U.S. patent application number 10/584255 was filed with the patent office on 2007-10-18 for embryonic stem cell line and method for preparing the same.
This patent application is currently assigned to Seoul National University Industry Foundation. Invention is credited to Woo-Suk Hwang, Youn-Young Hwang, Sung-Keun Kang, Soon-Woong Kim, Sun-Jong Kim, Ja-Min Koo, Dae-Kee Kwon, Hee-Sun Kwon, Byeong-Chun Lee, Eu-Gene Lee, Eul-Soon Park, Jong-Hyuk Park, Sung-Il Roh, Young-June Ryu, Hyun-Soo Yoon.
Application Number | 20070243610 10/584255 |
Document ID | / |
Family ID | 34737817 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070243610 |
Kind Code |
A1 |
Roh; Sung-Il ; et
al. |
October 18, 2007 |
Embryonic Stem Cell Line and Method for Preparing the Same
Abstract
An embryonic stem cell line derived from a nucleus-transferred
oocyte prepared by transferring a nucleus of a human somatic cell
into an enucleated human oocyte may differentiate into various
desired cell types.
Inventors: |
Roh; Sung-Il; (Seoul,
KR) ; Hwang; Woo-Suk; (Seoul, KR) ; Lee;
Byeong-Chun; (Seoul, KR) ; Kang; Sung-Keun;
(Seoul, KR) ; Ryu; Young-June; (Seoul, KR)
; Lee; Eu-Gene; (Seoul, KR) ; Kim; Soon-Woong;
(Seoul, KR) ; Kwon; Dae-Kee; (Seoul, KR) ;
Kwon; Hee-Sun; (Seoul, KR) ; Koo; Ja-Min;
(Seoul, KR) ; Park; Eul-Soon; (Chungcheongbuk-do,
KR) ; Hwang; Youn-Young; (Gyeonggi-do, KR) ;
Yoon; Hyun-Soo; (Seoul, KR) ; Park; Jong-Hyuk;
(Seoul, KR) ; Kim; Sun-Jong; (Gyeonggi-do,
KR) |
Correspondence
Address: |
DICKSTEIN SHAPIRO LLP
1825 EYE STREET NW
Washington
DC
20006-5403
US
|
Assignee: |
Seoul National University Industry
Foundation
San 4-2, Boncheon-dong, Gwanak-gu
Seoul,
KR
151-050
|
Family ID: |
34737817 |
Appl. No.: |
10/584255 |
Filed: |
December 30, 2004 |
PCT Filed: |
December 30, 2004 |
PCT NO: |
PCT/KR04/03528 |
371 Date: |
June 28, 2007 |
Current U.S.
Class: |
435/346 ;
435/404; 435/449 |
Current CPC
Class: |
C12N 15/87 20130101;
C12N 5/0606 20130101; C12N 2500/38 20130101; C12N 5/0619 20130101;
C12N 15/8776 20130101; C12N 2501/13 20130101; C12N 2501/115
20130101; C12N 2500/25 20130101; C12N 2501/235 20130101; C12N
5/0623 20130101 |
Class at
Publication: |
435/346 ;
435/404; 435/449 |
International
Class: |
C12N 5/28 20060101
C12N005/28; C12N 5/00 20060101 C12N005/00; C12N 5/02 20060101
C12N005/02; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 30, 2003 |
KR |
PCT/KR03/02899 |
Claims
1. An embryonic stem cell line derived from a nucleus-transferred
oocyte prepared by transferring a nucleus of a human somatic cell
into an enucleated human oocyte.
2. The embryonic stem cell line of claim 1, which is a cell line
deposited under the accession number of KCLRF-BP-00092.
3. A method for preparing a blastocyst derived from a human somatic
cell and a human oocyte, comprising: (1) culturing a human somatic
cell to prepare a nuclear donor cell; (2) enucleating a human
oocyte to prepare a recipient oocyte; (3) preparing a
nucleus-transferred oocyte by transferring a nucleus of the nuclear
donor cell into the recipient oocyte and fusing the nucleus of the
nuclear donor cell and the recipient oocyte; and (4) subjecting the
nucleus-transferred oocyte to reprogramming, activation and in
vitro culturing to form a blastocyst.
4. The method of claim 45, wherein the embryonic stem cell line is
a cell line deposited under the accession number of
KCLRF-BP-00092.
5. The method of claim 45, wherein the reprogramming in step (4) is
conducted for a time period of up to 20 hours.
6. The method of claim 45, wherein the reprogramming in step (4) is
conducted for a time period of up to 6 hours.
7. The method of claim 45, wherein the reprogramming in step (4) is
conducted for a time period of up to 3 hours.
8. The method of claim 45, wherein the reprogramming in step (4) is
conducted for a time period of about 2 hours.
9. The method of claim 45, wherein the activation in step (4) is
performed by treating the nucleus-transferred oocyte with a calcium
ionophore and subsequently with 6-dimethylaminopurine.
10. The method of claim 9, wherein the concentration of the calcium
ionophore ranges from 5 .mu.M to 15 .mu.M.
11. The method of claim 9, wherein the concentration of the calcium
ionophore is about 10 .mu.M.
12. The method of claim 9, wherein the concentration of
6-dimethylaminopurine ranges from 1.5 mM to 2.5 mM.
13. The method of claim 9, wherein the concentration of
6-dimethylaminopurine is about 2.0 mM.
14. The method of claim 45, wherein the in vitro culturing in step
(4) is performed by sequentially using at least two media, each
having a different composition from the other.
15. The method of claim 14, wherein the in vitro culturing is
performed by sequentially using two media having different
compositions each other.
16. The method of claim 15, wherein the in vitro culturing is
performed by sequentially using the G1.2 medium and the SNUnt-2
medium.
17. The method of claim 45, wherein step (4) is performed by
reprogramming the nucleus-transferred oocyte for a time period of
up to 20 hours, treating the nucleus-transferred oocyte with a
calcium ionophore at a concentration ranging from 5 .mu.M to 15
.mu.M and subsequently with 6-dimethylaminopurine at a
concentration ranging from 1.5 mM to 2.5 mM, and sequentially
culturing the nucleus-transferred oocyte in vitro in the G1.2
medium and the SNUnt-2 medium.
18. The method of claim 45, wherein the inner cell mass is isolated
from the blastocyst in step (5) by a process comprising the steps
of: (1) removing the zona pellucida or part thereof from the
blastocyst; and (2) isolating the inner cell mass by removing the
trophoblast from the resulting blastocyst.
19. The method of claim 45, wherein the inner cell mass is cultured
in step (5) on a feeder layer comprising a cell differentiated from
the embryonic stem cell line of claim 1.
20. A neuro progenitor differentiated from an embryonic stem cell
line derived from a nucleus-transferred oocyte prepared by
transferring a nucleus of a human somatic cell into an enucleated
human oocyte.
21. The neuro progenitor of claim 20, wherein the embryonic stem
cell line is a cell line deposited under the accession number of
KCLRF-BP-00092.
22. A method for preparing the neuro progenitor of claim 20,
comprising: (1) culturing the embryonic stem cell line to form an
embryoid body; (2) culturing the embryoid body in the presence of
an agent suitable for differentiating a cell of the embryoid body
into the neuro progenitor; and (3) selecting a cell expressing a
marker of the neuro progenitor and culturing the selected cell to
obtain the neuro progenitor.
23. The method of claim 22, wherein the embryonic stem cell line is
a cell line deposited under the accession number of
KCLRF-BP-00092.
24. The method of claim 22, wherein the agent employed in step (2)
is selected from the group consisting of retinoic acid; ascorbic
acid; nicotinamide; N-2 supplement; B-27 supplement; and a mixture
of insulin, transferrin, sodium selenite and fibronectin.
25. A medium for use in carrying out the in vitro culturing in step
(4) of claim 3, comprising: 95 to 110 mM NaCl; 7.0 to 7.5 mM KCl;
20 to 30 mM NaHCO.sub.3; 1.0 to 1.5 mM NaH.sub.2PO.sub.4; 3 to 8 mM
sodium lactate; 1.5 to 2.0 mM CaCl.sub.2.2H.sub.2O; 0.3 to 0.8 mM
MgCl.sub.2.6H.sub.2O; 0.2 to 0.4 mM sodium pyruvate; 1.2 to 1.7 mM
fructose; 6 to 10 mg/ml human serum albumin; 0.7 to 0.8 .mu.g/ml
kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5%
nonessential amino acids; 0.7 to 1.2 mM L-glutamine; and 0.3 to
0.7% a mixture of insulin, transferrin and sodium selenite.
26. The medium of claim 25 comprising: 99.1 to 106 mM NaCl; 7.2 mM
KCl; 25 mM NaHCO.sub.3; 1.2 mM NaH.sub.2PO.sub.4; 5 mM sodium
lactate; 1.7 mM CaCl.sub.2.2H.sub.2O; 0.5 mM MgCl.sub.2.6H.sub.2O;
0.3 mM sodium pyruvate; 1.5 mM fructose; 8 mg/ml human serum
albumin; 0.75 .mu.g/ml kanamycin; 2% essential amino acnonessential
amino aciL-glutamine; and mixture of insulin, transferrin and
sodium selenite.
27. The method according to claim 3, wherein the step (2) comprises
incising a part of zona pellucida of the oocyte and removing the
cytoplasm containing first polar body by pressing the oocyte.
28. The method according to claim 27, wherein the step (2) further
comprises removing surrounding cumulus cells from the oocyte before
the incising and the removing.
