4. Isolation of small cells from
TISSUES DERIVED FROM ALL THREE
GERM LAYERS
4.1 Introduction 63
4.2 Experimental - 63
4.2.1 Isolation of small cells from various tissues 64
4.2.2 Characterization of isolated small cells 64
m
4.2.3 Differentiation potential of sphere forming small cells 64
4.3 Results 65
4.3.1 Sphere formation from tissues derived from representative of the
three germ layers 65
4.3.2 Immature gene expression of spheres derived from tissues
representative of the three germ layers 66
4.3.3 Differentiation potential of Cells derived from tissues
representative of the three germ layers 67
4.3.4 Differentiation potential of cells in vivo 68
4.4 Summary of section 4 69
4.5 Discussion 69
4.6 References 76
6. Future prospects
6.1 General overview - - - - 95
6.2 Cell source for tissue engineering regenerative medicine 95
6.3 Future issues - 96
APPENDIX - 97
• Material and method
• Primer sequences
ACKNOWLEDGEMENTS - los
CURRICULUM VITAE
1.Background
1.1 General Introduction
• Importance of stem cells
Stem cells have the remarkable potential to develop into many different cell types
in the body during early life and growth. In addition, in many tissues they serve as
a sort of internal repair system, dividing essentially without limit to replenish other
cells as long as the person or animal is stiH alive. When a stem cell divides, each
new cell has the potential either to remain a stem cell or become another type of cell
with a more specialized function, such as a muscle cell, a red blood cell, or a brain
ceU.
Stem cells are distinguished from other cell types by two important characteristics.
First, they are unspeciaHzed cells capable of renewing themselves through cell
division, sometimes after long periods of inactivity. Second, under certain
physiologic or experimental conditions, they can be induced to become tissue- or
organ-specific cells with special functions. In some organs, such as the gut and bone
marrow, stem cells regularly divide to repair and replace worn out or damaged
tissues. In other organs, however, such as the pancreas and the heart, stem cells
only divide under special conditions.
Until recently, scientists primarily worked with two kinds of stem cells from
animals and humans^ embryonic stem cells and non-embryonic "somatic" or "adult"
stem ceUs. The functions and characteristics of these ceUs will be explained in this
section. Scientists discovered ways to derive embryonic stem cells from early mouse
embryos nearly 30 years ago, in 1981. The detailed study of the biology of mouse
stem ceUs led to the discovery, in 1998, of a method to derive stem ceUs from human
embryos and grow the cells in the laboratory. These cells are called human
embryonic stem cells. The embryos used in these studies were created for
reproductive purposes through in vitro fertilization procedures. When they were no
longer needed for that purpose, they were donated for research with the informed
consent of the donor. In 2006, researchers made another breakthrough by
identifying conditions that would allow some specialized adult cells to be
reprogrammed genetically to assume a stem cell-like state. This new type of stem
cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later part of
this section.
Stem ceUs are important for hving organisms for many reasons. In the 3* to
5-day-old embryo, called a blastocyst, the inner cells give rise to the entire body of
the organism, including all of the many specialized cell types and organs such as the
heart, lung, skin, sperm, eggs and other tissues. In some adult tissues, such as bone
marrow, muscle, and brain, discrete populations of adult stem cells generate
replacements for cells that are lost through normal wear and tear, injury, or
disease.
Given their unique regenerative abilities, stem cells offer new potentials for
treating diseases such as diabetes, and heart disease. However, much work remains
to be done in the laboratory and the clinic to understand how to use these cells for
cell-based therapies to treat disease, which is also referred to as regenerative or
reparative medicine.
Laboratory studies of stem cells enable scientists to learn about the cells' essential
properties and what makes them different from specialized cell types. Scientists are
already using stem cells in the laboratory to screen new drugs and to develop model
systems to study normal growth and identify the causes of birth defects.
