pre-B cell growth
stimulating factor, thrombopoetin, transforming growth factor
alpha, transforming growth factor-B1, transforming growth
factor-B2, transforming growth factor-B3, transforming
growth-factor-B5, tumor necrosis factor (alpha and B), and vascular
endothelial growth factor.
[0059] Differentiation agents according to the invention also
include hormones and hormone antagonists, such as 17B-estradiol,
adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte
stimulating hormone, chorionic gonadotropin, corticosteroid-binding
globulin, corticosterone, dexamethasone, estriol, follicle
stimulating hormone, gastrin 1, glucagon, gonadotropin,
hydrocortisone, insulin, insulin-like growth factor binding
protein, L-3,3',5'-triiodothyronine, L-3,3',5-triiodothyronine,
leptin, leutinizing hormone, L-thyroxine, melatonin, MZ-4,
oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone,
progesterone, prolactin, secretin, sex hormone binding globulin,
thyroid stimulating hormone, thyrotropin releasing factor,
thyroxine-binding globulin, and vasopressin.
[0060] In addition, differentiation agents according to the
invention include extracellular matrix components such as
fibronectin, proteolytic fragments of fibronectin, laminin,
thrombospondin, aggrecan, and syndezan.
[0061] Differentiation agents according to the invention also
include antibodies to various factors, such as anti-low density
lipoprotein receptor antibody, anti-progesterone receptor, internal
antibody, anti-alpha interferon receptor chain 2 antibody, anti-c-c
chemokine receptor 1 antibody, anti-CD 118 antibody, anti-CD 119
antibody, anti-colony stimulating factor-1 antibody, anti-CSF-1
receptor/c-fins antibody, anti-epidermal growth factor (AB-3)
antibody, anti-epidermal growth factor receptor antibody,
anti-epidermal growth factor receptor, phospho-specific antibody,
anti-epidernal growth factor (AB-1) antibody, anti-erythropoietin
receptor antibody, anti-estrogen receptor antibody, anti-estrogen
receptor, C-terminal antibody, anti-estrogen receptor-B antibody,
anti-fibroblast growth factor receptor antibody, anti-fibroblast
growth factor, basic antibody, anti-gamma-interferon receptor
chainl antibody, anti-gamma-interferon human recombinant antibody,
anti-GFR alpha-1 C-terminal antibody, anti-GFR alpha-2 C-terminal
antibody, anti-granulocyte colony-stimulating factor (AB-1)
antibody, anti-granulocyte colony-stimulating factor receptor
antibody, anti-insulin receptor antibody, anti-insulin-like growth
factor-1 receptor antibody, anti-interleukin-6 human recombinant
antibody, anti-interleukin-1 human recombinant antibody,
anti-interleukin-2 human recombinant antibody, anti-leptin mouse
recombinant antibody, anti-nerve growth factor receptor antibody,
anti-p60, chicken antibody, anti-parathyroid hormone-like protein
antibody, anti-platelet-derived growth factor receptor antibody,
anti-platelet-derived growth factor receptor-B antibody,
anti-platelet-derived growth factor-alpha antibody,
anti-progresterone receptor antibody, anti-retinoic acid
receptor-alpha antibody, anti-thyroid hormone nuclear receptor
antibody, anti-thyroid hormone nuclear receptor-alpha 1/Bi
antibody, anti-transferrin receptor/CD71 antibody,
anti-transforming growth factor-alpha antibody, anti-transforming
growth factor-B3 antibody, anti-tumor necrosis factor-alpha
antibody, and anti-vascular endothelial growth factor antibody.
[0062] Once subjected to the differentiation protocol, primitive
cells from a particular embryonic lineage can be isolated from the
differentiated inner cell mass derivatives by conventional
techniques. If desired, the isolated differentiated progenitor
cells can be expanded, for example, by cell culture or other
appropriate methods. By the present invention, the differentiated
progenitor cells are obtained through differentiated inner cell
mass cells without having to generate embryonic stem cells.
[0063] The differentiated progenitor cells can also be transfected.
Transfection can be performed during any appropriate stage during
the production of the differentiated progenitor cells. For example,
before blastocyst formation, differentiated cells used as nuclear
donors for formation of nuclear transfer units can be transfected.
Another possibility is to transfect the differentiated progenitor
cells after they have been isolated, e.g., transfection of
CD34+,CD38- cells of the hematopoietic system.
[0064] Any known method for inserting, deleting or modifying a
desired gene from a mammalian cell may be used for producing the
transfected differentiated progenitor cells. These procedures may
remove all or part of a gene, and the gene may be heterologous.
