U.S. patent application number 15/729038 was filed with the patent office on 2018-04-05 for replication of undifferentiated cells in a weightless environment, uses thereof and facility for such replication and the acceleration of the evolution of plants and animals.
The applicant listed for this patent is Zero Gravity Solutions, Inc.. Invention is credited to John Wayne Kennedy.
Application Number | 20180094237 15/729038 |
Document ID | / |
Family ID | 41449368 |
Filed Date | 2018-04-05 |
United States Patent
Application |
20180094237 |
Kind Code |
A1 |
Kennedy; John Wayne |
April 5, 2018 |
REPLICATION OF UNDIFFERENTIATED CELLS IN A WEIGHTLESS ENVIRONMENT,
USES THEREOF AND FACILITY FOR SUCH REPLICATION AND THE ACCELERATION
OF THE EVOLUTION OF PLANTS AND ANIMALS
Abstract
Manufacturing processes are described for biological replication
of undifferentiated plant and animal cells and tissue in a
weightless condition, including those systems used in current stem
cell research and development and use of undifferentiated
parenchyma in plants. Additionally, methods for adapting plants and
animals to survive outside their native environments are described.
In particular, undifferentiated cells from plants or animals are
replicated under weightless conditions in which cell replication or
proliferation is accelerated and sustained. Under such conditions,
the undifferentiated cells can be "forced" to express sets of genes
useful for survival in particular environmental conditions. In this
manner, cells surviving prolonged exposure to specific
environmental conditions can be selected for and cultivated to
produce an organism adapted to that particular environment in an
accelerated manner. Methods of identifying specific genes
associated with adaptation of a plant or animal to a specific
environment are also described.
Inventors: |
Kennedy; John Wayne;
(Stevensville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zero Gravity Solutions, Inc. |
Boca Raton |
FL |
US |
|
|
Family ID: |
41449368 |
Appl. No.: |
15/729038 |
Filed: |
October 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13183004 |
Jul 14, 2011 |
9816071 |
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15729038 |
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12473973 |
May 28, 2009 |
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13183004 |
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PCT/US2007/085821 |
Nov 28, 2007 |
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12473973 |
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PCT/US2009/034286 |
Feb 17, 2009 |
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12473973 |
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60867582 |
Nov 28, 2006 |
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61029053 |
Feb 15, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0602 20130101;
C12N 5/04 20130101; A01K 67/0273 20130101; A01K 67/00 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 5/04 20060101 C12N005/04; A01K 67/00 20060101
A01K067/00; A01K 67/027 20060101 A01K067/027 |
Claims
1. A method of adapting a plant to grow in a hostile environment,
the method comprising: providing replicating parenchyma cells to
produce a cell suspension culture comprising a plurality of
undifferentiated parenchyma cells; culturing the cell suspension
culture while simultaneously exposing the cell suspension culture
to both a microgravity condition and a stimuli corresponding to the
hostile environment to which the plant is to be adapted; and
harvesting cells that replicate during the step of culturing,
wherein the culturing simultaneously exposes the cell suspension
culture to both the microgravity condition and the stimuli
corresponding to the hostile environment to cause the plurality of
undifferentiated parenchyma cells to express genes that enable the
harvested cells to adapt to the hostile environment; and
sequentially removing the harvested cells from the microgravity
condition and cultivating the harvested cells to produce a mature
plant.
2. The method of claim 1 further comprising evaluating said mature
plant.
3. The method of claim 2, wherein said mature plant is evaluated
for at least one of length of survival, growth rate, reproductive
capability, cell structure, gene expression, or combinations
thereof.
4. The method of claim 1, wherein the parenchyma cells are obtained
from uniting a pollen and ovule of a plant prior to exposure to the
microgravity condition.
5. The method of claim 1, wherein the hostile environment is
selected from the group consisting of heat, cold, low barometric
pressure, excessive radiation, high carbon dioxide levels, low
oxygen levels, low humidity, high humidity, extreme salinity,
reduced or increased exposure to sunlight, and low water
conditions.
6. The method of claim 1, wherein the parenchyma cells are obtained
by uniting a pollen and an ovule in the microgravity condition,
wherein said parenchyma cells obtained from said union do not
develop into differentiated cells.
7. The method of claim 1, further comprising steps of: examining a
gene expression profile of the harvested cells in comparison to the
gene expression profile of control cells; and identifying genes
that have a change in expression level as compared to the gene
expression profile of the control cells; wherein said identified
genes are associated with adaptation to the hostile
environment.
8. The method of claim 7, wherein the change in expression level is
at least four fold in comparison to the expression profile of the
control cells.
9. A method of adapting a plant to grow in a cold environment, the
method comprising: providing replicating parenchyma cells to
produce a cell suspension culture comprising a plurality of
undifferentiated parenchyma cells; culturing the cell suspension
culture comprising the plurality of undifferentiated parenchyma
cells while simultaneously exposing the cell culture to both a
microgravity condition and a cold environment to which the plant is
to be adapted; and harvesting cells that replicate during the step
of culturing, wherein the culturing simultaneously exposes the cell
suspension culture to both the microgravity condition and the cold
environment, to cause the plurality of undifferentiated parenchyma
cells to express genes that enable the harvested cells to adapt to
the cold environment, where the plant is not a citrus plant.
10. The method as recited in claim 9, wherein the cold environment
is a temperature of about 25.degree. F. or less.
11. The method of claim 10, further comprising the sequential step
of (1) removing the harvested cells from the microgravity condition
and (2) cultivating the harvested cells to produce a mature
plant.
12. A method of adapting a plant to grow in a hostile environment,
the method comprising: providing replicating parenchyma cells to
produce cell suspension culture comprising a plurality of
undifferentiated parenchyma cells; culturing the cell suspension
culture comprising the plurality of undifferentiated parenchyma
cells while simultaneously exposing the cell culture to both a
microgravity condition and a hostile environment to which the plant
is to be adapted, wherein the hostile environment is selected from
the group consisting of heat, cold, low barometric pressure,
excessive radiation, high carbon dioxide levels, low oxygen levels,
low humidity, high humidity, extreme salinity, reduced or increased
exposure to sunlight, and low water conditions; and harvesting
cells that replicate during the step of culturing, wherein the
culturing simultaneously exposes the cell culture to both the
microgravity condition and the hostile environment to cause the
plurality of undifferentiated parenchyma cells to express genes
that enable the harvested cells to adapt to the hostile
environment, wherein said harvested cells do not develop into
differentiated cells during the step of culturing; sequentially
removing the harvested cells from the microgravity condition and
cultivating the harvested cells to produce a mature plant.
13. The method of claim 12, further comprising a step of excising
the parenchyma cells from a cellular sample, the step of excising
occurring prior to the step of replicating.
Description
BACKGROUND OF THE INVENTION
[0001] A weightless condition on space orbit has produced many
effects on visitors to that region. Gravity still is present on
orbit and through the course of interplanetary travel, but normal
plant and animal functions as is known on Earth do not function in
the same manner. If gravity acts as a dominant force in a
weightless condition, then the reproduction of cells would follow
the normal or close to normal pattern experienced on Earth.
Evidence indicates that it is weightlessness and not gravity that
drives the biology of both plants and animals on space orbit and
during interplanetary transits and human travel.
[0002] Weight is a physical attribute and force parameter. Weight
is a condition wherein the "escape force" [as a function of the
angular velocity of a mass (angular momentum)] around a
gravitational mass is less than the specific value of the
gravitational force. That is, when a body is at rest on the Earth
(stationary), it has an angular velocity due to the Earth's
rotation. This angular velocity opposes, to some extent, the
gravitational force that draws the mass to the center-of-gravity
point of Earth. As the angular velocity of the mass increases, the
inertial force that opposes the gravitational force increases, to
the extent that a sufficient increase will result in the
gravitational force equaling the inertial force.
[0003] This is the case for masses that have been launched from
Earth and are in an orbit of equilibrium around the Earth, i.e. "on
orbit". If the Earth had no angular velocity (i.e.--it did not
rotate), the weight of a mass would be greater than for a mass on a
rotating Earth. It should be noted that the Earth's rotation has
only a small effect on "gravity" (actually on "weight"), about
0.5%.
[0004] Weight is a condition that results from either (a) the
presence of a mass within a gravitational field and/or (b) a mass
that is subjected to an external force that accelerates that mass
(inertial gravity). Weight, as described in (a) or (b), results
from a "mass-acceleration" force so universal and common on Earth
that it is normally perceived as an ever-present attribute, state,
or condition and is not included in many patent process and
protocol parameter descriptions. "Weight" is a normal condition and
physical parameter that affects "Living Systems" on Earth.
Perceived weightlessness is experienced on orbit or in orbiting
spacecraft that have reached a constant velocity on orbit or
en-route to interplanetary destinations.
[0005] Biological organisms, and specifically most higher-ordered
biological organisms, including plants, animals, including humans,
hereafter referred to as "High Order Living Biological Systems"
(HOLBS) are adapted to Earth's gravity. The effects of
weightlessness on plants and animals are expressed by physiological
effects that alter the physiology and the morphology of the HOLBS,
causing deleterious, irreversible, compromising, and transmuting
effects from exposure to such conditions. For example, microgravity
has been shown to have an impact on an astronaut's body in space.
The effects of gravity in plants and animals and the biological
mechanisms involved in adapting to weightlessness may be studied
under real microgravity conditions. For example, research on
astronauts has shown that body function is disturbed in
microgravity. Space agencies are therefore continuing their
research in hope of eventually reducing or eliminating some of
these undesirable physical effects that appear during a stay in
space.
[0006] Generally, exposure of living systems to weightlessness
results in biological degradation. This degradation is a result of
biological processes that have been fundamentally altered due to
the absence of an essential force, gravity that is an essential
component of those biological processes. Prolonged exposures to a
weightless environment correlate to increased biological
alterations and degradations. It is the attribute of
"weightlessness" or "an apparent absence of gravity induced-force"
and/or the "absence of inertially-produced force" that is the
critical physical parameter which affects physiological process of
living systems in a weightless environment.
[0007] On-orbit environments, e.g., as would be found on the
International Space Station (ISS), are referred to as zero-g,
zero-gravity, and gravity-free environments. These terms are
misleading and incorrect. The term "weightless" is more correct and
can be equated to "micro-gravity" for our purpose.
[0008] The Earth's "gravitational field," whether in LEO, GEO or
other orbits (600 km altitudes, etc.), is still present, i.e.--90+%
of the gravitational field and gravitational force amplitude
remains. More precisely--static, on-orbit environments exhibit
"weight-free" conditions, wherein gravity forces remain substantial
as a consequence of a continuing presence of Earth's gravitation
field. For example: [0009] On Earth at Sea Level: Assumption:
Gravity=1.00, Weight=1.0. [0010] On Orbit* at 600 km: Then
Gravity=0.98, Weight=0.0. *Static environment: zero local
acceleration.
[0011] All mass, including the space vehicle and pay load will be
at zero weight on a earth bound scale that measures "weight".
However, it should be noted that gravity is still present. The
force of gravity between a spacecraft and Earth is directly
proportional to the product of their masses and indirectly
proportional to the square of the distance between them.
Acceleration of a mass into orbit overcomes the force of gravity
and the mass will enter what is considered to be a "free-fall"
effect. The mass in orbit may be a combination of many objects
(masses) that appear to be weightless in relation to the other
masses in an apparent weightless "free-fall" environment. Gravity
forces are still present, but the HOLBS are not able to function
properly without a force that mimics gravity.
[0012] As noted above, HOLBS exhibit marked physiological and
biological changes when resident on-orbit, e.g., given that this
environment is where Gravity=0.95-1.00 and Weight=0.00-0.10, it is
concluded that it is the attribute of "weightlessness" that links
these activities and processes, and that it may appear to be
constant without regard to gravity.
[0013] More specifically and to further clarify, it may be stated
that on Earth, all higher-order living systems, plants, animals,
including humans, proceed with biological processes under the
influence of a "constant acceleration of their mass." On Earth,
this constant acceleration is a result of Earth's gravity, and the
endless, largely constant, angular acceleration associated with
Earth's gravitational force. It is recognized that there are slight
(=<0.5%) variations in gravitational forces and Earth rotation
angular velocities that occur depending upon the location and
region the mass is on the Earth.
[0014] On orbit, in a static environment (no apparent inertial
acceleration is present and angular velocity is relatively
constant), near-weightlessness (commonly referred to as
zero-gravity or micro-gravity) conditions are achieved. These terms
are misleading and the terms should be: zero-weight or micro-weight
or some equivalents. The term "weightlessness" is being used herein
to collectively refer to these conditions. It is therefore evident
that it is the attribute of "weightlessness", not gravity, that is
critical to active biological processes and components of plants,
animals, humans, and higher-order living systems.
[0015] Furthermore, it is evident that commonly referred to
conditions such as "hyper-gravity," are misnomers, as the term has
been associated with the forces resulting from the use of
centrifuges that create "artificial gravity", when in actuality,
they produce centripetal, angular acceleration forces that are more
accurately "inertia produced angular accelerations," and may be
viewed as or termed "inertial gravity" or more accurately
"hyper-weight". Linear accelerations also apply here: in the form
of artificial gravity, especially on long space flights in the
acceleration and de-acceleration phases. In summary, an angular (or
linearly) induced acceleration of a mass will result in a force
upon that mass that causes that mass to possess "weight". Weight is
an attribute of acceleration of mass (evidenced elementarily by its
unit of measure being in--meters/per second/per second).
[0016] Thus, it is submitted that a linkage exists between
mass-acceleration (as a mass-energy function) and mass-density (as
a mass function), and that the linkage of these two attributes has
specific direct and indirect effects upon certain biological
processes that occur in plants and animals, including humans and
higher-order living systems (HOLBS), where these effects impact
functions of cellular replication, reproduction, regeneration,
creation, differentiation, specialization, function, cell
life-span, suspended animation, and cell death.
[0017] It is evident that specific, fundamental biological
processes possessed by plants, animals, humans and higher-order
organisms and living systems and their growth, development, and
life-cycles are affected by weightlessness, and, that these living
processes differ critically and profoundly when those processes
occur in a "weight" (mass-accelerating) environment versus a
"weightless" (mass-zero-accelerating) environment. Therefore it is
submitted that it is the attribute or characteristic of
"weightlessness" that determines and assures and drives certain
essential plant, animal (including humans) and higher-living system
processes and not the presence of gravity, nor the presence of a
gravitational field or its effects.
[0018] Single celled organisms such as bacteria reproduce with
little differentiation unless there is a mistake in the normal
cells development that may produce a new strain. Bacteria can
reproduce at amazing rates. Bacteria are protected by a cell wall
that surround the protoplasm. The asexual reproduction of the
bacteria continues until regulated by outside forces. Weightless
conditions do not appear to be a factor in the reproduction of
single celled bacteria over at least one generation.
[0019] The replication of undifferentiated cells from both plants
and animals may also follow the same model as the yeast if proper
preparation, transport and retrieval process are followed for the
production of undifferentiated cells on orbit.
[0020] Experiments and the data derived from visitors to
weightlessness indicate that weightlessness plays a role in the
development of human cells. See e.g., Longnecker et al., ed.,
"Review of NASA's Longitudinal Study of Astronaut Health," Jan. 20,
2004, The Institute of Medicine, which is herein incorporated by
reference in its entirety. The Earth's gravitational force
influences the developing cells to differentiate into specialized
cells, which in plants may be branches or roots and in animals the
brain or the legs. In weightlessness, the differentiation of the
primordial cells in both plants and animals cannot occur. The
continuous reproduction of the primordial tissues will continue
with the right nutrient system producing undifferentiated cells in
both plants and animals until terminated.
[0021] There are techniques that work on a limited basis for both
plants and animals in a gravity environmental condition. The
constraints are enormous and only a limited amount of primordial
cells are available. The use of a manufacturing process that can
produce an unlimited supply (based on logistics) of
undifferentiated plant and animal cells can be achieved using zero
gravity during the replication of undifferentiated tissues. The
present invention recognizes the effect of weightlessness on animal
and plant cells and utilizes this recognition to provide a method
for replicating large quantities of undifferentiated cells for both
animals and plants. Additionally, the present invention comprises a
space-based on-orbit manufacturing facility for creation,
replication, production, storage and ultimate transport of
undifferentiated stem cells from a weightlessness environment to a
gravity environment on Earth or at another facility. Currently used
space vehicles can be used for the replication of stem cells and
undifferentiated plant cells.