Research on stem cells continues to advance knowledge about how an organism
develops from a single cell and how healthy cells replace damaged cells in adult
organisms. Stem cell research is one of the most fascinating areas of contemporary
biology, but, as with many expanding fields of scientific inquiry, research on stem
cells raises scientific questions as rapidly as it generates new discoveries.
• The unique properties of all stem cells
Stem cells differ from other kinds of cells in the body. All stem cells—^regardless of
their source—^have three general properties^ they are capable of dividing and
renewing themselves for long periods," they are unspecialized; and they can give rise
to specialized cell types.
Stem cells are capable ofdividing and renewing themselves for longperiods. Unlike
muscle cells, blood cells, or nerve cells—^which do not normally replicate
themselves—stem cells may replicate many times, or proliferate. A starting
population of stem cells that proHferates for many months in the laboratory can
yield milHons of cells. If the resulting cells continue to be unspecialized, like the
parent stem cells, the cells are said to be capable of long-term self-renewal.
Scientists are trying to understand two fundamental properties of stem cells that
relate to their long-term self-renewal
why can embryonic stem cells proliferate for a year or more in the laboratory
without differentiating, but most non-embryonic stem cells cannot; and what are
the factors in hving organisms that normally regulate stem cell proHferation and
self-renewal?
Discovering the answers to these questions may make it possible to understand how
cell proliferation is regulated during normal embryonic development or during the
abnormal cell division that leads to cancer. Such information would also enable
scientists to grow embryonic and non-embryonic stem cells more efficiently in the
laboratory.
The specijSc factors and conditions that allow stem cells to remain unspeciaHzed are
of great interest to scientists. It has taken scientists many years of trial and error to
learn to derive and maintain stem cells in the laboratory without them
spontaneously differentiating into specific cell tj^es. For example, it took two
decades to learn how to grow human embryonic stem cells in the laboratory
following the development of conditions for growing mouse stem cells. Therefore,
understanding the signals in a mature organism that cause a stem cell population
to prohferate and remain unspecialized until the cells are needed. Such information
is critical for scientists to be able to grow large numbers of unspecialized stem cells
in the laboratory for further experimentation.
Stem cells are unspecialized. One of the fundamental properties of a stem cell is
that it does not have any tissue-specific structures that allow it to perform
specialized functions. For example, a stem cell cannot work with its neighbors to
pump blood through the body (like a heart muscle cell), and it cannot carry oxygen
molecules through the bloodstream (like a red blood cell). However, unspecialized
stem ceUs can give rise to specialized cells, including heart muscle cells, blood cells,
or nerve cells.
Stem cells can give rise to specialized cells. When unspecialized stem cells give rise
to specialized cells, the process is called differentiation. While differentiating, the
cell usually goes through several stages, becoming more specialized at each step.
Scientists are just beginning to understand the signals inside and outside cells that
trigger each stem of the differentiation process. The internal signals are controlled
by a cell's genes, which are interspersed across long strands of DNA, and carry
coded instructions for all cellular structures and functions. The external signals for
cell differentiation include chemicals secreted by other cells, physical contact with
neighboring cells, and certain molecules in the microenvironment. The interaction
of signals during differentiation causes the ceU's DNA to acquire epigenetic marks
that restrict DNA expression in the cell and can be passed on through cell division.
Many questions about stem cell differentiation remain. For example, are the
internal and external signals for cell differentiation similar for all kinds of stem
cells? Can specific sets of signals be identified that promote differentiation into
specific cell types? Addressing these questions may lead scientists to find new ways
to control stem cell differentiation in the laboratory, thereby growing cells or tissues
that can be used for specific purposes such as cell-based therapies or drug
screening.
Adult stem cells typically generate the cell types of the tissue in which they reside.
