U.S. patent application number 11/939397 was filed with the patent office on 2008-03-13 for isolation of inner cell mass for the establishment of human embryonic stem cell (hesc) lines.
This patent application is currently assigned to RELIANCE LIFE SCIENCES PVT LTD.. Invention is credited to FIRUZA RAJESH PARIKH, Shailaja Anupam Saxena, Satish Mahadoerao Totey.
Application Number | 20080064099 11/939397 |
Document ID | / |
Family ID | 23219502 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064099 |
Kind Code |
A1 |
PARIKH; FIRUZA RAJESH ; et
al. |
March 13, 2008 |
Isolation of Inner Cell Mass for the Establishment of Human
Embryonic Stem Cell (hESC) Lines
Abstract
A method of establishing a cell line from the inner cell mass of
a blastocyst, comprising: isolating a blastocyst having a zona
pellucida, a trophectoderm, and an inner cell mass; creating an
aperture in the blastocyst by laser ablation; isolating cells from
the inner cell mass from the blastocyst through the aperture; and
culturing the cells to establish a cell line, wherein the cells are
cultured under feeder free conditions.
Inventors: |
PARIKH; FIRUZA RAJESH;
(Mumbai, IN) ; Totey; Satish Mahadoerao; (Mumbai,
IN) ; Saxena; Shailaja Anupam; (Mumbai, IN) |
Correspondence
Address: |
VINSON & ELKINS, L.L.P.
1001 FANNIN STREET
2300 FIRST CITY TOWER
HOUSTON
TX
77002-6760
US
|
Assignee: |
RELIANCE LIFE SCIENCES PVT
LTD.
Navi Mumbai
IN
|
Family ID: |
23219502 |
Appl. No.: |
11/939397 |
Filed: |
November 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10226711 |
Aug 23, 2002 |
7294508 |
|
|
11939397 |
Nov 13, 2007 |
|
|
|
60314323 |
Aug 23, 2001 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
A61B 17/435 20130101;
C12N 5/0606 20130101; A61B 18/20 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/04 20060101
C12N005/04 |
Claims
1-20. (canceled)
21. A method of establishing a cell line from the inner cell mass
of a human blastocyst, comprising: (a) isolating a blastocyst
having a zona pellucida, a trophectoderm, and an inner cell mass;
(b) creating an aperture in the blastocyst by laser ablation; (c)
isolating cells from the inner cell mass from the blastocyst
through the aperture; and (d) culturing the cells to establish a
cell line, wherein the cells are cultured under feeder free
conditions.
22. The method of claim 21, wherein the aperture is through the
zona pellucida.
23. The method of claim 21, wherein the aperture is through the
zona pellucida and the trophectoderm.
24. The method of claim 21, wherein the laser ablation is achieved
using a non contact diode laser.
25. The method of claim 24, wherein the non contact diode laser is
a continuous 1.48 .mu.m diode laser.
26. The method of claim 21, wherein cells from the inner cell mass
are isolated by aspiration.
27. The method of claim 21, wherein isolating cells from the inner
cell mass is carried out in the absence of animal generated
antibodies and sera.
28. The method of claim 21, further comprising a micromanipulator
system comprising a microscope with a heating stage, a holding
pipette, an aspiration pipette, and an air syringe, wherein the
isolated blastocyst of step (a) is placed on the heating stage, the
micromanipulator system is adjusted so that the blastocyst is at
the center of the microscope field, and the blastocyst is secured
with the holding pipette by suction through the air syringe, so
that the inner cell mass is opposite the holding pipette.
29. The method of claim 21, wherein the feeder free conditions
comprise an extracellular matrix.
30. The method of claim 29, wherein the feeder free conditions
further comprise conditioned medium.
31. The method of claim 21, wherein the isolated blastocyst is the
product of in vitro fertilization.
32. A method of establishing a human embryonic stem cell line,
comprising: (a) isolating a human blastocyst comprising an inner
cell mass; (b) creating an aperture in the blastocyst by laser
ablation; (c) isolating cells from the inner cell mass of the
blastocyst through the aperture; and (d) culturing the cells under
feeder free conditions to obtain an isolated human embryonic stem
cell line.
33. The method of claim 32, wherein the aperture is through the
zona pellucida and the trophectoderm.
34. The method of claim 32, wherein the laser ablation is achieved
using a non contact diode laser.
35. The method of claim 32, wherein the cells of the inner cell
mass are cultured to produce inner cell mass-derived cell
masses.
36. The method of claim 35, wherein the inner cell mass-derived
cell masses are dissociated and re-plated on an extracellular
matrix.
37. The method of claim 32, wherein cells of the inner cell mass
are isolated by aspiration.