[0022] The plant studies described herein and conducted on the
space transportation system (STS)-118 mission demonstrate cell
replication rather than cell differentiation and that the cells
replicating in space demonstrate a greater mass than the control
cells on Earth, thereby demonstrating replication of
undifferentiated cell in weightlessness. These results allow a
further expansion of the basic logic to include the acceleration of
gene expression in undifferentiated cells on orbit and using the
protocol discussed herein to provide for a acceleration of the
natural selection process for plants and animals.
[0023] The accelerated and sustained proliferation of
undifferentiated cells on orbit provides an opportunity to "force"
the cells to express genes that will enable them to adapt to
specific environmental conditions. In other words, imposing
environmental constraints on the undifferentiated cells while they
are proliferating will result in a subset of cells expressing the
necessary genes which enable the cells to survive in that specific
environmental condition. One can then cultivate an organism from
these selected cells that will survive in that particular
environmental condition. For example, undifferentiated cells from a
species of citrus plant can be propagated at cooler temperatures on
orbit. Cells surviving the cool temperature conditions can be
returned to earth and cultivated to produce a citrus plant than can
thrive in cold temperature environments.
[0024] These techniques are applicable to both plant and animal
cells. "Hardy" organisms can be produced by selecting
undifferentiated cells expressing genes for survival in particular
environments, including extreme environments, such as the surface
of Mars. The present invention recognizes the advantages of
replicating primordial cells on orbit, and utilizes these
advantages to expand the range of cell function thus accelerating
the evolution of organisms. Therefore, the present invention
provides methods for adapting plants and animals to survive outside
their native environments.
SUMMARY OF THE INVENTION
[0025] In one aspect, the present invention provides a method of
using at least one undifferentiated cell as a result of uniting two
haploid cells to form a diploid single cell under conditions to
support fertilization in a weightless condition or environment,
wherein said the diploid cell can replicate itself continuously to
provide a mass of undifferentiated cells.
[0026] In one embodiment, the present invention comprises uniting a
sperm and egg from an animal or pollen and ovary from a plant to
form a single cell in a weightless condition.
[0027] In another embodiment, the present invention further
comprises uniting two haploid cells, such as a sperm and egg from
an animal or pollen and ovary from a plant to form a single cell in
a weightless condition or environment, and culturing the single
cell to replicate in a weightless condition or environment.
[0028] In yet another embodiment, the present invention
additionally comprises uniting two haploid cells, such as a sperm
and egg from an animal or pollen and ovary from a plant to form a
diploid single cell in a gravity condition or environment, such as
under normal Earth gravity, after unification or fertilization is
complete, immediately preserving cooling to below metabolic
activity) the resulting single cell so that no replication of the
single cell occurs, then under weightless conditions, reviving the
single cell, and culturing the single cell in a weightless
condition or environment to obtain replication of the single
cell.
[0029] In yet another embodiment, the present invention also
comprises a diploid single cell, such as a fertilized egg or single
cell produced by uniting a sperm and an egg in an animal or pollen
and ovary from a plant under conditions to support fertilization in
a weightless environment or condition.
[0030] In one embodiment, the present invention further comprises
one or more undifferentiated diploid cells produced by uniting two
haploid cells, such as a sperm and an egg from an animal or pollen
and ovary from a plant under conditions to support fertilization
resulting in a fertilized egg or diploid single cell in a
weightless environment or condition and by culturing the fertilized
egg in a weightless environment or condition resulting in the
replication of the fertilized egg so that one or more
undifferentiated cells are replicated.
[0031] In another embodiment, the present invention further
comprises the use of already undifferentiated tissues from liver,
kidney, etc. of animals (including humans) properly preserved
(cooling, etc.) for transport to on orbit for replication.
[0032] In yet another embodiment, the present invention encompasses
the use of undifferentiated plant tissues for the purpose of
replication of the undifferentiated cells on orbit en masse.
[0033] In another aspect, the present invention additionally
comprises a method of administration of the undifferentiated cells
to a subject or patient in need of treatment by the
undifferentiated stem cells, wherein the cells are produced by
uniting two haploid cells, such as a sperm and an egg from an
animal of the same blood type to support replication of the same
tissues or from replication of the undifferentiated tissues
produced on orbit.
[0034] In some embodiments, the present invention additionally
comprises known manufacturing, processes, biological processes,
cellular, organism, organ, and living-systems development,
creation, fabrication, harvesting, growth, replication,
reproduction, in-vivo, in-vitro, or equivalent activities,
suspended animation processes, development actions, or processes
that are undertaken in a weightless environment, or more
specifically in a mass-acceleration environment.
[0035] In another aspect, the present invention provides structures
and processes for on-orbit vehicles, systems, spacecraft, space
ships, and related habitats and containments used to transit to
and/or travel to distant destinations, wherein those structures and
processes resident or part of those structures are configured in a
fashion, such that they will lower, reduce, mitigate, or eliminate
the biological degradation of plants and animals (including humans)
in space, in weightless environments, on-orbit, during
inter-planetary transits or excursions, and when resident on sub-G
environments (Moon, Mars, etc.).
[0036] In yet another aspect, the present invention provides
methods of adapting a plant or animal to grow in a hostile
environment or an environment outside of the plant's or animal's
native environment. In a preferred embodiment, the method comprises
culturing undifferentiated cells from a plant or animal in a
weightless condition that mimics at least one element of the
hostile or non-native environment to which the plant or animal is
to be adapted; selecting the cells that replicate in said
condition; and cultivating said selected cells to produce plants or
animals, wherein the plants or animals are adapted to grow in said
hostile environment.
[0037] In one embodiment, the method further comprises evaluating
the resultant plants or animals in the hostile environment. The
organisms may be evaluated on various criteria including length of
survival, growth rate, reproductive capability, cell structure,
hardiness in hostile or non-native environments and other gene
expressions including but not limited to those enumerated
above.
[0038] Various environmental stimuli can be used in the methods of
the invention to induce the adaptation of the plant or animal to
the hostile environment. These stimuli may include excessive heat,
excessive cold, low barometric pressure, excessive radiation, high
carbon dioxide levels, low humidity, high humidity, drought
conditions and duration of sunlight exposure and other
environmental factors to mimic conditions in a climate other than
the present native climate of the plant or animal.
[0039] The present invention also provides methods of identifying
genes associated with adaptation of a plant or animal to a hostile
environment. In one embodiment, the method comprises culturing
undifferentiated cells from the plant or animal in a weightless
condition that mimics at least one element of the hostile
environment to which the plant or animal is to be adapted;
selecting the cells that replicate or proliferate in said
condition; examining the gene expression profile of the selected
cells in comparison to the gene expression profile of control
cells; and identifying genes that have a change in expression
level, wherein the identified genes are associated with adaptation
to the hostile environment. The selected genes that are
differentially expressed in the various environmental conditions
can be further used to produce transgenic plants and animals with
the desired adaptive characteristics by introducing these genes
into cells that mature into plants or animals. Through this
process, the adaptation and/or evolution of plants and animals can
be accelerated.
[0040] In another embodiment, the method can be used for the
production of vaccines to be used in animals and humans. By
stressing pathogenic microorganisms on orbit, modified strains of
microorganisms can be produced. These can include less virulent
and/or more virulent strains of bacteria and viruses, which can
then be utilized for the production of improved vaccines.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1. A 3-dimensional on-orbit manufacturing facility
useful for manufacturing stem cells and stem cell tissues in a
weightless environment.
[0042] FIG. 2. A robotic processing system for on-orbit
manufacturing of plant and animal undifferentiated cells useful in
the present invention.
[0043] FIG. 3. Gene expression profiles of Arabidopsis thaliana
seedlings on orbit and on Earth. The gene expression pattern from
orbit is plotted versus the gene expression pattern on Earth (panel
A). Panel B depicts a subset of the gene expression profile to
illustrate heat-shock and CAB (light) genes. The lines labeled with
4.times. and 10.times. illustrate four-fold and ten-fold threshold
levels of expression, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention provides manufacturing processes for
biological reproduction of plants and animals in a weightless
condition, including those systems used in current stem cell
research and development and use of undifferentiated parenchyma in
plants.
[0045] In one embodiment, the present invention provides a method
of replicating at least one undifferentiated cell comprising
uniting two haploid cells to form a diploid single cell under
conditions to support fertilization in a weightless condition or
environment, wherein said diploid single cell can replicate itself
forming another diploid cell and wherein said diploid single cells
will not develop into an embryo of differentiated cells and tissue.
The two haploid cells may be gametes, and more specifically the
gametes are of the opposite sex or mating types.
[0046] The present invention includes the use of the purest form of
undifferentiated tissues of organs such as, but not limited to
liver and kidneys that are replicated on orbit. The present
invention comprises uniting a sperm and egg from an animal or
pollen and ovule from the ovary from a plant to form a single
diploid cell, such as zygote, in a weightless condition or
environment as defined herein. The single cell will not divide to
ultimately produce differentiated cells with the capability of
developing into an embryo when created or produced or cultured
under a weightless environment. Then the replication of cells from
newly united sperm and egg or pollen and ovary in a weightless
condition provides an endless supply of undifferentiated cells and
tissues that can be used to reproduce cells that are identical to
the parent cells without transformation into multi-celled,
differentiated tissues. One or both of the haploid cells utilized
in the method may contain an incomplete haploid genome of the cell,
and could be missing genetic material or genes or fragments
thereof.
[0047] The present invention further comprises uniting two haploid
cells, such as a sperm and egg from an animal or pollen and ovary
from a plant to form a single diploid cell in a weightless
condition or environment, and culturing the single cell to
replicate into undifferentiated or primordial cell and tissue in a
weightless condition or environment. The method further comprises
culturing the diploid single cell to obtain multiple replications
of the replicated single cells to a suspension of undifferentiated
cells. The method further comprises harvesting the undifferentiated
cells from the suspension. Additionally, the method further
comprises changing the culture conditions of the suspension or
harvested undifferentiated cells to a state of suspended animation
or quiescence.
[0048] The present invention further intends to encompass a method
of producing or replicating at least one undifferentiated cell
comprising uniting two haploid cells to form another diploid single
cell under conditions to support fertilization in a gravity
condition or environment under normal earth gravity, after
unification is complete, preserving the resulting diploid single
cell so that no replication of the diploid single cell occurs,
reviving the diploid single cell under a weightless condition or
environment, and culturing the diploid single cell in culture
medium in a weightless condition or environment to obtain
replication of the diploid single cell that will not develop into
an embryo of differentiated or primordial cells and tissue. The two
haploid cells may be gametes, and more specifically the gametes are
of the opposite sex or mating types. Further, the gametes are a
sperm and an egg from an animal or a pollen and an ovule from an
plant. The diploid single cell can be a zygote. Additionally, at
least one of the haploid cells comprises an incomplete haploid
genome of said cell.
[0049] The method further comprises culturing the diploid single
cell to replicate in a weightless condition or environment.
Further, the method further comprises culturing the diploid single
cell to obtain multiple replications of the replicated singles
cells to a suspension of undifferentiated cells. The method further
comprises harvesting the undifferentiated cells from the
suspension, and optionally, changing the culture conditions of the
harvested undifferentiated cells to a state of suspended animation
or quiescence. Or alternatively, the method further comprises
changing the culture conditions of the suspension of
undifferentiated cells to a state of suspended animation or
quiescence. Also encompassed by this method, is an isolated diploid
single cell produced by the methods described herein. This single
cell can be a zygote. Additionally, a composition comprising more
than one single cell produced by the methods described herein is
disclosed that is optionally mixed with a pharmaceutically
acceptable carrier.
[0050] The present invention further comprises uniting two haploid
cells, such as a sperm and egg from an animal or pollen and ovule
from the ovary of a plant to form a single diploid cell in a
weightless condition or environment, and culturing the single cell
to replicate into undifferentiated or primordial cell and tissue in
a weightless condition or environment.
[0051] The method further comprises culturing the diploid single
cell to obtain multiple replications of the replicated single cells
to a suspension of undifferentiated cells. The method further
comprises harvesting the undifferentiated cells from the
suspension. Additionally, the method further comprises changing the
culture conditions of the suspension or harvested undifferentiated
cells to a state of suspended animation or quiescence.
[0052] The present invention also comprises a fertilized egg or
single cell produced by uniting a sperm and an egg in an animal or
pollen and ovary from a plant under conditions to support
fertilization in a weightless environment or condition. An isolated
diploid single cell produced by the method described herein, and
wherein the diploid single cell comprises a zygote which can be
replicated without development into further stages.
[0053] The present invention further comprises a method producing
at least one undifferentiated cell comprising uniting a sperm and
egg of an animal prior to culturing in a weightless condition or
environment to produce undifferentiated cells that replicate at a
higher rate than in a weight or gravity condition or
environment.
[0054] Also encompassed by the present invention is a composition
comprising more than one single cells (diploid) of produced by the
method described herein, and optionally a pharmaceutically
acceptable carrier. Such a diploid single cell comprises a zygote
that will not progress into a further stage of development unless
prompted to do so.
[0055] The present invention further comprises one or more
undifferentiated cells produced by uniting a sperm and an egg from
an animal or pollen and ovary from a plant under conditions to
support fertilization resulting in a fertilized egg or single cell
in a weightless environment or condition and by culturing the
fertilized egg in a weightless environment or condition resulting
in the replication of the fertilized egg so that one or more
undifferentiated cells are produced or replicated.
[0056] The present invention additionally comprises a method of
administration of an effective amount of the undifferentiated cells
produced by the methods of the present invention to a subject or
patient in need of treatment by the undifferentiated cells, wherein
the cells are produced by uniting two haploid cells, such a sperm
and an egg from an animal or pollen and ovary from a plant under
conditions to support fertilization to for a fertilized egg or
single cell in a weightless environment or condition and by
culturing the fertilized egg in a weightless environment or
condition resulting in the replication of the fertilized egg so
that one or more undifferentiated cells are produced or replicated.
The subjects treated may be in need of undifferentiated cells to
treat diseases and conditions, such a type I diabetes, Parkinson's
disease, Alzheimer's disease, blood diseases, such as leukemia,
different types of anemia, systemic lupus, autoimmune diseases and
deficiencies, heart tissue repair, bone and cartilage repair, eye
and skin repair to name a few of the conditions and diseases that
can benefit from the undifferentiated stem cells produced by the
present invention. The stem cells will be administered utilizing
known methods presently used for the administration of adult stem
cells to patients. Known pharmaceutically acceptable carriers can
be mixed with the composition comprising the undifferentiated stem
cells produced by the methods of the present invention under
weightlessness conditions or environments to form a pharmaceutical
compositions comprising the undifferentiated stem cells and
optionally a pharmaceutically acceptable carrier.
[0057] The present invention additionally comprises manufacturing
processes, biological processes, cellular, organism, organ, and
living-systems development, creation, fabrication, harvesting,
growth, replication, reproduction, in vivo, in vitro, or equivalent
activities, suspended animation processes, development actions, or
processes that are undertaken in a weightless environment, or more
specifically in a mass-acceleration environment wherein the
mass-velocity is near-constant and the mass-acceleration is
near-constant and is of a value which is equal and opposite to the
force-value of gravity present.
[0058] The present invention further comprises processes,
manufacturing, development, prototyping, activities and actions
that are directed to the development, replication, reproduction,
growth, maturation, or creation of new cellular materials, tissues,
cellular components, organs, living systems, and suspended
animation technologies--specifically when those activities are in a
near weightless or fully weightless condition.
[0059] The present invention also comprises a culture device for
replicating a diploid cell produced by the methods disclosed herein
and a method for manufacturing, processing, storing (including
suspended animation) and shipping cells created by the unification
of animal (sperm and egg) and plant (pollen and ovary) for the
purpose of replication of primordial also known as undifferentiated
cells and tissues, including, but not limited to animal, such as
human, stem cells and plants (undifferentiated tissues such as
apical meristems), and other tissues, specifically being
methodologies that utilize processing environments, wherein, the
forces of weigh are absent, specifically near-weightless and
fully-weightless environments and processing conditions.
[0060] The present invention additionally comprises those
structures and processes for on-orbit vehicles, systems,
spacecraft, space ships, and related habitats and containments used
to transit to and/or travel to distant destinations, wherein those
structures and processes resident or part of those structures are
configured in a fashion, such that they will lower, reduce,
mitigate, or eliminate the biological degradation of plants and
animals (including humans) in space, in weightless environments,
on-orbit, during inter-planetary transits or excursions, and when
resident on sub-G environments (Moon, Mars, etc.).
