Adult Stem Cells

New Therapies, No Cloning

by Wolfgang Lillge, M.D.

Most people around the world believe that embryonic stem cells offer the greatest
promise for developing new medical treatments: In reality, adult stem cells from the body
of the patient being treated, have shown a much greater potential for curing severe
diseases. One reason for this distorted public opinion is the pattern of media reports about
ongoing research with stem cells. Any new result, even if a very minor one, with
embryonic stem cells, is blown up in the media as a major breakthrough. But when
researchers announce even greater success using adult or alternative sources of stem
cells, there are generally only small notes in the remote corners of the science pages.

The same spin has an obvious political purpose, as it has a major influence on the current
cloning debate, both in the United States and abroad. There is a powerful lobby,
including biotechnology companies and investors, that is pushing hard to completely
open up scientific research to the "therapeutic" cloning of human embryos, disregarding
moral objections associated with it. While adult stem cell research continues—somehow
anonymously, but with remarkable success!—the public focus is clearly on embryonic
stem cell research. The current debate in the U.S. Congress on whether to allow cloning
of human embryos for research purposes is a reflection of that.

What is needed in the legislative process is a clear perspective about the consequences of
such decisions. Scientists cannot achieve progress by overriding basic human values;
here, this means developing treatments for diseases by killing human embryos in the
process. One must understand there is no qualitative difference between cloning a human
embryo for "therapeutic" means (to obtain embryonic stem cells) and for "reproductive"
means (to produce a complete human clone). The difference is only technical; those who
strongly favor therapeutic cloning should honestly admit to these consequences.

The legislation in the United States is still undecided: The House has passed a strict
cloning ban, which the President supports; many Senators are being lobbied and have
come out for human embryo cloning for "medical research only." In Germany, such
attempts remain banned, but the importation of stem-cell lines produced by this method
from other countries, has now been permitted. In Britain, no restrictions exist on attempts
at human cloning. What must be brought to bear is the informed moral stand of the
citizens, against a further moral decline of society.

The Use of Stem Cells

Reviewing the research results of the last couple of years, it becomes obvious that
embryonic stem cells involve severe problems when scientists try to use them as means
of treatment. Largely unmentioned is the high "price" paid for these very uncertain
results. To obtain embryonic stem cells, an embryo—a nascent human being—must be
killed in the morula phase of growth, when one can harvest "pluripotent" cells (i.e., those
still able to develop into many different cell types).

The general idea of therapy with stem cells, both embryonic and adult, is to use these
cells to regenerate or replace diseased tissue, as in Parkinson's disease or multiple
xclerosis patients. One of the major advantages of adult stem cells is that cells from the
patient's own body can be used, which removes the risk of immune reaction against
reimplanting these cells; whereas, in the case of embryonic stem cells, exogenous cells
are used which definitely will provoke a major immune response. The only future way
out of this immune dilemma would be to produce embryonic stem cells of the patient, by
way of "therapeutic cloning." For that, you take a body cell, extract the nucleus, insert
this nucleus into a denucleated egg cell, and stimulate its embryonic development. If it
reaches the morula phase, you kill the embryo extracting the stem cells, which should be
immunologically identical to the patient's cells. After culturing and transforming them
into the needed tissue, they will be retransplanted to the patient for treatment.

There has been, so far, no report of successful treatments of humans in this way. These
cells seem to be too "unripe"—also reflected by the fact, that embryonic stem cells have a
severe tumor risk. Of course, all these approaches are in a very early phase of research;
but why pursue a technique where such problems are combined with a major moral
handicap?

New Promising Results

The pattern of media twisting was visible recently when several important research
results were achieved. Anyone who relies just on television for news, or quickly scans
newspapers, would have missed them altogether. Using experimental procedures with
adult stem cells, patients with Parkinson's disease and multiple sclerosis have experienced
significant medical improvements. Comparable benefits using embryonic stem cell
treatments have not yet been demonstrated even in animal experiments.

The most recent case concerns a 57-year-old man, who was diagnosed at the age of 49
with Parkinson's disease, typified by a progressive loss of dopamine-producing cells in
the brain. The disease worsened progressively, leading to tremors and rigidity in the
patient's right arm. Traditional drug therapy did not help. As reported by neurosurgeon
Michel Levesque of Cedars-Sinai Medical Center in Los Angeles, at a conference in
Chicago in early April, stem cells were harvested three years ago from the patient's brain
using a routine brain biopsy procedure. They were cultured for several months in the
laboratory, growing to several million cells. About 20% matured into dopamine-secreting
neurons. In March 1999, these neurons were injected into the patient's brain. Within three
months the man's motor skills had improved by 37%, with an increase in dopamine
production of 55.6%. By March 2000, the patient's overall performance had improved by
83%, and as of March 2002, no symptoms of Parkinson's disease can be seen—and that,
without taking any other Parkinson's medication.
Even if this is yet only a single case, a treatment program on the basis of adult stem cells
could soon outdo other approaches, including recent therapeutic attempts using neural
cells of aborted fetuses. Levesque will now expand his program to 12 additional patients.

One can only assume, that had the treatment been conducted with embryonic stem cells,
the media would have spread the story all over the place.

There is also a potential new treatment for people with severe cases of multiple sclerosis
(MS). Recent research results were presented during the American Academy of
Neurology's 54th Annual Meeting in Denver, April 13-20, 2002. The new treatment
involves removing stem cells from the patient's blood, killing those cells that are working
against the body's immune system, and then returning the healthy stem cells back to the
body. Similar treatments have also been tried in advanced stages of cancer, where strong
chemotherapy would destroy the blood-producing system in the bone marrow.

"The hope is that these stem cells will eventually reconstitute into healthy immune
system cells and the disease process can be stopped," said study author George Kraft of
the University of Washington Medical Center in Seattle. There, 26 people with severe
MS underwent this treatment, called autologous stem cell transplantation. Their results
were followed for an average of 14.2 months. Conventional treatments had previously
been unsuccessful for all of the patients, either with no improvement or intolerable side
effects. After the stem cell transplant, 20 patients were stable, with no change in their
amount of disability. Six showed some degree of mild improvement by some measures.
"This is good news," said Kraft. "These patients had all been rapidly deteriorating over
the past year, so to get them to a point where they are stabilized is great progress."

Meanwhile in Canada, younger MS patients, at a less severe stage of the disease, have
shown even greater benefit from the same procedure. Six months after the first patient
was treated, she was found to have no evidence of the disease on MRI scans. Three other
patients have also received successful adult stem cell grafts, with no current evidence of
active disease. It is still too early to tell whether the Canadian patients have achieved
permanent remission or a cure, but there can be no question that the research is
significant.

We may add a short list of other advances made in adult-cell therapies and research in the
recent period:

* In Israel, doctors inserted white blood cells of a paraplegic patient into her severed
spinal cord, after which she regained bladder control and the ability to wiggle her toes
and move her legs.

* Immune systems destroyed by cancer were restored in children using stem cells from
umbilical-cord blood.

* Harvard Medical School researchers killed cells producing insulin in mice, inducing
diabetes; then, the animals' adult stem cells took over and regenerated missing cells
needed to produce insulin and eliminate the disease. This compares to an experiment in
which embryonic stem cells, injected into diabetic mice, achieved a 3% insulin
production rate, and all the mice died. "The permanent reversal of Type 1 diabetes in
mice may end the wrenching debate over harvesting stem cells from the unborn to treat
adult diseases," said the Harvard University Gazette of July 19, 2001, of this astonishing
success. "It should be possible to use the same method to reverse Type 1 diabetes in
humans," says Denise Faustman, the associate professor of medicine who leads the
research. Set-up has already begun for a trial for human patients at Massachusetts
General Hospital in Boston.

Nature Creates Doubts

Against the mounting evidence in favor of adult stem cell treatments, a note was
published in Nature magazine in March, implying that hopes for this research and
practice may be premature. The claim that adult stem cells are just as versatile as
embryonic ones may have been misleading, said the British publication's note; they may
be just fusing with other cells to form abnormal hybrids, which are mistaken for pristine
new tissues.

