[Pflienews] PharmFacts E-News Update: Ethical and Scientific Concerns About Induced Pluripotent Stem Cell Research -- Yamanaka and Thomson
PFLI PharmAid Center
pfli at pfli.org
Tue Jun 3 12:20:39 MDT 2008
*PharmFacts E-News Update -- 3 June 2008 AD #2
*
http://www.lifeissues.net/writers/irv/irv_127concerns.html
Ethical and Scientific Concerns About Induced Pluripotent Stem Cell
Research -- Yamanaka and Thomson
Dianne N. Irving, PhD
© June 1, 2008
Reproduced with Permission
[Note: This article is copyrighted and thus must be acknowledged when
using its original ideas and resources or quoting from it.]
------------------------------------------------------------------------
I. Introduction
In a valiant effort to resolve at last the obvious and highly divisive
ethical concerns surrounding the use of human embryonic and fetal "stem
cells", tissues and body parts in experimental and therapeutic research
and in patient "therapies", several researchers have recently released
their studies involving the production of "induced pluripotent stem
cells" (iPS cells). [See, e.g.: Yamanaka, Takahashi et al., "Induction
of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined
Factors", Cell (2007), doi:10.1016/j.cell.2007.11.019,
http://images.cell.com/images/Edimages/Cell/IEPs/3661.pdf; see also,
Thomson, Yu et al., "Induced Pluripotent Stem Cell Lines Derived from
Human Somatic Cells", Science DOI: 10.1126/science.1151526 (November 20,
2007), http://www.sciencemag.org/cgi/content/abstract/1151526]. As noted
by the authors in their iPS cell research studies, the advantages of
discovering and developing iPS cells include the facts that: no human
embryos (or fetuses?) are involved; it permits the creation of
patient-specific "stem cells" for transplant therapies; it allows for
the creation of models of specific diseases; the cells can be used,
e.g., for screening drugs, vaccines, chemical and biological agents,
etc.; and the techniques are very simple to do. Thus the techniques
could be easily copied by multiple research labs/clinics around the
world and lead to "therapies" for millions of sick patients.
Since most basic research studies realistically resolve some existing
concerns but not necessarily all, the researchers are generally open to
and intellectually honest about noting themselves some of the on-going
unresolved issues in their studies as well. For example, in the Yamanaka
and Thomson iPS cell studies, it is acknowledged that foreign genes
could end up in germ line cells (and thus be passed on through the
generations), that tumor markers on iPS cells were picked up in assays,
and that the possibility exists for the presence of unknown genetic
errors and mutations in iPS cells that could also be passed on to
patients. It is hoped that they and other researchers will also consider
some additional questions about the research as identified below.
The purpose of this article is thus to briefly take a closer ethical and
scientific look at these studies, as well as to raise serious concerns
about the possibility/probability of multiple scientific artifacts
inherent in them, thus rendering the data highly questionable and the
"therapeutic" application of iPS cells to patients dangerous. Coming
from a bench research background (career appointed biochemist/biologist,
NCI/NIH, including basic research in cancer and antigen/antibody
studies), as well as from an ethics background (Ph.D. in philosophy,
Georgetown Univ. and KIE), the scientific and ethical issues involved in
this research was of great interest to me. My approach will be to
consider these iPS cell studies (briefly) from the point of view of two
well-established ethical norms. The first is one that seems to have been
largely abandoned within the research community itself lately, i.e.,
that the /first /requirement of /ethical research /is that the science
used is as accurate and reliable as possible, and that the researcher(s)
are academically and practically qualified to perform the research.
Hence the "ethics" and the "science" are inherently intertwined. The
second is drawn from some of the universal and ageless norms of natural
law ethics, specifically that it is always wrong to intentionally kill
innocent human beings (e.g., those involved in any way in either
experimental or therapeutic research studies, or in clinical trials),
even if purportedly for the benefit of other human beings. Both of these
ethical norms have been universally accepted and concretized for many
decades in several international ethical research guidelines, e.g., the
Nuremberg Code and the Declaration of Helsinki (see Irving, "Biomedical
research with 'decisionally incapacitated' human subjects: legalization
of a defunct normative bioethics theory" (June 1998), at:
http://www.lifeissues.net/writers/irv/irv_70incapacitated1.html).
Unfortunately, upon much closer scrutiny of these iPS studies
themselves, it would also seem that both universal ethical norms have
been neglected, and too many questions about these research studies
remain unresolved or unaddressed.
II. Use of erroneous and vague scientific terms
As noted above, the first /ethical /consideration in doing research is
that /the science used is as accurate and reliable as possible/.