38. The method of claim 32, wherein the human embryonic stem cell
line is established in the absence of animal generated antibodies
and sera.
39. The method of claim 32, wherein the feeder free condition
comprises plating the cells of the inner cell mass on an
extracellular matrix.
40. A method of establishing a human embryonic stem cell line,
comprising: (a) isolating cells of an inner cell mass from a
blastocyst by creating an aperture in the blastocyst by laser
ablation and removing cells of the inner cell mass from the
blastocyst through the aperture; (b) culturing the cells of the
inner cell mass to produce inner cell mass derived masses; and (c)
culturing the inner cell mass derived masses under feeder free
conditions to produce an isolated human embryonic stem cell
line.
41. The method of claim 40, wherein the feeder free conditions
comprise an extracellular matrix.
42. The method of claim 40, further comprising mechanically
dissociating the inner cell mass derived masses of step (b) and
re-plating the mechanically dissociated cells of the inner cell
mass derived masses under feeder free conditions.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S.
application Ser. No. 10-226,711, filed Aug. 23, 2002, U.S. Pat. No.
7,294,508, which claims the benefit of priority of U.S. Provisional
Application Ser. No. 60/314,323 filed on Aug. 23, 2001, the
disclosures of both of which are incorporated herein in their
entirety by reference for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of isolation of
inner cell mass (ICM) derived from blastocyst stage mammalian
embryo for establishing human embryonic stem cell (hESC) lines,
using a non-contact diode laser technique.
BACKGROUND OF THE INVENTION
[0003] The isolation of human stem cells offers the promise of a
remarkable array of novel therapeutics. Biologic therapies derived
from such cells through tissue regeneration and repairs as well as
through targeted delivery of genetic material are expected to be
effective in the treatment of a wide range of medical conditions.
Efforts to analyze and assess the safety of using human stem cells
in the clinical setting are vitally important to this endeavor.
[0004] Embryonic stem (ES) cells are the special kind of cells that
can both duplicate themselves (self renew) and produce
differentiated functionally specialized cell types. These stem
cells are capable of becoming almost all of the specialized cells
of the body and thus, may have the potential to generate
replacement cells for a broad array of tissues and organs such as
heart, pancreas, nervous tissue, muscle, cartilage and the
like.
[0005] Stem cells have the capacity to divide and proliferate
indefinitely in culture. Scientists use these two properties of
stem cells to produce seemingly limitless supplies of most human
cell types from stem cells, paving the way for the treatment of
diseases by cell replacement. In fact, cell therapy has the
potential to treat any disease that is associated with cell
dysfunction or damage including stroke, diabetes, heart attack,
spinal cord injury, cancer and AIDS. The potential of manipulation
of stem cells to repair or replace diseased or damaged tissue has
generated a great deal of excitement in the scientific, medical
and/biotechnology investment communities.
[0006] ES cells from various mammalian embryos have been
successfully grown in the laboratory. Evans and Kaufman (1981) and
Martin (1981) showed that it is possible to derive permanent lines
of embryonic cells directly from mouse blastocysts. Thomson et al.
(1995 and 1996) successfully derived permanent cell lines from
rhesus and marmoset monkeys. Pluripotent cell lines have also been
derived from pre-implantation embryos of several domestic and
laboratory animal species such as bovines (Evans et al., 1990)
Porcine (Evans et al., 1990, Notarianni et al., 1990), Sheep and
goat (Meinecke-Tillmann and Meinecke, 1996, Notarianni et al.,
1991), rabbit (Giles et al., 1993, Graves et al., 1993) Mink
(Sukoyan et al., 1992) rat (Iannaccona et al., 1994) and Hamster
(Doetschman et al., 1988). Recently, Thomson et al (1998) and
Reubinoff et al (2000) have reported the derivation of human ES
cell lines. These human ES cells resemble the rhesus monkey ES cell
lines.
[0007] ES cells are found in the ICM of the human blastocyst, an
early stage of the developing embryo lasting from the 4th to 7th
day after fertilization. The blastocyst is the stage of embryonic
development prior to implantation that contains two types of cells
viz.
[0008] 1. Trophectoderm: outer layer which gives extra embryonic
membranes.
[0009] 2. Inner cell mass (ICM): which forms the embryo proper.
[0010] In normal embryonic development, ES cells disappear after
the 7th day and begin to form the three embryonic tissue layers. ES
cells extracted from the ICM during the blastocyst stage, however,
can be cultured in the laboratory and under the right conditions
proliferate indefinitely. ES cells growing in this undifferentiated
state retain the potential to differentiate into cells of all three
enthryonic tissue layers. Ultimately, the cells of the inner cell
mass give rise to all the embryonic tissues. It is at this stage of
embryogenesis, near the end of first week of development, that ES
cells can be derived from the ICM of the blastocyst.