What seemed a strong argument on first view, soon turned out not to be very convincing.
The conditions of the experiment reported were such, that no significant results could be
expected, and later the authors themselves admitted that fusion of this type is a very rare
event. However, the Nature warning was multiplied widely enough in leading non-
scientific media, to create a general doubt in public opinion.

In contrast, a small, but highly important note went almost unnoticed: Catherine
Verfaillie at the University of Minnesota reported, that she had found stem cells in the
bone marrow of adults, that can turn into every single tissue in the body. Until now, only
stem cells from early embryos were thought to have such "pluripotent" properties. If
further research confirms these findings, there would be no need to resort to "therapeutic
cloning" of human embryos in order to get matching stem cells.

Experiments so far seem to confirm that the cells—dubbed multipotent adult progenitor
cells, or MAPCs—have the same potential as embryonic stem cells. Verfaillie says that
her lab has reliably isolated the cells from about 70% of the 100 or so human volunteers
who donated marrow samples. She reports the cells seem to grow indefinitely in culture,
like embryonic stem cells; some cell lines have been growing for almost two years and
have kept their characteristics, with no signs of aging.

Therapeutic Cloning: An Unnecessary Risk

Characteristics, and a Comparison with Adult Stem Cell Research

Proponents of therapeutic cloning claim that this technique may offer a new mode of
treatment in the repair and regeneration of tissue and organs. Especially in the field of
organ transplantation, where immune rejection is common, therapeutic cloning is often
seen as a way of eliminating this problem of immune rejection. Critics, however, claim
that this view is incorrect, and that immune rejection still exists in therapeutic cloning,
along with a myriad of other complexities and problems.

In therapeutic cloning, stem cells are created from a donor for the main purpose of
providing tissue (such as for organ repair), in the event that the donor might need such
treatment at a future date. The way in which this is done is as follows.

A somatic (adult) cell from the donor is transferred into an enucleated egg (an egg from
which the nucleus has been removed), yielding a single celled cloned embryo. When the
egg has developed into a blastocyst, the inner cell mass is then removed and cultured into
embryonic stem cells, which are grown to produce the desired, healthy, "therapeutic"
cells (such as nerve cells, muscle cells, organ tissue, etc.). These new cells are then
transplanted back into the patient (who is presumed to be the same as the donor of the
original somatic cell, in order to avoid immune rejection).

A single cell, cultured in a dish by itself, will divide to form a population of identical
cells, known as cell clones. The resulting cloned embryo is therefore genetically identical
to the donor. Until very recently, this characteristic of identical genetic matching has
been considered the primary advantage of "therapeutic" cloning. Now, however, there is
evidence to prove otherwise.

Other names for therapeutic cloning include Somatic Cell Nuclear Transfer (SCNT,
which gave rise to Dolly the sheep), and Cell Replacement through Nuclear Transfer
(CRNT). Whichever name is used, therapeutic cloning requires the deliberate creation
and disaggregation (destruction, in other words) of a human embryo.

It is possible, however, that the cloning may be used for reproductive purposes instead of
for "therapeutic" purposes. For example, from an adult female, the DNA may be removed
from a harvested egg (thus "enucleating" the egg). Skin cells may then be removed from
an adult male, and the DNA of these cells may be transferred into the nucleus of the
woman's unfertilized egg, to produce an early stage embryo with the donor's DNA. From
this cloned embryo, human embryonic stem cells (hESCs) may be grown in the
laboratory, and then implanted into a surrogate woman who ultimately gives birth to a
clone of the man from whom the skin cells were derived. This is how Dolly the sheep
was produced. Although "reproductive cloning" and "therapeutic cloning" have different
objectives, the means by which they are conducted, and the controversies surrounding
such means, are inseparably linked.

According to Dr. Robert Lanza, "It is true that the techniques developed in CRNT
research can prepare the way scientifically and technically for efforts at reproductive
cloning." (Robert Lanza et al., "The ethical validity of using nuclear transfer in human
transplantation", JAMA, 284, 3175 - 3179, 12/27/2000). Needless to say, reproductive
cloning is already widely recognized to be an ethical can of worms.
Ethical controversies aside, however, David A. Prentice, Ph.D., of the Department of Life
Sciences at Indiana State University, adds that cloning is unsafe both for the clone and for
the surrogate mother. He points out that even apparently healthy clones have
abnormalities in gene expression. "A review of all the world's cloned animals suggests
that every one of them is genetically and physically defective," he says. He also cites Ian
Wilmut, who points out that, "There is abundant evidence that cloning can and does go
wrong and there is no justification for believing that this will not happen in humans."
(Quoted in "Gene defects emerge in all animal clones", Sunday Times of London,
4/28/02).

The success rate of reproductive cloning is extremely low, as 277 nuclear transfers were
required to enucleated the eggs from which Dolly the sheep was created. It has been
pointed out that even when animals are successfully cloned, every one of them, without
exception, suffers from numerous genetic abnormalities. Even Dolly the sheep was
"born" with incomplete epigenetic reprogramming (the heritable erasure and remarking
of genes that determines either normal or abnormal development). Currently, the highest
efficiency rate of SCNT cloning in any species is 7% (with pigs), and in most species the
success rate is below 1%. However, even when successful, from 10,000 genes that were
analyzed in cloned mice, approximately 400 of these genes were found to express genetic
abnormalities.

Dr. Prentice offers some further alarming statistics on the success rates (or the lack
thereof) of cloning in animals:

• Dolly the sheep, the first cloned animal: 1 live birth out of 277 cloned embryos.
Success rate = 0.4%.
• Cloned mice: 5 live births out of 613. Success rate = 0.8%
• Cloned pigs: 5 live births out of 72 cloned embryos implanted. Success rate = 7%.
• Cloned goats: 3 live births out of 85 cloned embryos implanted. Success rate =
3.5%.
• Cloned cattle: 30 live births out of 496 cloned embryos implanted. Success rate =
6%.
• Cloned cat: 1 live birth out of 188 cloned embryos. Success rate = 0.5%.
• Cloned gaur: 1 live birth out of 692 cloned embryos. Success rate = 0.1%.
• Cloned rabbits: 6 live births out of 1852 cloned embryos. Success rate = 0.3%.

One of the risks for the surrogate mother presented by cloning is what is known as "large
offspring syndrome", in which the cloned embryo develops into an abnormally, and
dangerously, large fetus by the time of birth. But there are also many other ways in which
cloning places women at risk. For example, in order to treat all of the 17 million people
in the U.S. who suffer from diabetes, Dr. Prentice has made some sobering calculations.
Allowing for 10 eggs harvested per donor, and allowing for a generous 20% cloning
efficiency to achieve the blastocyst stage, as well as a generous 10% efficiency at
initiating the embryonic stem cell culture, a minimum of 850 million eggs would be
required, which translates into 85 million women of childbearing age who would be
required as donors. This would be more than one-third the population of the United
States, who would be needed as egg donors for the treatment of a group of people
roughly one-sixteenth as large in population size.

While high dose hormone therapy and surgery have been developed to obtain eggs in
large numbers, such techniques nevertheless pose significant health risks by jeopardizing
the donor's immediate health and future reproductive success. Additionally, the
possibility for commercial exploitation puts economically disadvantaged women in
particular jeopardy.

Overall, Dr. Prentice concludes, therapeutic cloning may be judged as unsuccessful.

Transplantation remains one of its many problems, and Dr. Prentice cites W.M. Rideout
as having stated, "Our results raise the provocative possibility that even genetically
matched cells derived by therapeutic cloning may still face barriers to effective
transplantation for some disorders." (W.M. Rideout et al., "Correction of a genetic defect
by nuclear transplantation and combined cell and gene therapy," an online publication in
Cell, 3/8/2002).