For example, a blood-forming adult stem cell in the bone marrow normally gives
rise to the many types of blood cells. It is generally accepted that a blood-forming
cell in the bone marrow—^which is called a hematopoietic stem cell—cannot give rise
to the cells of a very different tissue, such as nerve cells in the brain. Experiments
over the last several years have purported to show that stem cells from one tissue
may give rise to cell tj^es of a completely different tissue. This remains an area of
great debate within the research community. This controversy demonstrates the
challenges of studying adult stem cells and suggests that additional research using
adult stem cells is necessary to understand their full potential as future therapies.
1.2 Adult stem cells
An adult stem cell is thought to be an undifferentiated cell, found among
differentiated cells in a tissue or organ that can renew itself and can differentiate to
yield some or all of the major specialized cell types of the tissue or organ. The
primary roles of adult stem cells in a hving organism are to maintain and repair the
tissue in which they are found. Scientists also use the term somatic stem cell
instead of adult stem cell, where somatic refers to cells of the body (not the germ
cells, sperm or eggs). Unlike embryonic stem cells, which are defined by their origin
(cells from the preimplantation-stage embryo), the origin ofadult stem cells in some
mature tissues is still under investigation.
Research on adult stem cells has generated a great deal of excitement. Scientists
have found adult stem cells in many more tissues than they once thought possible.
This finding has led researchers and clinicians to ask whether adult stem cells could
be used for transplants. In fact, adult hematopoietic, or blood-forming, stem cells
from bone marrow have been used in transplants for 40 years. Scientists now have
evidence that stem cells
exist in the brain and the heart. If the differentiation of
adult stem cells can be controlled in the laboratory, these cells may become the
basis of transplantation-based therapies.
• History of Adult stem cell research
The history of research on adult stem cells began about 50 years ago. In the 1950s,
researchers discovered that the bone marrow contains at least two kinds of stem
cells. One population, called hematopoietic stem cells, forms all the types of blood
cells in the body. A second population, called bone marrow stromal stem cells (also
called mesenchymal stem cells, or skeletal stem cells by some), were discovered a
few years later. These non-hematopoietic stem cells make up a small proportion of
the stromal cell population in the bone marrow, and can generate bone, cartilage,
fat, cells that support the formation of blood, and fibrous connective tissue.
In the 1960s, scientists who were studying rats discovered two regions of the brain
that contained dividing cells that ultimately become nerve cells. Despite these
reports, most scientists believed that the adult brain could not generate new nerve
cells. It was not until the 1990s that scientists agreed that the adult brain does
contain stem cells that are able to generate the brain's three major cell
types—astrocjrtes and oligodendrocytes, which are non-neuronal cells, and neurons,
or nerve cells.
• Function of Adult stem cells
Adult stem cells have been identified in many organs and tissues, including brain,
bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart,
gut, Hver, ovarian epithelium, and testis. They are thought to reside in a specific
area of each tissue (called a "stem cell niche"). In many tissues, current evidence
suggests that some types of stem cells are pericytes, cells that compose the
outermost layer of small blood vessels. Stem cells may remain quiescent
(non-dividing) for long periods of time until they are activated by a normal need for
more cells to maintain tissues, or by disease or tissue injury.
Typically, there is a very small number of stem cells in each tissue, and once
removed from the body, their capacity to divide is limited, making generation of
large quantities of stem cells difficult. Scientists in many laboratories are trying to
find better ways to grow large quantities of adult stem cells in cell culture and to
manipulate them to generate specific cell types so they can be used to treat injury or
disease. Some examples of potential treatments include regenerating bone using
cells derived from bone marrow stroma, developing insulin-producing cells for
type 1 diabetes, and repairing damaged heart muscle following a heart attack with
cardiac muscle cells.
1.3 Research methods for identifying adult stem cells
Scientists often use one or more of the following methods to identify adult stem
cells: (i) label the cells in a living tissue with molecular markers and then
determine the speciaHzed cell types they generate; (2) remove the cells from a living
animal, label them in cell culture, and transplant them back into another animal to
determine whether the cells replace (or "repopulate") their tissue of origin.