29. The method according to claim 3, wherein the step (2) comprises
holding said human oocyte with a holding pipette; incising a part
of zona pellucida of the oocyte with an incision pipette; removing
first polar body and nucleus from the oocyte supported by the
holding pipette through a hole made by the incision process by
pressing the oocyte with the incision pipette.
30. The method according to claim 3, wherein the step (2) comprises
removing part of cytoplasm containing first polar body
corresponding to 10 to 15 percent of total cytoplasm.
31. The method according to claim 3, wherein the reprogramming in
step (4) is conducted for a time period of up to 20 hours.
32. The method according to claim 3, wherein the reprogramming in
step (4) is conducted for a time period of up to 6 hours.
33. The method according to claim 3, wherein the reprogramming in
step (4) is conducted for a time period of up to 3 hours.
34. The method according to claim 3, wherein the reprogramming in
step (4) is conducted for a time period of up to 2 hours.
35. The method according to claim 3, wherein the activation in step
(4) is performed by treating the nucleus-transferred oocyte with a
calcium ionophore and subsequently with 6-dimethylaminopurine.
36. The method of claim 35, wherein the concentration of the
calcium ionophore ranges from 5 .mu.M to 15 .mu.M.
37. The method of claim 36, wherein the concentration of the
calcium ionophore is about 10 .mu.M.
38. The method of claim 35, wherein the concentration of
6-dimethylaminopurine ranges from 1.5 mM to 2.5 mM.
39. The method of claim 38, wherein the concentration of
6-dimethylaminopurine is about 2.0 mM.
40. The method of claim 3, wherein the in vitro culturing in step
(4) is performed by sequentially using at least two media, each
having a different composition from the other.
41. The method of claim 40, wherein the in vitro culturing is
performed by sequentially using two media having different
compositions from each other.
42. The method of claim 41, wherein the in vitro culturing is
performed by sequentially using G1.2 medium and SNUnt-2 medium.
43. The method of claim 3, wherein step (4) is performed by
reprogramming the nucleus-transferred oocyte for a time period of
up to 20 hours, treating the nucleus-transferred oocyte with a
calcium ionophore at a concentration ranging from 5 .mu.M to 15
.mu.M and subsequently with 6-dimethylaminopurine at a
concentration ranging from 1.5 mM to 2.5 mM, and sequentially
culturing the nucleus-transferred oocyte in vitro in G1.2 medium
and SNUnt-2 medium.
44. A blastocyst prepared by the method according to claim 3.
45. A method for preparing an embryonic stem cell comprising: (1)
culturing a human somatic cell to prepare a nuclear donor cell; (2)
enucleating a human oocyte to prepare a recipient oocyte; (3)
preparing a nucleus-transferred oocyte by transferring a nucleus of
the nuclear donor cell into the recipient oocyte and fusing the
nucleus of the nuclear donor cell and the recipient oocyte; (4)
subjecting the nucleus-transferred oocyte to reprogramming,
activation and in vitro culturing to form a blastocyst; and (5)
isolating an inner cell mass from the blastocyst and culturing the
inner cell mass in an undifferentiated state to establish the
embryonic stem cell line.
46. The method according to claim 45, wherein the step (2)
comprises incising a part of zona pellucida of the oocyte and
removing the cytoplasm containing first polar body by pressing the
oocyte.
47. The method according to claim 44, wherein the step (2) further
comprises removing surrounding cumulus cells from the oocyte before
the incising and the removing.
48. The method according to claim 45, wherein the step (2)
comprises holding said human oocyte with a holding pipette;
incising a part of zona pellucida of the oocyte with an incision
pipette; removing first polar body and nucleus from the oocyte
supported by the holding pipette through a hole made by the
incision process by pressing the oocyte with the incision
pipette.
49. The method according to claim 45, wherein the step (2)
comprises removing part of cytoplasm containing first polar body
corresponding to 10 to 15% of total cytoplasm.
50. A medium for use in carrying out the in vitro culturing in step
(4) of claim 45, comprising: 95 to 110 mM NaCl; 7.0 to 7.5 mM KCl;
20 to 30 mM NaHCO.sub.3; 1.0 to 1.5 mM NaH.sub.2PO.sub.4; 3 to 8 mM
sodium lactate; 1.5 to 2.0 mM CaCl.sub.2.2H.sub.2O; 0.3 to 0.8 mM
MgCl.sub.2.6H.sub.2O; 0.2 to 0.4 mM sodium pyruvate; 1.2 to 1.7 mM
fructose; 6 to 10 mg/ml human serum albumin; 0.7 to 0.8 .mu.g/ml
kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5%
nonessential amino acids; 0.7 to 1.2 mM L-glutamine; and 0.3 to
0.7% a mixture of insulin, transferrin and sodium selenite.
51. A stem cell line prepared by the method according to claim 3.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an embryonic stem cell line
and a method for preparing the same and, more particularly, to an
embryonic stem cell line prepared by transferring a nucleus of a
human somatic cell into an enucleated human oocyte, culturing the
resulting nucleus-transferred oocyte to form a blastocyst, and
culturing an inner cell mass isolated from the blastocyst, and a
method for preparing the same.
BACKGROUND OF THE INVENTION
[0002] A stem cell is normally taken to mean an undifferentiated
cell capable of differentiating into all types of mature functional
cells constituting a body. For example, a hematopoietic stem cell
can differentiate into various corpuscular cells. An embryonic stem
(ES) cell derived from an embryo has pluripotency to differentiate
and develop into all types of organs, tissues and cells that form a
body.
[0003] A mouse ES cell line constructed in 1981 has provided a
technique and paradigm for developing a human ES cell. The
development of the ES cell has been studied using a mouse
teratocarcinoma, a tumor that occurs in a gonad of a closely bred
mouse strain (Evans & Kaufman, Nature, 292:154-156 (1981)).
[0004] Bongso et al. reported a method for culturing and
maintaining cells isolated from a human embryo derived from in
vitro fertilization for a short-term period (Bongso et al., Human
Reproduction, 9:2110-2117 (1994)). The cells isolated by Bongso et
al. had a morphology expected in a pluripotent stem cell; however,
they could not be cultured for a long-term period apparently
because a proper feeder layer was not used.
[0005] Primate ES cells have been prepared from a blastocyst of a
rhesus monkey and a marmoset monkey. The primate ES cells are
diploid, and very similar to a human ES cell.
[0006] The study of ES cells prepared from a monkey and a human has
suggested that a pluripotent stem cell might be derived from a
human blastocyst, although the ES cells from the monkey and the
human are somewhat different from that of a mouse in terms of
phenotype (Thomson et al., Proc. Natl. Acad. Sci. USA, 92:7844-7848
(1995)).
[0007] The characteristic features of human pluripotent ES cells
developed by Thomson et al. in 1998 (Thomson et al., Science,
282:1145-1147 (1998)) are as follows:
[0008] (1) expression of stage-specific embryonic antigen-3
(SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), tumor
rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81
(TRA-1-81), and alkaline phosphatase;
[0009] (2) high telomerase activity;
[0010] (3) differentiation into three types of blastodermal cells
when injected into mice;
[0011] (4) dependency on feeder cells; and
[0012] (5) no response to a human leukemia inhibitory factor
(hLIF).
[0013] Thomson et al. obtained the above ES cells from a blastocyst
donated by a couple under sterility treatment. Specifically, a
trophectoderm known to inhibit the establishment of an ES cell was
removed immunosurgically, an inner cell mass (ICM) was plated on a
fibroblast feeder layer derived from a mouse embryo, and the ICM
was replated on another feeder layer after a short attachment and
expansion period. Thomson's method was not significantly different
from the mouse ES cell protocol in terms of the medium or culture
system; and yet a relatively high success rate was achieved.
[0014] The isolation of human pluripotent ES cells and
breakthroughs in somatic cell nuclear transfer (SCNT) in mammals
(Solter, Nat. Rev. Genet., 1:199-207 (2000)) have raised the
possibility of performing human SCNT to generate virtually
unlimited sources of undifferentiated cells for research, with
potential applications in tissue repair and transplantation
medicine. This concept, known as "therapeutic cloning," employs a
nuclear transfer of a somatic cell into an enucleated oocyte (Lanza
et al., Nat. Med., 5:975-977 (1999)). Previous studies on such
therapeutic cloning dealt with the production of bovine ES-like
cells (Cibelli et al., Nat. Biotechnol., 16:642-646 (1998)) and
mouse ES cells from ICMs of cloned blastocysts (Munsie et al.,
Curr. Biol., 10:989-992 (2000); Wakayama et al., Science,
292:740-743 (2001)) and development of cloned human embryos until 8
to 10 cell stages (Cibelli et al., J. Regen. Med., 2:25-31
(2001)).
[0015] Although several reports have indicated that an ES cell line
can be established by employing a non-human mammalian oocyte, no ES
cell line developed from a human oocyte utilizing the nuclear
transfer technology has been reported yet.
SUMMARY OF THE INVENTION
[0016] Through extensive research and development efforts, however,
the present inventors have successfully established an ES cell line
by culturing a nucleus-transferred human oocyte.
[0017] Accordingly, it is an object of the present invention to
provide an ES cell line derived from a nucleus-transferred oocyte
prepared by transferring a nucleus of a human somatic cell into an
enucleated human oocyte.