It has been proposed by a number of researchers that cloning is not able to provide the
claimed medical treatments, and Drs. James Thomson and Alan Trounson add that there
is a very low chance of success in the clinical use of therapeutic cloning. (Dr. James
Thomson, "Multilineage differentiation from human embryonic stem cell lines", Stem
Cells, 2001; Dr. Alan Trounson, "The derivation and potential use of human embryonic
stem cells", Reproduction, Fertility and Development, 2001). Additionally, Dr. Irving
Weissman of Stanford University, and Dr. John Gearhart have both stated before the
President's Council on Bioethics that transplant rejection will still occur, even though the
cells from the cloned embryos are considered "genetically identical" to the donor. (Dr.
Irving Weissman, 2/13/2002, before the President's Council on Bioethics; Dr. John
Gearhart, 4/25/2002, before the President's Council on Bioethics). Dr. Thomas Okarma,
CEO of Geron Corporation, has pointed out that cloning is not commercially viable,
stating that, "The odds favoring success are vanishingly small, and the costs are daunting.
It would take thousands of [human] eggs on an assembly line to produce a custom
therapy for a single person. The process is a nonstarter, commercially." (Quoted by
Denise Gellene in, "Clone Profit? Unlikely", Los Angeles Times, 5/10/2002).
Corroborating such a view, Drs. Odorico, Kaufman and Thomas have written, "The poor
availability of human oocytes, the low efficiency of the nuclear transfer procedure, and
the long population doubling time of human embryonic stem cells make it difficult to
envision this becoming a routine clinical procedure." (Odorico JS, Kaufman DS,
Thomson JA, "Multilineage differentiation from human embryonic stem cell lines," Stem
Cells, 2001).

Dr. Prentice adds, however, that it is unlikely that large numbers of mature human
oocytes would actually be available for the production of embryonic stem cells,
particularly if hundreds are required to produce each embryonic stem cell line. "The
technical capability for nuclear transfer would also need to be widely available, and this
is unlikely," he says. Dr. Alan Trounson adds,
"In addition, epigenetic remnants of the somatic cell used as the nuclear donor can cause
major functional problems in development, which must remain a concern for embryonic
stem cells derived by nuclear transfer. Although it is possible to customize embryonic
stem cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that
these strategies will be used extensively for producing embryonic stem cells compatible
for transplantation." (Alan O. Trounson, "The derivation and potential use of human
embryonic stem cells," Reproduction, Fertility and Development, 2001).

As Dr. Irving Weissman of Stanford University stated in his testimony before the
President's Council on Bioethics,

"I should say that when you put the nucleus in from a somatic cell, the mitochondria still
come from the host. And in mouse studies it is clear that those genetic differences can
lead to a mild but certainly effective transplant rejection, and so immunosuppression,
mild though it is, will be required for that." (Dr. Irving Weissman, 2/13/02, before the
President's Council on Bioethics).

Dr. Alan Trounson, the Australian embryonic stem cell expert and a globally recognized
leader in the field, has stated that cloning has now become "unnecessary and obsolete".
He says that stem cell research has advanced so rapidly, just in the past few months
alone, that therapeutic cloning is now unnecessary. "My view," he states, "is that there are
at least three or four other alternatives that are more attractive already." In light of this
realization, he has abandoned his own work in therapeutic cloning, turning his attention
instead to the more promising field of stem cell research.

Emphasizing the point that therapeutic cloning faces too many "logistical problems," and
that other techniques show "greater promise" and offer "better options," Dr. Trounson
adds, "I can't see why, then, you would argue for therapeutic cloning in the long term,
because it is so difficult to get eggs and you've got this issue of [destroying] embryos as
well." ("Stem cell cloning not needed, says scientist", The Age [Melbourne], 7/29/2002;
"Stem cell research outpaces cloning", The Australian, 7/29/2002; "Therapeutic cloning
no longer necessary: expert", AAP Newsfeed, 7/29/2002).

Many experts are now extolling the promise of regenerative medicine through stem cells,
instead of through therapeutic cloning. Even the previously assumed multipotency and
monopotency of adult stem cells is now being challenged by more recent data, which
have demonstrated that some adult stem cells are capable of exhibiting pluripotency. The
versatility of adult stem cells is thus much greater than originally thought.

Arguments against human cloning include:

• There is no evidence that cloning is necessary or useful for medical treatments.

• Cloning research will divert resources away from other, more worthy areas of
research, and delay cures.

• Banning only implantation (reproductive cloning) is unenforceable.

 • Cloning creates a class of human beings who exist only as a means to achieve the
ends of others.

• Cloning risks the health, safety and possible exploitation of women.

• Cloning may possibly lead to the commodification and commercialization of

human life.

• Cloning is the "gateway" to genetic manipulation and control of human beings.

Additionally, a report issued in February of 2004 from South Korea highlights the
difficult logistics of human cloning. The report describes the first successful attempt to
create a hES cell line by SCNT. Researchers at the South Korea Seoul National
University, in a study led by Dr. Hwang Woo Suk, were the first to successfully clone a
human embryo, for purposes of growing "customized" stem cells for replacement tissue
in the treatment of disease. Sixteen (unpaid) women voluneteers had been recruited for
participation in the study, and they were then given hormone injections to induce
superovulation, which resulted in the production of 242 eggs that were used to produce
the single hES cell line. Each volunteer also donated some cells directly from one of her
ovaries. From the cumulus cells surrounding the developing oocyte (the immature egg
cell), the nuclei were transplanted to the egg of the same individual, using the same
process as that used in the cloning of animals. Donor and recipient were therefore the
same, presumably eliminating the risk of immunological rejection. However, only one-
fourth of the SCNT eggs successfully reached the blastocyst stage (at which point the
inner cell mass was harvested to create hES cells), despite the fact that additional
chemicals were used to "jump start" the cellular division. From a total of 30 blastocysts,
20 inner cell masses were harvested, but only one ES cell line was successfully obtained.
The cells in this ES cell line are genetically identical to the donor, and began forming
muscle, bone and other tissues in test tubes and when implanted into mice. The results
were published in the U.S. Journal Science.

Citizen's rights activists and bioethicists complained of the lack of transparency

surrounding the recruitment of the egg donors, and they raised questions over how
rigorously Hwang and his colleagues had followed the ethical guidelines imposed upon
their research. A further complication is the fact that it is not yet fully understood how to
control the direction of hESC growth, so researchers are still trying to determine the
specific types of tissue into which the cells will grow, a goal which continues to remain
elusive.

Even Alan Colman, one of the experts on Dolly the sheep, was quoted as saying, "I do
not welcome this." Cloned embryos, if transferred into a woman's uterus, could,
theoretically, grow into cloned babies. The bioethics of such research has triggered
debate among scientists and politicians worldwide.

It was noted, however, that attempts by the South Koreans to clone male cells failed.
Even beyond the ethical questions, there still remain concerns that therapeutic cloning is
too inefficient and too expensive.
In summary, therapeutic cloning may correctly be viewed as unsafe, unethical, and
unnecessary.

The greatest hope for clinical regenerative medicine may therefore be found in postnatal
and adult stem cell research.

The question of stem cells is currently the dominant subject in the debate over biotechnology and human gen
use embryonic stem cells or adult stem cells for future medical therapies? Embryonic stem cells are taken fro
embryo at the blastocyst stage, destroying the embryo, a developing human life. Adult stem cells, on the othe
found in all tissues of the growing human being and, according to latest reports, also have the potential to tran
themselves into practically all other cell types, or revert to being stem cells with greater reproductive capacity
stem cells have not yet been used for even one therapy, while adult stem cells have already been successfully
numerous patients, including for cardiac infarction (death of some of the heart tissue).

Stem cells are of wide interest for medicine, because they have the potential, under
suitable conditions, to develop into almost all of the different types of cells. They should
therefore be able to repair damaged or defective tissues (for example, destroyed insulin-
producing cells in the pancreas). Many of the so-called degenerative diseases, for which
there are as yet no effective therapies, could then be alleviated or healed.

It is remarkable that in the debate–often carried on with little competence–the potential of

embryonic stem cells is exaggerated in a one-sided way, while important moral questions
and issues of research strategy are passed over in silence. Generally, advocates of
research with embryonic stem cells use as their main argument that such research will
enable us to cure all of the diseases that are incurable today–cancer, AIDS, Alzheimers,
multiple sclerosis, and so forth. Faced with such a prospect, it is supposed to be
"acceptable" to "overlook" a few moral problems.