Importantly, it must be demonstrated that a single adult stem cell can generate a
line of genetically identical cells that then gives rise to all the appropriate
differentiated cell tj^es of the tissue. To confirm experimentally that a putative
adult stem cell is indeed a stem cell, scientists tend to show either that the cell can
give rise to these genetically identical cells in culture, and/or that a purified
population of these candidate stem cells can repopulate or reform the tissue after
transplant into an animal.
As indicated above, scientists have reported that adult stem cells occur in many
tissues and that they enter normal differentiation pathways to form the specialized
cell types of the tissue in which they reside.
• Normal differentiation pathways of adult stem cells.
In a living animal, adult stem cells are available to divide, when needed, and can
give rise to mature ceU types that have characteristic shapes and specialized
structures and functions of a particular tissue. Hematopoietic stem cells give rise to
all the types of blood cells' red blood cells, B lymphocytes, T lymphocytes, natural
killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.
Mesenchymal stem cells give rise to a variety of cell types' bone cells (osteocytes),
cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective
tissue cells such as those in tendons.
Neural stem cells in the brain give rise to its three major cell types- nerve cells
(neurons) and two categories of non-neuronal cells—astrocytes and
oligodendrocytes.
Epithelial stem cells in the hning of the digestive tract occur in deep crypts and give
rise to several ceD types: absorptive cells, goblet cells, paneth cells, and
enteroendocrine cells.
Skin stem cells occur in the basal layer of the epidermis and at the base of hair
follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the
surface of the skin and form a protective layer. The follicular stem cells can give rise
to both the hair follicle and to the epidermis.
• Transdifferentiation.
A number of experiments have reported that certain adult stem cell t5^es can
differentiate into cell t5TDes seen in organs or tissues other than those expected from
the cells' predicted lineage (i.e., brain stem cells that differentiate into blood cells or
blood-forming cells that differentiate into cardiac muscle cells, and so forth). This
reported phenomenon is called transdifferentiation.
Although isolated instances of transdifferentiation have been observed in some
vertebrate species, whether this phenomenon actually occurs in humans is under
debate by the scientific community. Instead of transdifferentiation, the observed
instances may involve fusion of a donor cell with a recipient cell. Another possibility
is that transplanted stem cells are secreting factors that encourage the recipient's
own stem cells to begin the repair process. Even when transdifferentiation has been
detected, only a very small percentage of cells undergo the process.
In a variation of transdifferentiation experiments, scientists have recently
demonstrated that certain adult cell t5T)es can be "reprogrammed" into other ceU
tjT^es in vivo using a weU-controUed process of genetic modification (see Section VI
for a discussion of the principles of reprogramming). This strategy may offer a way
to reprogram available cells into other cell types that have been lost or damaged due
to disease. For example, one recent experiment shows how pancreatic beta cells, the
insulin-producing ceUs that are lost or damaged in diabetes, could possibly be
created by reprogramming other pancreatic ceUs. By "re-starting" expression of
three critical beta-cell genes in differentiated adult pancreatic exocrine cells,
researchers were able to create beta ceU-like cells that can secrete insulin. The
reprogrammed cells were similar to beta cells in appearance, size, and shape;
expressed genes characteristic of beta cells." and were able to partially restore blood
sugar regulation in mice whose own beta cells had been chemically destroyed. While
not transdifferentiation by definition, this method for reprogramming adult cells
may be used as a model for directly reprogramming other adult cell types.
In addition to reprogramming ceils to become a specific ceD type, it is now possible
to reprogram adult somatic cells to become like embryonic stem cells (induced
pluripotent stem cells, iPSCs) through the introduction of embryonic genes. Thus, a
source of cells can be generated that are specific to the donor, thereby avoiding
issues of histocompatibility, if such cells were to be used for tissue regeneration.