[0018] It is another object of the invention to provide a method
for preparing an ES cell line, comprising the steps of:
[0019] (1) culturing a human somatic cell to prepare a nuclear
donor cell;
[0020] (2) enucleating a human oocyte to prepare a recipient
oocyte;
[0021] (3) preparing a nucleus-transferred oocyte by transferring a
nucleus of the nuclear donor cell into the recipient oocyte and
fusing the nucleus of the nuclear donor cell and the recipient
oocyte;
[0022] (4) subjecting the nucleus-transferred oocyte to
reprogramming, activation and in vitro culturing to form a
blastocyst; and
[0023] (5) isolating an ICM from the blastocyst and culturing the
ICM in an undifferentiated state to establish the ES cell line.
[0024] It is a further object of the invention to provide a medium
suitable for an in vitro culturing of a nucleus-transferred oocyte
prepared by transferring a nucleus of a human somatic cell into an
enucleated human oocyte.
[0025] It is still another object of the invention to provide a
nerve cell or a neuro progenitor differentiated from an ES cell
line derived from a nucleus-transferred oocyte prepared by
transferring a nucleus of a human somatic cell into an enucleated
human oocyte.
[0026] It is a still further object of the invention to provide a
method for preparing a neuro progenitor differentiated from an ES
cell line derived from a nucleus-transferred oocyte prepared by
transferring a nucleus of a human somatic cell into an enucleated
human oocyte, comprising the steps of:
[0027] (1) culturing the ES cell line to form an embryoid body;
[0028] (2) culturing the embryoid body in the presence of an agent
suitable for differentiating a cell of the embryoid body into the
neuro progenitor; and
[0029] (3) selecting a cell expressing a marker of the neuro
progenitor and culturing the selected cell to obtain the neuro
progenitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, in which:
[0031] FIG. 1 shows photographs of an undifferentiated colony of ES
cells derived from a nucleus-transferred oocyte in accordance with
the present invention (A: x100, B: x200);
[0032] FIG. 2 represents a photograph of a fluorescence-stained
neuro progenitor differentiated from an undifferentiated colony
obtained in accordance with the present invention by adding a
mixture of insulin, transferrin, sodium selenite and fibronectin
(x400);
[0033] FIG. 3 depicts the incision process of the zona pellucida of
an oocyte (3) with a holding pipette (1) and an incision pipette
(2);
[0034] FIG. 4 presents a photograph showing the removal of the
first polar body and the nucleus of the oocyte (3) with the holding
pipette (1) and the incision pipette (2);
[0035] FIG. 5 offers a photograph showing the transfer of a nuclear
donor cell into an enucleated recipient oocyte (3) with the holding
pipette (1) and a transfer pipette (4);
[0036] FIGS. 6A to 6D diagrammatically summarize the results of a
karyotype analysis of an ES cell line derived from a
nucleus-transferred oocyte prepared in accordance with the present
invention and that of a somatic cell obtained from a female, said
somatic cell providing the nucleus used for establishing the ES
cell line;
[0037] FIG. 7 illustrates three types of blastodermal cells
identified within a teratoma formed by injecting an
undifferentiated cell colony obtained in accordance with the
present invention into a gonad of an immune deficiency mouse (A:
cartilage, B: intestinal tract, C: neural tube (A,B,C: x200));
and
[0038] FIG. 8 provides photographs confirming the formation of an
embryoid body from an ES cell line in accordance with the present
invention (A,B,C: endoderm; D,E,F: mesoderm; G,H,I,: ectoderm; A:
alpha-1-fetoprotein; B: cytokeratin; C: HNF-2-alpha; D: BMP-4; E:
Myo D; F: desmin; G: neurofilament; H: S-100; and I: NCAM).
DETAILED DESCRIPTION OF THE INVENTION
[0039] The term "nuclear transfer" as used herein means a process
of transferring a nucleus of a somatic cell (or referred to as
"nuclear donor cell") into an enucleated oocyte (or referred to as
"recipient oocyte"). The resulting cell obtained by the nuclear
transfer is referred to as a "nucleus-transferred oocyte" or
"nuclear transfer oocyte." The term "somatic cell" as used herein
means any cell constituting a body that has two sets of chromosomes
(2n), excluding a germ cell that has a single set of chromosomes
(n).
[0040] The term "autologous nucleus-transferred oocyte" used herein
means a nucleus-transferred oocyte obtained by transferring a
nucleus of a somatic cell into an enucleated oocyte where the
somatic cell is isolated from a human who is expected to receive a
stem cell derived from the nucleus-transferred oocyte, or a
specific cell or tissue differentiated from the stem cell.
[0041] Accordingly, one of the salient advantages or benefits to be
derived from the present invention resides in the fact that the
person who receives a specific cell or tissue derived from the
autologous nucleus-transferred oocyte would not exhibit
immunorejection or suffer adverse reaction since such cell or
tissue is to carry the genetic characteristics of the person.
[0042] The term "embryonic stem cell (ES cell)" means an
undifferentiated cell derived from an embryo, which has the
capability of differentiating into various types of mature cells.
Here, "embryo" means a fertilized egg up to eight (8) weeks after
its fertilization or a nucleus-transferred oocyte in the
corresponding developmental stage. An embryo is created by a
repetitive division of such fertilized egg or nucleus-transferred
oocyte, and comprises a blastocyst containing an ICM and an outer
trophectoderm.
[0043] The term "ES cell line derived from an autologous
nucleus-transferred oocyte" or "autologous nucleus-transferred ES
cell line" means a stem cell line derived from an ICM isolated from
an autologous nucleus-transferred oocyte.
[0044] The term "neuro progenitor" refers to cells to be
differentiated into nerve cells including neurons and glia such as
astrocytes, oligodendrocytes, schwann cells, satellite cells,
ependymal cells and microglia.
[0045] In accordance with one aspect of the present invention,
there is provided a method for preparing an ES cell line,
comprising the steps of:
[0046] (1) culturing a human somatic cell to prepare a nuclear
donor cell;
[0047] (2) enucleating a human oocyte to prepare a recipient
oocyte;
[0048] (3) preparing a nucleus-transferred oocyte by transferring a
nucleus of the nuclear donor cell into the recipient oocyte and
fusing the nucleus of the nuclear donor cell and the recipient
oocyte;
[0049] (4) subjecting the nucleus-transferred oocyte to
reprogramming, activation and in vitro culturing to form a
blastocyst; and
[0050] (5) isolating an ICM from the blastocyst and culturing the
ICM in an undifferentiated state to establish the ES cell line.
[0051] Hereinafter, the method for preparing an ES cell line in
accordance with the present invention will be described in
detail.
[0052] Step 1: Preparation of Nuclear Donor Cell
[0053] A human somatic cell is cultured to function as a nuclear
donor cell.
[0054] A somatic cell from a human is amenable for such nuclear
donor cell, and a nucleus thereof is transferred into an enucleated
human oocyte.
[0055] There is no limitation on the type or source of the somatic
cell as long as it is obtained from a human, and it is also
possible to use a somatic cell obtained from an institute storing
human cells for commercial purposes. Preferred exemplary somatic
cells include a dermal cell, a nerve cell, a cumulus cell, an
oviduct epithelial cell, and the like.
[0056] In case of preparing an autologous nucleus-transferred
oocyte in accordance with the present invention, the nuclear donor
cell is taken from an individual who is expected to receive a stem
cell derived from the nucleus-transferred oocyte, or a specific
cell or tissue differentiated from the stem cell.
[0057] The somatic cell can be cultured to establish a cell line by
using the Mather and Barnes method (Animal Cell Culture Methods:
vol. 57 of Methods in Cell Biology (Mather & Barnes eds.,
Academic Press, 1998)).
[0058] In accordance with a preferred embodiment of the present
invention, a uterus fluid and a phosphate buffered saline (PBS)
containing P/S antibiotic (penicillin 10,000 IU, streptomycin 10
mg) are added to a somatic cell. Such somatic cell is centrifuged
and washed, and cultured in a DMEM medium containing human serum,
nonessential amino acids (NEAAs) and the P/S antibiotic at, e.g.,
39.degree. C. in 5% CO.sub.2 atmosphere.
[0059] Especially, in case of using a cumulus cell as a nuclear
donor cell, the cumulus cell can be prepared by treating a
cumulus-oocyte complex with hyaluronidase to isolate a cumulus cell
layer surrounding an oocyte, adding a trypsin-EDTA solution to the
cumulus cell layer and placing the resulting solution at, e.g.,
39.degree. C. in 5% CO.sub.2 atmosphere under saturated humidity.
After centrifuging and washing, the collected cumulus cells can be
cultured under the same condition described above.
[0060] Step 2: Preparation of Recipient Oocyte
[0061] A recipient oocyte as used in the present invention means an
oocyte that lacks its own nucleus and receives a foreign nucleus
from a human somatic cell.
[0062] A mature oocyte may be prepared by collecting a
superovulated oocyte from a human ovary or obtaining an oocyte from
an institute storing human oocytes for commercial purposes and
culturing the oocyte using a method known in the art (Yuzpe et al.,
J. Reprod. Med., 34:937-942 (1989)). For example, an oocyte may be
matured by culturing the oocyte in the G1.2 medium, marketed by
Vitro Life of Goteborg, Sweden, supplemented with 5% human serum
albumin (HSA) under the condition of, e.g., 5% CO.sub.2 for 4
hours.
[0063] Next, an enucleated recipient oocyte is prepared by removing
the surrounding cumulus cells from the oocyte, and eliminating part
of the zona pellucida and the cytoplasm containing the first polar
body.
[0064] In accordance with a preferred embodiment of the present
invention, the enucleation process is performed as follows.