On closer inspection, however, the much extolled vision of the future turns out to be a
case of completely empty promises: Given the elementary state of research today, it is by
no means yet foreseeable, whether even one of the hoped-for treatments can be realized.
Basically, such promised cures are a deliberate deception, for behind the mirage of a
coming medical wonderland, promoted by interested parties, completely other research
objectives will be pursued that are to be kept out of public discussion as much as
possible.

Perfect candor should rule in stem cell research. This requires that the scientist himself
clearly establish the moral limits of his activity and declare what the consequences of
research with embryonic stem cells really are. In the process, no one can escape the fact
that, should one wish to use embryonic stem cells for "therapeutic purposes," the very
techniques will be developed that will also be used for the cloning of human beings, the
making of human-animal hybrids, the manipulation of germ lines, and the like–thus for
everything other than therapeutic purposes. Any coverup or hypocrisy in this matter will
very quickly reflect upon the research as a whole.
What Are Stem Cells, Exactly?
It is appropriate here to sketch the characteristics of stem cells, and the overthrow of
some dogmas of developmental biology. Broadly speaking, a stem cell is one that–in the
course of cell division and increase in the numbers of cells–is able to reproduce itself and
also mature into various specialized types of cells. The stem cell with the greatest
potential (totipotential) is the fertilized egg cell, which is capable of developing into a
complete organism.

According to the usual–but actually very doubtful–explanation, the fertilized egg cell has
totipotential up to the stage of division into eight cells, and in later stages the cells retain
only "pluripotential." That is, they can form many different types of tissues, but not the
complete organism. Embryonic stem cells–that is, those 50 cells within a blastocyst,
which then continue to develop into the embryo proper–have this pluripotential. In the
course of further specialization, stem cells of individual tissues are formed, such as that
of the bone marrow, from which all the other kinds of blood cells develop.

Behind this description lies the conception that a linear process of differentiation is
played out, in the development of the individual, toward increasingly "mature,"
specialized cells in the individual tissues, from totipotentiality to tissue specificity. This
process is supposed to run only forward, but never backward. That is, as soon as a cell
has reached a certain degree of "maturity," the way back to earlier stages of development
is closed off. So it is evident that a stem cell’s capacity to perform is increasingly limited
to specific functions, and it loses, correspondingly, the manifold capabilities still present
in earlier developmental stages.

According to latest reports, however, this dogma of developmental biology does not hold.
Evidently, tissue-specific stem cells have the ability–as has been impressively
demonstrated in experiments with animals–to "transdifferentiate" themselves when in a
different environment–that is, to take on the cell functions of the new tissue. Thus,
neuronal stem cells of mice have transformed themselves into blood stem cells and
produced blood cells. Indeed, there are indications of another capability of adult stem
cells: Apparently they have the potential to be "reprogrammed." Not only can they adjust
to the specific conditions of a new tissue environment, but they can even assume more
generalized, earlier levels of development, so that it even appears possible that they
become totipotent again.
 Laboratory Virola in Ukraine has demonstrated that bone marrow stromal cells
in culture are pluripotent–that is, they are able to differentiate into cells of liver, bone,
fat, cartilage, and so on. Researchers at this laboratory have developed techniques to
differentiate in vitro mouse bone marrow stromal cells into different types of neuronal
and glial cells. The laboratory is seeking funds to develop similar methods for human
bone marrow stromal cells.

Problems of ‘Therapeutic Cloning’

Until now, talk of a possible source of human replacement tissue has centered on
embryonic stem cells, the production of which has been extremely controversial. They
are a typical product of "consuming embryonic research," so called, because in obtaining
them from a human embryo produced by artificial fertilization in vitro, the embryo is
destroyed.

The most important research technique for which such embryos are obtained is
"therapeutic cloning." In principle, a human egg cell is denucleated, that is, the DNA is
removed, and in its place is put the nucleus of a somatic (body) cell. The egg cell is
stimulated with a short electrical pulse, and it then develops into the blastocyst, from
which stem cells can be removed. These are identical with those of the donor of the
somatic cell nucleus.

Normally it goes unmentioned, that it is only a small step from this so-called "therapeutic
cloning" (because, it is claimed, in this way a therapy for diseases can be developed) to
what is called "reproductive cloning." The only difference is that the development of the
embryo is not interrupted in the early blastocyst stage; instead the embryo is implanted in
a uterus and a complete organism develops–an exact genetic copy of the donor. "Dolly,"
the first cloned sheep, was produced by this method, and here is the basis for the
widespread fear that the same method that is used for "therapeutic cloning" can also be
used for the selective breeding of humans.

In addition to the obvious moral consideration, there are still other serious disadvantages
that make this path to the development of human "replacement parts" appear to be
untenable.

The danger of tumors. So far there has been no solution to the problem of developing in
the laboratory an unmistakable identifier for stem cells that can distinguish them
unequivocally from cancer cells. For this reason, it is also not possible to produce
sufficiently pure cell cultures from stem cells. So far, with embryonic mouse stem cells, a
purity of only 80 percent has been achieved. That is in no way sufficient for cell
transplantation as a human therapy. In a cell culture for therapeutic purposes, there must
not be a single undifferentiated cell, since it can lead to unregulated growth, in this case
to the formation of teratomas, a cancerous tumor derived from the germ layers. This
problem would not be expected with adult stem cells, because of their greater
differentiation.
Genetic instability. Only recently a further problem has emerged. Fundamental doubt of
the suitability of embryonic stem cells for transplantation has come to the surface because
of the genetic instability of cloned cells.

Cloned animals like Dolly give the outward appearance of full health, but the probability
of their having numerous genetic defects is very high. Moreover, the entire cloning
procedure is extremely ineffective. Most cloned animals die before birth, and of those
born alive, not even half survive for three weeks. In the best case, there is a success rate
of 3 to 4 percent.

One of the reasons for this high failure rate has now been discovered by the German
scientist Rudolf Jaenisch at the Institute for Biomedical Research at the Massachusetts
Institute of Technology, and his colleague, Ryuzo Yanagimachi. Their conception is that
in cloning–that is, when the nucleus of a somatic cell is inserted into a denucleated egg
cell–the reprogramming of the genes does not proceed properly, so that not all of the
genes that are necessary to the early phase of embryonic development, are activated.
Even when cloned animals survive at all, probably every clone would have subtle genetic
abnormalities that would frequently become noticeable only later in life.

Jaenisch performed his experiments with mice that had been cloned using embryonic
stem cells in place of the somatic cells, which produces better results. But to his surprise,
the reprogramming of the inserted genetic material by the embryonic cells proceeded in a
very unregulated way. There were no two clones in which the same pattern of gene
activation was found, and Jaenisch is convinced that the use of embryonic stem cells was
clearly responsible.

What consequences follow from this for the therapeutic use of human embryonic
stem cells–consequences that will in fact be multiplied through cloning–are not yet
foreseeable.
Whoever Would Cure, Must Use Adult Stem Cells
It has been known for about 30 years that stem cells are present in the tissue of the adult,
but it was assumed that they could only form cells of a particular tissue. That is,
reprogramming them was considered impossible. In recent years, however, pluripotent
stem cells were discovered in various human tissues–in the spinal cord, in the brain, in
the mesenchyme (connective tissue) of various organs, and in the blood of the umbilical
cord. These pluripotent stem cells are capable of forming several cell types–principally
blood, muscle, and nerve cells. It has been possible to recognize, select, and develop
them to the point that they form mature cell types with the help of growth factors and
regulating proteins.

This shows that in tissues of the body, adult stem cells possess a much greater potential
for differentiation than previously assumed. This knowledge must be brought into the
public consciousness with all possible emphasis. If stem cell research were really only
meant for therapeutic uses, which it most obviously should be, adult stem cells would
promise a very productive research field–and beyond that, a possibility, without moral
objection, to discover fundamentals of the dynamics of tissue differentiation.
It has become clear from transplantation experiments with animals, that stem cells of a
particular tissue can develop into cells of a completely different kind. Thus, bone marrow
stem cells have been induced to become brain cells, but also liver cells.