[0011] The ability to isolate ES cells from blastocysts and grow
them in culture seems to depend in large part on the integrity and
condition of the blastocyst from which the cells are derived. In
short, the blastocyst that is large and has distinct inner cell
mass tends to yield ES cells most efficiently. Several methods have
been used for isolation of inner cell mass (ICM) for the
establishment of embryonic stem cell lines. The most common methods
are as follows:
1. Natural Hatching of the Blastocyst:
[0012] In this procedure blastocyst is allowed to hatch naturally
after plating on the feeder layer. The hatching of the blastocyst
usually takes place on day 6. The inner cell mass (ICM) of the
hatched blastocyst develops an outgrowth. This outgrowth is removed
mechanically and is subsequently grown for establishing embryonic
stem cell lines. However, this procedure has few disadvantages.
First, trophectoderm cells proliferate very fast in the given
culture conditions and many times, suppress the outgrowth of inner
cell mass. Second, while removing the outgrowth of the inner cell
mass mechanically, there is a chance of isolating trophectoderm
cells. Third, the percentage of blastocysts hatching spontaneously
in humans is very low.
2. Microsurgery:
[0013] Another method of isolation of inner cell mass is mechanical
aspiration called microsurgery. In this process, the blastocyst is
held by the holding pipette using micromanipulator system and
positioned in such a way that the inner cells mass (ICM) is at 9
o'clock position. The inner cell mass (ICM) is aspirated using a
bevel-shaped biopsy needle and is inserted into the blastocoel
cavity. This procedure too is disadvantageous as the possibility to
isolate the complete inner cell mass is low and many times cells
get disintegrated. It is a very tedious procedure and may cause
severe damage to the embryo. The operation at the cellular level
requires tools with micrometer precision, thereby minimizing damage
and contamination.
3. Immunosurgery:
[0014] Immunosurgery a commonly used procedure to isolate inner
cell mass (ICM). The inner cell mass (ICM) is isolated by
complement mediated lysis. In this procedure, the blastocyst is
exposed either to acid tyrode solution or pronase enzyme solution
in order to remove the zona pellucida (shell) of blastocyst. The
zona free embryo is then exposed to human surface antibody for
about 30 min to one hour. This is followed by exposure of embryos
to guinea pig complement in order to lyse the trophectoderm. The
complement mediated lysed trophectoderm cells are removed from
inner cell mass (ICM) by repeated mechanical pipetting with a
finely drawn Pasteur pipette. All the embryonic stem cell lines
reported currently in the literature have been derived by this
method. However, this method has several disadvantages. First the
embryo is exposed for a long time to acid tyrode or pronase causing
deleterious effects on embryo, thereby reducing the viability of
embryos. Second, it is a time consuming procedure as it takes about
1.5 to 2.0 hours. (Narula et al., 1996). Third, the yield of inner
cell mass (ICM) per blastocyst is low. Fourth, critical storage
conditions are required for antibody and complement used in the
process. Last, it involves the risk of transmission of virus and
bacteria of animal origin to humans, as animal derived antibodies
and complement are used in the process. In this process, two animal
sera are used. One is rabbit antihuman antiserum and the other is
guinea pig complement sera.
[0015] The human cell lines studied to date are mainly derived by
using a method of immunosurgery, where animal based antisera and
complement was used.
[0016] Other possible disadvantages of the existing cell lines are
as follows:
[0017] 1. Use of feeder cells for culturing the human embryonic
stem cell (hESC) lines produces mixed cell population that require
the Embryonic stem cells (ESC) to be separated from feeder cell
components and this impairs scale up.
[0018] 2. Embryonic stem cells (ESC) get contaminated by
transcripts from feeder cells and cannot be used on a commercial
scale. It can be used only for research purposes.
[0019] Geron established a procedure where human Embryonic Stem
Cell (hESC) line was cultured in the absence of feeder cells (XU
et.al 2001). The hESC were cultured on an extracellular matrix in a
conditioned medium and expanded in this growth environment in an
undifferentiated state. The hESC contained no xenogenic components
of cancerous origin from other cells in the culture. Also, the
production of hESC cells and their derivatives were more suited for
commercial production. In this process, there was no need to
produce feeder cells on an ongoing basis to support the culture,
and the passaging of cells could be done mechanically. However, the
main disadvantage of this procedure is that the inner cell mass
(ICM) is isolated by immunosurgery method, wherein the initial
derivation of Embryonic Stem Cells is carried out using feeder
layer containing xenogenic components. This raises the issue of
possible contamination with animal origin viruses and bacteria.
[0020] In order to simplify the procedure of inner cell mass
isolation and to make it safe, the scientists of the present
invention have come out with a novel method of isolation of the
inner cell mass using a non-contact laser, wherein, the use of
animal based antisera and complement have been eliminated.