[0065] A mature oocyte is placed in a washing solution containing
hyaluronidase, and the cumulus cell is physically removed. Next,
the mature oocyte is washed with the G1.2 medium. Subsequently, the
zona pellucida of the oocyte is penetrated to form a small hole
therein. The oocyte is enucleated by removing part of the cytoplasm
containing the first polar body corresponding to 10 to 15% of the
total cytoplasm through the small hole. After this removal, the
enucleated oocyte is washed with the G1.2 medium and placed in the
G1.2 medium for culturing.
[0066] The enucleation can be confirmed by investigating cytoplasm
stained with Hoechst 33342 (Sigma Co., St. Louis, Mo., U.S.A.)
using a UV detector.
[0067] Step 3: Preparation of Nucleus-Transferred Oocyte and
Electrofusion
[0068] The nuclear donor cell prepared by step 1 is transferred
into the enucleated recipient oocyte obtained in step 2, and the
nucleus-transferred oocyte is treated with electrofusion.
[0069] The nuclear transfer of a somatic cell into a recipient
oocyte may be realized by transferring either the nucleus of the
somatic cell or the whole somatic cell into the recipient
oocyte.
[0070] In accordance with a preferred embodiment of the present
invention, the nuclear transfer and electrofusion are performed as
follows.
[0071] First, the enucleated oocyte is washed with the G1.2 medium.
The nuclear donor cell is injected into the enucleated oocyte in a
phytohemagglutin-P (PHA-P) solution via a small hole formed in the
zona pellucida using a transfer pipette to produce a
nucleus-transferred oocyte. Next, the resulting nucleus-transferred
oocyte is washed with the G1.2 medium and placed in the same
medium.
[0072] Subsequently, the nucleus-transferred oocyte is treated with
electrofusion with the aid of a cell manipulator. A mannitol
solution is added to the G1.2 medium containing the
nucleus-transferred oocyte. The resulting mannitol solution
containing the nucleus-transferred oocyte is placed between two
electrodes of the cell manipulator and is positioned such that the
nuclear donor cell faces the (+) electrode. The nucleus-transferred
oocyte is electrofused by treating it with a direct current ranging
from 0.75 to 2.00 kV/cm for 10 to 15 .mu.s, 1 to 5 times at an
interval of, e.g., 1 second.
[0073] The fused nucleus-transferred oocyte is washed with a
mannitol solution and the G1.2 medium. The mannitol solution used
in this step is prepared by dissolving bovine serum albumin (BSA)
and mannitol in a 4-(2-hydroxyethyl)-1-perazine ethanesulfonic acid
(HEPES) buffer at a pH ranging from 7.2 to 7.4
[0074] Step 4: Reprogramming, Activation and In Vitro Culturing of
Nucleus-Transferred Oocyte
[0075] In order to allow the nucleus-transferred oocyte prepared in
step 3 to undergo a same developmental procedure as a normal
fertilized oocyte formed as a result of fusion between a sperm and
an oocyte, several critical factors, such as reprogramming time,
activation method and in vitro culturing conditions, should be
judiciously chosen.
[0076] The present invention provides unique fertilization and
development procedures conducive for activating and culturing the
nucleus-transferred oocyte. Specifically, the nucleus-transferred
oocyte prepared by electrofusion in step 3 is subjected to
reprogramming, activation, and in vitro culturing to form a
blastocyst.
[0077] The reprogramming time means the time lapsed between the
electrofusion and the activation, and the length of the
reprogramming time may affect the developmental capacity (in
particular, the blastocyst formation rate) of the
nucleus-transferred oocyte. This reprogramming time is required to
allow the gene expression pattern of the somatic cell to turn into
one that is appropriate and necessary for the development of the
nucleus-transferred oocyte. Such reprogramming time plays a
critical role in chromatin remodeling, and it is known to determine
the developmental competence in vivo and in vitro of the
nucleus-transferred oocyte.
[0078] The reprogramming time of the present invention may be 20
hours or below, preferably, 6 hours or below, more preferably 3
hours or below, and, most preferably, about 2 hours.
[0079] After the reprogramming, the nucleus-transferred oocyte may
be activated by various chemical, physical and mechanical stimuli,
such as calcium ionophore, ionomycin, ethanol, Tyrode's solution
(Sigma-Aldrich, St. Louis, Mo., U.S.A.) puromycin, and the like. In
the present invention, it is preferable to treat the
nucleus-transferred oocyte with calcium ionophore for its
activation. It is more preferable to treat the nucleus-transferred
oocyte with calcium ionophore and then with 6-dimethylaminopurine
(6-DMAP). Specifically, the calcium ionophore may be used at a
concentration ranging from 5 to 15 .mu.M, and, preferably, about 10
.mu.M. In addition, said 6-DMAP may be employed at a concentration
ranging from 1.5 to 2.5 mM, and, preferably, about 2.0 mM. If the
concentrations of the calcium ionophore and the 6-DMAP are within
the above respective ranges, the nucleus-transferred oocyte may be
activated effectively. Both calcium ionophore and 6-DMAP are
preferably dissolved in an in vitro culture medium.
[0080] A representative example of the in vitro culture medium is
the G1.2 medium (Vitro Life, Goteborg, Sweden) comprising NaCl,
KCl, NaHCO.sub.3, NaH.sub.2PO.sub.4, CaCl.sub.2, sodium lactate,
glucose, phenol red, BSA, kanamycin, essential amino acids (EAAs),
NEAAs, and glutamine.
[0081] Further, for an efficient in vitro culturing of the
nucleus-transferred oocyte, it is preferable to supplement the
culture medium with various energy substrates known in the art or
employ a sequential culturing system using at least two media
having different compositions suitable for each stage of the
embryonic development. The sequential culturing system useful in
the present invention may be any one of commercially available
culturing systems. Preferably, said in vitro culturing is performed
by sequentially using two media having different compositions each
other, such as the G1.2 and the G2.2 media (Vitro Life, Goteborg,
Sweden).
[0082] Such in vitro culture medium preferably contains a human
modified synthetic oviductal fluid with amino acids (hmSOFaa),
which has been designated as "SNUnt-2 medium." The hmSOFaa is
prepared by supplementing a modified synthetic oviductal fluid with
amino acids (mSOFaa) (Choi et al., Theriogenology, 58:1187-1197
(2002)) with HSA and fructose instead of BSA and glucose,
respectively. The mSOFaa medium has been widely used for culturing
bovine embryos.
[0083] In particular, the SNUnt-2 medium comprises 95 to 110 mM
NaCl; 7.0 to 7.5 mM KCl; 20 to 30 mM NaHCO.sub.3; 1.0 to 1.5 mM
NaH.sub.2PO.sub.4; 3 to 8 mM sodium lactate; 1.5 to 2.0 mM
CaCl.sub.2.2H.sub.2O; 0.3 to 0.8 mM MgCl.sub.2.6H.sub.2O; 0.2 to
0.4 mM sodium pyruvate; 1.2 to 1.7 mM fructose; 6 to 10 mg/ml HSA;
0.7 to 0.8 .mu.g/ml kanamycin; 1.5 to 3% EAAs; 0.5 to 1.5% NEAAS;
0.7 to 1.2 mM L-glutamine; and 0.3 to 0.7% a mixture of insulin,
transferrin and sodium selenite. Preferably, the SNUnt-2 medium
comprises the ingredients as listed in Table 1. TABLE-US-00001
TABLE 1 Ingredient Concentration NaCl 99.1.about.106 mM KCl 7.2 mM
NaHCO.sub.3 25 mM NaH.sub.2PO.sub.4 1.2 mM sodium lactate 5 mM
CaCl.sub.2.cndot.2H.sub.2O 1.7 mM MgCl.sub.2.cndot.6H.sub.2O 0.5 mM
sodium pyruvate 0.3 mM fructose 1.5 mM HSA 8 mg/ml kanamycin 0.75
.mu.g/ml EAAs 2% NEAAs 1% L-glutamine 1 mM ITS* 0.5% *ITS: a
mixture of 1.0 g/L insulin, 0.55 g/L transferrin and 0.67 mg/L
sodium selenite
[0084] The sequential culturing system of the present invention may
employ any combination of the different media. For example, in the
two-step culturing system, the first culturing may be conducted in
the G1.2 medium, and the second culturing, in the SNUnt-2
medium.
[0085] Step 5: Removal of Zona Pellucida or Part Thereof
[0086] In order to obtain an ES cell derived from the blastocyst
obtained in step 4, the zona pellucida or part thereof has to be
removed from the blastocyst. This removal may be carried out by
using one of the methods known in the art, e.g., pronase treatment,
incubation in acidic Tyrode's solution, or a physical method such
as laser dissection. It is preferable to use pronase dissolved in a
suitable medium such as PBS, G2 medium (Vitro Life, Goteborg,
Sweden) or S2 medium (Scandinavian IVF Sciences, Goteborg, Sweden).
In a preferred embodiment, pronase is dissolved in a mixture of PBS
and the S2 medium at equal volumes. The blastocyst is treated with
0.1% pronase for about 1 to 2 minutes, preferably 1 to 1.5 minutes,
to remove the zona pellucida therefrom.
[0087] Step 6: Removal of Trophoblast and Isolation of ICM
[0088] Once the zone pellucida is removed from the blastocyst as
described above, the trophoblast is exposed. It is preferable to
completely separate the trophoblast from the ICM. The trophoblast
may be separated from the ICM using one of the methods known in the
art, such as an immunosurgical method employing an antibody or a
mechanical method using a pipette.
[0089] In a preferred embodiment, the trophoblast is removed by an
immunosurgical method that treats the trophoblast with an antibody
responsive to an epitope located on a surface of the trophoblast.