Adult stem cells obviously have a universal program for division that is common to all
the kinds of tissue stem cells, and makes them mutually interchangeable. This was
discovered by Alexei Terskikh at Stanford University School of Medicine in California.
He was able to prove that adult stem cells of blood-forming tissues, and of the brain,
activate the same genes, in order to preserve their status as stem cells.

In May 2001, a further, spectacular experiment was reported, which was carried out on
mice by scientists at Yale University. The researchers obtained stem cells from the bone
marrow of male mice, and injected it into females whose own marrow had been
destroyed by radioactive irradiation. Eleven months later, the male stem cells
(identifiable through the male Y-chromosome) were found not only in the females’ bone
marrow, but also in their blood, and in their gut, lung, and skin tissues.

If these observations are correct and are confirmed by other teams of scientists, science
should concentrate on research with adult stem cells and renounce further experiments
with the embryonic.

Human Treatments
Moreover, very promising treatments of serious diseases with adult stem cells have
already been tried. The special advantage is that there are no rejection reactions, because
the cells are from the same body.

Of longer standing is treatment with bone marrow stem cells. The treatment comes into
play when, for example, a patient has lost his or her blood-forming tissue through
radiation or high-dose chemotherapy. Previously removed bone marrow stem cells are
then retransplanted, and are able to resume the formation of blood cells.

In 2001, however, a team of doctors at the Duesseldorf University Clinic carried out a
treatment of very far-reaching consequences. For the first time, they treated a cardiac
infarct patient with stem cells from his own body. The cardiologist, Prof. Bodo Eckehard
Strauer, is sure that the stem cells from the patient’s bone marrow, after injection into the
infarct zone, autonomously converted to heart muscle. The functioning of the severely
damaged heart clearly improved within a few weeks.

Four days after the infarction, the doctors took bone marrow from the patient’s pelvis
using local anesthesia. The stem cells in the marrow were concentrated outside of the
body and implanted in the infarct area the next day with a special technique via a
coronary artery. However, the doctors could not yet take cardiac tissue to prove
definitively that the implanted blood stem cells had converted to heart muscle cells. But,
according to Strauer, there is no other way to explain the marked improvement in the
patient’s condition. After this first successful operation, six more patients have already
been treated with their own stem cells, with similarly positive results.
There are also reports of successful treatments with adult stem cells in cases of Crohn’s
disease (a chronic infection of the gut), thalassemia (a blood disease), and a rare skin
disease. And–despite the fact that basic research with adult stem cells is in its earliest
beginnings and is in no way being promoted with urgency–there have been a growing
number of reports lately of experiments with animals, from which it emerges that adult
stem cells can successfully transform themselves into differentiated cells of organs of
many kinds.

In contrast, reports of successful conversions of embryonic stem cells are very infrequent
and cautious. Thus, we find in Science of Dec. 1, 2000 (Vol. 290, pp. 1672-1674): "In
contrast, the human embryonic stem cells and fetal germ cells that made headlines in
November 1998 because they can, in theory, develop into any cell type have so far
produced relatively modest results. Only a few papers and meeting reports have emerged
from the handful of labs that work with human pluripotent cells. . . . The work suggests
that it will not be simple to produce the pure populations of certain cell types that would
be required for safe and reliable cell therapies. . . ."

This is the restrained language used by established science to describe a truly disastrous
set of results.

There are, of course, still substantial problems to be overcome, even with adult stem
cells: They are relatively rare, and are hard to find with the techniques used so far. They
are also not very easy to culture outside of the body. It was therefore an important
advance that Australian researchers of the Walter and Eliza Hall Institute of Medical
Research have now found a way to isolate nerve stem cells with "extreme purity" from
the brains of mice. In Nature of August 16, 2001 (Vol. 412, pp. 736-739), they reported
obtaining a culture of 80 percent purity, compared to a previous rate of 5 percent at best.

It is now urgently necessary to tackle the research in precisely this direction, in order to
find out the exact conditions under which the differentiation of stem cells comes about
and how, in detail, it proceeds. Only by this morally unassailable route will it be possible
to develop new therapies for serious, heretofore incurable diseases, and beyond that, to
improve our understanding of the development of life itself.

What is Vet-Stem Regenerative Medicine?

Regenerative medicine uses a concentrated form of autologous adipose-derived adult

stem cells to treat traumatic and degenerative diseases, including bowed tendons,
ligament injuries, osteoarthritis, and osteochondral defects in horses and dogs.

Success in human clinical trials and animal models

Despite its infancy, regenerative medicine is not new. Success in numerous animal
models of disease and emerging success in human clinical trials for Crohn's fistulas1 and
stroke2, along with hundreds of ongoing clinical trials (See sidebar) support the rationale
for stem cell use, and now success, in veterinary medicine. Vet-Stem collaborative and
clinical research demonstrate positive results in treating horses with tendon and ligament
injuries, osteochondral defects, and osteoarthritis.3-6

Stem cells are multipotent and can differentiate into tendon, ligament, bone, cartilage,
cardiac, nerve, muscle, blood vessels, fat, and liver tissue22, 23 (see figure below). The
stromal fraction that is harvested from adipose tissue is a heterogeneous mixture of
regenerative cells (see below).

Vet-Stem Technology: Summary

Vet-Stem Regenerative Cell Therapy is based on a clinical technology originally licensed

from Artecel Inc., Sunnyvale, CA. Original patents from University of Pittsburgh and
Duke University.

• Rationale based on consistent therapeutic success in numerous animal models of

disease (see sidebar)
• Adipose-derived adult stem cells (Vet-Stem Regenerative Cells: VSRC™)
• Autologous cell therapy
• Currently used in horses with bowed tendons, ligament injuries, and fractures, and
in dogs with osteoarthritis
• More than 2,000 horses treated since 2003
• No systemic adverse events reported and < 0.5% local tissue reactions.3-6
• Demonstrated efficacy with VSRC therapy in horses and dogs
o Cornell University double-blind, placebo controlled study5
o Retrospective studies3,4
 o Case studies6

Why use adipose-derived regenerative cells rather than regenerative cells derived
from bone marrow?

Adipose-derived regenerative cells are:

• Readily available source

• Can be collected in far greater concentrations than those from bone marrow24
• Able to differentiate into multiple lineages implicating their potential in bone,
cartilage, and cardiac repair23 (See figure above)
• Fractions isolated from adipose tissue contain a heterogeneous mixture of
regenerative cells, including:23
o Mesenchymal stem cells
o Endothelial progenitor cells
o Pericytes
o Immune cells
o Fibroblasts
o Other growth factor-secreting bioactive cells

Differences in Regenerative Medicine compared to traditional medicine;

• Does not rely on a single target receptor or a single pathway for its action
• Regenerative cell mixture is delivered either directly to the traumatic wound (e.g.:
tendonitis, desmitis, fracture) or are delivered systemically (e.g.: liver disease,
renal disease)
• Regenerative cells can differentiate into many tissue types, induce repair, and
stimulate regeneration22
• Regenerative cells "communicate" with the cells of their local environment
through paracrine and autocrine modalities, creating the optimal environment for
natural healing25
• Regenerative cells produce a variety of both secreted and cell surface substances
that regulate tissue growth, integrity, and function25

Stem Cells as Vehicles for Gene Therapy

Stem cells can be classified as embryonic or adult, depending on their tissue of origin.
The role of adult stem cells is to sustain an established repertoire of mature cell types in
essentially steady-state numbers over the lifetime of the organism. Although adult tissues
with a high turnover rate, such as blood, skin, and intestinal epithelium, are maintained
by tissue-specific stem cells, the stem cells themselves rarely divide. However, in certain
situations, such as during tissue repair after injury or following transplantation, stem cell
divisions may become more frequent. The prototypic example of adult stem cells, the
hematopoietic stem cell, has already been demonstrated to be of utility in gene
therapy.4,5 Although they are relatively rare in the human body, these cells can be readily
isolated from bone marrow or after mobilization into peripheral blood. Specific surface
markers allow the identification and enrichment of hematopoietic stem cells from a
mixed population of bone marrow or peripheral blood cells.