[0061] All of the manufacturing processes, biological process, and
related actions and activities conducted on Earth, are subject to
and include a "mass-acceleration" component. A forcing component
that results in a condition or characteristic known as
"weightlessness" is encompassed by the present invention.
[0062] Gravity is not the direct force or condition that drives
biological processes in a weightless environment. Gravity on Earth,
for example, is the condition that results in forces that drive the
biological universe on Earth. Gravity is not absent on-orbit.
Substantial gravitational fields exist on-orbit and in space. The
gravity on orbit is approximately 95% the gravity on Earth and
approximately 0% weight. The rules regarding reproduction of cells
change in a weightless condition. Both plant and animal cells do
not replicate in a weightless condition according to the rules on
Earth. On Earth, gravity is a condition that ties to specific
forces that govern the reproduction of cells.
[0063] Gravity is a condition which may contribute to
"weightlessness" or "weight" depending upon the presence of other
forces, i.e.--other conditions, energies, or forces which result in
the acceleration of mass present, and which may add to or oppose
the effects of mass-acceleration that result from the presence of
mass within a gravitational field. In a weightless condition (where
the acceleration of mass is generally equal to and opposite the
force that exists from gravitational conditions), gravity is a
null-component of the system and does not have a unique, essential,
critical, or driving effect upon the biological processes described
and claimed herein.
[0064] To clarify the present invention with respect to gravity,
gravity forces and gravitational fields, it is recognized that
gravitational fields and forces exist to varying degrees and at
varying amplitudes on Earth, in the Earth, at altitudes above the
Earth, on-orbit around the Earth, between Earth and Moon, and
inter-planetary in the Solar System and beyond. The gravitational
field forces in these regions vary from approximately 1 G on Earth,
to 0.4 G, to 0.6 G on Mars, etc., and at other varying amplitudes
throughout the Solar System. It is recognized that these
gravitational force scalars (values) remain, whether or not other
inertial forces are present. It is recognized that gravitational
forces at any particular point within the Solar System, vary
temporally given the motions of the planets and heavenly bodies
present within the System. It is recognized that gravitational
forces present on local systems, e.g.--Earth, Moon, Mars, are
relatively constant and that local gravitational field amplitudes
in these point regions are relatively constant in amplitude over
time.
[0065] The present invention recognizes that an essential and
critical constellation of processes associated with the
reproduction of cells is uniquely and profoundly different in a
weightless condition, than where those processes are conducted or
performed or executed in an environment where (a) gravity that
results in mass-acceleration exists, (b) on Earth, (c) in
artificial gravity environments [i.e.--inertial gravity
environments e.g.--centrifuges, so-called hyper-gravity, more
correctly termed hyper-weight) systems, units, devices, or
facilities.] The nature of all reproduction processes are
influenced by gravity and cells continue to replicate under the
influence of the gravitational force, following a genetic code
prescribed in the genes that respond to the force of gravity. A
fertilized egg replicates, and begins forming unions and congregate
into a mass that expands exponential fashion (1, 2, 4, 8, etc.) to
eventually form an embryo, then differentiated cells and tissue.
The present invention is different from the development of the
cells in the gravity environment, in that the weightless conditions
result in the replication of cells to form cultures of
undifferentiated cells that do not develop into an embryo. In
weightless conditions, a fertilized egg can be replicated
indefinitely to form cultures of undifferentiated cells that do not
develop into an embryo.
[0066] Methods of preparing such undifferentiated cell cultures in
weightless environments and their use are described herein. Plant
experiments on the STS-118 mission demonstrated that plants
replicate in weightlessness and since both plants and animals
follow the Kreb's Cycle, animal cells similarly replicate in
weightlessness.
[0067] The present invention further comprises the biological and
living-system processes, manufacturing, cellular, replication,
reproduction, creation, duplication, harvesting, development,
maturation, and growth, processing storage (including suspended
animation and shipping) that are conducted or performed, whether
through human or autonomous-control, in (process) environments that
have zero-acceleration of mass in orbit or in interplanetary travel
and/or where the acceleration of mass is equal and opposite the
gravity induced force present.
[0068] To further clarify those process environments where there is
no mass-acceleration force present and/or where the acceleration of
mass is equal and opposite the gravity induced force present, no
mass-acceleration force is included as part of the biological,
physiological, replication, reproduction, growth, harvesting,
manufacturing, or production operation on orbit or in
interplanetary travel unless artificially produced.
[0069] The present invention further comprises all "replication"
processes where plants and/or animals including humans, and
higher-order organisms and living systems "HOLBS" and the cellular
processes of which they are based-upon duplicate their cellular
constituents, most specifically and critically, those cellular
aspects and types which are primordial in structure and biological
configuration.
[0070] The present invention comprises the "replication processes"
as contrasted to normally occurring "reproductive processes" in
orbit or during interplanetary travel. Wherein those cellular
activities that progress through normal maturation stages and
cycles are known as "reproductive" on Earth, however, on orbit,
cellular activities that "duplicate" and "replicate" copies of
cells that are identical in structure and function to their
originating predecessors is "replication" and the process, storage
(including suspended animation) and shipping is encompassed by the
present invention.
[0071] Wherein, specifically, the creation of, processing, and
manufacturing of "primordial stem cells" from animals,
undifferentiated parenchyma from plants and from other primordial
tissues are central to the present invention, and where these
processes are defined and termed as being "replication", not
reproductive.
[0072] The present invention further comprises processes that occur
or are performed in zero-acceleration of mass environments and/or
where the acceleration of mass is equal and opposite the gravity
induced force present. In one embodiment, the present invention
comprises processes where the production of "undifferentiated
tissues, stem cells--of any stage or type or maturation, of
primordial cells, of zygotes, sperm, egg, pollen, are part of said
processes, and particularly wherein the objective is the
manufacture, creation, growth, development, harvesting or
production of these cellular masses, tissues, organisms, and/or
similar living systems.
[0073] The present invention comprises processes that enable the
"replication" of a single cell from newly united sperm and egg or
pollen and ovary in a weightless condition that under continued
weightless conditions will provide an endless supply of
undifferentiated cells or tissues that can be used to reproduce
cells that are identical to the parent cells without transformation
into multi-celled tissues or differentiated tissues.
[0074] In animals and plants, the sexual stage of the organism is
one celled for the male (sperm or pollen) or female (egg or ovary).
Both the sexual stage of plants and animals are also protected by
cell walls. Such cells can be transported into orbit without
interference for use in the replication phase of undifferentiated
cells. The selection of the cells being used for replication must
be conducted with great care to ensure the germ plasma will be a
match with the use intended. For example, matching blood types from
a donor cell with the blood types of an eventual recipient is a
criteria. The plant and animal single-celled male and female sexual
stages are also single celled and are not effected by gravity in
limited experimentation. Formation of a zygote occurs after the
union of the egg and sperm and exists up until the point that the
cell replication begins. It is at this stage that the cells in
either plants or animals can differentiate into any part of the
plant or animal.
[0075] Furthermore, the present invention comprises all
manufacturing processes for biological replication and reproduction
of undifferentiated cells of both plants and animals in a
weightless condition.
[0076] The present invention also comprises all processes and
systems used in current stem cell research, development,
processing, and manufacture, but with the additional step or
condition of performing or conducting these processes in a
"weightless" (zero-acceleration of mass) environment as defined in
the present invention and as understood by persons skilled in the
art of gravitational theories and sciences, and/or where the
acceleration of mass is equal and opposite the gravity induced
force present.
[0077] By way of further clarification, the above-described
invention comprises all stem cell, cellular, tissue, biological
entity, and living organism processes and processing and all
processes and process patents for same, applied for, pending,
filed, and issued that predicate their disclosed inventions on
processes that are conducted either (a) on Earth or (b) in an
environment wherein there is an energy, force, or equivalent
process that includes a significant "mass-accelerating" component
or free-fall effect as previously described. However, the present
invention distinguishes its embodiments from these known processes
by performing all of the steps and processes including cultivation
of or culturing the resulting replicated cells in a weightless
environment. In other words, the disclosed or known process
elements, contents and activities are conducted in an environment
where the constituents (cells, tissues, organs, organisms) have
weight (and where the origins or causes of that weight may be from
either gravitational or inertial energy sources), such as performed
on Earth in its gravitational environment.
[0078] In a further embodiment, the present invention comprises the
use of undifferentiated parenchyma in plants and undifferentiated
stem cells in animals as a unique process component for plant and
animal replication for mitigation of diseases or arthropods.
[0079] Biological systems concerning the replication of cells
adjacent to reproductive tissues in mature animals or plants in a
weightless condition do not follow the rules for a gravity
condition, but rather follow a non-descript process that provides
for replication of primordial tissues without differentiating into
a conglomeration of cells to form a plant or animal. Space visitors
have reproduction of cells that mimic but do not follow the normal
reproduction into a functional cell. The primordial cells adjacent
to bone, muscle, nerve and other types of differentiated cells and
the mass of cells that produces blood and hormones, do not function
as in a gravity or other force field (accelerated mass), and form
non-functional cells. Space visitors experience this effect and
indicate that cell reproduction is not normal. It is believed that
the cells being reproduced are undifferentiated with minimal
influence from the adjoining tissues with a result being
non-functional tissue.
[0080] The primary cell to be used for stem cell replication is
formed by the union of a sperm and egg in animals and can be
conducted in a weightless (in orbit) environment. In plants, the
apical meristem cells (undifferentiated parenchyma) resulting from
the union of pollen and an ovary in a weightless condition can
provide an unlimited supply of identical cells that do not
differentiate at the time of replication. The present invention is
intended to encompass all processes currently used and known by
persons skilled in the art for the reproduction of plants and
animals in a gravity driven environment for use to replicate cells
at the telophase stage of mitosis in a weightless condition.
[0081] The present invention is based on the premise that the
biological systems including plants, animals, humans, and
high-order living systems function in accordance with a newly
coined term "K-Law," which states that primordial cells and living
tissues will "replicate," which is defined as producing an exact
copy in a "zero-acceleration of mass" environment. The "K-Law"
states that primordial cells formed in weightlessness will
self-replicate, but will not proceed to the next step of cellular
and tissue replication resulting in cellular differentiation into
recognizable plants or animals, i.e.--progressive maturation and
differentiation along a recognizable life-cycle path. Stem cell
staging, for example, will progress from primordial tissues to
adult stem cells, but will not combine with other stem cells to
form a zygote.
[0082] With regard to the present invention, the "K-Law" is that
which is defined by the conditions wherein primordial cells, in a
zero-acceleration of mass environment are allowed to develop and
that said development is not reproductive, but rather these cells
only replicate. The "K-Law" applies and is effective only in
"zero-acceleration of mass" environments, or where there is a
"net-zero-acceleration of mass" and/or where the acceleration of
mass is equal and opposite the gravity induced force present. The
present invention further encompasses all processes wherein the
"K-Law" applies.
[0083] Furthermore, the present invention comprises those processes
wherein the primary cell formed by the union of a sperm and egg (in
animals, including man) that are processed, produced or created in
a weightless environment, including manufacturing, handling,
storage (including suspended animation) and shipping of the final
product. And wherein, the process for plants is that which employs
the apical meristem cells (undifferentiated parenchyma) resulting
from union of pollen and an ovary in a weightless condition, such
that the result is an unlimited supply of identical cells that do
not differentiate at the time of replication into specialized cells
and tissue.
[0084] The present invention also comprises all processes currently
used and known for the reproduction of plants and animals in a
gravity driven environment for use to replicate cells at the
telophase stage of mitosis in a weightless condition, storage
(including suspended animation), handling and shipping of the final
product. Further, primordial cells created or produced as a result
of the unification of an egg and sperm that have or have not been
compromised by another force (example, Earth's gravity) will
reproduce identical cells (given mistakes in genetic code) in a
weightless environment. Additionally, primordial cells resulting
from apical meristem tissue or from the germination of a seed
and/or new tissues from another source (new supplies) will
reproduce the same undifferentiated cells during mitosis in a
weightless environment. The present invention encompasses all known
processes that are Earth bound to reproduce cells at the telophase
stage of mitosis for the purposes of stem cell replication and/or
other purposes but for use in a weightless environment.
[0085] The present invention further comprises all Earth bound
processes for the replication and manufacturing of undifferentiated
tissues from plants and/or animals or both for use in a weightless
and/or reduced "gravity" condition. Such conditions include the
replication of undifferentiated tissues on the Moon, Mars, etc., or
other reduced gravity conditions.
[0086] Additionally, the present invention also comprises an
orbiting craft, for example, Space Station, to provide a weightless
condition. Gravitational forces are still present on such a craft.
However, the biological process is not chained to gravity; but the
condition of weightlessness. The extensive literature describes the
maladies suffered by space visitor, especially in prolonged stays.
The present invention provides a unique method of transfer of
plants and animals from a weightless condition to a device that
mimics the force of gravity on Earth and back again to
weightlessness. This method of producing a condition that mimics
gravity in an orbiting vehicle will allow animals and plants to
survive long periods of space flight.
[0087] Long-term visitors to weightlessness are afflicted by many
deficiencies during the stay in this environment, especially
"wasting" of tissues on a Bell Curve--the longer the stay, the more
"wasting" of the body and functions. Exercise machines, etc. have
been incorporated into procedures to "condition" visitors to the
weightless environment. All have failed to a lesser or greater
degree. A "Wheel" circulating around a central unit was impractical
because the whole system would rotate. However, the present
invention provides a "Clutch" system in which the hub of the system
will be at a weightless condition, a secondary "Clutch" that can be
stopped at the hub (weightless condition), allowing personnel
and/or equipment to be off-loaded and then gain speed to the
circulating "Wheel" at intervals to allow for proper balance. The
present invention comprises such a vehicle comprising the above
described "Wheel" and "Clutch" that will allow for weightlessness
experimentation and/or allow for long periods of orbital and/or
space travel to distance objective (Mars, etc.). The "clutch" and
wheel can be composed of composite section that can be transported
to orbit and assembled. The "clutch" and wheel can also be
constructed of puncture-proof inflatable composites that can be
transported to orbit and inflated in two sections for the "clutch"
and the wheel. (attachment). The number of "spokes" in the wheel
can vary, but weight distribution and balance must be of
concern.
[0088] The present invention is also intended to encompass a system
to be used to develop artificial gravity while still maintaining a
weightless condition for the reproduction of undifferentiated
tissues of both plants and animals.
[0089] The present invention further comprises the system as
described can be "mated" with the current space station that can
act as a haven for the crew of the Space Station. A universal
"mating" system to allow transfer of supplies, personnel and
experiments will be incorporated. The space tourism possibilities
could also utilize the use of the "clutch" and wheel concept,
especially if the space visitor will be in orbit for some time.
[0090] By way of further explanation of the present invention,
replication of cells in a weightless environment will not reproduce
in a normal fashion because a gravitational force must be present
to align the components of the primordial cells. For example,
germinating seeds on Earth align the roots to go down and the stem
to go upward due to the force of gravity. Newly united sperm and
egg form zygotes. The present invention is premised upon the theory
that cells that are not influenced by a force that is required to
properly align the components of a reproductive system will not act
according to known understanding of common cell replication. Cells
that replicate according to the laws that govern replication will
be replaced by a new set of rules (K Law) based on a "weightless"
condition that allows a primordial cell to reproduce itself over
and over again.
[0091] Space visitors have suffered numerous maladies in direct
proportion to the time spent in orbit. The visitors are
experiencing the result of primordial cell replication adjacent to
existing cells where the "new" cells are dysfunctional because
there is no force, such as gravity or an artificial gravity-like
force, to provide alignment according to the "gravity" instruction.
Cells adjacent to muscles, bone, etc. will attempt to reproduce the
same cells, but instead produce a cell the looks like the original
cell, but does not function properly because the gravity like force
was not there to align the components of the cell in proper fashion
(K Law). The cells that replicate in space visitors are primordial
in nature, and cannot provide tissues adjacent to the tissue (bone,
muscle, nerve, blood cells etc.) which resemble the same tissues in
a Earth environment. Instead the cells produced in a weightless
environment will follow the instructions of the adjacent cell, but
not reproduce totally viable cells that function as the cells would
if on Earth. The references sited attest to the maladies that our
space visitors have encountered and are consistent with the (K
Law).