However, like embryonic stem cells, determination of the methods by which iPSCs
can be completely and reproducibly committed to appropriate cell Hneages is still
under investigation
1.4 Pluripotent stem cells
• The similarities and differences between embryonic and adult stem cells
Human embryonic and adult stem cells each have advantages and disadvantages
regarding potential use for cell-based regenerative therapies. One major difference
between adult and embryonic stem cells is their different abilities in the number
and type of differentiated ceU types they can become. Embryonic stem cells can
become all cell types of the body because they are pluripotent. Adult stem cells are
thought to be limited to differentiating into different cell types of their tissue of
origin.
Embryonic stem cells can be grown relatively easily in culture. Adult stem cells are
rare in mature tissues, so isolating these cells from an adult tissue is challenging,
and methods to expand their numbers in cell culture have not yet been worked out.
This is an important distinction, as large numbers of cells are needed for stem cell
replacement therapies.
Scientists believe that tissues derived from embryonic and adult stem cells may
differ in the likeHhood of being rejected after transplantation. We don't yet know
whether tissues derived from embryonic stem cells would cause transplant rejection,
since the first phase 1 cHnical trial testing the safety of cells derived from hESCS
has only recently been approved by the United States Food and Drug
Administration (FDA).
Adult stem cells, and tissues derived from them, are currently believed less likely to
initiate rejection after transplantation. This is because a patient's own cells could be
expanded in culture, coaxed into assuming a specific cell tj^e (differentiation), and
then reintroduced into the patient. The use of adult stem cells and tissues derived
from the patient's own adult stem cells would mean that the cells are less lilcely to
be rejected by the immune system. This represents a significant advantage, as
immune rejection can be circumvented only by continuous administration of
immunosuppressive drugs, and the drugs themselves may cause deleterious side
effects
• Embryonic stem cells
A. What stages of early embryonic development are important for generating
embryonic stem cells?
Embryonic stem cells, as their name suggests, are derived from embryos. Most
embryonic stem cells are derived from embryos that develop from eggs that have
been fertilized in \dtro—^in an in vitro fertilization clinic—and then donated for
research purposes with informed consent of the donors. They are not derived from
eggs fertilized in a woman's body.
A. Establish of embryonic stem cells grown in the laboratory
Growing cells in the laboratory is known as cell culture. Human embryonic stem
cells (hESCs) are generated by transferring cells from a preimplantation-stage
embryo into a plastic laboratory cultm'e dish that contains a nutrient broth known
as culture medium. The cells divide and spread over the surface of the dish. The
inner surface of the culture dish is typically coated with mouse embryonic skin cells
that have been treated so they will not di\dde. This coating layer of cells is called a
feeder layer. The mouse cells in the bottom of the culture dish provide the cells a
sticky surface to which they can attach. Also, the feeder cells release nutrients into
the culture medium. Researchers have devised ways to grow embryonic stem cells
without mouse feeder cells. This is a significant scientific advance because of the
risk that viruses or other macromolecules in the mouse cells may be transmitted to
the human cells.
The process of generating an embryonic stem cell line is somewhat inefficient, so
Hnes are not produced each time cells from the preimplantation-stage embryo are
placed into a culture dish. However, if the plated cells survive, divide and multiply
enough to crowd the dish, they are removed gently and plated into several fresh
culture dishes. The process of re-plating or subculturing the cells is repeated many
times and for many months. Each cycle of subculturing the cells is referred to as a
passage. Once the cell line is estabhshed, the original cells yield millions of
embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for
a prolonged period of time without differentiating, are pluripotent, and have not
developed genetic abnormalities are referred to as an embryonic stem cell line. At
any stage in the process, batches of cells can be frozen and shipped to other
laboratories for further culture and experimentation.
B. Tests for identifying embryonic stem cells
At various points during the process of generating embryonic stem cell lines,
scientists test the cells to see whether they exhibit the fundamental properties that
make them embryonic stem cells. This process is called characterization.