It is more preferable to carry out the immunosurgical method
together with a complement treatment. In this case, an antibody and
a complement may be used independently or simultaneously. A
preferred combination between the antibody and the complement may
include anti-placental alkaline phosphatase antibody (anti-AP) and
baby rabbit complement, or anti-human serum antibody and guinea pig
complement.
[0090] The antibody and the complement may be diluted with a
suitable medium such as SNUnt-2, G2.2 or S2 medium. Preferably, the
anti-AP may be diluted with the S2 medium at the ratio of 1:20; and
other antibodies and complements, at the ratio of 1:1.
[0091] It is preferable to treat the zona pellucida-removed
blastocyst with an antibody and then with a complement. Preferably,
the blastocyst may be treated with the antibody for about 30
minutes, washed with a suitable medium, e.g., SNUnt-2, G2.2 or S2
medium, and then, treated with the complement for about 30
minutes.
[0092] Moreover, the trophoblast or part thereof may be removed
from the blastocyst by washing the blastocyst with a suitable
medium such as SNUnt-2, G2.2 or S2. In such case, the trophoblast
may be removed by a mechanical method known in the art, e.g.,
pipetting a solution containing the blastocyst using a pipette
having a small bore.
[0093] Through these steps, the trophoblast is removed from the
blastocyst; and the ICMs, i.e., the remaining part thereof, are
obtained.
[0094] Step 7: Culturing of ICMs on Fibroblast Feeder Layer
[0095] ICMs isolated in step 6 are cultured on a fibroblast feeder
layer since ICMs maintain their undifferentiated state when
cultured on the fibroblast feeder layer. Sometimes, hLIF has been
suggested for maintaining the undifferentiated morphology of ICMs
instead of the feeder layer. However, it is practically impossible
for a human cell to remain in its undifferentiated state without
using a fibroblast feeder layer. Accordingly, the condition that
does not induce extraembryonic differentiation and apoptosis in the
ES cells generally requires culturing on a fibroblast feeder
layer.
[0096] It is preferable to employ a mouse- and/or a human-derived
fibroblast for preparing the fibroblast feeder layer. They may be
used alone or in a mixture. It is more preferable to use cells
differentiated from the ES cells derived from an autologous
nucleus-transferred oocyte of a human as a feeder layer (this
feeder layer has been designated as "auto feeder layer"). It is
most preferable to use the fibroblasts differentiated from the ES
cells derived from an autologous nucleus-transferred oocyte of an
individual. The use of such feeder layer can prevent other foreign
cells from contaminating the ES cells.
[0097] Such human-derived fibroblasts are capable of inducing an
optimum growth and differentiation inhibition of the ES cells when
appropriately mixed with mouse-derived fibroblasts.
[0098] The cell density in the fibroblast feeder layer may affect
its stability and capability. In case of using a mixture of mouse
and human fibroblasts, it is preferable to maintain the human
fibroblasts at a density of, e.g., 2.5.times.10.sup.4
cells/cm.sup.2 and the mouse fibroblasts at a density of, e.g.,
7.0.times.10.sup.4 cells/cm.sup.2. In case of using the mouse
fibroblasts alone, it is preferable to use the same at a density
ranging from 7.5.times.10.sup.4 to 1.0.times.10.sup.5
cells/cm.sup.2. It is preferable to establish such feeder layer 6
to 48 hours before the addition of ES cells thereon.
[0099] Further, it is preferable to use mouse or human fibroblasts
having a low passage number. Quality of the fibroblasts may affect
the capability of supporting the ES cells. It is preferable to use
the fibroblasts isolated from an embryo. The mouse fibroblasts are
preferably obtained from 13.5-day old fetus, and the human
fibroblasts, from an embryo or a fetal tissue. These fibroblasts
can be cultured by using a cell culturing method known in the
art.
[0100] In handling the mouse embryonic fibroblasts, it is important
to minimize the use of trypsin and inhibit overcrowding. Otherwise,
the mouse embryonic fibroblasts cannot support the growth of
undifferentiated ES cells. Each batch of the mouse embryonic
fibroblasts so prepared has to be tested first to confirm whether
it is suitable for supporting and maintaining the ES cells.
[0101] Between fresh primary embryonic fibroblasts and fibroblasts
having undergone a freezing-thawing treatment, the former is
normally considered more suitable for supporting renewal of the ES
cells. However, certain batches may show their capability of
supporting the ES cells even after repeated freezing-thawing.
[0102] Certain mouse strains can produce embryonic fibroblasts more
suitable for supporting the ES cells than other strains. For
example, it has been demonstrated that the fibroblasts derived from
the mice produced by inbreeding of 129/Sv or CBA strain or by
crossbreeding of 129/Sv and C57/B16 strains are more suitable for
supporting the ES cells.
[0103] In addition, it is preferable to inhibit the growth of
feeder cells by using any one of the methods known in the art,
including irradiation and chemical treatment. In a preferred
embodiment, such cells are treated with mitomycin C.
[0104] The fibroblast feeder layer thus prepared is cultured on a
petri dish coated with gelatin, preferably 0.1% gelatin.
[0105] The fibroblast feeder layer may be maintained in an ES
medium. A suitable ES medium is the DMEM/F12 medium comprising 20%
serum replacement, 0.1 mM .beta.-mercaptoethanol, 1% NEAAs, 2 mM
glutamine, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin,
and 4 ng/ml human recombinant fibroblast growth factor (FGF).
[0106] Further, such ES medium may be supplemented with a soluble
growth factor capable of stimulating growth or survival of the stem
cells or inhibiting differentiation thereof. Representative
examples of the growth factor are human pluripotent stem cell
factor, ES cell renewal factor, and the like.
[0107] The isolated ICMs may be cultured for 6 days or longer, and
cell colonies are generated therefrom. The colonies typically
comprise undifferentiated stem cells. The undifferentiated stem
cells may be isolated by using one of the methods known in the art.
It is preferable to use a micropipette for isolating the
undifferentiated stem cells. Such mechanical isolation may be
supplemented with a treatment of a Ca.sup.2+/Mg.sup.2+-free PBS
medium or an enzyme helpful for cell dissociation such as
dispase.
[0108] Step 8: Subculturing of ES Cells
[0109] The ES cells cultured in step 7 are detached from the feeder
layer and transferred to a fresh feeder layer. Then, the ES cells
may be further cultured to propagate in a morphologically
undifferentiated state.
[0110] In this case, it is preferable to culture the ES cells for 5
to 7 days. Undifferentiated stem cell colonies start to be observed
by about the second day of culturing. The stem cells are
morphologically identified by a high ratio in nucleus to cytoplasm,
clear nucleoli, condensed colony formation and distinctive cell
boundary.
[0111] Propagation of the undifferentiated stem cells is initiated
by isolating an undifferentiated stem cell clump from the stem cell
colony. Such isolation may be carried out by using one of the
methods known in the art, such as a chemical or mechanical method.
Preferably, the stem cells are isolated from the colony by washing
with a Ca.sup.2+/Mg.sup.2+-free PBS medium, a mechanical method, or
a combination thereof. It is more preferable to mechanically
isolate the stem cells from the colony.
[0112] In a preferred embodiment, the Ca.sup.2+/Mg.sup.2+-free PBS
medium may be used for reducing intercellular adhesive power. After
incubation in the above medium for about 15 to 20 minutes, the
cells begin to detach themselves gradually from the feeder layer,
and, finally, are isolated as a clump having a desired size. In
case such isolation of the cells proves to be insufficient, a
mechanical method using a sharp edge of a micropipette may be more
effectively employed for isolating and cutting the clump.
[0113] A chemical method employing an enzyme may be also used. The
enzyme, preferably, dispase, may be used alone or in combination
with a mechanical method.
[0114] In another preferred embodiment, it is possible to isolate
clumps from the colony by treating with dispase after mechanical
cutting of the colony. Cutting of the colony is carried out in a
Ca.sup.2+/Mg.sup.2+-containing PBS medium. The colony can be
mechanically cut into clumps, each clump containing about 100
cells, with the aid of a sharp edge of a micropipette. As soon as a
clump is isolated, it is picked up with a micropipette having a
wider bore, washed with the Ca.sup.2+/Mg.sup.2+-containing PBS
medium, and transferred to a fresh fibroblast feeder layer.
[0115] It is necessary to confirm whether the stem cells maintain
their undifferentiated state during these culturing processes.
Undifferentiated stem cells can be identified by examining their
typical morphological characteristic features as described above.
Such stem cells can be also identified by detecting a cell marker
or measuring the gene expression specific for a pluripotent
cell.
[0116] Representative examples of genes specific for a pluripotent
cell or a typical lineage include, but are not limited to, alkaline
phosphatase, Octamer-4 (Oct-4), SSEA-3 and SSEA-4 which may be used
as stem cell markers. Other exemplary genes specific for stem cells
may include genesis, GDF-3 and cripto. The expression profile of
these genes can be analyzed by using one of the methods known in
the art, including reverse transcription-polymerase chain reaction
(RT-PCR), a differentiation gene expression method, a microarray
assay, and the like.
[0117] Preferably, the stem cells can be identified by an
immunological reaction with a human pluripotent stem cell marker
such as SSEA-4, germ cell tumor marker-2 (GCTM-2) antigen,
TRA-1-60, or the like. In particular, the stem cells may express
Oct-4 as a transcription factor and maintain a normal diploid
karyotype.