Use of Laser Technique in Assisted Reproduction:
[0021] With the advent of assisted reproductive technologies (ART),
several methods have been used for improving fertilization,
facilitating blastocyst hatching (Cohen et al, 1990) and performing
blastomere biopsy (Tarin and Handyside, 1993). Commonly used
methods are chemical (Gordon and Talansky 1986), mechanical
(Depypere et al., 1988) and laser (Feichtinger et al., 1992) so as
to produce holes in the zona pellucida (Gordon, 1988). Recently, an
infrared 1.48 .mu.m diode laser beam focused through a microscope
objective was shown to allow rapid, easy and non-touch
microdrilling of mouse and human zona pellucida and high degree of
accuracy was maintained under conventional culture conditions (Rink
et al., 1994). The drilling effect was shown due to a highly
localized heat-dependent disruption of the zona pellucida
glycoprotein matrix (Rink et al., 1996). Contrary to the
detrimental effect on compacted mouse embryos induced by the 308 nm
xenon-chlorine excimer laser (Neev et al., 1993), the drilling
process in the infrared region did not affect embryo survival in
mice (Germond et al., 1995) or in humans (Antinori et al.,
1994).
[0022] Currently, lasers are being investigated as a tool to aid
fertilization and in assisted hatching. Recent reports show that
use of 1.48 .mu.m diode laser for microdrilling mouse zona
pellucida is highly safe and does not affect neuro-anatomical and
neurochemical properties in mice and also improves fertilization
(Germond et al., 1996). Obruca and colleagues first reported the
success of laser-assisted hatching in human IVF in 1994. In this
study, a 20- to 30-micron hole was made in the zona pellucida (ZP)
when the embryos were at the two- to four-cell stage, and embryos
were transferred immediately. Patients with previous IVF failures
from two separate centers were included in this study. There was a
higher implantation rate per embryo in the laser-assisted hatching
group (14.4%) versus the control group (6%). Pregnancy rates per
transfer were also improved (40% versus 16.2%).
[0023] In a separate study, Er:YAG laser was used to thin the ZP of
embryos derived from patients undergoing repeated IVF. Using a
laser for thinning the ZP, embryologists are able to achieve
accurate reduction of the ZP by 50%, which is very difficult with
acidic Tyrode's solution. Presence of Acid Tyrode's solution near
the embryo may also be detrimental. The rate of clinical
pregnancies in the laser-hatched group was 42.7%, as compared to
23.1% in the control unhatched group. Since this data looked
promising, the indication of laser-assisted hatching was extended.
Women undergoing IVF for the first time yielded 39.6% clinical
pregnancy rate in the laser-treated group versus a 19% rate in the
control unhatched group (Parikh et al 1996).
[0024] During the last decade there has been ongoing research on
the isolation of inner cell mass (ICM), as it is useful in
establishing embryonic stem cell lines which in turn have the
ability to develop into most of the specialized cells in the human
body including blood, skin, muscle and nerve cells. They also have
the capacity to divide and proliferate indefinitely in culture.
[0025] The present invention involves the isolation of inner cell
mass (ICM), using laser ablation technique without undergoing the
cumbersome procedure of immunosurgery. Hence, in the present
invention, the use of animal derived antibodies or sera are
eliminated and the procedure is safe, simple, rapid, and
commercially viable.
[0026] The present invention, obviates the shortcomings associated
with the conventional methods of isolation of inner cell mass
(ICM). The inner cell mass (ICM) isolated by the present invention
is found to be intact without causing any destruction or damage to
the cells. The present invention thus provides a quick reliable and
non-invasive method for isolation of inner cell mass (ICM). It also
completely ruptures the trophectoderm thereby minimizing the
contamination of inner cell mass (ICM), thus ensuring the purity of
inner cell mass (ICM).
REFERENCES
[0027] 1. Antinori S, Versaci C, Fuhrberg P et al (1994). Seventeen
live birth after the use of erbium-yytrium aluminum garnet laser in
the treatment of male factor infertility. Hum Reprod. 9:
1891-1896.
[0028] 2. Cohen J, Elsner C, Kort H et al (1990). Impairment of the
hatching process following IVF in the human and improvement of
implantation by assisting hatching using micromanipulation. Hum
Reprod. 5: 7-13
[0029] 3. Depypere H T, McLaughlin K J, Seamark R F et al (1988).
Comparison of zona cutting and zona drilling as techniques for
assisted fertilization in the mouse. J. Reprod Fertil. 84:
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[0030] 4. Doetschman T, Williams P and Maeda N (1988) Establishment
of hamster blastocyst derived embryonic stem (ES) cell.