Included is the technique of homologous recombination, which allows
the insertion, deletion or modification of a gene or genes at a
specific site or sites in the cell genome.
[0065] A retroviral high-throughput screening procedure can also be
used to identify genes involved in lineage determination in
embryonic stem cells. Those lineage-determinant genes can then be
inserted and/or induced in morula-derived cells or inner cell mass
cells used to produce differentiated progenitor cells. An example
of retroviral-mediated screening is as follows.
[0066] A. Construction of Retroviral Library
[0067] A retroviral cDNA library is constructed from a
differentiated tissue (e.g., neurons, heart muscle cells,
hematopoietic stem cells (CD34+) cells or other differential
cells). These libraries can be built using any Moloney-based vector
system. Commercially available examples are pBabe and pLIB vectors
(Clontech). Already constructed libraries from muscle, liver and
brain can also be obtained from commercial sources (Clontech).
Furthermore, construction of specialized libraries can be simply
achieved.
[0068] B. Development of the Functional Screen in ES Cells
[0069] Primate or mammalian ES cells are transfected with a
reporter gene construct which is composed of the promoter of a
tissue specific gene, for example, a CD34 promoter linked to GFP
protein. This promoter reporter construct is then transfected into
ES cells. Clones are selected that have minimum background activity
and a positive control cell line (KG-1) would be used to ensure
expression of the promoter construct.
[0070] C. Packaging of the Retroviral Library
[0071] The retroviral cDNA library is subsequently efficiently
transfected (>80% frequency) into 293 based EcoPack or AmphoPack
cell lines (Clontech). These cell lines express the Gag-pol and
Envelope proteins required for efficient packaging. The high
(>10.sup.6) titre production of virus in these cell lines makes
them ideal for library representation. In case of problems
associated with these lines, an alternative system can be
constructed by stable transfection of Gag-pol and Env genes into
293 cells.
[0072] D. The Screen
[0073] The ES-Rep (reporter) cells are subsequently infected with
the packaged retroviruses at the multiplicity of infection of 1
(MOI=1). Upon infection, the ES-Rep cells are allowed to recover
and divide. These cells are then subjected to Fluorescent Activated
Cell Sorting (FACs) and the GFP+ cells are selected, re-cultured
and allowed to recover. Genomic DNA is prepared from these cells
and subjected to PCR amplification using primers which amplify the
retroviral cDNA insert. These inserts are then individually
sequenced and tested for their ability to upregulate GFP in ES-Rep
cells. Alternatively the cDNAs can be recovered as a pool and then
be infected into virgin ES-Rep cells to enrich for the event of
interest (GFP+). The individual cDNA clones are then further
characterized for their ability to elicit the desired phenotype,
i.e., their ability to turn ES cells into neuronal, muscle or other
desired lineages. See Deiss, L. P. et al., 1995, Genes Dev. 9:15;
Whitehead, I. et al, 1995, MCB 15:704; Rayner, J. R. et al, 1994,
Ibid. 14:880; Goldfarb, M. et al, 1982, Nature 296:404; Gudkov, A.
V. et al, 1993, PNAS 90:3231; and Deiss, L. P. & Kimchi, A.,
1991, Science, 252:117; the contents all of which are hereby
incorporated by reference.
[0074] Transplanted embryonic stem cells are known to be capable of
not only forming benign teratomas, but malignant tumors as well. To
eliminate the risk of both benign and malignant tumors in the
process of the present invention, it is useful to introduce or
delete genes from cells (e.g., cells used as nuclear donors for
nuclear transfer) that prevent the growth of undifferentiated
embryonic cells in culture. For example, an inducible promoter such
as the MMTV promoter can be introduced into cells, followed by
induction with dexamethasone to drive the expression of a gene that
blocks the growth of undifferentiated cells, or induces their
differentiation. Another possibility is to introduce a promoter for
a gene that is germ line-specific to drive the expression of a cell
cycle blocker or an apoptosis gene. Alternatively, undifferentiated
embryonic stem cells can be identified and eliminated.
[0075] A preferred method of making differentiated progenitor cells
comprises obtaining a human embryo by in vitro fertilization or by
nuclear transfer, and culturing the embryo until the blastocyst
stage in G1.2/G2.2 culture media. Zona pellucida is removed from
the embryo using mild digestion with pronase. Trophoblastic cells
are removed from the embryo by immunosurgery. Inner cell mass cells
are induced to differentiate with or without cytokines by any of:
a) flat adhesive environment; b) 3D adhesive environment; c)
microgravity; d) generation of teratomas in immunodeficient mice;
or e) formation of teratomas from encapsulated inner cell mass
cells in isogenic or allogenic humans. Differentiated progenitor
cells of a particular embryonic lineage are then isolated from the
differentiated inner cell mass derivatives.