Primordial or Undifferentiated Replication in Weightlessness
[0092] However, the bad news can be the good news, and knowing the
problem will allow a solution. As described herein, in a weightless
condition, both plants and animals, cells can replicate to the
telophase stage of mitosis and remain undifferentiated. Therefore,
a weightless condition allows the production of undifferentiated
cells that can be harvested en masse. Without the influence of a
gravitational force, it is submitted that cells do not replicate
properly beyond the telophase stage of mitosis to form a plant or
animal. Cells at the telophase or final stage of mitosis in a
weightless condition only replicate the original cell at the
telophase stage. Cells produced to the telophase stage of mitosis
can be harvested on a continuous basis and immediately stored in a
state of suspended animation for transport back to Earth or an
out-post where a suitable condition exists for reproduction.
Space Visitors
[0093] In a weightless environment, formation of cells adjacent to
tissues in a space visitor (red blood cells, bone, muscle, etc.)
will form the same cell adjacent to the parent cell, but will be
dysfunctional. A different system, but an analogy, is a cell or
cells in the body that become a different, but similar cell,
because of lack of sufficient oxygen and because of not being
oxygenated properly, the cells become anaerobic. Those cells follow
a fermentation process and do not follow the "Krebs" cycle, and
although the cells will form, they will not be functional. There is
a high probability that those cells can become cancerous.
[0094] Cells adjacent to normally reproducing tissues (skin, bone,
etc.) in a space visitor in a weightless condition produce
undifferentiated cells that would produce normal cells by
instructions by the adjacent tissue and gravity. However, a
weightless environment will produce cells that have all the genetic
make-up of the adjacent cells (muscle, skin, etc.), but will not be
functional.
[0095] The basic problem with the reproduction of the primordial
cells for commercial purposes of both plant and animals has been a
system that compensates for replication of the multi-celled higher
plants and animals, but will not replicate cells that reach only
mitosis (union of the egg and sperm to formation of a reproductive
cell) regardless of the process used. Production of the primordial
or undifferentiated cells in both plants and animals in a
weightless environment can produce the same tissues during mitosis
present in the original union of egg and sperm in animals or
plants, and possibly the extraction of apical meristem resulting in
the germination of a seed in zero gravity and use of the
undifferentiated parenchyma cells.
[0096] Current procedures cannot reproduce progeny of the
originally united single cell at mitosis. There is a need to
develop a process of manufacturing of the primordial cells also
known as undifferentiated cells into a source of germ plasma for
plants or stem cells for animals (including man). The force of
gravity has prevented the establishment of a process that would
allow the primordial tissue for replication of both plants and
animals in a more efficient and productive manner. Currently the
only source of cells at the telophase stage of mitosis is the
harvesting of fetus and associated tissues, including the cord
tissue and blood from miscarriages obtained from hospitals, clinics
and other facilities.
[0097] A proposed method is presented herein for reproduction of
the cells in mitosis in both multi-celled plants and animals in a
weightless environment. The methods used for the reproduction of
the cells at the telophase stage of mitosis will be adjusted as
necessary by persons skilled in the art, but taken from current
technology with modifications to adjust for a weightless condition
or reduced gravity, including possible simplification of the
process. Gravitational forces may govern the reproduction of
animals in a weightless condition, but this fact has provided the
opportunity for the replication of cells during mitosis is
weightlessness.
[0098] The scientific literature describes the differences in the
success in the reproductive rate, and more specifically, the
longevity of individuals that live at sea level vs. the tallest
mountains (note: the criteria is longevity, not mortality). The
data only suggests that gravity may be a factor in the reproduction
of the human species. A greater success rate for reproduction of
the human species is indicated by a greater gravitational force. An
overlay of the greatest population centers coincides with those
regions with the highest gravity. Gravity on Mars may present a
problem for reproduction of the human species as well since the
humanoid is not adapted to the reduced gravitational force.
[0099] The understanding of cell reproduction in zero gravity
provides a further benefit beyond the union of "egg and sperm" in
animals and the union of pollen (or equivalent) with a plant ovary.
The final stage or telophase of mitosis occurs and replication of
the undifferentiated cells results in a population explosion of
cells that can be processed and stored in suspended animation, such
as freezing in liquid nitrogen or other low temperature
environments or other techniques, to ensure suspension of
replication, for shipment to a gravitational force for use as
totally undifferentiated stem cells in animals and undifferentiated
parenchyma (or like tissues) in plants.
[0100] The literature supports the premise that compromised"
tissues exposed to gravity at conception (produced as a result of
gravity at conception) for plants and animals will not reproduce
the parent tissue faithfully and indicates serious concerns for
space travel for long periods of time. The literature and
observations of long-term visitors to zero gravity provide such
evidence.
[0101] The present invention is premised upon the theory that
mitosis occurs in cells from dedicated tissues (bone, muscle, etc.)
that does not produce the cells of origin (bone, muscle, etc.) but
will follow reproduction into cells of origin that may be
influenced by, but not in control of the parent cell (bone, muscle,
etc.). Such cells are not the replication of the parent tissue, nor
new undifferentiated tissues, but a "K" cell (new terminology) that
has no purpose, but can function. Such factors must be included to
incorporate an artificial gravity technology that must be required
for any long-term space travel or colonization of the Moon, Mars or
any other body with a gravity force of less than one "G".
Methods
[0102] Current practices on Earth for unification of egg and sperm
in higher order living biological systems (HOLBS) are commonly
practiced in the reproduction of man and other animals. These same
procedures are useful for impregnation of an egg with a sperm in a
weightlessness environment according to the present invention. All
procedures used on Earth for impregnation of egg and sperm in
HOLBS, and other animals are useful for the production of
undifferentiated cells for the production of tissues or for other
purposes, such as reproduction of endangered species.
[0103] The procedures include, but are not limited to the use of a
preserved egg and sperm held in a suspended animation phase that
are revitalized using current techniques in a weightlessness
environment and allowed to unite to form a single cell. The current
techniques and advanced robotic processing are used to unify the
sperm and egg to produce a cell capable of replication in the
weightless environment described herein. The united egg and sperm
preserved immediately upon the unification, can replicate itself
without forming a zygote that will develop into an embryo of
differentiated cells and tissue, and can be utilized for the
purposes discussed (tissue reproduction, reproduction, etc.). The
use of the procedure of using preserved egg and sperm of HOLBS and
lower order animals in a weightlessness environment when and eggs
and sperm are transported to an on orbit facility, processed and
unified in weightlessness, allowed to reproduce in a contained
space, processed through a transitional production phase that may
include, but is not limited to and not necessarily in this order of
the coating of the individual cells, to prevent "grouping" (if
necessary), gradual cool-down to suspended animation, chemical
treatment (if necessary), into a vessel where the replicated cells
can be stored in suspended animation. The replicated cells can then
be transported back to Earth for uses described.
[0104] Additionally, the replication and production as described
herein for the unification of pollen (sperm) and ovary (egg) of the
equivalency of HOLBS and lower plant life in a state of
weightlessness condition for the purpose of production of
undifferentiated cells is described herein. The procedure includes,
but is not limited to routine isolation of the pyramid cells
(undifferentiated tissues) in the apical meristem and culturing the
tissues. However, the weightlessness environment will provide an
exponential growth of the undifferentiated tissues, yielding high
quantities of the cells that may be processed using a coating to
preserve hydration and other life-sustaining parameters, a
suspended animation process and packaging for transport back to
Earth.
[0105] The principle is the same whether the cells being replicated
for production are animal or plant cells. The cells being
replicated are the result of the male and female cell union from
animals or plants. This union can be accomplished in weightlessness
with great success using the current state of the art used on
Earth. Alternatively, the union can be achieved by uniting a female
and male cell on Earth under gravity conditions, placing the single
cell in suspended animation prior to any cell division of the
single formed cell, and transporting the stored cell to orbit or a
similar environment for replicated production in weightless
conditions.
[0106] The single cells produced by union of the female and male
(egg and sperm from animals; pollen and ovary from plants) can be
replicated as single cells in a production mode that will yield
cells at an exponential rate, providing cells for use in tissues
for body parts or branches or roots, depending on the progeny.
Plants
[0107] The present methods of reproduction of plants include
cloning and seeds. Reproduction and use of apical cell reproduction
has greatly increased the numbers of plants in a vegetative
reproduction process. The process depends on the isolation of the
reproducing cells at the tip of a plant or plant part (root,
branch, etc.) known as the meristem and successful cloning of the
limited number of cells at the undifferentiated stage of
development at the tip of the plant or other actively growing
portions of the plant (root, cambium, etc.). The process is
efficient for herbaceous plants, but the "woody" plants cannot be
reproduced in high numbers because of the limited availability of
apical cells. Cells in mitosis in quantity would speed the
introduction of new varieties and species, including endangered
species. The commercial advantage would be saving years of time
between discovery and introduction of the plant to the market.
[0108] The production of an unlimited number of the
undifferentiated cells has not been possible because gravitation
forces "pull" cell matter into "layers" (starch, etc.) that will
start the processing of the undifferentiated cell into specific
(differentiated) cells. The process in the presence of gravity
allows the cells to differentiate into a stem or a root and guide
the plant using gravity to form the stems, leaves, flowers, etc.
that have been programmed over billions of years to form such
structures given the "keys" that that are needed by the genes in
the genetic code. However, the plant will perform "naturally" at
only one "G" or more to another limit.
[0109] Forces exerted by artificial gravity induced have been
demonstrated by a few zero gravity experiments where plants were
subjected to a "spinning" in the experiment to produce an
artificial gravity. The experiment demonstrated that such means of
plant production cannot be produced using limited experimentation.
The plants were deformed, due to the migration of molecules within
the cells (starches). The plant was conditioned to formation of the
root vs. stem, etc. in a one "G" situation, the only sequence the
plant was acclimated to over a few million years. The plants were
also subject to the effects of gravity prior to being sent to zero
gravity, and the effect of the "G" factor on the plants may have
also contributed to the failure of the experiment. Otherwise
designed with the germination of the seeds in a spinning device
that would mimic one "G" may have produced a different result, and
healthy plants may be produced using this technique.
[0110] The plants sent into zero gravity had already been
compromised as a result of the initial introduction to gravity
after germination and the additional force of the "G" forces from
take-off of the mission and had already programmed genetically the
plant to produce roots in one direction and stems in another. But
it is possible to replicate and produce undifferentiated parenchyma
resulting in the unification of pollen (sperm) and egg (ovary) in
plants that are unified on Earth, preserved prior to any division
of the united single cell, and transported immediately to orbit for
the purpose of producing undifferentiated cells capable of
replicating identical cells for production of tissues used for
parts of plants, and the plant itself, including, but not limited
to stems, roots, flowers, seeds, fruits, and other tissues. Such
tissues are preserved in a suspended animation state from orbit to
use in the field using the current technologies now present on
Earth.
[0111] The present invention is premised upon the theory that a
union of pollen and ovary (egg) in zero gravity will produce a cell
that will go to mitosis and then reproduce that cell continually en
masse or until a genetic break down in the cell(s) may occur that
would disrupt the exponential reproduction of the same mitosis. The
newly formed cells can be harvested through an exudation process
and stored (including suspended animation) until the cells are
shipped back to Earth or some other base camp with sufficient
gravity. The cells can then be incorporated using current
techniques for use of undifferentiated tissues. The apical cells of
plants produced in orbit that reproduce cells in mitosis that will
faithfully reproduce the identical cells can be used for multiple
purposes.
[0112] The current practice for the production of plants by uniting
the pollen and ovary on Earth is time-consuming and difficult, but
using the undifferentiated growing cells produced in a weightless
environment can produce the same results with exponential numbers
in comparison. New growth from plant cells and tissue in
weightlessness can provide a source of the undifferentiated cells
for the manufacture of such cells in an exponential fashion using
the techniques described and/or other technologies that exist or to
be discovered.
[0113] Once produced in a zero gravity environment, the
undifferentiated cells at mitosis can be stored and conditioned
fora storage, and preparations for distribution of the manufactured
cells organized for transportation to gravity and utilized as stock
for plants. Undifferentiated plant tissue en masse could be used in
the production of fruit tree scions on stock already established.
For example, the insertion of the undifferentiated cells from
several varieties for a compatible nature (example: Rose
Family--apples, pear, peaches, plums, etc.) can be strategically
placed on a sturdy stock (Rose Family). It would be possible to
reproduce several varieties of fruit, all strategically placed on
that stock to allow a "checker-board square (all fruit of a certain
variety) facing along lines for harvest. The farmer would harvest
the crop of plants facing into a row on both sides of the trees.
The next maturing crop would be on another ninety-degree direction
depending on the sun and shade requirements of each species on the
root stock. This would allow the production of at least four
varieties of fruit from the rose Family to be harvested depending
on the maturation of the fruit. Apples, pears, peaches, plums, etc.
All have different maturation times and the same stock used to
impregnate each of the species would make that rootstock more
productive. The same care (fertilization and pest control) would
only be given to one plant while producing four different Species,
varieties etc., from the same plant. As described several varieties
from the same species may be harvested from the same root stock if
maturity of the varieties will not interfere with pollination.
[0114] The same process can be used for stocks from other fruit or
nut bearing Families and species. The production of these varieties
en masse at an almost exponential rate would provide an industry
with a prolonged season with the same care of only the standard
number of plants. It would be advantageous to back yard gardeners
to have a season-long production of fruit from the same tree.
[0115] The technique for introduction of the undifferentiated
parenchyma from each individual stock would be less time consuming
than the present method of introducing an already vegetative active
branch from an existing plant. The method will be to make an
incision on the sturdy stock in each of the areas where the
specific variety will be positioned and introduce the
undifferentiated cells with the necessary nutrient mix and
patch.
[0116] Most plants will reproduce new growth if a limb is severed.
There is reproduction of parenchyma cells at or near the severed
limb ant the undifferentiated cells reproduce a new limb with all
the characteristics of the missing limb. The new limb will follow
all the rules set by that plant for a gravity environment,
including the production of undifferentiated cells spontaneously in
normal gravity when new growth or trauma occurs. New
undifferentiated cells can be introduced at such sites or be
artificially induced to provide the same results.
[0117] If the union of pollen and ovary for a plant occur in a
weightless state, the plant would not have any instructions to
reproduce anything but undifferentiated cells (parenchyma). The use
of apical undifferentiated cells may produce the same results in
regard to exponential growth of cells for purposes of harvesting
undifferentiated tissues and that tissue can be replicated at an
almost exponential rate.
[0118] Specific varieties can be selected for the use as scions.
The amount of a new variety produced in zero gravity can be
enormous, and provide a load-time of the introduction of a new
variety by years. The use of this technology for plants, especially
woody plants with a long reproductive cycle could be tremendous
considering the long time frame between discovery of a new variety,
etc. of a plant and introduction to the industry. This is
especially important in the efforts to save endangered plants or
when a need for a massive number a specific species is required for
the detoxification of a region (radiation, etc.) is required.
[0119] The time between the discovery of a new and remarkable
variety of a plant and the market place for plants can vary based
on the life cycle of the plant. There are exceptions such as
herbaceous plants like tomatoes that can be reproduced by seed and
self-pollinated using standard processes without too much of a
problem. However, woody plants that have life cycles that may be
years long cannot be reproduced in any numbers and consequently the
timeline for introduction into the market place may be years after
discovery. When a plant that has a unique property (including
endangered species) and has a reproductive cycle that may be more
than a few years the use of zero gravity become evident. The seed
or the use of pollen with an ovary may be an optimum vehicle in
spite of being exposed to gravity during formation and needs to be
studied because any material transported to zero gravity may have
already been exposed to the elements that will prevent cell
reproduction of the primordial cells in bulk. The undifferentiated
cells from a growing plant placed in a weightless environment may
be one of the sources of undifferentiated cells that can reproduce
in an exponential fashion. The cells in the primordial tissues or
undifferentiated parenchyma of apical meristems can be redirected
to the replication of the undifferentiated cells in a manufacturing
process.
Animals
[0120] The current method of harvesting stem cells for a useful
purpose in animals is complex. The use of all relatively
non-differentiated tissues from after birth has become the only
source of "stem cells" under that definition. It is known that even
such tissues have merits. Problems involving the "harvesting" of
the placenta and related tissues obtained from humans and
distribution of such "refined" tissues using the current state of
the art have become a political issue because the stem cells
harvested are programmed in a genetic sense to become zygotes (new
beings).
[0121] All mitosis in an Earth-bound reproduction will lead to
reproduction of cells leading to a zygote and beyond. Current
methods of obtaining stem cells include using the unborn, the
placenta, and forcing a further reproduction of such cells. This
procedure usually ends with the straining of "stem" cells in a
selective method based on the molecular size of a zygote versus a
more advanced cell that can be eliminated based on size produced in
a gravity environment. There are no real quality assurances with
such a procedure. Reproduction of pure undifferentiated stem cells
on a commercial basis is not available in a gravity-based
system.