Developmental Biology 127: 224-227.
[0031] 5. Evans M J and Kaufman M H (1981). Establishment in
culture of pluripotential cells from mouse embryo. Nature 292:
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[0032] 6. Evans M J, Notarianni E, Laurie S and Moor R M (1990)
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[0033] 7. Feichtinger W, Strohmer H, Fuhrberg P et al (1992).
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[0034] 8. Gordon J W (1988). Use of micromanipulation for
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Microdissection of mouse and human zona pellucida using 1.48
microns diode laser beam: efficacy and safety of the procedure.
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[0037] 11. Germond M, Nocera D, Senn A, Rink A et al (1996).
Improved fertilization and implantation rates after non-touch zona
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laser beam. Hum Reprod. 11: 1043-1048
[0038] 12. Giles J R, Yang X, Mark X and Foot R H (1993).
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[0039] 13. Graves K H and Moreadith R W (1993). Derivation and
characterization of putative pluripotential embryonic stem cells
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[0040] 14. Iannaccone P M, Taborn G U, Garton R L et al (1994).
Pluripotent embryonic stem cells from the rat are capable of
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[0041] 15. Martin G R (1981) Isolation of pluripotent cell lines
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[0043] 17. Narula A, Taneja, Totey S M (1996) Morphological cells
to trophectoderm inner cell mass of in vitro fertilized and
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assisted fertilization and hatching. Hum Reprod. 9:1723-1726.
[0046] 20.Parikh F R, Kamat S A, Nadkarni S et al (1996). Assisted
hatching in an in vitro fertilization program. J Reprod Fertil
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A (2000) Embryonic stem cell lines from human blastocysts: Somatic
differentiation in vivo. Nat Biotechnol. 18:299-304.
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Proceedings SPIE. 2134A, 412-422.
[0049] 23. Rink K, Delacretaz G, Salathe R P et al (1996).
Non-contact microdrilling of mouse zona pellucida with an objective
delivered 1.48 microns diode laser. Lasers Surg Med. 18:52-62.
[0050] 24. Sukoyan M A, Golublitsa A N, Zhelezova A I et al (1992)
Isolation and cultivation of blastocyst derived stem cell lines
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strategies for preimplantation diagnosis. Fertil Steril
59:943-952.
[0052] 26. Thomson J A, Itskovitz-Eldor J, Shapiro S S. et al.
(1998). Embryonic stem cell lines derived from human blastocyst.
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Pluripotent cell line derived from common marmoset blastocyst.
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OBJECTS OF THE INVENTION
[0054] 1. It is an object of the present invention, to develop a
process of isolation of inner cell mass, using laser ablation
technique, without undergoing the cumbersome procedure of
immunosurgery.
[0055] 2. It is another object of the present invention to isolate
ICM using laser ablation technique without using any animal
generated antibodies and sera, thereby preventing the possibility
of transmission of animal organism to human and thus can be used on
commercial scale.
[0056] 3. It is another object of the present invention to isolate
inner cell mass (ICM) from blastocyst stage of a mammalian embryo
using a non-contact diode laser.
[0057] 4. It is another object of the present invention to isolate
inner cell mass (ICM) by simple, shorter and easily feasible way
without affecting/destroying the inner cell mass (ICM).
[0058] 5. It is still another object of the present invention to
ensure the purity of inner cell mass (ICM) by rupturing completely
trophectoderm thereby minimising the contamination of inner cell
mass (ICM).
[0059] 6. It is still another object of the present invention to
isolate inner cell mass (ICM) of high yield and purity as compared
to the inner cell mass (ICM) isolated by the conventional
methods.
[0060] These and other objects of the invention will become more
readily apparent from the ensuing description.
DETAILS OF INVENTION
[0061] The present invention relates to isolation of inner cell
mass, using laser ablation of zona pellucida (ZP) and trophectoderm
(TE) and aspiration of inner cell mass for establishing embryonic
stem cell lines. In the present invention, the non contact diode
laser used is highly accurate and reliable tool for cellular
microsurgery. The system incorporates the latest in fiber optic
technology to provide the most compact laser system currently
available. The 1.48 .mu.m diode non-contact Saturn Laser System is
mounted/implanted via the epifluorescence port to inverted
microscope fitted with micromanipulators. A pilot laser is used to
target the main ablation laser and a series of LEDs inform the user
when the laser is primed and is ready to fire. A
two-second-operation window is used to reduce the possibility of
accidentally firing the laser. The spot diameter of the laser can
be varied according to the hole size required.