[0076] The resultant differentiated progenitor cells of the present
invention, preferably human differentiated progenitor cells, have
numerous therapeutic and diagnostic applications. Most especially,
such differentiated progenitor cells may be used for cell
transplantation therapies. Human differentiated progenitor cells
have application in the treatment of numerous disease
conditions.
[0077] The subject differentiated progenitor cells may be used to
obtain any desired differentiated cell type. Therapeutic usages of
such differentiated human cells are unparalleled. For example,
human hematopoietic stem cells may be used in medical treatments
requiring bone marrow transplantation. Such procedures are used to
treat many diseases, e.g., late stage cancers such as ovarian
cancer and leukemia, as well as diseases that compromise the immune
system, such as AIDS. Hematopoietic stem cells can be obtained,
e.g., by fusing adult somatic cells of a cancer or AIDS patient,
e.g., epithelial cells or lymphocytes with an enucleated oocyte,
obtaining inner cell mass cells as described above, and culturing
such cells under conditions which favor differentiation, until
hematopoietic stem cells are obtained. Such hematopoietic cells may
be used in the treatment of diseases including cancer and AIDS.
[0078] Alternatively, adult somatic cells from a patient with a
neurological disorder may be fused with an enucleated oocyte, human
inner cell mass cells obtained therefrom, and such cells cultured
under differentiation conditions to produce neural cell lines.
Specific diseases treatable by transplantation of such human neural
cells include, by way of example, Parkinson's disease, Alzheimer's
disease, ALS and cerebral palsy, among others. In the specific case
of Parkinson's disease, it has been demonstrated that transplanted
fetal brain neural cells make the proper connections with
surrounding cells and produce dopamine. This can result in
long-term reversal of Parkinson's disease symptoms.
[0079] The great advantage of the subject invention is that it
provides an essentially limitless supply of isogenic or syngenic
human cells suitable for transplantation. Therefore, it will
obviate the significant problem associated with current
transplantation methods, i.e., rejection of the transplanted tissue
which may occur because of host-vs-graft or graft-vs-host
rejection. Conventionally, rejection is prevented or reduced by the
administration of anti-rejection drugs such as cyclosporine.
However, such drugs have significant adverse side-effects, e.g.,
immunosuppression, carcinogenic properties, as well as being very
expensive. The present invention should eliminate, or at least
greatly reduce, the need for anti-rejection drugs.
[0080] Other diseases and conditions treatable by isogenic cell
therapy include, by way of example, spinal cord injuries, multiple
sclerosis, muscular dystrophy, diabetes, liver diseases, i.e.,
hypercholesterolemia, heart diseases, cartilage replacement, burns,
foot ulcers, gastrointestinal diseases, vascular diseases, kidney
disease, urinary tract disease, and aging related diseases and
conditions.
[0081] This methodology can be used to replace defective genes,
e.g., defective immune system genes, cystic fibrosis genes, or to
introduce genes which result in the expression of therapeutically
beneficial proteins such as growth factors, lymphokines, cytokines,
enzymes, etc. For example, the gene encoding brain derived growth
factor may be introduced into human inner cell mass cells, the
cells differentiated into neural cells and the cells transplanted
into a Parkinson's patient to retard the loss of neural cells
during such disease.
[0082] Previously, cell types transfected with BDNF varied from
primary cells to immortalized cell lines, either neural or
non-neural (myoblast and fibroblast) derived cells. For example,
astrocytes have been transfected with BDNF gene using retroviral
vectors, and the cells grafted into a rat model of Parkinson's
disease (Yoshimoto et al., Brain Research, 691:25-36, (1995))
[0083] This ex vivo therapy reduced Parkinson's-like symptoms in
the rats up to 45% 32 days after transfer. Also, the tyrosine
hydroxylase gene has been placed into astrocytes with similar
results (Lundberg et al., Develop. Neurol., 139:39-53 (1996) and
references cited therein).