After in vitro manipulation, these cells may be retransplanted into patients by injection
into the bloodstream, where they travel automatically to the place in the bone marrow in
which they are functionally active. Hematopoietic stem cells that have been explanted,
in vitro manipulated, and retransplanted into the same patient (autologous
transplantation) or a different patient (allogeneic transplantation) retain the ability to
contribute to all mature blood cell types of the recipient for an extended period of time
(when patients' cells are temporarily grown quot;outside the bodyquot; before being
returned to them, the in vitro process is typically referred to as an quot;ex vivoquot;
approach).

Another adult bone marrow-derived stem cell type with potential use as a vehicle for
gene transfer is the mesenchymal stem cell, which has the ability to form cartilage, bone,
adipose (fat) tissue, and marrow stroma (the bone marrow microenvironment).6
Recently, a related stem cell type, the multipotent adult progenitor cell, has been isolated
from bone marrow that can differentiate into multiple lineages, including neurons,
hepatocytes (liver cells), endothelial cells (such as the cells that form the lining of blood
vessels), and other cell types.7 Other adult stem cells have been identified, such as those
in the central nervous system and heart, but these are less well characterized and not as
easily accessible.8

The traditional method to introduce a therapeutic gene into hematopoietic stem cells from
bone marrow or peripheral blood involves the use of a vector derived from a certain class
of virus, called a retrovirus. One type of retroviral vector was initially employed to show
proof-of-principle that a foreign gene (in that instance the gene was not therapeutic, but
was used as a molecular tag to genetically mark the cells) introduced into bone marrow
cells may be stably maintained for several months.9 However, these particular retroviral
vectors were only capable of transferring the therapeutic gene into actively dividing cells.
Since most adult stem cells divide at a relatively slow rate, efficiency was rather low.
Vectors derived from other types of retroviruses (lentiviruses) and adenoviruses have the
potential to overcome this limitation, since they also target non-dividing cells.

The major drawback of these methods is that the therapeutic gene frequently integrates
more or less randomly into the chromosomes of the target cell. In principle, this is
dangerous, because the gene therapy vector can potentially modify the activity of
neighboring genes (positively or negatively) in close proximity to the insertion site or
even inactivate host genes by integrating into them. These phenomena are referred to as
quot;insertional mutagenesis.quot; In extreme cases, such as in the X-linked SCID gene
therapy trials, these mutations contribute to the malignant transformation of the targeted
cells, ultimately resulting in cancer.

Another major limitation of using adult stem cells is that it is relatively difficult to
maintain the stem cell state during ex vivo manipulations. Under current suboptimal
conditions, adult stem cells tend to lose their stem cell properties and become more
specialized, giving rise to mature cell types through a process termed
quot;differentiation.quot; Recent advances in supportive culture conditions for mouse
hematopoietic stem cells may ultimately facilitate more effective use of human
hematopoietic stem cells in gene therapy applications.10,11

Embryonic Stem Cell: "The Ultimate Stem Cell"

Embryonic stem cells are capable of unlimited self-renewal while maintaining the
potential to differentiate into derivatives of all three germ layers. Even after months and
years of growth in the laboratory, they retain the ability to form any cell type in the body.
These properties reflect their origin from cells of the early embryo at a stage during
which the cellular machinery is geared toward the rapid expansion and diversification of
cell types.

Murine (mouse) embryonic stem cells were isolated over 20 years ago,12,13 and paved the
way for the isolation of nonhuman primate, and finally human embryonic stem cells.14
Much of the anticipated potential surrounding human embryonic stem cells is an
extrapolation from pioneering experiments in the mouse system. Experiments performed
with human embryonic stem cells in the last couple of years indicate that these cells have
the potential to make an important impact on medical science, at least in certain fields. In
particular, this impact includes: a) differentiation of human embryonic stem cells into
various cell types, such as neurons, cardiac, vascular, hematopoietic, pancreatic, hepatic,
and placental cells, b) the derivation of new cell lines under alternative conditions, c) and
the establishment of protocols that allow the genetic modification of these cells.

The Potential of Human Embryonic Stem Cells for Gene Therapy

Following derivation, human embryonic stem cells are easily accessible for controlled
and specific genetic manipulation. When this facility is combined with their rapid growth,
remarkable stability, and ability to mature in vitro into multiple cell types of the body,
human embryonic stem cells are attractive potential tools for gene therapy. Two possible
scenarios whereby human embryonic stem cells may benefit the gene therapy field are
discussed below.

First, human embryonic stem cells could be genetically manipulated to introduce the
therapeutic gene. This gene may either be active or awaiting later activation, once the
modified embryonic stem cell has differentiated into the desired cell type. Recently
published reports establish the feasibility of such an approach.15 Skin cells from an
immunodeficient mouse were used to generate cellular therapy that partially restored
immune function in the mouse. In these experiments, embryonic stem cells were
generated from an immunodeficient mouse by nuclear transfer technology. The nucleus
of an egg cell was replaced with that from a skin cell of an adult mouse with the genetic
immunodeficiency. The egg was developed to the blastula stage at which embryonic stem
cells were derived. The genetic defect was corrected by a genetic modification strategy
designated quot;gene targeting.quot; These quot;curedquot; embryonic stem cells were
differentiated into hematopoietic quot;stemquot; cells and transplanted into
immunodeficient mice. Interestingly, the immune function in these animals was partially
restored. In principle, this approach may be employed for treating human patients with
immunodeficiency or other diseases that may be corrected by cell transplantation.

However, significant advances must first be made. The levels of immune system
reconstitution observed in the mice were quite modest (<1% of normal), while the
methodology employed to achieve hematopoietic engraftment is not clinically feasible.
This methodology involved using a more severely immunodeficient mouse as a recipient
(which also had the murine equivalent of the human X-linked SCID mutation) and
genetically engineering the hematopoietic engrafting cells with a potential oncogene prior
to transplantation.

Embryonic stem cells may additionally be indirectly beneficial for cellular gene therapy.
Since these cells can be differentiated in vitro into many cell types, including presumably
tissue-specific stem cells, they may provide a constant in vitro source of cellular material.
Such quot;adultquot; stem cells derived from embryonic stem cells may thus be utilized
to optimize protocols for propagation and genetic manipulation techniques.16 To acquire
optimal cellular material from clinical samples in larger quantities for experimental and
optimization purposes is usually rather difficult since access to these samples is limited.

Genetic Manipulation of Stem Cells

The therapeutic gene needs to be introduced into the cell type used for therapy. Genes
may be introduced into cells by transfection or transduction. Transfection utilizes
chemical or physical methods to introduce new genes into cells. Usually, small
molecules, such as liposomes, as well as other cationic-lipid based particles are employed
to facilitate the entry of DNA encoding the gene of interest into the cells. Brief electric
shocks are additionally used to facilitate DNA entry into living cells. All of these
techniques have been applied to various stem cells, including human embryonic stem
cells. However, the destiny of the introduced DNA is relatively poorly controlled using
these procedures. In most cells, the DNA disappears after days or weeks, and in rare
cases, integrates randomly into host chromosomal DNA. in vitro drug selection strategies
allow the isolation and expansion of cells that are stably transfected, as long as they
significantly express the newly introduced gene.

Transduction utilizes viral vectors for DNA transfer. Viruses, by nature, introduce DNA
or RNA into cells very efficiently. Engineered viruses can be used to introduce almost
any genetic information into cells. However, there are usually limitations in the size of
the introduced gene. Additionally, some viruses (particularly retroviruses) only infect
dividing cells effectively, whereas others (lentiviruses) do not require actively dividing
cells. In most cases, the genetic information carried by the viral vector is stably integrated
into the host cell genome (the total complement of chromosomes in the cell).