[0118] Growth progress of the stem cells and maintenance status of
their differentiated or undifferentiated state can be monitored by
quantitatively measuring the proteins specific for the stem cells
excreting into the medium or analyzing fixed cell preparations with
enzyme-linked immunosorbent assay. Representative examples of the
proteins specific for the stem cells are a soluble type of CD
antigen and GCTM-2 antigen, and these proteins can be monitored by
detecting a cell marker or measuring the gene expression.
[0119] In accordance with another aspect of the present invention,
a nerve cell or a neuro progenitor is differentiated from an ES
cell line derived from a nucleus-transferred oocyte prepared by
transferring a nucleus of a human somatic cell into an enucleated
human oocyte.
[0120] In accordance with a further aspect of the present
invention, there is provided a method for preparing a neuro
progenitor differentiated from an ES cell line derived from a
nucleus-transferred oocyte prepared by transferring a nucleus of a
human somatic cell into an enucleated human oocyte, which comprises
the steps of:
[0121] (1) culturing the ES cell line to form an embryoid body;
[0122] (2) culturing the embryoid body in the presence of an agent
suitable for differentiating a cell of the embryoid body into the
neuro progenitor; and
[0123] (3) selecting a cell expressing a marker of the neuro
progenitor and culturing the selected cell to obtain the neuro
progenitor.
[0124] Hereinafter, the inventive method for preparing the neuro
progenitor from the ES cell line is described in detail.
[0125] Step A: Preparation of an Embryoid Body
[0126] The first step for differentiating the ES cells derived from
the nucleus-transferred oocytes (referred to as "nuclear transfer
embryonic stem cells" or "ntES cells") into neuro progenitors is to
generate an embryoid body by culturing the ES cells. The embryoid
body can be prepared from the ES cells by using one of the methods
known in the art (Zhang et al., Nat. Biotechnol., 19:1129-1133
(2001)).
[0127] In a preferred embodiment, the embryoid body is obtained by
transferring cultured ntES cell colonies into a non-adhesive
culture dish containing the DMEM/F12 medium supplemented with a 20%
serum replacement and culturing them for 3 to 5 days. Typically,
floating embryoid bodies start to appear about one day after the
beginning of the culturing (about 40 to 60 embryoid bodies/dish).
At this point, it is preferable to transfer such embryoid bodies to
a new dish while removing any remaining feeder cells. Then, the
embryoid bodies are plated on an adhesive dish coated with
polyornithine/laminin.
[0128] Step B: Inducement of Differentiation into Neuro Progenitors
by an Agent
[0129] Representative agents which may be employed in the present
invention for inducing differentiation of the embryoid bodies
obtained in step A into neuro progenitors include, but are not
limited to, retinoic acid; ascorbic acid; nicotinamide; N-2
supplement (100.times., 17502-048; Gibco, Grand Island, N.Y.,
U.S.A.); B-27 supplement (50.times., 17504-044, Gibco, Grand
Island, N.Y., U.S.A.); and a mixture of insulin, transferrin,
sodium selenite and fibronectin (ITSF). Neuro progenitors
differentiated from the ntES cells can be obtained by culturing the
embryoid bodies in a medium supplemented with such agent and
inducing their expansion and differentiation.
[0130] In a preferred embodiment, the embryoid bodies prepared in
step A are further cultured for 1 day followed by culturing in the
DMEM/F12 medium supplemented with ITSF, i.e., insulin (about 25
.mu.g/ml), transferrin (about 100 .mu.g/ml), sodium selenite (about
30 nM) and fibronectin (about 5 .mu.g/ml) for 5 to 10 days, thereby
inducing differentiation of the ntES cells into the neuro
progenitors.
[0131] Step C: Selection and Culturing of Cells Expressing a Neuro
Progenitor Marker
[0132] The neuro progenitors differentiated from the ntES cells may
be obtained by selecting cells expressing a neuro progenitor marker
such as nestin among the differentiated cells obtained in step B
and culturing them.
[0133] Further, the obtained neuro progenitors may be
differentiated into desired specific type of nerve cells. The
differentiation into the nerve cells can be carried out through
conventional methods such as induction with chemicals, etc.
[0134] In a preferred embodiment, the cells exhibiting a positive
signal for a neuro progenitor marker are selected; their expansion
is induced by culturing the selected cells in the DMEM/F12 medium
supplemented with the N-2 supplement, laminin and basic fibroblast
growth factor (bFGF) for 5 to 7 days; and, then, they are further
cultured in the DMEM/F12 medium supplemented with only the N-2
supplement and laminin for 8 to 14 days.
[0135] It is well known that ES cells are capable of
differentiating into almost any type of cells. Accordingly, the ES
cell line of the present invention may be a good source providing
various types of cells. For instance, the ES cells may be induced
to differentiate into hematopoietic cells, nerve cells, beta cells,
muscle cells, liver cells, cartilage cells, epithelial cells, etc.,
by culturing them in a medium under conditions suitable for cell
differentiation. Such medium and conditions are well known in the
art.
[0136] Accordingly, the ES cell line of the present invention may
have numerous therapeutic and diagnostic applications. Especially,
such ES cell line may be used in cell transplantation therapies for
the treatment of numerous diseases, e.g., diabetes, Parkinson's
disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS),
cerebral palsy and cancer. Further, the ES cell line derived from
the autologous nucleus-transferred oocyte can be advantageously
used in the cell transplantation therapies since no adverse
immunorejection reaction may occur during and after the treatment
procedure.
[0137] The following Examples are intended to further illustrate
the present invention without limiting its scope.
[0138] The G1.2 medium or G1 ver.3 medium (Vitro Life, Goteborg,
Sweden) used in these Examples are supplemented with 5% HSA unless
indicated otherwise.
EXAMPLE 1
Preparation of Oocyte and Nuclear Donor Cell
[0139] Voluntary oocyte donors were screened carefully through
physical and mental examinations, and administered with follicle
stimulation hormone (FSH) to induce superovulation.
[0140] About 36 hours after the administration of human chorionic
gonadotropin (hCG) to the donors, cumulus-oocyte complexes (COCs)
were recovered and cultured for 40 minutes in the G1.2 medium using
an incubator maintained at 37.degree. C., 5% CO.sub.2 and saturated
humidity. Such COCs were treated with 0.1% (w/v) hyaluronidase
(Sigma Co., St. Louis, Mo., U.S.A.) for 1 hour to disperse cumulus
cells.
[0141] The oocytes were obtained by separating such cumulus cells
from the COCs. The separated cumulus cells were isolated through a
mouth pipette and washed with the G1.2 medium. Those cumulus cells
having a modal diameter ranging from 10 to 12 mm were selected as
nuclear donor cells.
EXAMPLE 2
Enucleation of Oocyte and Cell Fusion
[0142] One of the oocytes obtained in Example 1 was cultured in the
G1.2 medium for 1 to 2 hours in order to induce the maturation of
its nucleus. Thereafter, enucleation, nuclear transfer and
electrofusion thereof were performed as follows.
[0143] (2-1) Enucleation of Oocyte and Nuclear Transfer from
Somatic Cell
[0144] The oocyte washed once with the G1.2 medium. Such oocyte was
transferred to a hyaluronidase solution prepared by mixing 1 ml of
the G1.2 medium with 111 .mu.l of a solution, wherein 0.05 g of
hyaluronidase was dissolved in 5 ml of the G1.2 medium, and
adjusted to a 0.1% (w/v) hyaluronidase concentration. The oocyte
was stripped of any remaining cumulus cells, washed three times
with the G1.2 medium and placed in the same medium. Then, the
oocyte was transferred to a cytochalasin B solution prepared by
mixing 1 ml of the G1.2 medium supplemented with 10% fetal bovine
serum (FBS) with 1 .mu.l of a solution wherein cytochalasin B was
dissolved in dimethyl sulfoxide to a concentration of 7.5 .mu.g/ml.
The zona pellucida of the oocyte was incised by a micromanipulator
to form a small hole, and the oocyte was enucleated by removing
part of the cytoplasm containing the first polar body thereof and
corresponding to 10 to 15% of the total cytoplasm through the small
hole.
[0145] FIG. 3 shows the incision process of the zona pellucida of
the oocyte (3) by employing a holding pipette (1) and an incision
pipette (2). FIG. 4 shows the enucleation process removing the
first polar body and the nucleus from the oocyte where the oocyte
(3) having the small hole vertically positioned was supported by
the holding pipette (1) positioned beneath the oocyte and then
lightly pressed by the incision pipette (2) to enucleate the same.
Such enucleated oocyte washed three times with the G1.2 medium and
placed in the same medium.
[0146] Subsequently, a nuclear donor cell in a 4 .mu.l drop of PBS
supplemented with 1% BSA was transferred, using a holding pipette
and a transfer pipette, into the enucleated oocyte in a 4 .mu.l
drop of a solution prepared by mixing 400 .mu.l of the G1.2 medium
with 100 .mu.l of a PHA-P solution wherein 5 mg of PHA-P was
dissolved in 10 ml of the G1.2 medium. The drops containing the
nuclear donor cell and the enucleated oocyte were coated with a
mineral oil to prevent the evaporation of the drops.
[0147] FIG. 5 describes the process used to transfer the nuclear
donor cell into the enucleated oocyte. As can be seen from FIG. 5,
the enucleated oocyte (3) was fixed to a holding pipette (1), a
transfer pipette (4) was injected through the small hole into the
enucleated oocyte (3) and, then, the nuclear donor cell was
injected into the oocyte (3) to obtain a nucleus-transferred
oocyte. Such nucleus-transferred oocyte washed three times with the
G1.2 medium and placed in the same medium.