Scientists who study human embryonic stem cells have not yet agreed on a
standard battery of tests that measure the cells' fundamental properties. However,
laboratories that grow human embryonic stem cell lines use several kinds of tests,
including-
Growing and subculturing the stem cells for many months. This ensures that the
cells are capable of long-term growth and self-renewal. Scientists inspect the
cultures through a microscope to see that the cells look healthy and remain
undifferentiated.
Using specific techniques to determine the presence of transcription factors that are
typically produced by undifferentiated cells. Two of the most important
transcription factors are Nanog and Oct4. Transcription factors help turn genes on
and off at the right time, which is an important part of the processes of cell
differentiation and embryonic development. In this case, both Oct 4 and Nanog are
associated with maintaining the stem cells in an undifferentiated state, capable of
self-renewal.
Using specific techniques to determine the presence of paricular cell surface
markers that are typically produced by undifferentiated cells.
Examining the chromosomes under a microscope. This is a method to assess
whether the chromosomes are damaged or if the number of chromosomes has
changed. It does not detect genetic mutations in the cells.
Determining whether the cells can be re-grown, or subcultured, after freezing,
thawing, and re-plating.
Testing whether the human embryonic stem cells are pluripotent by l) allowing the
cells to differentiate spontaneously in cell culture.' 2) manipulating the cells so they
will differentiate to form cells characteristic of the three germ layers; or 3) injecting
the cells into a mouse with a suppressed immune system to test for the formation of
a benign tumor called a teratoma. Since the mouse's immune system is suppressed,
the injected human stem cells are not rejected by the mouse immune system and
scientists can observe growth and differentiation of the human stem cells.
Teratomas t5T)ically contain a mixture of many differentiated or partly
differentiated cell types—an indication that the embryonic stem cells are capable of
differentiating into multiple cell types. As long as the embryonic stem cells in
culture are grown under appropriate conditions, they can remain undifferentiated
(unspecialized). But if cells are allowed to clump together to form embryoid bodies,
they begin to differentiate spontaneously. They can form muscle cells, nerve cells,
and many other cell tjTpes. Although spontaneous differentiation is a good
indication that a culture of embryonic stem cells is healthy, it is not an efficient way
to produce cultures of specific cell types.
So, to generate cultures of specific types of differentiated cells—^heart muscle cells,
blood cells, or nerve cells, for example—scientists try to control the differentiation of
embryonic stem cells. They change the chemical composition of the culture medium,
alter the surface of the culture dish, or modify the cells by inserting specific genes.
Through years of experimentation, scientists have established some basic protocols
or "recipes" for the directed differentiation of embryonic stem cells into some specific
cell types.
If scientists can reliably direct the differentiation of embryonic stem cells into
specific cell types, they may be able to use the resulting, differentiated cells to treat
certain diseases in the future. Diseases that might be treated by transplanting cells
generated from human embryonic stem cells include Parkinson's disease, diabetes,
traumatic spinal cord injury, Duchenne's muscular dystrophy, heart disease, and
vision and hearing loss.
• Induced pluripotent stem ceUs
Induced pluripotent stem cells (iPSCs) are adult ceUs that have been genetically
reprogrammed to an embryonic stem cell-like state by being forced to express genes
and factors important for maintaining the defining properties of embryonic stem
cells. Although these cells meet the defining criteria for pluripotent stem cells, it is
not known if iPSCs and embryonic stem cells differ in clinically significant ways.
Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in
late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem
cells, including expressing stem cell markers, forming tumors containing cells from
all three germ layers, and being able to contribute to many different tissues when
injected into mouse embryos at a very early stage in development. Human iPSCs
also express stem cell markers and are capable of generating cells characteristic of
all three germ layers.
Although additional research is needed, iPSCs are already useful tools for drug
development and modeHng of diseases, and scientists hope to use them in
transplantation medicine. Viruses are currently used to introduce the
reprogramming factors into adult cells, and this process must be carefully
controlled and tested before the technique can lead to useful treatments for humans.