[0084] However, such ex vivo systems have problems. In particular,
retroviral vectors currently used are down-regulated in vivo and
the transgene is only transiently expressed (review by Mulligan,
Science, 260:926-932 (1993)). Also, such studies used primary
cells, astrocytes, which have finite life span and replicate
slowly. Such properties adversely affect the rate of transfection
and impede selection of stably transfected cells. Moreover, it is
almost impossible to propagate a large population of gene targeted
primary cells to be used in homologous recombination techniques. By
contrast, the difficulties associated with retroviral systems
should be eliminated by the use of differentiated progenitor
cells.
[0085] Genes which may be introduced into the subject
differentiated progenitor cells include, by way of example,
epidermal growth factor, basic fibroblast growth factor, glial
derived neurotrophic growth factor, insulin-like growth factor (I
and U), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic
factor, AFT-1, cytokine genes (interleukins, interferons, colony
stimulating factors, tumor necrosis factors (alpha and beta),
etc.), genes encoding therapeutic enzymes, etc.
[0086] In addition to the use of human differentiated progenitor
cells in cell, tissue and organ transplantation, the present
invention also includes the use of non-human cells in the treatment
of human diseases. Thus, differentiated progenitor cells of any
species may be used in the treatment of human disease conditions
where cell, tissue or organ transplantation is warranted. In
general, differentiated progenitor cells according to the present
invention can be used within the same species (autologous, syngenic
or allografts) or across species (xenografts).
[0087] Also, the subject differentiated progenitor cells,
preferably human cells, may be used as an in vitro model of
differentiation, in particular for the study of genes which are
involved in the regulation of early development.
[0088] Also, differentiated cell tissues and organs using the
subject differentiated progenitor cells may be used in drug
studies.
[0089] The present invention also provides a method of creating
lineage-defective human embryonic stem cells. Such cells are
derived from a human pre-embryo produced by nuclear transfer. If
the hypothetical case of transferring such an embryo into the
uterus of a woman, it would never develop into a human being.
[0090] To produce a lineage-defective embryonic stem cell,
preferably lineage-defective human embryonic stem cell a somatic
cell is genetically engineered to be incapable of differentiation
into a particular cell lineage. Such genetic modifications include
knockout of selected genes, or expression of appropriate antisense
nucleic acids or ribozymes. Examples of knockout genes or genes not
allowed to be expressed are serum response factor (SRF) (Arsenian
et al, EMBO J, 17(2.1):6289-6299, 1998), MESP-1 (Saga, Mech Dev,
75(1-2):53-66, 1998), HNF-4 (Chen et al, Genes Dev,
8(20):2466-2477, 1994), beta1 integrin (Rohwedel et al, Dev Biol,
201(2):167-184, 1998) and MSD (Holdener et al, Development,
120(5):1335-1346, 1994), for mesoderm; GATA-6 (Morrisey et al,
Genes Dev, 12(22):3579-3590, 1998) and GATA-4 (Soudais et al,
Development, 121(11):3877-3888, 1995), for endoderm; and RNA
helicase A (Lee et al, Proc Natl Acad Sci USA, 95(23):13709-13713,
1998) and H beta 58 (Radice et al, Development, 111(3):801-811,
1991), for ectoderm. The invention further embraces the
introduction of one or more genetic modifications that prevent
differentiation of particular cell lineages, e.g. neural cells.
Methods and vectors for effecting gene knockout are subject of
numerous patents, including U.S. Pat. Nos. 5,110,735, 6,074,853,
5,998,144, 5,948,653, 5,945,339, 5,925,544, 5,869,718, 5,830,698,
5,780,296, 5,614,396, 5,612,205, 5,468,629, 5,093,257, all of which
are incorporated by reference in their entirety herein.
[0091] Other genes that my be deleted or inactivated that are
involved in specific cell lineages include by way of example ICSBP,
hedgehog, Cbfal, VASA, HESXI, transcription factors, among many
others. Recently, Sato, et al., Mol. Reprod. Devel. 56(1): 34-44
(2000) reported a genetic approach for identifying genes involved
in specific cell lineages that uses the Cre-LoxP_system, that is
incorporated by references in its entirety herein.
[0092] The genetically engineered cells are then used as nuclear
donors for generation of nuclear transfer units. Human oocytes or
any other mammalian eggs, e.g., bovine, may be used as the
recipient of the nuclear donor. The nuclear transfer units are
allowed to development to blastocysts as described above, and
lineage-defective human embryonic stem cells are derived therefrom.