[0148] (2-2) Preparation of Nucleus-Transferred Oocyte by
Electrofusion
[0149] The nucleus-transferred oocyte was subjected to
electrofusion through a BTX-electro cell manipulator (BTX Inc., San
Diego, Calif., U.S.A.).
[0150] A 20 .mu.l drop of a mannitol solution prepared by
dissolving 0.1 mM MgSO.sub.4, 0.05% BSA and 0.28 mM mannitol in a
0.5 mM HEPES buffer (pH 7.2), a 20 .mu.l drop of a mixing solution
containing 10 .mu.l of the G1.2 medium and 10 .mu.l of the mannitol
solution, and a 20 .mu.l drop of the G1.2 medium were prepared.
[0151] First, the nucleus-transferred oocyte obtained in Example
(2-1) was incubated in the 20 .mu.l drop of the mixing solution for
1 minute. Next, the nucleus-transferred oocyte was transferred to
the 20 .mu.l drop of the mannitol solution via a mouth pipette and
incubated therein for 1 minute. Subsequently, the
nucleus-transferred oocyte was transferred to a mannitol solution
having the above composition and placed between two electrodes
connected to the BTX-electro cell manipulator and was positioned
such that the nuclear donor cell faced the (+) electrode. The
nucleus-transferred oocyte was electrofused by applying a direct
current of 1 kV/cm for 15 .mu.s twice, at an interval of 1
second.
[0152] The fused nucleus-transferred oocyte was incubated in the 20
.mu.l drop of the mixing solution for 1 minute, transferred to the
20 .mu.l drop of the G1.2 medium and then washed with the G1.2
medium three times.
EXAMPLE 3
Reprogramming, Activation and In Vitro Culturing of
Nucleus-Transferred Oocyte
[0153] Since a sperm-mediated activation, which is one of the major
factors for a normal embryonic development, was absent in case of
the nucleus-transferred oocyte obtained in Example 2, an artificial
stimulus was needed instead. In order to determine the optimum
conditions for artificial embryogenesis, therefore,
nucleus-transferred oocytes were reprogrammed, activated and in
vitro cultured under various conditions as shown in Tables 2 to
4.
[0154] First, to examine the effect of the reprogramming time on
the rate of blastocyst formation, the reprogramming times were set
at about 2, 4, 6 and 20 hours, respectively, while applying the
same conditions for activation and in vitro culturing as can be
seen from Table 2. As a result, the highest rate of blastocyst
formation was obtained when the reprogramming time was about 2
hours. TABLE-US-00002 TABLE 2 In vitro culture No. of
nucleus-transferred condition oocytes developed to Reprogramming
1.sup.st 2.sup.nd No. of 2-cell blasto- time (hour) Activation
condition medium medium oocytes stage morula cyst 2 10 .mu.M 2.0 mM
G1.2 SNUnt-2 16 16 4 4 ionophore* 6-DMAP 4 10 .mu.M 2.0 mM G1.2
SNUnt-2 16 15 1 0 ionophore* 6-DMAP 6 10 .mu.M 2.0 mM G1.2 SNUnt-2
16 15 1 1 ionophore* 6-DMAP 20 10 .mu.M 2.0 mM G1.2 SNUnt-2 16 9 1
0 ionophore* 6-DMAP *calcium ionophore A23187
[0155] Next, to find the optimal activation condition for
blastocyst formation, nucleus-transferred oocytes subjected to
about 2 hour-reprogramming time were treated for 5 minutes with
calcium ionophore A23187 (5 or 10 .mu.M; Sigma Co., St. Louis, Mo.,
U.S.A.) or ionomycin (5 or 10 .mu.M; Sigma Co., St. Louis, Mo.,
U.S.A.) in the G1.2 medium at 37.degree. C. as can be seen from
Table 3. Such nucleus-transferred oocytes were washed several times
with the G1.2 medium, transferred to the G1.2 medium containing 2.0
mM 6-DMAP (Sigma Co., St. Louis, Mo., U.S.A.) and, then, cultured
at 37.degree. C., 5% CO.sub.2, 5% O.sub.2 and 90% N.sub.2 for 4
hours. After these activation steps, the nucleus-transferred
oocytes were in vitro cultured under the same condition. As can be
seen from Table 3, the highest rate of blastocyst formation was
observed when the oocyte was sequentially treated with 10 .mu.M
calcium ionophore and 2.0 mM 6-DMAP. TABLE-US-00003 TABLE 3 In
vitro culture No. of nucleus-transferred condition oocytes
developed to Reprogramming 1.sup.st 2.sup.nd No. of 2-cell blasto-
time (hour) Activation condition medium medium oocytes stage morula
cyst 2 5 .mu.M 2.0 mM G1.2 SNUnt-2 16 11 0 0 ionophore 6-DMAP 2 10
.mu.M 2.0 mM G1.2 SNUnt-2 16 16 5 3 ionophore* 6-DMAP 2 5 .mu.M 2.0
mM G1.2 SNUnt-2 16 9 0 0 ionomycin* 6-DMAP 2 10 .mu.M 2.0 mM G1.2
SNUnt-2 16 12 0 0 ionomycin 6-DMAP *calcium ionophore A23187
[0156] Finally, the optimal in vitro culture condition was
determined as follows: The nucleus-transferred oocytes subjected to
the above optimal reprogramming and activation conditions were
washed vigorously with the G1.2 medium and cultured for 48 hours in
a 10 .mu.l drop of the G1.2 medium or SNUnt-2 medium at 37.degree.
C. in 5% CO2, 5% O2 and 90% N2 atmosphere. After such culturing,
the nucleus-transferred oocytes were transferred to a fresh SNUnt-2
medium or G2.2 medium and cultured further for 6 days. A
representative example of the in vitro culture medium is the G2.2
medium (Vitro Life, Goteborg, Sweden) comprising Alanine,
Alanyl-glutamine, Arginine, Asparagine, Aspartic acid, Calcium
chloride, Calcium pantothenate, Choline chloride, Cystine, Folic
acid, Glucose, Glutamic acid, Glycine, Histidine, Human serum
albumine, Inositol, Isoleucine, Leucine, Lysine, Magnesium
sulphate, Methionine, Nicotinamide, Penicillin G, Phenylalanine,
Potassium chloride, Proline, Pyridoxal HCL, Riboflavin, Serine,
Sodium bicarbonate, Sodium chloride, Sodium dihydrogen phosphate,
Sodium lactate, Sodium pyruvate, Thiamine, Threonine, Tryptophan,
Tyrosine, Valine and water.
[0157] As indicated in Table 4, the highest rate of blastocyst
formation was detected when the oocyte was first cultured in the
G1.2 medium and subsequently in the SNUnt-2 medium. TABLE-US-00004
TABLE 4 In vitro culture No. of nucleus-transferred condition
oocytes developed to Reprogramming 1.sup.st 2.sup.nd No. of 2-cell
blasto- time (hour) Activation condition medium medium oocytes
stage morula cyst 2 10 .mu.M 2.0 mM G1.2 SNUnt-2 16 16 4 3
ionophore* 6-DMAP 2 10 .mu.M 2.0 mM G1.2 G2.2 16 16 0 0 ionophore*
6-DMAP 2 10 .mu.M 2.0 mM SNUnt-2 SNUnt-2 16 16 0 0 ionophore*
6-DMAP *calcium ionophore A23187
[0158] Based on the above results, an optimal embryogenesis of a
nucleus-transferred oocyte was achieved by subjecting the oocyte to
2-hour reprogramming, activation through a serial treatment with 10
.mu.M calcium ionophore and 2.0 mM 6-DMAP, and a sequential
culturing in the G1.2 medium and the SNUnt-2 medium.
[0159] Under the above optimal conditions, additional 66
nucleus-transferred oocytes were reprogrammed, activated and in
vitro cultured to thereby yield 19 blastocysts (equal to 29%). This
percentage of the nucleus-transferred oocytes developed to
blastocysts in accordance with the present invention is comparable
to those observed in established SCNT methods in cattle (about 25%)
(Kwun et al., Mol. Reprod. Dev., 65:167-174 (2003)) and pigs (about
26%) (Hyun et al., Biol. Reprod., 69:1060-1068 (2003); Kuhholzer et
al., Biol. Reprod., 64:1635-1698 (2004)).
EXAMPLE 4
Removal of Zona Pellucida and Trophoblast, and Isolation of
ICMs
[0160] The blastocyst obtained in Example 3 was treated with 0.1%
pronase (Sigma Co., St. Louis, Mo., U.S.A.) for 1 minute to remove
its zona pellucida. Then, it was treated with 100% anti-human serum
antibody (Sigma Co., St. Louis, Mo., U.S.A.) for 20 minutes, and
was exposed to 10 .mu.l of guinea pig complement (Life
Technologies, Rockville, Md., U.S.A.) at 37.degree. C., 5% CO.sub.2
for 30 minutes to remove its trophoblast and isolate ICMs
therefrom.
EXAMPLE 5
Culturing of ICMs
[0161] The ICMs isolated in Example 4 were cultured in a tissue
culture dish coated with 0.1% gelatin, which contained a feeder
layer (7.5.times.10.sup.4 cells/cm.sup.2) of mitomycin
C-inactivated primary mouse (C57BL breed) embryonic fibroblasts.
DMEM/F12 medium (Life Technologies, Rockville, Md., U.S.A.)
comprising 20% serum replacement, 0.1 mM .beta.-mercaptoethanol, 1%
NEAAs, 2 mM glutamine, 100 units/ml penicillin, and 100 .mu.g/ml
streptomycin, and 4 ng/ml bFGF (Life Technologies, Rockville, Md.,
U.S.A.) was used as the culture medium.