[0122] There is an ethical question regarding a "formed" animal
zygote versus the use of undifferentiated cells that cannot
reproduce into an animal unless there is an intervention that would
require extraordinary steps to produce an animal. The cells
produced in a weightless environment will not be used for
reproduction (although theoretically possible) during this process
and will be manufactured for the purpose of saving, preserving and
healing animals (including man). The cells will be single cells
without any direction on production of an individual or even a
specific organ. Undifferentiated tissues re-construct the cells in
plants and animals continuously until death. The use of pure
undifferentiated cells for the purpose of healing tissues is a gift
for animals, including man. Creation of the "perfect" animal is not
an objective, but should be carefully monitored.
[0123] The method of reproduction of animal cells at mitosis will
be in regard to current practices. The process must be modified to
take into account that the cells will be reproducing in a
three-dimensional environment. Current practice only allows for a
two dimensional expansion. The container for the expansion of the
cells produced, storage and other manufacturing concerns will be
addressed. Cells that reach telophase of mitosis will continue to
expand in numbers (exponential) and can be confined, exuded through
a special process, preserved (including suspended animation) and
packaged for distribution in an efficient manner through the use of
robotics. The present method utilizes the current technology for
the extraction and reproduction to provide a mechanized system for
the manufacture of primordial or undifferentiated tissues from both
plants and animals (including humans) in zero gravity.
[0124] The present invention also encompasses a method of producing
stem cells comprising obtaining purified stem cells from an embryo
of new-born animal and culturing said stem cells in a weightless
condition or environment resulting in the cell replication of
identical stem cells. Further included in the present invention is
a method of producing undifferentiated cells from an animal, such
as cells from liver, kidney, heart, skin, and other cells from the
animal body including organs for the purpose of replication of
undifferentiated cells including obtaining the undifferentiated
cell from the animal, and culturing the undifferentiated cell in a
weightless condition or environment resulting in the cell
replication of identical undifferentiated cells. Additionally, the
undifferentiated cells may be a subcutaneous skin cell obtained by
harvested from the animal, wherein the culturing results in near
exponential replication the cells overtime.
[0125] Further, the present invention encompasses a plant or animal
or undifferentiated cell thereof produced by any of the methods
described herein and in any of the priority documents, wherein said
plant, animal or undifferentiated cell thereof comprises at least
one identified gene that has a change in expression level as
compared to the gene expression profile of control cell, wherein
the identified genes are associated with adaptation to the hostile
environment.
[0126] More specifically, the egg and sperm are united using
standard in vitro fertilization (IVF) techniques for harvesting
human or animal eggs, collecting sperm and inseminating the egg
with the sperm in a laboratory dish in IVF culture medium. The dish
is then placed in an incubator at a controlled temperature which
should be the same temperature as the female species' body. It
generally takes 18 hours for fertilization of the egg to be
complete.
[0127] If the fertilization or the union of the egg and sperm
occurs in weightless conditions then the single fertilized cell can
be maintained in the incubator with the change of medium over time.
If the fertilization occurs in a gravity condition or environment,
then after fertilization is complete, and before the fertilized egg
or zygote divides, the fertilized egg should be immediately
transferred to a weightless condition for culturing. However, if
there will be a delay until the culturing in weightless conditions
can occur, the fertilized egg should be placed in suspended
animation before it divides into two cells, such as freezing the
egg. At a later time, when the fertilized egg or zygote can be
cultured in a weightless environment, it should be revitalized from
its suspended animation, and cultured under the conditions to
expand the fertilized egg into a culture of undifferentiated stem
cell using known stem cell culture techniques that a publicly
available in scientific publications, patents and by disclosed
on-line methods and media.
[0128] For example, a medium useful for the isolation of embryonic
stem cells is "ES medium." ES medium consists of 80% Dulbecco's
modified Eagle's medium (DMEM; no pyruvate, high glucose
formulation, (InVitrogen or Signma), with 20% fetal bovine serum
(FBS; Hyclone), 0.1 mM 13-mercaptoethanol (Sigma), 1% non-essential
amino acid stock (Sigma or other known sources). Preferably, fetal
bovine serum batches are compared by testing clonal plating
efficiency of a low passage mouse ES cell line. FBS batches must be
compared because it has been found that batches vary dramatically
in their ability to support embryonic cell growth, but any other
method of assaying the competence of FBS batches for support of
embryonic cells will work as an alternative. But any known media
for culturing the replicating stem cells can be used and tested by
the scientists performed these experiments to select the
appropriate medium to obtain optimum results. Appropriate plant
cell culture media known to skilled persons can be selected to
culture undifferentiated plant cells according to the present
invention.
[0129] The cells are cultured in 3-dimensions by simply suspending
the cells in a closed culture vessel in the weightless environment
which will keep the cells suspended without the need for any
agitation as the cell will not settle to the bottom of the vessel
that they would in a gravity environment. Any know methods of
3-dimensional cell culture can be used to culture the replicating
undifferentiated stem cells, which could include culturing methods
from Mina Bissell's laboratory, such as for example disclosed in J.
Cell. Sci., 2003 June 15; 116(Pt 12):2377-88.
[0130] Somatic embryogenesis has been the model for mass clonal
propagation of a diverse array of higher plants, and also a model
for studies of embryo development and cell differentiation. For
certain species, differentiation into somatic embryo requires cells
to undergo a transitional stage, whereby embryo-like structures are
formed. These embryo-like structures are comprised mainly of
undifferentiated parenchyma cells. The conversion of these
structures into embryos is regulated by a number of different genes
under normal conditions. However, when submitted to microgravity
conditions, the cells in embryo-like structures may behave
differently. The principal objective of the experiments of the
present invention is to evaluate whether cells of a
monocotyledonous and a dicotyledonous plant develop normally from
the transitional stage into normal somatic embryos under space
(microgravity) conditions. Histological (structural) and genetic
analyses will be performed to assess the cells.
[0131] Additionally, suspension cultures have been widely used for
tissue culture and mass clonal propagation of a diverse array of
higher plants, and also as models for studies of cell development
and differentiation. Analysis of these cells will verify structural
and genetic changes in plant cells submitted to the effects of
microgravity. In addition, cell growth and replication will be
assessed visually. Structural changes will be performed through
histological analyses, including light microscopy, transmition
electron microscopy (TEM), and if feasible, scanning electron
microscopy (SEM). Genetic analyses will be performed to evaluate
differential gene expression under microgravity.
MATERIAL AND METHODS
Plant Material
[0132] Monocotyledonous model species: Encyclia plicata, an
endangered orchid
[0133] Dicotyledonous model species: Arabidopsis thaliana, a small
flowering plant that is widely used as a model organism in plant
biology and its genome has been completely sequenced, providing
extensive genetic and physical maps [0134] Dicotyledonous model
species: Caesalpinia puicherrima, a tropical flowering tree.
[0135] Dicotyledonous model species: Tabebuia aurea, a tropical
flowering tree.
Ground Cell Suspension
[0136] Cell suspension cultures are initiated for all species above
mentioned, using callus tissue. Callus is comprised of
undifferentiated parenchyma cells, with no pre-determined growth
pattern.
[0137] For the orchid suspension cultures, the MS culture medium
(Murashige and Skoog, 1962), modified with 1 mg/L 2,4-D
(2,4-dichlorophenoxic acetic acid), an auxin, and 0.25 mg/L 6-BA
(benzyladenine), a cytokinin is utilized. Cultures are multiplied
and built up in 125-ml Erlenmeyer flasks, under agitation in an
orbital shaker at 109 RPMs. Arabidopsis and tree c ell suspensions
are cultured on MS medium modified with 1 mg/L 2,4-D. Once a
significant amount of cells are produced, they are transferred to
10-ml opticells. Also WPM (woody plant culture medium) medium
(Lloyd and McCown, 1986) modified as the MS medium above may be
used for woody species of plant.
[0138] OptiCell.TM. is a sterile, sealed cell culture environment
between two optically clear gas-permeable growth surfaces in a
standard microliter plate-sized plastic frame with specially
designed ports for access to the contents. OptiCell allows an ideal
environment for cell growth, microscopy, treatment, selection,
separation, harvest, storage, and shipping. Optically clear
gas-permeable growth surfaces allow diffusion of oxygen and carbon
dioxide for optimal cell growth and permit microscopic examination
at any stage of any cell process. OptiCell is compatible for use
with standard, phase contrast, confocal, and high-resolution
time-lapse video microscopes and takes up a fraction of the space
of conventional cell culture devices. Access ports allow aseptic
access to the interior and its contents.
Space Hardware
[0139] Each opticell contains about 10-12 ml of cell suspension.
Opticells are maintained in quiescent conditions for both ground
and space environments and are evaluated periodically through
visual observations for cell growth and development. Under
microgravity (space) conditions, opticells are arranged in a C-Hab
hardware developed by Bioserve, University of Colorado, comprised
of 6 individual Opticell cell culture systems, peristaltic pumps
and a control circuit board. The C-Flab hardware allows the
transfer of 1 ml of suspension from one opticell to the next during
transfer of cells to fresh medium. An aluminum base and an extruded
aluminum outer box with a clear optical window provide the second
level of containment. Visual evaluations in space are performed
with the aid of video cameras. The C-Hab is associated with CSI
camera modules. Each of the camera modules contains up to three
analog color video cameras, fitted with either microscope adaptors
or standard lenses for macroscopic view. This allows the
observation of cell growth and replication throughout the period of
experimentation in space. Still images (jpegs) are fed to the
ground periodically during the entire period of the experiment,
thus generating a time lapse for cell growth and replication. The
hardware and related control software are tested and evaluated
previous to launch.
Evaluations of Cell Suspensions
[0140] Samples from suspension cells maintained in opticells, under
both ground and space conditions are collected and compared for
histological and genetic analysis: [0141] 1. Histological analyses:
Cell suspensions are prepared for light and electron microscopy.
Opticells are compatible for use with standard, phase contrast,
confocal, and high-resolution time-lapse video microscopes. Cells
are examined microscopically on either optical growth surface or in
between. Oil immersion lenses (up to 100x) are used on the membrane
without disruption or contamination. The membrane is sectioned for
small scale staining and microscopy. Additional samples are removed
and fixed in Glutaraldehyde for subsequent evaluation of cell
ultrastructure through TEM and SEM. These analyses are performed
for all species. [0142] 2. Genetic analysis: Gene expression
analyses is performed to evaluate possible genes that are either
turned on and/or off under microgravity. Suspension cultures
maintained in space are fixed in RNAlater (Ambion) liquid
preservative through the Kennedy space center fixation tube (UT),
hardware designed to provide proper containment of fixatives for
biological samples in space placed inside the C-hab environment.
RNA is isolated and compared for both ground and space suspension
cultures to evaluate gene expression. Molecular biology techniques
for reverse transcriptase polymerase chain reaction (RT-PCR) and/or
copy-DNA amplified fragment length polymorphism (cDNA-AFLP) and gel
electrophoresis according to standard known techniques are used for
gene expression analyses in ground (TREC lab). Microarray analysis
of gene expression is performed. [0143] 3. Additional evaluations:
Preliminary experiments in ground environment (on earth as opposed
to in space) are useful to address details and to generate
practical solutions for the successful establishment of experiments
in space. Evaluations for both earth and space experiments provide
additional information and data specific for the Opticell cell
culture system environment and include specific conditions for the
opticell cell culture system environment, such as cell growth
rates, cell densities, subculture frequency, and size and condition
(live vs. fixed) of samples for histological and genetic
analyses.
Experimental Design and Statistical Analyses
[0141] Controls are comprised of 10 ml of suspension cultures
maintained in 6 opticells under ground-laboratory conditions.
Treatments are comprised of 10 ml of suspension cultures maintained
in 6 opticells in microgravity. The C-Hab environment has been
accommodated into modular racks inside modules provided by
Spacehab, placed in the Space Shuttle Endeavour, launched on Aug.
8, 2007, Mission STS-118. The experiment ran fora period of three
months approximately, whereas the controls are maintained in ground
and the treatments are maintained in the International Space
Station (ISS). Treatments were returned to ground through another
space shuttle mission on Nov. 8, 2007. Analysis of Variance (ANOVA)
are performed using SAS statistical package for analyses of
results. Means for treatments will be compared for all parameters
evaluated using the Tukey's test.
[0142] Data were collected regarding plant growth characteristics
and the results of these studies showed that the cells grown in the
weightless environment on the ISS replicated to a greater mass than
the control cells grown in a laboratory under the earth's gravity
with all other culture conditions the same. One of the two tree
species demonstrated significant growth on orbit in the weightless
environment as compared to the ground controls.
[0143] The studies support that cells would replicate rather than
duplicate and that exponential growth of the cells will occur as
long as the undifferentiated cells are nurtured and maintained in a
weightless environment. The results show that that cell growth on
the ISS in three of the species of plants demonstrated higher
growth than the controls on Earth. The third experiment
(Moncot--orchid species) was contaminated with a yeast infestation
and is being examined to determine the species and information
regarding gene expression and replication.
Effect of Weightless on Growth of Replacement Cells in Visitors
Maintained in a Weightless Environment
[0144] As noted above, there have been reported studies of humans
who lived in a weightless environment, such as on the Space
Station, that show that these individuals lost body mass during
their time in this weightless environment even though these
individuals exercised during their stay in this weightless
environment. In a weightless environment, even established cells
produce undifferentiated cells immediately adjacent to the existing
cells that "mimic" the cell, but do not perform properly and do not
produce the tissue of origin (bone, brain). The tissues that have
the greatest amount of circulation will be effected first, such as
blood and brain, and these tissues will be compromised. All long
term orbital and interplanetary missions should take under
consideration the loss of body mass regardless of the precautions
incorporated into a space mission, including exercise, supplements
and the like to maintain body mass. The rule concerning exercise in
a weightless condition may be harmful because of the degradation of
the body and replacement of the destroyed cells by a cell that will
not be a replicate of the original cell. The cell reproduced in
normal cell reproduction in one "G" does not have the influence of
the one "G" and replicates into a "K" cell which functions, but is
not the replicate needed for normal function in the tissue. For
example, a new cell in a muscle tissue should function as a normal
muscle cell but it does not.
[0145] All long-term missions should be reconsidered until there is
an evaluation of this concept and examination of the information
presented herein regarding the formation of non-differentiated
cells and unlimited reproduction of such cells. Thus, the risk to
individuals who spend any extended time in a weightless environment
is based on the premise that undifferentiated tissues can reproduce
as "pure" undifferentiated cells produced by a male and female
union in weightlessness.
[0146] The effects of long-term exposure to weightless conditions
is governed by the knowledge that the reproduction of existing
cells in a visitor will not be faithfully reproduced to provide
replicates of the generating tissue. Not wishing to be bound by a
specific theory, the present invention is based on the theory that
the cells that continually are produced do not form into tissues
(bone, muscle, blood), and will not develop or mature into the
differentiated cells from the original cells. The cells will not
differentiate properly into the parental cell, but under the
weightless environment will form a cell not functional to the
specific task intended for the cell, such as bone, muscle or blood.
The term "K" cell' is only a designation, but is being used herein
to describe a cell not yet studied, but present in all long-term
space visitors.
[0147] In an effort to solve the loss of body mass in weightless
environments, the experiment concerning the use of embryonic
tissues in the "South African" experiment confirmed the theory that
the reproduction of new cells does not follow a normal growth
pattern. The tissues derived from animals within hours of lift-off
in Russia provided information concerning the techniques that may
be used in future missions. The South African mission did provide
information including the reproduction of tissues in a weightless
environment NOT reproduced with a sperm and egg in zero gravity, or
a "new" seed or egg of a plant recently pollinated. The expansion
of cell growth did not take on the normal pattern and instead
became an unrecognized pattern. Again, the experiments provide
information that lead to the confirmation of the theory that all
cells derived from tissues in weightless environment are not
orientated to reproduce the same type of tissues adjacent to those
tissues.
[0148] The cells reproduced in likely become "inert" cells meaning
that the cells do not have the same specialized function as the
adjacent differentiated cells in the tissue from which these cells
originate and are reproduced. In other words, the longer an
individual stays in space, the higher the probability that the body
will degrade, and may do so at a higher rate in the individual who
stays in a weightless state. In fact, such cells may be anaerobic
in character and pose a future cancer risk since the cells do not
follow the "Krebs cycle," resulting in fermentation rather than an
aerobic cycle.