For methods relating to generation of human embryonic stems cells,
see for example, Thomson et al, Science, 282:1145-1147, 1998 and
U.S. Pat. No. 5,843,780. Upon induction of differentiation, the
lineage-defective human embryonic stem cells will not differentiate
into at least one of the embryonic germ layers (mesoderm, endoderm
or ectoderm). If desired, the lineage-defective human embryonic
stem cells can also be engineered to be "mortal", for example by
expression of an antisense or ribozyme telomerase gene.
[0093] While the foregoing aspect of the invention has been
described with respect to lineage-defective human embryonic stem
cells, the present invention also includes lineage-defective
embryonic stem cells for any mammalian species.
[0094] In order to more clearly describe the subject invention, the
following examples are provided. Such examples are provided by way
of illustration and not by way of limitation.
EXAMPLE 1
[0095] The following example illustrates that only a fraction of
the inner cell mass cells cultured are capable of developing into
embryonic stem cell-like cells. Thus, there are pluripotent inner
cell mass cells which cannot, or do not, develop into embryonic
stem cells.
[0096] Explant Derivation and Culture
[0097] A. Sample Retrieval
[0098] 1) Thoroughly clean the section of tissue to be removed,
such as by use of an iodine or ethanol solution.
[0099] 2) Remove sample with an ear notcher or scissors and
immediately place the tissue in an antibiotic solution (Solution 1
or equivalent). Swirl solution to remove any remaining iodine.
[0100] 3) Transfer to a 50 ml conical tube of fresh Solution 1.
[0101] 4) Store or ship overnight at 4.degree. C.
[0102] B. Tissue Sectioning and Culture
[0103] 1) Remove sample from conical tube and place in fresh warm
Solution 1 in a 100 mm culture dish.
[0104] 2) Trim any hair using sterile forceps and scissors and
transfer to another dish of Solution 1.
[0105] 3) Use forceps and a sterile scalpel to carefully cut very
thin sections of tissue. Thinner sections will yield more
cells.
[0106] 4) Place the sections flat on the bottom center of tissue
culture dishes and cover with sterile glass slides.
[0107] 5) Flood the dishes with .about.10 ml normal culture media
and place in an incubator at 5% CO.sub.2 and 37.degree. C.
[0108] 6) Culture explant samples for 10 days, changing media
once.
[0109] 7) Remove media, rinse with DPBS and add 5 ml trypsin
solution (0.08% trypsin and 0.02% EDTA)
[0110] 8) Place on a warming plate until cells loosen.
[0111] 9) Remove solution from plates and place in a 50 ml conical
tube with an equal amount of warm culture media (10% FBS).
[0112] 10) Spin down cells.
[0113] 11) Remove supernatant and re-suspend cell pellet in normal
media.
[0114] 12) Culture.
[0115] Solution 1
1 DPBS (Biowhittaket, 04-479Y) 200 ml Ciprofloxacin IICL
(Mediatech, 61-277) 2.33 mg Fungizone (Gibco, 15295-017) 1.5 ml
[0116] In Vitro Maturation of Bovine Oocytes
[0117] Ovaries are recovered at a slaughterhouse, placed in warm
PBS (34.degree. C.) and brought to the laboratory within 8 hours.
Each follicle of more than 2 mm in diameter is aseptically
aspirated with an 18 G needle. Search of oocytes is performed in
modified Tyrode's medium (TL Hepes). Oocytes with a homogeneous
cytoplasm, considerable perivitelline space and intact cumulus
cells are placed in maturation medium M199 (GIBCO), 10% FCS, 5
.mu.l/ml bFSH (Nobl), 5 .mu.l/ml bLH (Nobl) and 10 .mu.l/ml
Penstrep (Sigma) for 22 h at 38.5.degree. C. and 5% CO.sub.2.
[0118] Nuclear Transplantation
[0119] Eighteen hours post maturation, oocytes are placed in a 100
.mu.l drop of TL HECM-Hepes under mineral oil (Sigma). Oocyte
enucleation (extraction of chromosomes) is performed using a
beveled glass pipette of 25 .mu.m diameter. Evaluation of
enucleation is done by exposure of individual oocytes previously
cultured for 15 min in 1 .mu.g/ml of bisBENZIMIDE (Hoechst 33342,
Sigma) in TL HECM-Hepes under UV light. Donor cells are placed in
the perivitelline space and fused with the egg's cytoplasm at 23
hours post maturation.
[0120] Embryo Culture
[0121] During the first 3 days after fertilization, embryos are
cultured in 500 .mu.l well plates with mouse embryonic fibroblast
(MF) feeder layers in ACM media with 10% fetal calf serum. On day
4, embryos were transferred to 500 .mu.l well plates with mouse
fibroblast (MF) feeder layers, and fresh ACM media with 10% FCS
until blastocyst stage.