[0062] Couples undergoing in vitro fertilization (IVF) treatment
voluntarily donate surplus human embryos. These embryos are used
for research purposes after taking the written, voluntary consent
from these couples. In the present invention, blastocyst stage
embryos are taken for the isolation of inner cell mass. The
blastocyst is placed in a 35 mm petridish in a 50 micro litre
droplets of Ca++/Mg++ free embryo biopsy medium and is covered with
mineral oil. The micromanipulator is set up to perform the embryo
biopsy procedure. The blastocyst is placed in embryo biopsy medium
and the petridish containing the blastocyst is placed on the
heating stage of the microscope. The blastocyst is positioned at
the center of the field. The blastocyst is immobilized on to the
holding pipette in such a way that the inner cell mass is at 3
o'clock position. The zona pellucida and trophectoderm close to
inner cell mass is positioned on the aiming spot of the laser beam.
A small portion of zona pellucida and trophectoderm is laser
ablated. Biopsy pipette is then gently inserted through the hole in
the zona pellucida and trophectoderm and the inner cell mass is
gently aspirated. After isolation of the complete inner cell mass,
the cells are given several washes with embryonic stem cell (ESC)
medium. The cells are then plated on to feeder layer with embryonic
stem cell medium for establishing embryonic stem cell lines. The
embryonic stem cells were then characterized for cell surface
markers such as SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4
and alkaline phosphatase. The embryonic stem cell lines are also
karyotyped.
a) Development of Blastocyst in Vitro:
[0063] Institutional Ethics Committee approval has been obtained
before initiation of this study. Prior written consent was taken
from individual donor for the donation of surplus embryos for this
study after completion of infertility treatment.
[0064] Protocol generally used for infertility patients for
obtaining viable embryo is as follows:
[0065] The ovarian superovulation began with gnRH agonist analog
suppression daily starting in the mid-luteal phase and administered
in doses of 500-900 mgs for about 9-12 days. Ovarian stimulation
was started after adequate ovarian suppression with human
menopausal gonadotropins (hMG) or recombinant follicle stimulating
hormone (FSH) (Gonal-F, Recagon) in appropriate doses depending on
the age and ovarian volume. The dose was also adjusted as necessary
to produce controlled ovarian stimulation. Serum beta-estradiol
(E2) measurements were carried out as required. Vaginal ultrasound
was performed daily from cycle day 7 onward. Human Chorionic
gonadotropin 5000-10000 I.U. was administered when three or more
follicles were at least 17 mm in largest diameter. Transvaginal
aspiration was performed 34-36 h later. Oocytes were then subjected
to intracytoplasmic sperm injection.
[0066] A glass holding pipette 40-60 .mu.m in diameter was used to
secure the egg. Motile sperm were placed in a drop of polyvinyl
pyrolidone (PVP) solution and overlaid with mineral oil. An
injection needle with an outer diameter of roughly 5-6 .mu.m and
inner diameter 3-4 .mu.m was used to pierce the zona pellucida at
about 3 o'clock position. The selected spermatozoon was immobilized
by cutting the tail with the injection micropipette. The holding
pipette secured the oocyte and spermatozoon was injected directly
into the center of the oocyte.
[0067] Oocytes were checked after 16-18 hours of culture for
fertilization. At this point the fertilized oocyte had pro-nuclei
(also called one cell embryo). One-cell embryos were then
transferred into pre-equilibrated fresh ISM-1 medium and incubated
at 37.degree. C. in a 5% CO.sub.2 in air. The next day embryos were
transferred into ISM-2 medium. Every alternate day embryos were
transferred into fresh ISM-2 medium. From day 5 onward embryos were
checked for the blastocyst development. After the treatment is
over, the surplus blastocysts were donated by the couples for this
research work.
b) Setting up of the Laser:
[0068] The present invention relates to describing a unique method
for inner cell mass isolation for establishment of embryonic stem
cells using the non-contact diode laser. The laser is highly
accurate and reliable tool for cellular microsurgery. The system
incorporates the latest in fiber optic technology to provide the
most compact laser system currently available. The 1.48 .mu.m diode
non-contact Saturn Laser System was mounted via the epifluorescence
port to Zeiss inverted microscope fitted with
micromanipulators.
[0069] A pilot laser was used to target the main ablation laser and
a series of LED's, inform the user when the laser is primed and
ready to discharge the laser beam. A two-second-operation window
was used to reduce the possibility of accidentally firing the
laser. The spot diameter of the laser can be varied according, to
the ablation size required.
c) Laser Ablation and Isolation of inner Cell Mass.