[0149] Individuals in orbit weightlessness have been experiencing
loss of bone and muscle mass, and other tissue. The areas of the
body with the least amount of circulation (bone, muscle, tendons,
fat,) will experience the most trauma because of the circulation to
those areas of the body.
[0150] Intense exercise in zero gravity has not been able to slow
the loss of tissues from the body and may be counter-productive to
long-term intact cell survival, since cells are created and
destroyed at much higher rate. It is known that the body is
reproducing new cells on a continuous basis and the more exercise
is performed, the more cells that become exposed to the
reproductive process and the more undifferentiated cells that are
produced. The reason for the loss of mass in the bone, muscle and
other tissues is that the reproducing primordial (stem) cells that
do not have a direction (bone vs. muscle, etc.) die or form a cell
we are not familiar with unless we look. A lock at the space
traveler in a weightless environment without the proper devises to
allow for an artificial gravity may create a major problem.
[0151] Given that the human body continuously reproduces specific
differentiated cells for each organ and that such cells may only be
primordial with no direction, there is a strong possibility that
the primordial or undifferentiated cells do not develop into the
differentiated tissues intended (such as bone, muscle, or other
specialized cells) and may degenerate or possibly form a cell that
we are not familiar with in the reason of normal thinking. It is
known that there is a loss of tissue and all orbital and
interplanetary visitors should be examined for necrotic or other
cells in the areas of the body that have low growth, including the
brain and other organs studied.
[0152] Many technologies have been applied, including vigorous
exercise, to slow or prevent this loss of tissue. The latter may in
fact be harmful, since exercise promotes the life and death cycle
of cells, and death of the cells does occur, but the new cells
produced are undifferentiated with no program to produce the
differentiated cells which they are to replace. Even the new
reproductive tissues of visitors become undifferentiated when
produced and either transform into the tissue itself (with a "lag"
time) into the bone, etc. or die or become an "inert" tissue with
no specialized function.
On-Orbit Manufacturing Facilities for Stem Cell Production
[0153] The use of an on-orbit manufacturing facility or "wheel" in
space using new technology will allow production of primordial or
undifferentiated cells and tissues from plants and animals and
provides an environment for those that harvest the cell crop (FIG.
1).
[0154] The reproduction of the original cell obtained from the
union of the sperm and egg in animals or the pollen and ovary in
plants occurs in a 3-dimensional fashion in a culture vessel or
container, since no gravity is involved, the cells will replicate
and remain in suspension floating in the media in the culture
vessel. The cells will continue expanding in the culture vessel
until the "yoke" of the process is expended until the size of the
cell population has reached its maximum capacity in the container.
Then, the newly formed cells can be transferred or exuded from the
culture vessel into a container that will provide a suspended
animation system for storage and shipment to Earth. A system will
be described for the manufacture of undifferentiated tissues in a
weightless site, and the cells will be cultured in the weightless
chamber in the on-orbit manufacturing facility shown in FIG. 1 or
in a similar type chamber in another space vessel or station.
Manufacturing
[0155] Capturing the reproducing cells from plant and animal
primordial tissues will be a mechanized procedure. Cells
(individual or two or more) can be captured and pulled through a
mechanism to encapsulate the cell(s) in a sheath that mimics the
cells walls of single celled organisms such as bacteria. The
storage of the undifferentiated encapsulated cells must be stored
in a weightless system where the metabolism and physiology of the
cells proceeds into a state of suspended animation. Temperatures
and storage conditions can be adjusted to provide an optimal
condition that allows the harvested cells to be stored. Slam
freezing is used to preserve semen specimens for cattle now and can
be used. The process of the cells must be slowed to a level that
will allow storage and eventual handling of the cells when returned
to a gravity environment.
[0156] Yeast may represent a model for replication of both plant
and animal undifferentiated tissues since yeast provides the
preparation (substrate), innoculum (undifferentiated cells and/or
tissues), cultivation (providing ideal conditions), harvesting
(separation of cells from the process), cooling and storage of the
harvest at temperatures not to interfere with gravity.
[0157] The aerobic respiration (presence of O2) expected in the
culturing processes with both plants and animals will be interfered
with by anerobic respiration (lack of O2) due to the containment of
the production and the difficulty of feeding the entire nutrient
system with adequate oxygen, especially near the end of the
exponential growth phase of the manufacturing process. Aerobic
respiration has both the substrate level and oxidative
phosphorylation while anerobic respiration has only substrate
phosphorylation. Both use hexose as a fuel (glycolysis). A problem
in the manufacture of the undifferentiated cells is that during the
anerobic respiration expected in the process, the final electron
acceptor through alcohol will be other organic molecules such as
pyruvate, acetaldehyde, etc. that will be toxic to the total mass
of undifferentiated cells. In the aerobic respiration, the final
acceptor is oxygen and presents no problems.
[0158] Respiration resulting from undifferentiated cell replication
occurs in both plant and animal cell cultures. The exponential
expansion of cells follows the general guidelines for plants and
animals: [0159] 1. Sugars (Hexose in plants) is the fuel. [0160] 2.
Zymase in plants is an oxidizing/reduction enzyme to act as a
catalytic agent to covert sugar into labile intermediate products.
[0161] 3. Animal conversion during the metabolic process follow the
same general format (Kreb's Cycle).
Plant Model
[0162] The expansion of the cells will produce by-products
depending on the oxygenation of the system but in containment, both
aerobic and anerobic respiration will take place at the same time,
producing toxic substances:
##STR00001##
[0163] The amount of byproducts accumulates and alcohol with
aldehydes, etc. requires removal from the manufacturing system
through the use of filters.
[0164] The production of undifferentiated cells requires the
filtering of the toxic byproducts and the injection of oxygen into
the system. The production of cells requires careful consideration
of the "critical mass" determined by the maximum numbers of cells
that can be produced by the available nutrients, the supplied
oxygen and the removal of wastes. A "flow" system is highly
probably where a reaction chamber allows a mix of the nutrient,
"seed" undifferentiated cells and oxygen can produce a continual
flow of the nurtured "seed" cells with a proper oxygen environment
would expand into a container vessel that has environmental
controls. However a more simplistic model is useful that can be
well-adapted to a robotic system, for example, such as disclosed in
FIG. 2. The configuration of the system and its manufacture for
each container is based on the following parameters: [0165] 1.
Volume of reactor [0166] 2. Amount of substrate (sugars). [0167] 3.
Amount of innoculum required. [0168] 4. Filter system for toxic
byproducts [0169] 5. Injection of Oxygen [0170] 6. System for
cooling reaction during progression of reaction and cooling for
storage
[0171] If the efficiency of the "Volatiles Filter" proves to be
inefficient, a separator may be necessary in the system. The
manufacturing unit is preferably designed toward robotic
manufacturing with the following considerations: [0172] 1. The
manufacturing unit is at a pre-determined size to contain the
initial substrate and also serve as the storage unit for the
processed cells return to Earth. [0173] 2. The substrate
environment is carefully monitored for parameters that will insure
a proper environment for the injection of undifferentiated cells
(or tissues) into the substrate. [0174] 3. An oxygen injection
system that provides levels of oxygen to keep the process aerobic
and evenly distributed within the nutrient. [0175] 4. A
re-circulation system with a exit into a filtering system for
removal of toxic volatiles also assists in the distribution of
oxygen. The system is preferably designed to prevent any high
movements of nutrients and cells to maintain a "non-gravity"
condition. [0176] 5. The exponential expansion of the
undifferentiated tissues will cease because of the amount of
nutrient, size of the container and/or [0177] 6. The toxic
materials that may result. [0178] 7. The unit can then be reduced
in temperature to below the level required for respiration and
shipped back to Earth.
[0179] The payload of undifferentiated cells will be retrieved much
like the Get-Away-Special Program in the 1980's and the SpaceHab
system and replated with another "factory" Such factories can
replicate the primordial or undifferentiated cells and tissues of
both plant and animal on a commercial scale. The advancement in
technology will allow for better efficiency with each mission.
Production
[0180] The primary cells used to replicate identical cells in
weightlessness will produce cells in an undifferentiated state much
like a "seed" in crystallization in minerals or super-saturated
solutions. Biological processes may follow different patterns, but
the replication of the seed cell will produce undifferentiated
cells in an exponential fashion. Therefore, there is a need for a
device to control the replicated "seed" primordial tissues to
produce encapsulated cells.
[0181] Biological "factories" such a female termites can be used as
model for the replication in zero gravity. The primordial tissue in
an exponential explosion needs to be controlled as with the queen
termite. The process involves the direction of the undifferentiated
tissues through a processing chamber where a protective coat is
applied. The process would allow a continuous flow of the
primordial cells into the encapsulation process. The cells would
then be directed into a chamber that is designed to provide a
conditioning process for processing the cells into a state of
suspended animation. The processing of the undifferentiated cells
can be conducted using current technology with modification known
to persons skilled in the art of cell culture and manipulation. Use
of ultra-light components of a non-metallic nature may be essential
to provide not only a gravity force, but also an electro-magnetic
influence. Containment of the production in a shielded environment
to protect the production and maintain the sterility of the
cultures will be necessary.
[0182] Selection of the egg and sperm is of great importance. The
animals must be selected to provide the egg and sperm at an early
age. The break down of the genetic code in latter years is well
known for both egg and sperm Even the generation prior to the
donors must be considered. The telophase of mitosis of the union of
the egg and sperm will provide stem cells for the individuals in a
particular blood type and other tissue matching characteristics to
match the donor with potential animals of like character. Banks of
eggs and sperm in the case of animals (including man) and pollen,
plant female organs and undifferentiated cells, such as parenchyma
can be stored in suspended animation for future generations. It is
expected that individuals will "bank" sperms and eggs in the early
years of their sexual years to insure the capability of
regenerating or even replacing body parts. The bank of sperm and
eggs can be held in suspended animation. If a situation arises
where there is a future need for stem cells, the "pure" stem cells
will be available.
[0183] The present invention comprises the methods and facilities
so that pure stem cells can be reproduced in a factory mode. The
cells will only proceed to mitosis and will not produce
undifferentiated cells so that no embryo will develop from the
original single cell. The union of a sperm and egg in zero gravity
will not result in the production of a zygote capable of developing
into an embryo and then into a normal life form. This premise is
based on the observations of maladies that occur to space visitors
from both the US and Russia who spend some time in a weightless
condition. The present invention is based on the theory that the
undifferentiated cells produced by specific tissues (bone, muscle)
did not differentiate into the intended differentiated, specialized
tissues (bone, muscle) and became necrotic (Reference). The
additional non-productive cells produced in a weightless condition
did not have the "gravity" message required to reproduce in a
gravity environment and are imbedded in the normal tissue and
interfere with the "normal" cells surrounding the tissue.
[0184] The additional data provided by the South African space
attempts also lead to a conclusion that primordial cells
(undifferentiated) can and will reproduce in a 3-dimensional
fashion. The cells produced were the result of already compromised
(by gravity) cells that did indeed reproduce in the 3-dimensional
environment. However, the instructions on reproduction (genes) were
not only compromised by the union of egg and sperm under gravity,
but also lift-off that provided many more gravity forces that
certainly migrated the starches, etc. and effected the experiment.
The production of undifferentiated cells in volume can be achieved
using the present technology in a weightless condition.
Design and Function of a Space Craft Using a "Clutch"
Technology
[0185] The current visitors to space in orbit and destined for
other planets are subjected to a weightless environment where
extensive documentation points toward degradation of the human
body. Long space voyages to Mars, for example would not be
practical and even suicidal. A method of providing an artificial
gravity has been under study for decades, but docking of a space
craft, conducting experiments in weightlessness, and still
providing an environment where a simulation of gravity are present
have not been solved until now.
[0186] The present invention comprises a space craft (as shown by
the diagram in FIG. 1) that can be assembled in orbit using a
"tinker toy" method of construction using capacity figures for the
space shuttle or more advanced vehicles. A puncture-proof
inflatable two-piece ("clutch" and wheel) can be put in place
around the stationary central weightless chamber. The central
"weightless" chamber can be used for docking and experimentation.
An adjacent outer segment (wheel within a wheel") is a "Clutch"
that will be at neutral or rotate with the outer wheel. The neutral
condition will always exist in inner "Hub". The "clutch" can be
slowed to "Neutral" and air-locked" to allow the transfer of
personnel and/or supplies into the "clutch". The "clutch" system is
sealed and begins to rotate to catch up with the outer spinning
wheel. Once it "catches" the speed of the outer rotating wheel, it
can mate with the wheel and transfer of the personnel can take
place. The rotation of the "clutch" allows for transfer from a
neutral docking zone and laboratory that allows experimentation in
a weightless environment to the outer wheel that mimics Earth's
gravity. The wheel may be approximately 100 meters or more in
diameter and include at least four to eight "spokes". The vehicle
can be "mated" to the existing space station.
[0187] The present invention further comprises an electromagnetic
"track" system that will interface between the inner capsule
(docking, laboratory, control area, and other compartments), the
"clutch," and the outer wheel to provide a friction-less interlace.
A ball bearing system can be engaged during transfers between the
"capsule" (weightless condition), the "clutch" and the wheel during
transfer of goods and personnel and act as a back-up for the
electromagnetic system. The rotation between the "Neutral
(Capsule), the "Clutch" and the "Wheel" can all be interfaced with
electromagnets that will allow for the production of
electricity.
[0188] The present invention additionally comprises a computerized
system of weights and balances that are necessary to adjust the
entire system. Transfers of personnel and equipment will require a
weight and balance to this relatively sensitive system and can be
accomplished by pumping fluids or movement of weights in the spokes
of the "wheel" to counteract the movement of supplies and personnel
to the "rim" of the "wheel".
[0189] The present invention also comprises the spokes of the outer
wheel that will act as the elevators to the outer region of the
wheel and to provide ballast for balance of the system. The "rim"
of the wheel will circulate at a speed that creates a centripetal
force that will mimic Earth's gravity and allow the inhabitants and
plants requires for subsistence to exist in a near-nor mal
condition. The "rim" will require adjustment by visitors, but there
some individuals that do adapt to such an environment and function.
The sleeping "cocoons" can be arranged to allow for maximum benefit
of the centripetal force. Plants can also be propagated in such an
environment.
[0190] The mathematics to calculate the circumference of the wheel
to create a near Earth simulated gravity is well known. Examples
are given in a publication "Artificial Gravity and the Architecture
of Orbital Habitats" by Theodore W. Hall, presented at the 1.sup.st
International Symposium on Space Tourism, Bremen, Germany, 1997 No
definitive calculations will allow for a prediction of comfort in a
space habitat, but the circumference of the wheel will be
determined by the adaptation of individuals to the physical
conditions and stamina in such conditions. A selection process may
be necessary to determine personnel for such missions.
[0191] Providing an artificial environment that mimics gravity is
also necessary based on review of the data and understanding the
principals of cell reproduction in weightlessness. A space craft
traveling at a constant velocity in interplanetary flight will
create a weightless environment and be subject to the same science
already discussed concerning orbital missions. A "wheel" and
"Clutch" arrangement must be incorporated into any vehicle
attempting a long interplanetary flight, including a voyage to
Mars.
Spacecraft Structures and Electromagnetic Sub-System
[0192] The Earth is protected from the Sun's radiation and other
particles by the Earth's ionic shield. Mars and the Moon do not
have such a field and is cited as a reason for the inability of
these bodies to maintain life on our scale. The Earth's ionic
shield is the result of the expulsion of a positive charge from the
Northern polar region that forms a electromagnetic force that forms
a spherical force field covering the planet and terminating at the
Southern polar region. The system that provides the electromagnetic
shield is powered by the motion of the various metals (primarily
Iron) in the heated core as described by current science.
[0193] The present invention further comprises the space craft
described herein which will have a abundance of electrical power
developed by the interface of the "clutch" system using simple
electromagnets. The weightless inner portion of the hub must remain
stable for the purpose of the docking of a visiting space craft,
experiments, and other functions. The "clutch" will also remain in
a position adjacent to the main facility. However, the movement of
the outer wheel will always provide a field production at the
interface of the clutch and the wheel. The continuous electric
power produced can be used to produce a electromagnetic field based
on the same principles that protect Earth from the damaging
radiation, etc. from the Sun. The portion of the space craft
providing the positive charge (as the North Pole) will be directed
toward the sun and a receiver will be placed at the opposite end of
the space craft. The positive charge may be on an extension boon to
provide a suitable profile for deflection of harmful radiation and
other harmful effects from the Sun. As with Earth as a model, a
proper profile for the amount of power required to achieve
protection from harmful radiation can be determined. The amount of
power to accomplish a protective shield will be low because the
power supply at the positive side will be reduced to a certain
degree, but retrieved at the negative pole. There are no ground
cables in space. The principles that protect the Earth are
applicable in space.