In animal studies, the virus used to introduce the stem cell factors sometimes
causes cancers. Researchers are currently investigating non-viral delivery
strategies. In any case, this breakthrough discovery has created a powerful new way
to "de-differentiate" cells whose developmental fates had been previously assumed
to be determined. In addition, tissues derived from iPSCs wiU be a nearly identical
match to the ceU donor and thus probably avoid rejection by the immune system.
The iPSC strategy creates pluripotent stem cells that, together with studies of other
types of pluripotent stem cells, will help researchers learn how to reprogram cells to
repair damaged tissues in the human body.
1.5 Possibility of existence of adult pluripotent stem cells
Many important questions about adult stem cells remain to be answered. They
include:
How many kinds of adult stem cells
exist, and in whichtissues do they exist?
How do adult stem cells evolve during development and how are they maintained in
the adult? Are they "leftover" embrj'^onic stem cells, or do they arise in some other
way?
As described above, stem cells were discovered by studies of development. In
development, germ-layer differentiation is a critical point. All adult stem cells
discovered until today are known to be generated after germ-layer differentiation.
By this reason, adult stem cells cannot differentiate crossing germ layers.
Whereas, some scientists suggested that "left over" embryonic cells still reside in
adult body.
For example, MAPC, spore-like stem cells, MIAMI cells, VSESC and MUSE ceUs.
They suggest those theories because some phenomena can not be explained by
knowledge from development study. Adult stem cells have generally been felt to be
Hmited to multipotency and unable to cross germ layer lineages as they develop.
However, first, a part of mesenchymal stem ceUs were derived firom ectoderm not
mesoderm. Second, a part of mesenchymal stem cells can differentiate into cells
derived firom ectoderm. Thus, some of adult stem cells cross the germ layers. If the
origin of germ layer is critical for determining stem cells' fate, these phenomena can
not be explained. This is the reason why researchers suggest that "left over"
embryonic cells still reside in adult body.
Especially spore-like stem cell research suggested a very unique theory.
• Spore-like stem cells (Figure 2)
While the existence of adult stem cells has been reported for more than a decade,
our research team has advanced the theory that common adult stem cells reside in
all body tissues, they possess small figure and stress-tolerant property. These cells
were named spore-like stem cells.
1.6 Hypothesis of this research
• Sphere formation
Sphere formation is recognizes as one of isolation method of adult stem cells.
Because, stem cells should hold a strong proliferative potential and self-renewal
potency, sphere formation is recognized as a result of those potencies.
Interestingly, as presented by neurospheres, some sphere forming stem cells show
gene expression over lapping with ES cells.
• Immature adult stem ceUs
If stem cells which can cross the germ layer lineages
existing in native organisms. ES cells are artificial.
• Hypothesis
Considering all the various factors together.
1. Adult stem cells stem which can cross germ layers (very
immature adult stem cells) may exist.
2. They are very small, and stress-tolerant
3. They form spheres
In this study, we characterized cells isolated from three adult tissues (lung, muscle
and spinal cord) representative of the three different germ layers (endoderm,
mesoderm and ectoderm) and from bone marrow. The cells were triturated to break
mature cells and propagated as non-adherent clusters or spheres in a serum-free
culture medium. We found that cells from each source, initially expressed many of
the markers associated with ESCs and demonstrated differentiation potential into
all three germ layers at a time that neural Hneage markers had not yet been
expressed. Ultimately cells from each tissue, differentiated into cells representative
of all three germ layers in \itro. The isolation initially contained a significant
amount of floating debris, non-adherent cells, insoluble proteins or fibers, and other
extraneous materials, all of which appeared to participate in the formation of
non-adherent spherical clusters that contained the cells. The cellular make-up of
individual spheres was not identical; that is, spheres were composed of
heterogeneous populations of cells, even when the spheres were generated from
cells procured fi'om the same tissue at the same time. Similarities or differences
seen in the cell content of different spheres, were believed to be secondary to the
environment in which they were cultured.