[0122] ES-Like Cell Culture
[0123] Blastocysts are placed in a 32 mm plate (Nunc) with a
mitotically inactivated MF feeder layer and ES medium (DMEM-high
glucose, 15% fetal calf serum, 4 .mu.l/ml antibiotic-antimycotic,
2.8 .mu.l/ml 2-mercaptoethanol, 0.3 mg/ml L-glutamine, 10 .mu.l/ml
of non-essential amino acids and 1 .mu.l/ml tylosin tartrate)
equilibrated a day in advance at 38.5.degree. C. and 5% CO.sub.2.
Using a 22 G needle, the zona pellucida and trophoblast cells of
the blastocyst are mechanically removed. The remaining inner cell
mass (ICM) is placed on top of the MF. After one to two weeks in
culture, ES-like cells are passaged to a fresh mitotically
inactivated MF. Inactivation of MF is performed by exposing them to
gamma radiation (2956 rads). ES-like cells are passaged by cutting
a small piece (50 to 100 cells) of the colony and placing it on top
MF feeder layers using a pulled Pasteur pipette.
[0124] By the above procedure, 41 bovine embryos were reconstructed
from transgenic adult somatic cells. Of those 41 embryos, 15 (or
37%) generated embryonic stem cell-like cells when inner cell mass
cells were cultured under the above conditions. However, 26 of the
embryos could not produce embryonic stem cell-like cells in spite
of culturing inner cell mass cells under ideal conditions for stem
cell generation.
[0125] It is unclear if the timing of embryo development, intrinsic
differences of individual embryos, or the particular culture
conditions (e.g., the presence or absence of various growth
factors) might have been responsible for only a fraction of the
inner cell masses developing into embryonic stem cell-like cells.
Regardless, these results indicate that there are pluripotent inner
cell mass cells which cannot, or do not, develop into embryonic
stem cells. These inner cell mass cells which do not develop into
embryonic stem cells should also be of therapeutic value. It should
be noted that Thompson et al (Science, 282:1145-1147, 1998)
reported the production of human embryonic stem cells in which only
S of 14 (or 36%) inner cell mass cells developed into ES cell
lines. Thus, the existence of inner cell mass cells which do not
develop into embryonic stem cells appears to be a widespread
phenomenon among mammalian species.
EXAMPLE 2
[0126] Three adult Holstein steers approximately 8-10 months old
(weighing approximately 500-1000 lbs) were purchased from Thomas
Morris, Inc., Maryland, and shipped to the South Deerfield Farm at
the University of Massachusetts, Amherst. To obtain fibroblasts for
nuclear transfer, skin biopsies were obtained from each of the
animals by ear notch. A plasmid which expresses a reporter gene
encoding enhanced green fluorescent protein (eGFP), was transfected
into the cells, and transfected cells were selected with neomycin.
Purified cells, analyzed by PCR and/or FISH, were used for nuclear
transfer as described previously in Nature (2998) Biotechnol. 16:
642-646, herein incorporated by reference.
[0127] Isolated inner cell mass cells generated from bovine
blastocysts are then injected into the paralumbar fascia of the
donor steers (three sites with inner mass cells from three 10 day
embryos, three sites with inner mass cells from three 12 day
embryos, and three sites with inner mass cells from three 14 day
embryos, per animal). The inner cell masses are derived from the
same animal into which they are injected and, thus, the inner cell
masses are immune compatible with the steers. After two months, the
muscle is examined for teratoma formation. Any tumors identified
are removed for histological analysis.
[0128] The procedure is performed on the standing animal using 20
mg Xylazine/8 mg Butorphanol Tatrate administered IV in the tail
vein. The paralumbar fascia area is clipped and surgically
prepared, using 100 ml of 2% Lidocaine as a local anesthetic
administered as a paralumbar block. The animals should be given
antibiotics for three days post-surgically as a precautionary
measure (Ceftlofur Hcl 50 mg/cc @ 1 cc/100 pounds). Immediately
following surgery a single injection of Flunixin Meglumine @ 1
cc/100 pounds may be given to control pain and swelling at the
surgical site. If teratoma formation does not occur at the
paralumbar fascia, other sites may be analyzed, i.e.,
subcutaneously.
[0129] It is expected that cells from all three germ layers, i.e.,
ectoderm, mesoderm, and endoderm, will be observed in
teratomas.
[0130] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
* * * * *