Acceleration of Adaptation and/or Evolution
[0194] When in a weightless and/or near-weightless environment,
typically in space and on-orbit, but also in any and all such
equivalent non-weight and weightless circumstances, there exists
cellular materials and wherein a process or processes are performed
in those environments, specifically in the absence of a
"weight-forcing condition" (a circumstance where weightlessness
prevails), specific genetic attributes will be altered, i.e.,
either switched on-from-off or switched off-from-on, and that
certain of these gene characteristics may be identified, as to
their function and nature, and may consequently and purposefully
selected and altered in order to achieve specific, valuable, and
useful outcomes, to derive targeted cellular characteristics and
accordingly, behavior, performance, and function, and/or that
result in the securing, development, and/or enhancement of certain
preferable and valuable attributes of said cells, whether as part
of a cell, a cellular mass, a cellular volume, or a living
organism, in whole or in part.
[0195] Wherein these cellular components and sub-components
including cellular elements, genes, DNA, RNA, enzymes, hormones,
etc., may be tied individually or collectively in structure,
process, and function, such that they act to produce
characteristics, attributes and capabilities both cellular and when
in-complex and mass as may be observed with a living organism or
organisms, such that they express living behaviors which include,
but are not limited to increased or decreased or modulated
metabolic rate, i.e. respiration, transpiration, anabolic and
catabolic activity levels or both, up and/or down regulated,
increased, decreased, altered or modulated nutrient uptake,
increased, decreased, altered, or modulated waste and byproduct
output, modification or alteration of water and fluid uptake or
release, alteration of structure, i.e., modification, adaptation,
or variation of cell wall thickness, structure, or composition,
alternation of character as relates to external environmental pH
(acid/base) conditions, salinity and mineral presence, ionics and
salts, water, temperature variations, both high and low extremes as
well as cellular character, performance and viability in the
presence of rapid temperature excursions, or combinations of
external chemical, thermal, physical, energy, electromagnetic,
radiation, solar, vibrational, acoustic, magnetic, paramagnetic,
gravitational, weight-forcing conditions. In other words, all
external conditions which may affect or interact with the cells,
cellular mass or volumes, components thereof, or organisms in whole
or in part may be modulated.
[0196] Also contemplated by this invention is the alteration of
those biological and cellular processes, activities reactions,
motions, behaviors, process induced reactions and behaviors and the
like which are deliberately conducted or affected or driven or
occur in a "zero-weight-force" (so-called microgravity)
environment. This includes, but is not limited to, the alteration
of genes, DNA, RNA, enzymes, hormones, and all biologics known to
those competent in the art of biology and cellular sciences, which
result in the creation of and/or development of cellular
components, cells, cellular masses and volumes such that they or
their complexed resulting cellular organisms, may be altered,
modified, and enhanced to yield valuable and useful new cellular
types and variant organisms including, but not limited to, cows
resistant to mastoid staphylococcus disease, cattle resistant to
mad cow disease, wheat resistant to wheat rust, soybean crops
resistant to soybean rust, agriculture crops resistant to
ralstonia, citrus capable of growing in Northern latitudes,
Jatropha, Camelina, and other third generation biofuel crops
capable of being grown in the Continental US latitudes, citrus
resistant to canker and greening, interbreeding of Tamarix to
overgrown existing invasives in the Western US to eliminate water
losses, tailored halophyte production and growth, minimization of
waterway invasives through retailoring of hydrilla, water hyacinth
and egeria species, modification of grapevines to eliminate grape
blight, modification of corn, soy, wheat, rye, rice and multiple
other types of food crops enabling them to be grown and viable in
drought conditions, in harsh wind and climate environments, or in
adverse soils and a wide variety of other biological and cellular
and organism modifications, alterations, and restructurings of
similar usefulness and value and nature.
[0197] The present invention recognizes the value of the
accelerated and sustained proliferation or replication of
undifferentiated cells from plants and animals in weightless
conditions as an opportunity to select for organisms adapted to
specific, even harsh, environments. By exposing the proliferating
or replicating undifferentiated cells to one or more "non-natural"
environmental conditions while in orbit (or weightlessness), one
can "force" the cells to express genes that enable the cells to
adapt and survive in these abnormal environments. The selected
cells can be cultivated to develop into an organism that would be
adapted to the specific environmental conditions to which its
primordial cells were exposed.
[0198] The present invention comprises "replication processes" that
occur in a weightless environment as contrasted to the normally
occurring reproductive processes that result in progress or
development through normal maturation stages and cycles resulting
in the differentiation of cells which are known as "reproductive
processes" on Earth. However, on orbit in weightless or
microgravity conditions, cellular activities occur and are referred
to as "replication" which can also be referred to as "duplication"
or "proliferation" of copies of cells that are identical in
structure and function to their originating predecessors. This
process is known as replication and results in an undifferentiated
cell replicating or duplicating itself without differentiating into
a more specialized cell with a predetermined function. Thus, the
process allows for the production of large amounts of
undifferentiated plant and animal cells in the weightless or
microgravity environment.
[0199] As used herein, the term "undifferentiated" means a
primordial state of a cell or cells capable of differentiation and
proliferation to produce progeny cells that can be physiologically,
biochemically, morphologically, anatomically, immunologically,
physiologically, or genetically distinct from the primordial
state.
[0200] As described above, the present invention provides methods
of adapting plants and animals to survive in a hostile environment,
wherein the method comprises culturing undifferentiated cells from
the plant or animal in a weightless condition that mimics at least
one element of the hostile environment to which the animal or plant
is to be adapted; selecting the cells that proliferate in said
condition; and cultivating said selected cells to produce plants
and animals that are adapted to grow in that particular hostile
environment.
[0201] Any suitable means for achieving reduced gravity or
microgravity conditions can be used for performing the method. In
one embodiment, the method is performed under reduced gravity or
microgravity conditions in space, e.g., aboard the Space Shuttle,
the Space Station, a sounding rocket, or a satellite. In another
embodiment, the method is performed under reduced gravity or
microgravity conditions simulated on Earth using a machine or other
device suitable for this purpose.
[0202] As used herein, the term "hostile environment" is used
interchangeably with "non-natural environment" and means an
environment in which the plant or animal does not normally exist or
survive. By way of example, a hostile environment for a banana
plant would be the Arctic Circle. Another example of a hostile or
non-natural environment for almost any plant or animal would be the
surface of Mars.
[0203] Many elements of the particular hostile environment of
interest can be chosen as the selection pressure to "force" the
undifferentiated cells from the plant or animal to express a subset
of genes that will enable them to adapt to the hostile environment.
As used herein, the term "force" means to apply a selection
pressure to the population of proliferating undifferentiated cells
to obtain cells that survive in the condition of interest. Some
environmental elements suitable for use include, but are not
limited to, temperature, such as excessive heat or excessive cold,
high or low concentrations of carbon dioxide, barometric pressure,
radiation levels, humidity levels, oxygen concentration, low
sunlight exposure, extreme drought, extreme salinity, and the
presence of environmental toxins.
[0204] In one embodiment, the present invention provides a method
of adapting a plant to grow in a hostile environment, wherein the
method comprises culturing undifferentiated cells from the plant in
a weightless condition that mimics at least one element of the
hostile environment to which the plant is to be adapted; selecting
the cells that proliferate in said condition; and cultivating said
selected cells to produce mature plants, wherein the mature plants
are adapted to grow in said hostile environment. The method can be
similarly applied to adapting an animal to grow in a hostile
environment.
[0205] In one embodiment, the method further comprises evaluating
the mature plants in the hostile environment. The plants may be
evaluated on several criteria including, but not limited to, length
of survival, growth rate, reproductive capability, cell structure,
and gene expression. Further, the undifferentiated cells that are
cultured can be obtained from uniting a pollen and ovule of a plant
or a sperm and egg of an animal prior to culturing in a weightless
condition or environment or alternatively, can be united in a
gravity condition and preserved to be cultured in a weightless
environment.
[0206] In one embodiment, plants suitable for use in the methods of
present invention include dicotyledons. In certain exemplary
embodiments, the dicotyledons may include leguminous plants and
other large seed dicots, e.g., peanuts, soybeans, common beans,
squash, zucchini, peppers, melons, cucumbers and others. Other
dicots for use in the invention include potatoes, tomatoes,
alfalfa, canola, apples, and pairs. In certain other embodiments, a
plant suitable for use in the invention can be a woody dicot,
including pome fruits, citrus crops, and vegetable crops.
[0207] In other embodiments, plants suitable for use in the methods
of present invention include be monocotyledons. In certain
exemplary embodiments, the monocotyledons include may include corn
("maize"), rice, wheat, barley, sorghum, rye, banana, plantains,
and other grasses.
[0208] In another embodiment, the plant may be from the genus
Jatropha. Jatropha is a genus of approximately 175 succulent
plants, shrubs and trees (some are deciduous, like Jatropha curcas
L.), from the family Euphorbiaceae. The hardy Jatropha is resistant
to drought and pests, and produces seeds containing up to 40% oil.
When the seeds are crushed and processed, the resulting oil can be
used in a standard diesel engine, while the residue can also be
processed into biomass to power electricity plants and jet engines.
Thus, Jatropha curcas is a promising candidate for future biofuel
and energy production. Therefore, expanding the range of habitats
in which it can survive is of great interest and importance.
[0209] In another embodiment, the organism may be a lichen. Lichens
are composite organisms consisting of a symbiotic association of a
fungus (the mycobiont) with a photosynthetic partner (the
photobiont or phycobiont), usually either a green algae or
cyanobacterium. The morphology, physiology and biochemistry of
lichens are very different to that of the isolated fungus and alga
in culture. Lichens occur in some of the most extreme environments
on Earth--arctic tundra, hot deserts, rain forests, rocky coasts
and toxic slag heaps.
[0210] In another embodiment, the organism may be an algae or
fungus by themselves. In other embodiments, the algae or fungus may
be associated with other primitive organisms, such as lower plants,
including, but not limited to, Thallophytes, Chlorophyceae (for
example, green algae, spirogyra, or vaucheria) and Phycomycetes
(for example, algae fungi, bread mold, or water mold).
[0211] In another embodiment, the undifferentiated cells may be
from a plant that has been genetically modified to result in a
specific phenotype. There are numerous examples of plants that have
been transformed with specific genes so that the resulting
transgenic plants exhibit a particular characteristic, such as
resistance to a particular pathogen or increased size of fruit. For
instance, herbicide resistant plants as disclosed in U.S. Pat. No.
7,169,970, plants that have enhanced nitrogen assimilation as
disclosed in U.S. Pat. No. 6,107,547, and tomatoes with a delayed
ripening phenotype as disclosed in U.S. Pat. No. 5,952,546 are just
a few of the various examples of genetically-modified plants that
have been created. Undifferentiated cells may be obtained from any
of the many varieties of transgenic plants for use in the methods
of the present invention.
[0212] In some embodiments, certain plant species known to be
suitable for use as biofuels may be modified, tailored, altered,
enhanced, by on-orbit, weightless processing of the cells and/or
cellular components, such that higher-energy by weight or by volume
biofuel product may be produced from said plant seeds and
cultivars, and that these species may include the known, 1st, 2nd,
3rd, and 4th generation biofuels, including but not limited to
corn, soy, sugar cane, sugar beet, sweet sorghum, maize, palm,
pinnata, switchgrass, rapeseed, miscanthus, hemp and other known
suitable biofuels of the 1st generation, as well as Jatropha (as
described above), Camelina, Manihot, and algae, including in
particular marine algae, as third generation biofuels, including
halophytes, particularly Salicornia. This embodiment further
contemplates modifications, alterations, and optimizations of these
biofuel species as a result of weightless (microgravity) cellular
processes executed in the zero-force weightless environment
on-orbit, as claimed herein, that enable and result in the
production of useful and valuable genetically altered and tailored
biofuel and crop outputs and end-products.
[0213] Methods of obtaining undifferentiated plant cells are well
known in the art. A mass of undifferentiated plant cells may be
obtained by aseptically removing a small piece of plant tissue from
a selected organ, such as from the root, stem, etc., and placing it
in a sterile medium containing appropriate nutrients. Such a tissue
explant will grow and proliferate into a large number of the same
type of plant cells or of related plant cells, without
specialization of these cells to form specific plant organs such as
roots or leaves, etc. These cells may be referred to as a
heterogeneous population or colony of undifferentiated plant cells
comprised of single cells as well as aggregates of cells. This type
of uninterrupted cell growth and multiplication without the
formation of specific plant organs is known as undifferentiated
cell growth.
[0214] In one embodiment, the undifferentiated plant cells to be
used in methods of the invention may be obtained from the
undifferentiated parenchyma from the apical meristems of the plant.
Reproduction and use of apical cell reproduction has greatly
increased the numbers of plants in a vegetative reproduction
process. The process depends on the isolation of the reproducing
cells at the tip of a plant or plant part (root, branch, etc.)
known as the meristem and successful cloning of the limited number
of cells at the undifferentiated stage of development at the tip of
the plant or other actively growing portions of the plant (root,
cambium, etc.). Suspension cultures of undifferentiated cells may
be prepared from meristem isolates.
[0215] Alternatively, the undifferentiated plant cells suitable for
use in the methods of the invention may be obtained by
proliferation or replication of diploid cells formed by the union
of pollen (sperm) and ovule (egg) from the particular plant species
of interest under weightless conditions as described herein. It is
possible to replicate and produce undifferentiated parenchyma
resulting from the unification of pollen (sperm) and egg (ovary) in
plants that are unified on Earth, preserved prior to any division
of the united single cell, and transported immediately to orbit for
the purpose of producing undifferentiated cells capable of
replicating identical cells for production of tissues used for
parts of plants, and the plant itself, including, but not limited
to stems, roots, flowers, seeds, fruits, and other tissues. The
union of pollen and ovary (egg) in zero gravity will produce a cell
that will go to mitosis and then reproduce that cell continually en
masse or until a genetic break down in the cell(s) may occur that
would disrupt the exponential reproduction of the same mitosis. The
newly formed cells can subsequently be used in the methods of the
present invention.
[0216] The following example outlines an experiment for adapting a
species of citrus plant to grow in colder climates. This example is
for illustration purposes only and in no way limits the scope of
the invention. As described above, various types of plants may be
used in the methods of the invention. Similarly, many different
elements of a hostile environment may be used as selective
pressures to adapt the plants.
EXAMPLE
Method of Adapting a Citrus Plant to Thrive in Cold Climates
[0217] Suspension cultures have been widely used for tissue culture
and mass clonal propagation of a diverse array of higher plants,
and also as models for studies of cell development and
differentiation. Analysis of these suspension cultures determine
structural and genetic changes in undifferentiated plant cells
submitted to the effects of environmental elements, such as
abnormal temperatures. In addition, cell growth and replication are
assessed visually. Structural changes are performed through
histological analyses, including light microscopy, transmission
electron microscopy (TEM), and if feasible, scanning electron
microscopy (SEM). Genetic analyses is performed to evaluate
differential gene expression under the specific environmental
condition.
[0218] Cell suspension cultures are initiated for a variety of
citrus tree (e.g. Citrus sinensis) that has superior fruit, but is
not cold tolerant below 28.degree. F. Cultures are prepared by
excising the undifferentiated parenchyma cells from the apical
meristems of the plant about one day before space shuttle launch.
The cell suspensions are cultured on MS medium modified with 1 mg/L
2,4-D. Once a significant amount of cells are produced, they are
transferred to 10-ml opticells and cultured as described above.
[0219] On orbit, in the opticells the experimental cell suspension
cultures are subjected to temperatures of 25.degree. F. for a
predetermined period of time, such as for several weeks or about
three months or more on the International Space Station (ISS). Also
on orbit, corresponding control cell suspension cultures would be
exposed to the optimal growing temperature for that species of
citrus. Cells exposed to each growing temperature would be returned
to Earth and a portion used for further analyses (see below). The
other portion would immediately be separated into individual cell
containers with agar and cultured to determine which cells
survived. The cells that survived can be nurtured to mature trees
and then subjected to temperatures of 25.degree. F. to determine
the level of cold tolerance achieved. Other parameters of the
mature trees would also be measured, including yield of the trees,
length of survival, and growth.
[0220] Samples from suspension cells maintained in opticells, under
both experimental (25.degree. F.) and control (greater than
28.degree. F.) temperature conditions are collected and compared
for histological and genetic analysis. For histological analyses,
cell suspensions are prepared for light and electron microscopy.