An important parameter that must be carefully monitored is the random integration into
the host genome, since this process can induce mutations that lead to malignant
transformation or serious gene dysfunction. However, several copies of the therapeutic
gene may also be integrated into the genome, helping to bypass positional effects and
gene silencing. Positional effects are caused by certain areas within the genome and
directly influence the activity of the introduced gene. Gene silencing refers to the
phenomenon whereby over time, most artificially introduced active genes are turned off
by the host cell, a mechanism that is not currently well understood. In these cases,
integration of several copies may help to achieve stable gene expression, since a subset of
the introduced genes may integrate into favorable sites. In the past, gene silencing and
positional effects were a particular problem in mouse hematopoietic stem cells.17 These
problems led to the optimization of retroviral and lentiviral vector systems by the
addition of genetic control elements (referred to as chromatin domain insulators and
scaffold/matrix attachment regions) into the vectors, resulting in more robust expression
in differentiating cell systems, including human embryonic stem cells.18

In some gene transfer systems, the foreign transgene does not integrate at a high rate and
remains separate from the host genomic DNA, a status denoted quot;episomalquot;.
Specific proteins stabilizing these episomal DNA molecules have been identified as well
as viruses (adenovirus) that persist stably for some time in an episomal condition.
Recently, episomal systems have been applied to embryonic stem cells.19

An elegant way to circumvent positional effects and gene silencing is to introduce the
gene of interest specifically into a defined region of the genome by the gene targeting
technique referred to previously.20 The gene targeting technique takes advantage of a
cellular DNA repair process known as homologous recombination.21 Homologous
recombination provides a precise mechanism for defined modifications of genomes in
living cells, and has been used extensively with mouse embryonic stem cells to
investigate gene function and create mouse models of human diseases. Recombinant
DNA is altered in vitro, and the therapeutic gene is introduced into a copy of the genomic
DNA that is targeted during this process. Next, recombinant DNA is introduced by
transfection into the cell, where it recombines with the homologous part of the cell
genome. This in turn results in the replacement of normal genomic DNA with
recombinant DNA containing genetic modifications.

Homologous recombination is a very rare event in cells, and thus a powerful selection
strategy is necessary to identify the cells in which it occurs. Usually, the introduced
construct has an additional gene coding for antibiotic resistance (referred to as a
selectable marker), allowing cells that have incorporated the recombinant DNA to be
positively selected in culture. However, antibiotic resistance only reveals that the cells
have taken up recombinant DNA and incorporated it somewhere in the genome. To select
for cells in which homologous recombination has occurred, the end of the recombination
construct often includes the thymidine kinase gene from the herpes simplex virus. Cells
that randomly incorporate recombinant DNA usually retain the entire DNA construct,
including the herpes virus thymidine kinase gene. In cells that display homologous
recombination between the recombinant construct and cellular DNA, an exchange of
homologous DNA sequences is involved, and the non-homologous thymidine kinase
gene at the end of the construct is eliminated. Cells expressing the thymidine kinase gene
are killed by the antiviral drug ganciclovir in a process known as negative selection.
Therefore, those cells undergoing homologous recombination are unique in that they are
resistant to both the antibiotic and ganciclovir, allowing effective selection with these
drugs (see Figure 4.2).

Figure 4.2. Gene targeting by homologous recombination.

Gene targeting by homologous recombination has recently been applied to human

embryonic stem cells.22 This is important for studying gene functions in vitro for lineage
selection and marking. For therapeutic applications in transplantation medicine, the
controlled modification of specific genes should be useful for purifying specific
embryonic stem cell-derived, differentiated cell types from a mixed population, altering
the antigenicity of embryonic stem cell derivatives, and adding defined markers that
allow the identification of transplanted cells. Additionally, since the therapeutic gene can
now be introduced into defined regions of the human genome, better controlled
expression of the therapeutic gene should be possible. This also significantly reduces the
risk of insertional mutagenesis.

Future Challenges for Stem Cell–Based Gene Therapy

Despite promising scientific results with genetically modified stem cells, some major
problems remain to be overcome. The more specific and extensive the genetic
modification, the longer the stem cells have to remain in vitro. Although human
embryonic stem cells in the culture dish remain remarkably stable, the cells may
accumulate genetic and epigenetic changes that might harm the patient (epigenetic
changes regulate gene activity without altering the genetic blueprint of the cell). Indeed,
sporadic chromosomal abnormalities in human embryonic stem cell culture have been
reported, and these may occur more frequently when the cells are passaged as bulk
populations. This observation reinforces the necessity to optimize culture conditions
further, to explore new human embryonic stem cell lines, and to monitor the existing
cell lines.23,24 Additionally undifferentiated embryonic stem cells have the potential to
form a type of cancer called a teratocarcinoma. Safety precautions are therefore
necessary, and currently, protocols are being developed to allow the complete depletion
of any remaining undifferentiated embryonic stem cells.25 This may be achieved by
rigorous purification of embryonic stem cell derivatives or introducing suicide genes that
can be externally controlled.

Another issue is the patient's immune system response. Transgenic genes, as well as
vectors introducing these genes (such as those derived from viruses), potentially trigger
immune system responses. If stem cells are not autologous, they eventually cause
immuno-rejection of the transplanted cell type. Strategies to circumvent these problems,
such as the expression of immune system-modulating genes by stem cells, creation of
chimeric, immunotolerable bone marrow or suppression of HLA genes have been
suggested.25 In this context, nuclear transfer technology has been recently extended to
human embryonic stem cells.26* Notably, immune-matched human embryonic stem cells
have now been established from patients, including an individual with an
immunodeficiency disease, congenital hypogammaglobulinemia.27* Strategies that
combine gene targeting with embryonic stem cell-based therapy are thus potential novel
therapeutic options.
Figure 4.3. Strategies for Delivering Therapeutic Transgenes into Patients.

© 2006 Terese Winslow

The addition of human embryonic stem cells to the experimental gene therapy arsenal
offers great promise in overcoming many of the existing problems of cellular based gene
therapy that have been encountered in clinic trials (see Figure 4.3). Further research is
essential to determine the full potential of both adult and embryonic stem cells in this
exciting new field.

According to Patel, special editor for this issue, suitable sources of cells for cardiac
transplant will depend on the types of diseases to be treated. For acute myocardial
infarction, a cell that reduces myocardial necrosis and augments vascular blood flow will
be desirable. For heart failure, cells that replace or promote myogenesis, reverse
apoptopic mechanisms and reactivate dormant cell processes will be useful.
“Very little data is available to guide cell dosing in clinical studies,” says Patel. “Pre-
clinical data suggests that there is a dose-dependent improvement in function.”

Patel notes that the availability of autologous (patient self-donated) cells may fall short.

Determining optimal delivery methods raise issues not only of dose, but also of timing.
Also, assessing the fate of injected cells is “critical to understanding mechanisms of
action.”

Will cells home to the site of injury? Labeling stem cells with durable markers will be
necessary and new tracking markers may need to be developed.

Improved cell survival drugs

Adult bone marrow-derived mensenchymal stem cells (MSCs) have shown great
signaling and regenerative properties when delivered to heart tissues following a
myocardial infarction (MI). However, the poor survival of grafted cells has been a
concern of researchers. Given the poor vascular supply after a heart attack and an active
inflammatory process, grafted cells survive with difficulty. Transmyocardial
revasularization (TMR), a process by which channels are created in heart tissues by laser
or other means, can enhance oxygenated blood supply.

“We hypothesized that using TMR as a scar pretreatment to cell therapy might improve
the microenvironment to enhance cell retention and long-term graft success,” said Amit
N. Patel, lead author of a study titled Improved Cell Survival in Infarcted Myocardium
Using a Novel Combination Transmyocardial Laser and Cell Delivery System. “TMR
may act synergistically with signaling factors to have a more potent effect on myocardial
remodeling.”

Patel and colleagues, who used a novel delivery system to disperse cells in the TMR-
generated channels in an animal model, report significant cell survival in the TMR+Cell
group versus Cells or TMR alone. The researchers speculated that there was an increase
in local production of growth factors that may have improved the survival of transplanted
cells.

Stem cells depolarize

Recent studies have suggested that there are stem cells in the heart. In this study,
researchers engineered mesenchymal stem cells (MSC) to over express stromal cell-
derived factor-1 (SDF-1), a chemokine.