[0070] The blastocyst stage embryo was individually placed in a 50
.mu.l drop of biopsy medium (Ca.sup.++/Mg.sup.++ free) in a 35-mm
petri dish. The embryo was immobilized on to the holding pipette in
such a way that the inner cell mass remained at 3 o'clock position
and the zona pellucida and trophectoderm close to inner cell mass
positioned on the aiming spot. A continuous 1.48 .mu.m diode laser
was used to aperture the Zona Pellucida (ZP), which is a
glycoprotein layer protecting the oocyte. At this wavelength, the
hole was induced by a local thermo-dissolution of the glycoprotein
matrix. Once the zona pellucida was dissolved, trophectoderm cells
were ablated by giving 3 pulses to cause photolysis. After ablation
of both zona pellucida and trophectoderm, the aspiration pipette
was introduced through laser-ablated hole and ICM was removed by
gentle aspiration, having an internal diameter of 30-35
microns.
d) Culturing of Human Embryonic Stem Cells (hESC)
[0071] Prior to culturing, the aspirated ICM was washed thoroughly
in ES medium, which medium was found to be preferred for isolation
of embryonic stem cell lines. Given below is the procedure when the
invention was carried out using feeder layer. In this process, the
inner cell mass was cultured in 96 well plate in the presence of
mouse inactivated embryonic fibroblast feeder layer. Embryonic
fibroblast feeder layer was preferably obtained from 12.5 to 13.5
day old C57BL/6 mice or C57L/6XSJL F-1 mice or out bred CD1 mice or
from human amniotic fluid-and used as a feeder layer. Embryonic
fibroblast feeder layer was inactivated by gamma irradiation (3500
rads). The mouse embryonic fibroblast feeder layer was cultured on
0.5% gelatin coated plate with ES medium consisting of Dulbecco's
modified Eagle's medium without Sodium pyruvate with high glucose
contain (70-90%), Fetal bovine serum (10-30%), beta-mercaptoethanol
(0.1 mM), non-essential amino acids (1%), L-Glutamine 2 mM, basic
fibroblast growth factor (4 ng/ml). Inner cell mass was then plated
on mouse inactivated embryonic fibroblast. After 4-7 days, ICM
derived masses were removed from outgrowth with sterile fire
polished pipette and were dissociated mechanically and plated on
fresh feeder cells. Further dissociation was carried out with 0.5%
trypsin-EDTA supplemented with 1% chicken serum.
[0072] Established cell lines were karyotyped and characterized for
several surface markers such as SSEA-1, SSEA-3, SSEA-4, OCT-4,
Alkaline phosphatase, TRA-1-81, TRA-1-60 as described by Thomson et
al., (1998), Reubinoff et al., (2000).
EXAMPLES
[0073] The following examples are intended to illustrate the
invention but do not limit the scope thereof.
Example 1
[0074] Total of 24 blastocyst stage human embryos were used for the
isolation of inner cell mass. Embryos were washed several times in
blastocyst culture medium (ISM-2 medium, Medicult, Denmark).
Individual blastocyst was then placed in the 50 .mu.l drop of
Ca++/Mg++ free embryo biopsy medium (EB 10 medium, Scandinavian).
Micro drops were covered with mineral oil. Micromanipulator was set
up. A glass holding pipette with outer diameter 75 .mu.m and inner
diameter 15 .mu.m was used to secure the embryo. Biopsy pipette
with an outer diameter of roughly 49 .mu.m and inner diameter 35
.mu.m was used for aspiration of inner cell mass. A pilot laser was
used to target the main ablation laser. Embryo was immobilized on
to the holding pipette in such a way that inner cell mass remained
at 3 o'clock position and the zona pellucida and trophectoderm
close to inner cell mass positioned to the aiming spot. The hole
was induced by a local thermo-dissolution of the zona.
Trophectoderm cells were ablated by giving 3 pulses to cause
photolysis. After ablation of both the zona pellucida and
trophectoderm, the biopsy pipette was introduced through laser
ablated hole and inner cell mass was removed. Inner cell mass was
then washed several times in ES medium and placed in 96 well dish
in the presence or absence of feeder cells. The following data is
presented in the tabular form. TABLE-US-00001 TABLE 1 Summary of
hESC lines developed using Laser ablation Technique of the present
invention with the use of mouse feeder cells. With mouse feeder
cells No. of blastocysts used Total inner cell No. of No. of ES
cell lines for laser ablation mass removed ICM used established 24
18 14 4
[0075] Similarly, an experiment was conducted with conventional
method of isolation of inner cell mass i.e. using immunosurgery and
may be reported as follows:
Example 2
[0076] The objective was to determine efficiency of isolation of
inner cell mass with conventional method, i.e. immunosurgery, and
compared with newly invented laser ablated method.
[0077] Twenty-one blastocyst stage human embryos were used for
isolation of inner cell mass. Embryos were washed several times
with blastocyst culture medium (ISM-2 medium) and followed by ES
medium. Individual blastocyst stage embryo was then placed in 50
.mu.l microdrops of 1:50 anti-human antibody (Sigma) for 30 minutes
at 37.degree. C. and 5% CO.sub.2 in air. Blastocyst stage embryos
were then washed four times after incubation with ES medium.