Opticells are compatible for use with standard, phase contrast,
confocal, and high-resolution time-lapse video microscopes. Cells
are examined microscopically on either opticell growth surface or
in between. Oil immersion lenses (up to 100.times.) are used on the
membrane without disruption or contamination. The membrane is
sectioned for small scale staining and microscopy. Additional
samples are removed and fixed in glutaraldehyde for subsequent
evaluation of cell ultrastructure through TEM and SEM.
[0221] By the term "control" or "control cells" or "microorganisms"
within the meaning of the present invention, is meant cells or
microorganisms grown on earth or in a gravity environment as
compared to the cells or microorganisms grown in a weightless or
microgravity environment as described herein or on the Space
Station. Additionally, control or control cells or microorganisms
can also mean those cells or microorganisms grown under a normal
non-stressed environment, as compared to the stress environment
factors and stimuli, as set forth herein.
[0222] Gene expression analyses are performed to evaluate possible
genes that are either up-regulated or down-regulated in response to
the colder temperature. Suspension cultures maintained in space are
fixed in RNAlater (Ambion) liquid preservative through the Kennedy
Space Center fixation tube (UT), hardware designed to provide
proper containment of fixatives for biological samples in space
placed inside the C-hab environment. RNA is isolated and compared
for suspension cultures in both temperature conditions to evaluate
gene expression. Molecular biology techniques for reverse
transcriptase polymerase chain reaction (RT-PCR) and/or copy-DNA
amplified fragment length polymorphism (cDNA-AFLP) and gel
electrophoresis are performed according to well-known techniques to
those skilled in the art and are used for gene expression analyses.
Microarray analysis of gene expression is performed. Results of the
microarray data identify the genes involved in the tolerance factor
for cold. Genes involved in cold-tolerance adaptation will
typically show at least a four-fold change in expression compared
to the control cells exposed to the normal growing temperature.
[0223] Additional evaluations of the cell suspensions may also be
conducted including, but not limited to, cell growth rates, cell
densities, subculture frequency, and size and condition of
cells.
[0224] The above-described techniques are also applicable to
adapting animals to hostile environments. In certain embodiments,
the animal is a mammal. As used herein, the term "mammal" refers to
any mammal. Nonexclusive examples of such mammals include, but are
not limited to, animals such as dogs, cats, horses, cattle, sheep,
and goats. In other embodiments, the animal may be a bird. In yet
other embodiments, the animal may be an aquatic species.
[0225] In one embodiment, the invention provides a method of
adapting an animal to grow in a hostile environment, wherein an
element of the hostile environment is selected from the group
consisting of heat, cold, barometric pressure, excessive radiation,
high carbon dioxide levels, low humidity, high humidity, chemical
pollutants, disease, extreme salinity, reduced or increased
exposure to sunlight, low water and drought conditions, excess
water conditions or combinations thereof, including high and low
levels or concentrations of any of the described conditions.
[0226] In some embodiments, the undifferentiated cells from animals
suitable for use in the methods of the invention can be embryonic
stem cells. Methods for isolating embryonic stem cells are well
known to those of skill in the art, including, but not limited to,
somatic nuclear transfer, cell fusion, and genetic manipulation
techniques that create totipotent cells that are capable of
generating all the tissues of the entire animal.
[0227] Alternatively, the undifferentiated animal cells can be
obtained by methods comprising forming a diploid cell by uniting
two haploid cells and proliferating the diploid cell in a
weightless condition, wherein the diploid cell replicates itself
but does not differentiate into specialized cells and tissues. More
specifically, the egg and sperm are united using standard (IVF)
techniques as described herein.
[0228] Culture conditions and media for culturing the
undifferentiated cells according to the methods of the invention
are well known to the skilled artisan as described above.
[0229] Various methods for culturing stem cells, e.g., embryonic
stem cells (ESCs), may be used with the present invention.
Typically, ESCs are grown in adherent culture systems such as on
tissue culture plates. In certain aspects, culture plates for use
in the invention may comprise a gel matrix such as a collagen or
hydrogel matrix (e.g., a MATRIGEL.TM.). In various embodiments,
culture plates may be coated with, e.g., collagen IV, fibronectin,
laminin, and vitronectin in combination may be used to provide a
solid support for embryonic cell culturing and maintenance, as
described in Ludwig et al. (2006). Matrix components which may be
used with the present invention to coat tissue culture plates
includes a collagen such as collagen IV, laminin, vitronectin,
Matrigel.TM., gelatin, polylysine, thrombospondin (e.g., TSP-1, -2,
-3, -4 and/or -5), and/or ProNectin-F.TM.. Three dimensional
support matrices for use in tissue culture have been previously
described for example in U.S. Publication Nos. 20060198827 and
20060210596, each incorporated herein by reference. The skilled
artisan will recognize that in certain aspects adherent tissue
culture cells may be defined by the cell density or confluency.
Thus, in some cases, methods of the invention involve expansion of
proliferating cells from a high density to a lower density to
facilitate further cell proliferation. For example, methods for
expanding cells according to the invention may involve a first
population of embryonic stem (ES) cells that is between about 50%
and 99% confluent. For example, in certain aspects the first
population of ES cells may be about or less than about 60%, 70%,
80%, 90% or 95% confluent. Furthermore, in certain aspects
expansion or passage of adherent ES cells may involve seeding
separated cells in fresh growth media. As used herein the term
"seeding" cells means dispersing cells in growth media such that
the resultant cell culture(s) are of approximately uniform density.
Thus, seeding of cells may involve mixing separated cells with
fresh growth media and/or spatially dispersing separated cells over
the surface of a tissue culture plate.
[0230] Undifferentiated propagation of adherent colonies of ESCs
may be accomplished with a Knockout (KO) serum-free culture system
without the use of feeders by plating and growing the colonies on
extracellular matrices (ECM) within a feeder-conditioned KO-DMEM
medium supplemented with KOSR and fibroblast growth factor 2
(FGF2). Media available from commercial sources, such as Gibco
Invitrogen Corporation, Grand Island, N.Y. Furthermore, it has been
suggested that feeder conditioning may be replaced by substituting
the medium with high concentrations of FGF2 and noggin.
Alternatively, feeder conditioning was replaced by transforming
growth factor-1 and human leukemia inhibitory factor (LIF) (in
addition to FGF2) and growing the cells on human fibronectin, or by
serum-free media supplemented with soluble factors including FGF2,
activin A, transforming growth factor-.beta.1 (TGF-(.beta.1),
pipecolic acid, GABA, LiCL and culturing the cells on ECM
components. In general, a key limitation of ESC culture systems is
that they do not allow the propagation of pure populations of
undifferentiated stem cells and their use typically involves some
level of background differentiation. The stem cells most commonly
follow a default pathway of differentiation into an epithelial cell
type that grows either as a monolayer of flat squamous cells or
form cystic structures. Most probably, this form of differentiation
represents differentiation of human ESC (hESC) into extraembryonic
endoderm.
[0231] In these adherent culture systems of colonies, the ESCs are
most commonly propagated (mechanically and/or by using enzymatic
digestion) as clusters, on a small scale. These culture systems are
labor-intensive, highly variable, may contain undefined factors,
and do not provide steady-state operating conditions. Most
importantly, they do not typically allow for large scale production
of standardized homogenous undifferentiated ESCs needed for the
aforementioned uses.
[0232] Suspension culture bioreactors offer several advantages over
the conventional use of static monolayer cultures. These systems
facilitate the large-scale expansion of the cells in a homogeneous
culture environment, thus decreasing the risk of culture
variability. They are also less labor-intensive to operate and
offer the possibility of computer control and monitoring of the
culture conditions. Although bioreactors have been used to expand
neural stem cells, mouse ES cells and differentiating hESCs within
embryoid bodies (EBs), only recently some progress has been made
towards the development of protocols for the feeder-free expansion
of undifferentiated hESCs in suspension systems (see US20070212777,
or J. Biotechnology, Vol. 132 (2), 227-236 (2007), which are herein
incorporated by reference in its entirety).
[0233] The present invention also provides methods of identifying
genes associated with adaptation of a plant or animal to a hostile
environment. Previous experiments demonstrated that seedlings from
Arabidopsis thaliana grown under microgravity (weightless
condition) exhibited a change in expression of select genes
compared to their counterpart controls grown on Earth. The
microarray data (FIG. 3) from these experiments showed that 182
genes were differentially expressed with at least a four-fold
change in expression. Some of the differentially expressed genes
were identified as heat-shock and/or CAB (light) genes. Using the
same principles on which these experiments are based, the present
invention provides methods for identifying specific genes
differentially expressed between a control condition and a
particular environmental condition. These identified genes may play
a role in the adaptation of the plant or animal to that particular
environment.
[0234] In one embodiment of the invention, the method comprises
culturing undifferentiated cells from a plant or animal in a
weightless condition that mimics at least one element of the
hostile environment to which the plant or animal is to be adapted;
selecting the cells that proliferate in said condition; and
examining the gene expression profile of the selected cells in
comparison to the gene expression profile of control cells; and
identifying genes that have a change in expression level, wherein
the identified genes are associated with adaptation to the hostile
environment.
[0235] The change in expression level is at least 2-fold, at least
4-fold, at least 6-fold, at least 10-fold, at least 15-fold, or at
least 20-fold. The change in expression level could be an increase
in expression or a decrease in expression. Thus, particular
environmental stimuli may produce both up-regulation and
down-regulation of specific genes.
[0236] In one embodiment, the selected genes that are
differentially expressed in the various environmental conditions
can be further used to produce transgenic plants and animals with
the desired adaptive characteristics by introducing these genes
into cells that mature into plants or animals.
[0237] Therefore, the present invention further encompasses a plant
or animal or undifferentiated cell thereof produced by the methods
described herein, wherein said plant, animal or undifferentiated
cell thereof comprises at least one identified gene that has a
change in expression level as compared to the gene expression
profile of control cell, wherein the identified genes are
associated with adaptation to the hostile environment.
[0238] In one embodiment, a transgenic plant tolerant to
environmental stresses, such as low temperature, freezing, and
dehydration stresses, can be produced by introducing DNA encoding
the protein of the interest into a host plant using genetic
engineering techniques. Methods for introducing the gene into a
host plant include indirect introduction such as the Agrobacterium
infection method and direct introduction such as the particle gun
method, polyethylene glycol method, liposome method, and
microinjection method.
[0239] In the present invention, while the host for the
transformant is not particularly limited, it is preferably a plant.
The plant may be any cultured plant cells, the entire plant body of
a cultured plant, plant organs (such as leaves, petals, stems,
roots, rhizomes, or seeds), or plant tissues (such as epidermis,
phloem, parenchyma, xylem, or vascular bundle). Plants may be
monocotyledonous plants such as rice, maize, and wheat. When a
cultured plant cell, plant body, plant organ or plant tissue is
used as the host, the Agrobacterium infection method, particle gun
method, or polyethylene glycol method can be employed to introduce
the DNA encoding the protein of the present invention to transform
this host plant by introducing a vector into plant sections.
Alternatively, a vector can be introduced into a protoplast by
electroporation to produce a transformed plant.
[0240] For example, when a gene is introduced into Arabidopsis
thaliana by the Agrobacterium infection method, the step of
infecting the plant with an Agrobacterium containing a plasmid
comprising the gene of interest is essential. This step can be
performed by the vacuum infiltration method [CR Acad. Sci. Paris,
Life Science, 316:1194 (1993)]. Specifically, Arabidopsis thaliana
is grown in a soil composed of equivalent portions of vermiculite
and perlite. The Arabidopsis thaliana is immersed directly in a
culture fluid of an Agrobacterium, containing a plasmid comprising
the gene of interest, placed in a desiccator, and then sucked with
a vacuum pump to 65-70 mmHg. Then, the plant is allowed to stand at
room temperature for 5-10 min. The plant pot is transferred to a
tray, which is covered with a wrap to maintain humidity. On the
next day, the wrap is removed. The plant is grown in that state to
harvest seeds.
[0241] Subsequently, the seeds are sown on MS agar medium
supplemented with appropriate antibiotics to select those
individuals which have the gene of interest. Arabidopsis thaliana
grown on this medium are transferred to pots and grown there. As a
result, seeds of a transgenic plant into which the gene of the
interest has been introduced can be obtained. Generally, the genes
are introduced into the genome of the host plant in a similar
manner. However, due to differences in the specific locations on
the genome into which the genes have been introduced, the
expression of the introduced genes varies. This phenomenon is
called "position effect." By assaying transformants with DNA
fragments from the introduced gene as a probe by Northern blotting,
it is possible to select those transformants in which the
introduced gene is expressed more highly.
[0242] The confirmation that the gene of interest is integrated in
the transgenic plant into which the gene of the present invention
has been introduced and in the subsequent generation thereof can be
made by extracting DNA from cells and tissues of those plants and
detecting the introduced gene by PCR or Southern analysis, which
are conventional methods in the art.
[0243] The expression level and expression site of a gene in a
transgenic plant into which the gene of the present invention has
been introduced can be analyzed by extracting RNA from cells and
tissues of the plant and detecting the mRNA of the introduced gene
by RT-PCR or Northern analysis, which are conventional methods in
the art. Alternatively, the expression level and expression site
can be analyzed directly by Western blotting or the like of the
gene product of the present invention using an antibody against the
above product.
[0244] In one embodiment, the gene of interest encodes a
transcription factor. Because stress responses such as drought
tolerance involve coordinated changes in many genes, the ability to
affect many changes with one gene is an attractive proposition.
Transcription factors can activate cascades of genes that function
together to enhance stress tolerance. Transcription factors refer
to a class of genes that control the degree to which other genes in
a cell are activated. Transcription factors are able to recognize
and bind to regions of DNA that have a specific sequence in the
promoters of the genes they regulate. Thus, if a dozen genes all
have that region of DNA somewhere in their promoters, they will all
be regulated by the same transcription factor. Because
transcription factors are key controlling elements of biological
pathways, altering the expression levels of one or more
transcription factors can control a variety of genes involved in
the stress response.
[0245] In another embodiment, the gene of interest encodes a
stress-induced protein. In an exemplary embodiment, the gene of
interest encodes a heat-shock protein.
[0246] The control cells of the present invention to which the
experimental cells are compared would include undifferentiated
cells proliferating in a weightless condition exposed to the normal
or native environmental element of that which is being varied in
the experimental condition. For example, in a method to adapt a
plant to grow in arid conditions, the undifferentiated cells from
the plant would be exposed to a low humidity environment in the
experimental condition and an environment having normal humidity
for that particular plant species in the control condition.
[0247] In another embodiment, the method can be used for the
production of vaccines to be used in animals and humans. Strains of
Salmonella on orbit have previously been shown to demonstrate
increases in virulence. See Wilson et al., PLoS One 12(3): e3923:
1-10.
[0248] In one embodiment, the invention provides a method of
adapting a pathogenic microorganism to grow in a hostile,
non-native environment comprising culturing the microorganism in a
weightless condition that mimics at least one element of the
hostile environment to which the microorganism is to be adapted;
selecting the microorganisms that replicate in said condition;
cultivating said selected microorganism to produce a modified
strain of microorganism, wherein the microorganism is are adapted
to grow in said hostile environment; and optionally evaluating the
microorganism to determine the properties of said modified
microorganism.
[0249] In another embodiment, the virulence of the modified
microorganism is different than the virulence of the original
non-adapted microorganism.
[0250] The technology described in this invention can be used to
"force" the expression of genes on orbit to produced
microorganisms, such as a bacteria or virus, with desired
properties for vaccine production. By stressing, for example, a
pathogenic microorganisms on orbit, modified strains of
microorganisms can be produced. These modified microorganisms can
include less virulent and/or more virulent strains of bacteria and
viruses, which can then be utilized for the production of improved
vaccines. The vaccines produced by this method can be applied to
treatment and/or prevention of many animal and human diseases,
including, but not limited to, hoof and mouth disease and
brucellosis.
[0251] The foregoing detailed description has been given for
clearness of understanding only and no unnecessary limitations
should be understood therefrom as modifications will be obvious to
those skilled in the art.
[0252] It is understood that the present invention is not limited
to the particular methods and components, etc., described herein,
as these may vary. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention. It must be noted that as used herein, the
singular forms "a," "an," and "the" include the plural reference
unless the context clearly dictates otherwise.
[0253] All publications, patents, and patent applications
identified above are incorporated herein by reference in their
entirety. Although this invention has been described in relation to
certain specific embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
* * * * *