“Our study suggests that the prolongation of SDF-1 expression at the time of an acute
myocardial infarction (AMI) leads to the recruitment of what may be an endogenous stem
cell in the heart,” says Marc Penn, MD, PhD, director of the Skirball Laboratory for
Cardiovascular Cellular Therapeutics at the Cleveland Clinic Foundation. “These cells
may contribute to increased contractile function even in their immature stage.”
In the study titled SDF-1 Recruits Cardiac Stem Cell Like Cells that Depolarize in Vivo,
researchers concluded that there is a natural but inefficient stem cell-based repair process
following an AMI that can be manipulated through the expression of key molecular
pathways. The outcome of this inefficient repair can have a significant impact on the
electrical and mechanical functions of the surviving myocardium.

Grafting bioartifical myocardium for myocardial assistance

While the object of cell transplantation is to improve ventricular function, cardiac cell
transplantation has had limited success because of poor graft viability and low cell
retention. In a study carried out by a team of researchers from the Department of
Cardiovascular Surgery, Pompidou Hospital, a matrix seeded with bone marrow cells
(BMC) was grafted onto the infarcted ventricle to help support and regenerate post-
ischemic lesions.

“Our study demonstrated that bone marrow cell therapy associated with the surgical
implantation onto the epicardium of a cell-seeded collagen type 1 matrix prevented
myocardial wall thinning, limited post-ischemic remodeling and improved diastolic
function,” says Juan Chachques, MD, PhD, lead author for Myocardial Assistance by
Grafting a New Bioartificial Upgraded Myocardium (MAGNUM Clinical Trial): One
year follow-up.

“The use of the biomaterial appears to create a micro atmosphere where both exogenous
and endogenous cells find an optimal microenvironment to repair tissues and maintain
low scar production,” explains Chachques.

According to Chachques, the favorable effects may be attributed to several mechanisms.

The BMC seeded in the collagen matrix may be incorporated into the myocardium
through epicardial channels created at the injection sites. Too, the cell-seeded matrix may
help prevent apoptosis.

“This biological approach is attractive because of its potential for aiding myocardial
regeneration with a variety of cell types,” concluded Chachques.

Those cell types include skeletal myoblasts, bone marrow-derived mensenchymal stem
cells, circulating blood-derived progenitor cells, endothelial and mesothelial cells,
adipose tissue stem cells and, potentially, embryonic stem cells.

Our clinic offers advanced patented methods of stem cell treatment for different diseases
and conditions. The fetal stem cells we use are nonspecialized cells able to differentiate
(turn) into any other cell types forming different tissues and organs. Fetal stem cells have
huge potential for differentiation and proliferation and are not rejected by the recipient’s
body more...

Stem cell therapy has proven to be effective for organs and tissues restoration, and for
fight against the incurable and obstinate diseases. We treat patients with various diseases,
such as diabetes mellitus, multiple sclerosis, Parkinson’s disease, Duchenne muscular
dystrophy, cancer, blood diseases and many others, including rare genetic and hereditary
diseases. Among our patients there are also people willing to undergo anti-aging
treatment. Stem cell treatment allows for achieving effects that are far beyond the
capacity of any other modern method more...

For over 17 years, we have performed more than 6,000 transplantations of fetal stem cells
to people from many countries, such as the USA, Italy, Germany, China, Poland, Russian
Federation, Greece and Cyprus, etc. Our stem cell treatments helped to prolong life and
improve life quality to thousands of patients including those suffering from the incurable
diseases who lost any hope for recovery.

Thalassemia is among the most common genetic diseases worldwide. Thalassemia is a

blood disorder passed down through families (inherited) in which the body makes an
abnormal form of hameoglobin, the protein in red blood cells that carries oxygen. The
disorder results in excessive destruction of red blood cells and causes severe anemia that
can occur within months after birth. The diseased person has to undergo monthly blood
transfusion. If left untreated, severe anemia can result in insufficient growth and
development, as well as other common physical complications that can lead to a
dramatically decreased life-expectancy.

A newborn sibling’s umbilical cord blood provides a better chance of HLA matching as
there is a 25% chance for a perfect match, and 50% chance for a partial match. Also
using a sibling’s cord blood for transplant lowers the chances of donor rejection, and is
therefore considered as a preferred source for transplantation compared to using stem
cells from a non-related source.

Dr. Revathy Raj who examined Thamirabharuni, had recommended her to start on iron
reducing medication initially. She had asked Mr. Senthil Kumar and his wife to consider
another pregnancy and go for umbilical cord blood stem cell banking. With a pre-natal
test it was also confirmed that the foetus was not affected with Thalassemia.

“I came to know about my daughter’s condition when she was one-and-half-years old.
Being a carpenter by profession, bearing the medical expenses was really difficult for us.
We approached various societies and LifeCell International, for financial assistance.
LifeCell helped us to preserve my son’s Pugazhendhi, cord blood stem cells free of cost
at their center in Chennai. Today, with the guidance of the Doctors and with the aid of
LifeCell International, Pugazhendhi has become a means for his sister’s survival from
Thalassemia. I would like to thank all the kind souls- my friends, doctors, well wishers,
anonymous donors who helped and guided me at different stages,” says Mr. Senthil
Kumar- Thamirabharuni’s father.

For a year, the extracted cord blood stem cells were preserved under specific conditions.
HLA test was done which proved that the tissues of both the children matched and the
treatment could proceed. The first step was to destroy all the existing bone marrow cells
for which chemotherapy was used. Then the donor’s stem cells were injected in the
patient body. The procedure requires no surgery! explains Dr Revathy Raj.

In March 2009, the stem cells transplantation was done by Dr. Revathy Raj at the Apollo
Hospital and it helped Thamirabharuni get rid of Thalassemia. The stem cells that were
transplanted came from her brother’s cord blood and his bone marrow as there was
deficient amount of stem cells in Pugazhendhi cord blood.

When they used the needles for blood transfusion it would hurt… and sometimes they
would never get the vein but still I had to go for blood transfusion every month. My
Brother’s cord blood saved me or I knew I wouldn’t have been alive for long. I love my
brother, Says Thamirabharuni.

Mr. Mayur Abhaya, President and Executive Director, LifeCell International says,
“We at LifeCell would like to congratulate Mr. Senthil Kumar and his family on

Thamirabharuni’s triumph over Thalassemia. This success I am sure will give new hope
to thousands of families whose children are unfortunately the victim of this dreadful
disease. It feels very fulfilling that LifeCell could make an impact in a child’s life… we
are very happy to be a part of this success story.”

Every year 10,000 children with Thalassemia major are born in India, which constitutes
10% of the total number in the world, and one out of every 8 carriers of Thalassemia
worldwide lives in India .Thanks to this revolutionary development in the stem cell
industry, several life threatening diseases like Thalassemia, Leukaemia etc., can now be
treated completely with no surgery involved.

About LifeCell International: LifeCell is India’s first & largest umbilical cord blood
stem cell bank to bring the revolutionary concept of banking a baby’s umbilical cord
blood stem to the country. LifeCell facilitates the cryogenic preservation of stem cells in
technological collaboration with Cryo-Cell International Inc, USA - the world’s largest
and oldest stem cell bank with more than 16 years of expertise in stem cell banking.

LifeCell in the past 4 years has positioned itself as leaders in the Industry and was
recently accredited by AABB (American Association of Blood Banks) for adopting their
international standards and today LifeCell is the “First AABB Accredited Stem Cell Bank
in India” and recognized by DSIR, Govt. of India for R&D. Today LifeCell has over
thousands of members who have preserved their baby’s cord blood stem cells and has
over 50 centers across India and abroad. The company will soon be launching another
revolutionary service in Stem Cell Banking - Menstrual Blood Stem Cell Banking, for
every woman to preserve her stem cells and potentially secure her future from life-
threatening diseases. With addition of this service LifeCell will become the first & only
comprehensive stem cells solutions provider in the world to offer a complete spectrum of
services in stem cells through multi-service banking, R&D, Clinical Trials and Stem Cell
Therapy.