[0162] At an early stage of culturing the ES cells in the ICMs, the
medium was supplemented with a hLIF (100 units/ml; Chemicon,
Temecula, Calif., U.S.A.). The culturing was conducted for more
than 6 days until the colonies of undifferentiated ntES cells
appeared. The ntES cells were mechanically isolated from the
colonies by using a micropipette every five or seven days after
such colony formation.
[0163] The ntES cell line thus obtained from the
nucleus-transferred oocyte prepared by transferring a nucleus of a
female somatic cell into an enucleated human oocyte was designated
"hntES" and deposited with the Korean Cell Line Research Foundation
(KCLRF; Address: Cancer Research Institute, College of Medicine,
Seoul National University, 28, Yongon-dong, Chongno-gu, Seoul
110-744, Republic of Korea) on Dec. 29, 2003 under the accession
number of KCLRF-BP-00092, in accordance with the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure.
TEST EXAMPLE 1
Identification of Human ntES Cells Obtained in Example 5 by
Karyotype Analysis
[0164] The colonies of the undifferentiated ntES cells obtained in
Example 5 were washed with PBS containing 0.1 mM Ca.sup.2+ and 0.1
mM Mg.sup.2+, fixed with citrate-acetone-formaldehyde (the mixing
ratio in volume was 25:65:8) at 4.degree. C. for 1 hour, and washed
again with PBS containing 0.1 mM Ca.sup.2+ and 0.1 mM Mg.sup.2+.
The alkaline phosphatase activity of the ntES cells was determined
by AP kit (Sigma Co., St. Louis, Mo., U.S.A.). Further, an
immunohistochemical assay was performed in order to identify
specific surface antigens on the ntES cells, by employing
monoclonal antibodies Oct-4 (SC-5279) purchased from Santa Cruz
Biotechnology (Santa Cruz, Calif., U.S.A.); SSEA-1 (MC480), SSEA-3
(MC631) and SSEA-4 (MC-813-70) purchased from Developmental Studies
Hybridoma Bank (Iowa City, Iowa, U.S.A.); and TRA-1-60 and TRA-1-80
purchased from Chemicon (Temecula, Calif., U.S.A.) as primary
antibodies. Such primary antibodies were detected by using a
Vectastatin ABC kit (Vector laboratory, Burlingame, Calif., U.S.A.)
containing a biotinylated secondary antibody and an
avidin-horseradish peroxidase conjugate.
[0165] DNA fingerprinting analysis was performed with regard to the
genomic DNA and human short tandem repeat (STR) marker using a STR
AMP FLSTR PROFILER kit (Applied Biosystems, Foster City, Calif.,
U.S.A.) with an automated ABI 310 Genetic Analyzer (Applied
Biosystems, Foster City, Calif., U.S.A.). The results are shown in
FIGS. 6A to 6D.
[0166] As shown in FIGS. 6A to 6D, it was observed that the
karyotype of the ntES cells derived from the nucleus-transferred
oocyte prepared in accordance with Examples 1 to 5 above was
identical to that of the nuclear donor cell. This result
demonstrates that the ntES cells of the present invention have been
indeed derived from the nucleus-transferred oocyte prepared by
transferring a nucleus of a female somatic cell into an enucleated
human oocyte, not from a parthenogenetically activated oocyte.
TEST EXAMPLE 2
Identification of Human ntES Cells by Teratoma Analysis
[0167] 100 colonies of the undifferentiated ntES cells obtained in
Example 5 were isolated from their culture dish, injected into a
testis of a SCID mouse (Korea Research Institute of Bioscience and
Biotechnology, Korea) using a 1 ml syringe and cultured for 8
weeks. Teratomas thus formed were paraffin-fixed and examined by an
immunohistochemical assay to check whether three dermal cells were
formed. The result is shown in FIG. 7.
[0168] As indicated in FIG. 7, it was found that the ntES cells
obtained in Example formed three dermal cells (cartilage (A):
endoderm; intestinal tract (B): mesoderm; neural tube (C):
ectoderm) in the testis. This result demonstrates that such ntES
cells are pluripotent ES cells having the ability to differentiate
into various tissues.
TEST EXAMPLE 3
Examination of Embryoid Body Formation Through Immunohistochemical
Assay
[0169] Colonies of the human ntES cells obtained in Example 5 were
treated with 0.1% trypsin/1 mM EDTA to isolate the ntES cells,
which were then transferred to a plastic petri dish. The human ntES
cells were cultured for 14 days in the DMEM/DMEM F12 medium devoid
of hLIF and bFGF. For paraffin fixation, such ntES cells were
transferred to 1% low-melting temperature agarose dissolved in PBS
and cooled to 42.degree. C. The resulting solidified agarose
containing the ntES cells was fixed by 4% paraformaldehyde
dissolved in PBS and embedded in paraffin. Each 6-mm section of the
paraffin-embedded cells was placed on a slide and subjected to an
immunohistochemical analysis. As primary antibodies,
alpha-1-fetoprotein (18-0003), cytokeratin (18-0234), desmin
(18-0016), neurofilament (18-0171) and S-100 (18-0046) purchased
from Zemed (South San Francisco, Calif., U.S.A.) and HNF-2-alpha
(SC-6556), BMP-4 (SC-6896), Myo D (SC-760) and NCAM (SC-7326)
purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.,
U.S.A.) were employed. A biotinylated anti-rabbit, anti-mouse or
anti-goat antibody was used as a secondary antibody, and the
reaction was detected by streptavidin-conjugated horseradish
peroxidase and diaminobenzidine chromagen. The result is shown in
FIG. 8.
[0170] As shown in FIG. 8, it was confirmed that such ntES cells
could form embryoid bodies based on the fact that the marker
proteins of endoderm (i.e., alpha-1-fetoprotein (A), cytokeratin
(B), and HNF-2-alpha (C)), the marker proteins of mesoderm (i.e.,
BMP-4 (D), Myo D (E), and desmin (F)) and the marker proteins of
ectoderm (i.e., neurofilament (G), S-100 (H), and NCAM (I)) were
expressed in the ntES cells obtained in Example 5. This result
demonstrates that the cells obtained in the present invention fall
within the scope of an ES cell.
EXAMPLE 6
Differentiation into Neuro Progenitors
[0171] (6-1) Expansion of Undifferentiated ES Cells
[0172] The human undifferentiated ntES cells obtained in Example 5
were cultured at 37.degree. C. in 5% CO.sub.2 atmosphere on a mouse
embryonic fibroblast feeder layer with inactivated cell division,
contained in a culture plate coated with 2% gelatin. The culture
medium was composed of DMEM/F12 (1:1), 20% knock-out serum
replacement, 0.1 mM NEAAs, 0.1 mM .beta.-mercaptoethanol, 1 mM
L-glutamine, 100 U/ml penicillin G, 100 .mu.g/ml streptomycin, and
4 ng/ml bFGF; and was changed everyday.
[0173] (6-2) Formation of Embryoid Body
[0174] Colonies of the ntES cells cultured as above were collected
and cultured on a non-adhesive culture dish at 37.degree. C. in 5%
CO.sub.2 atmosphere. The culture medium was identical to that of
Example (6-1) except that 4 ng/ml bFGF was omitted therefrom. After
one day, such colonies began to grow as floating embryoid bodies
(about 50 embryoid bodies/dish). At that point, the embryoid bodies
were transferred to a new dish, while removing any remaining feeder
cells completely. After further culturing for 4 days, embryoid
bodies thus formed were plated on an adhesive dish coated with
polyornithine/laminin.
[0175] (6-3) Selection of Nestin-Positive Cells
[0176] After 1-day culturing on the adhesive dish, embryoid bodies
in the process of differentiation were transferred to the DMEM/F12
medium supplemented with insulin (25 .mu.g/ml), transferrin (100
.mu.g/ml), sodium selenite (30 nM) and fibronectin (5 .mu.g/ml) and
cultured at 37.degree. C. for 6 days. The resulting cells were
cultured at 37.degree. C. for 40 minutes in a solution wherein
anti-nestin antibody (Chemicon, Temecula, Calif., U.S.A.) was
diluted 1000 folds with a solution containing 0.01M PBS, 1% BSA and
5 mM EDTA. Such cells were washed with the DMEM/F12 medium, treated
with phycoerythrine (PE)-conjugated secondary antibody (Chemicon,
Temecula, Calif., U.S.A.) for 30 minutes and then washed three
times with the DMEM/F12 medium, thereby selecting the
nestin-positive cells.
[0177] (6-4) Expansion of Nestin-Positive Cells
[0178] The nestin-positive cells selected in Example (6-3) were
cultured at 37.degree. C. in the DMEM/F12 medium supplemented with
the N-2 supplement, laminin (1 ng/ml) and bFGF (10 ng/ml) for 6
days to expand those cells.
[0179] (6-5) Differentiation into Neuro Progenitors
[0180] The nestin-positive cells expanded in Example (6-4) were
cultured for 10 days at 37.degree. C. in the DMEM/F12 medium
supplemented with the N-2 supplement and laminin (1 ng/ml) but
devoid of bFGF to induce their differentiation into neuro
progenitors.
[0181] FIG. 2 shows the neuro progenitors differentiated from the
nucleus-transferred oocyte prepared by transferring a nucleus of a
female somatic cell into an enucleated human oocyte.
[0182] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made to the invention by those
skilled in the art which also fall within the scope of the
invention as defined by the appended claims.
Indications Relating to Deposited Microorganism or Other Biological
Material
* * * * *