Blastocysts were then again placed in 50 .mu.l of microdrops of
guinea pig complement at the concentration of 1:10 for 10 minutes
at 37.degree. C. and 5% CO.sub.2 in air. After incubation
blastocyst stage embryos were washed several times in ES medium
using fine bore glass pipette in order to remove trophectoderm.
Isolated inner cell mass was then washed with ES medium and
cultured in 96 well plate in the presence or absence of feeder
cells. Data are presented in the table: TABLE-US-00002 TABLE 2
Summary of hESC lines developed using immunosurgery with/without
the use of mouse feeder cells. With mouse feeder Without mouse No.
of cells feeder cells blastocyst Total No. No. No. used inner of of
ES of No. of ES for laser cell ICM cell lines ICM cell lines
ablation mass removed used established used established 21 14 12 3
2 0
[0078] Although the isolation of inner cell mass using both the
methods did not show any significant difference, however, of
isolation of inner cell mass by laser ablation has distinct
advantage. This method will eliminate the use of antibodies and
sera of animal origin. Isolation of inner cell mass by laser
ablation method can be further cultured in the presence or absence
of feeder layer. However, culturing of inner cell mass in a feeder
free condition will further eliminate the possibilities of
contamination of ES cell lines with animal viruses or bacteria and
can be commercially utilized for human transplantation studies. In
the current experiments, efforts were made to establish ES cell
line in the absence of feeder cells.
[0079] A preferred embodiment of the invention is illustrated in
the accompanying drawings.
[0080] FIG. 1(a) to 1(g) of the present invention, pertains to the
isolation of inner cell mass (ICM) from the blastocyst of one
embryo and FIG. 2(a) to 2(g) pertains to the isolation of inner
cell mass (ICM) from the blastocyst of another embryo. FIGS. 3, 4,
and 5 pertains to culturing of ICM on feeder cells at different
stages.
[0081] FIG. 1 (a) is a scanned image of human blastocyst, secured
with glass holding pipette such that the ICM is at 3 o'clock
position.
[0082] FIG. 1 (b) is a scanned image wherein part of zona pellucida
and trophectoderm ablated with laser (arrow).
[0083] FIG. 1 (c) is a scanned image of aspiration pippette close
to the blastocyst following zona and trophectoderm ablation.
[0084] FIG. 1 (d) is a scanned image of beginning of aspiration of
ICM with aspiration pipette.
[0085] FIG. 1 (e) is a scanned image of large portion of ICM in the
aspiration pipette during aspiration process.
[0086] FIG. 1 (f) is a scanned image of the ICM after removing from
the blastocyst.
[0087] FIG. 1 (g) is a scanned image of the remaining trophectoderm
and zona pellucida remaining after ICM isolation.
[0088] FIG. 2 (a) is a scanned image of another human blastocyst,
secured with glass holding pipette such that the ICM is at 3
o'clock position.
[0089] FIG. 2 (b) is a scanned image of slight protrusion of inner
cell mass after zona and trophectoderm is laser ablated.
[0090] FIG. 2 (c) is a scanned image of the aspiration pipette
being position close to the ICM after ablating the zona and
neighboring trophectoderm cells with laser.
[0091] FIG. 2 (d) is a scanned image of ICM being aspirated with
the aspiration pipette by gentle suction.
[0092] FIG. 2 (e) is a scanned image of large portion of ICM in the
aspiration pipette.
[0093] FIG. 2 (f) is a scanned image of the ICM after removing from
the blastocyst.
[0094] FIG. 2 (g) is a scanned image of the trophectoderm and zona
pellucida left after the isolation of ICM from the blastocyst.
[0095] FIG. 3 (a) is a scanned image of isolated inner cell mass in
culture seeded on primary mouse embryonic fibroblast feeder cells
(day 3).
[0096] FIG. 3 (b) is a scanned image of isolated inner cell mass in
culture on primary mouse embryonic fibroblast feeder cells (day
7).
[0097] FIG. 4 is a scanned image of isolated ICM in culture on the
primary mouse embryonic fibroblast feeder cells (day 5) another
embryo.
[0098] FIG. 5 is a scanned image of embryonic stem cell line
derived from inner cell mass isolated by laser ablation method.
[0099] One skilled in the art will appreciate that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned therein above. The instant invention
has been shown and described herein in what is considered to be the
most practical and preferred embodiment. It is recognized that
departures may be made from within the scope of the invention. It
is to be understood that the invention is not limited to the
particulars disclosed and extends to all equivalents within the
scope of the scope of the claims.
* * * * *