However, generally, one notices the use of certain erroneous and vague
scientific terms in the Yamanaka and Thomson studies which could easily
be misleading. For example, the scientific term "pluripotent" is always
used to refer to both normal human embryonic stem cells and to iPS
cells. However, the well-established scientific fact is that the
blastomeres of the early human zygote and human morula are mostly
totipotent (a range of totipotency). Even the cells of the inner cell
mass of the human blastocyst - those at least referred to as "stem
cells" - are a mixture of totipotent and pluripotent cells (also, a
range of potencies). (See Irving, "Framing the Debates on Human Cloning
and Human Embryonic Stem Cells: Pluripotent vs. Totopotent" (July 23,
2005), at:
http://www.lifeissues.net/writers/irv/irv_100debatecloning1.html).
Therefore, by falsely defining all of these cells scientifically as only
"pluripotent", and by thus labeling tests and assays for "pluripotency"
rather than for "totipotency", they thereby by-pass any ethical
considerations of the purposeful production of innocent living human
embryos (human beings) and their necessary destruction in order to
retrieve their stem cells. They also thereby by-pass the fact that
totipotent stem cells retrieved from living human embryos could possibly
undergo "regulation" and revert to new living human embryos themselves
(and be used as well in destructive research).
The studies also use the scientific term "undifferentiated" to refer to
all human embryonic and iPS stem cells. However, early human embryonic
stem cells are always differentiated to some degree - otherwise why do
they function differently than cells derived from earlier human embryos,
and how would they "know" how to differentiate later on into /more
specific /cells, tissues and organs desired by the researchers?
Even the position that each totipotent cell (blastomere) assumes in the
developing embryo (in the morula, as well as in the blastocyst) requires
specific differentiation. (See Bruce M. Carlson, Human Embryology and
Developmental Biology (St. Louis, MO: Mosby, 1999, p. 41). So there is
no such thing as "undifferentiated" early human embryonic stem cells, as
if they were just superfluous and uninteresting non-entities, just
biological "stuff".
Again, the use of the scientific phrase "immortal cell lines" that can
reproduce indefinitely as applied to human embryonic and to iPS cells is
rather "wishful thinking". There are no such things as "immortal cell
lines", if by that phrase is meant that once such cells are produced and
cultured they would continue to supply the researchers forever. As all
lab technicians know, cell lines fairly quickly begin to genetically
mutate and die off (called "senescence").
One also notices that the harder one attempts to identify the "smaller"
details of the "materials and methods" sections of these studies, the
vaguer they become. For example, an objective observer could not
determine from the initials and other labels employed if any innocent
living human embryos or fetuses were used in any way in these studies in
any media, feeder cells, DNA-chips, assays, etc., that had been bought
from commercial sources. If they were, then those commercial products
themselves involved the destruction of human embryos and fetuses (i.e.,
innocent living human beings in their embryonic or fetal periods of
development).
And it goes without saying that such research studies silently drag
along with them all the baggage of false scientific terms used lately in
other studies by their scientific colleagues to mis-define the early
human embryo in order to render pressing ethical questions mute, e.g.,
"scientific" claims that the embryo is not an organism, a living human
being, but rather just a "bunch of stem cells" or "a ball of cells".
(See Irving, "What Human Embryo? Funniest Mental Gymnastics from
Medicine and Research" (Oct. 14, 2004), at:
http://www.lifeissues.net/writers/irv/irv_82whathumanembryo1.html).
III. Do the scientific claims match the scientific details of their
studies?
That said, it is perhaps more instructive to turn to the stated
scientific and therapeutic claims of the researchers themselves to
determine if they actually match the given details as found in those
same studies.
A. No human embryos involved
The claim is made that iPS cell research solves all the divisive ethical
issues because no human embryos are involved in the research. However,
consider the following observations: (1) In order to prove that iPS
cells are like human embryonic stem cells (and thus are viable
substitutes for them), they must use human embryonic stem cells in the
studies so that the two cell types can be accurately compared. Such
"normal" human embryonic stem cells must have of necessity been derived
by destroying innocent living human embryos and deriving the stem cells
from them. Note too that both Thomson and Yamanaka used human embryonic
or fetal cells (and/or components) (2) in culture and other media, (3)
as "feeder cells", (4) for screening, (5) for "controls", (6) for
material parts of technical assays (e.g., DNA chips), and (7) as sources
of transcription factor genes themselves. Thomson also (8) used fetal
cells as /actual subjects of his transduction process /(e.g., IMR90
fetal cells). He admits the inherent requirement for human
embryos/fetuses and their components in a press comment: "Well, what I
hope will not happen is that everybody says, 'See? We don't have to do
embryonic stem cell research now.' ... In our research, we actually used
human embryonic stem cells as part of the screening process.
So the research itself on human embryonic stem cells led to the next
finding about pluripotent cells,"
[http://www.lifenews.com/nat3479.html]. Yamanaka notes also (9) that
"with the exception of cells at the edge", the assay for _embryonic
_surface antigens are negative, and the assay for embryonic _stem cell
_surface antigens are positive. Does his exception statement about
"cells at the edge of the colonies" indicate that transformed human
embryos were produced there, and that is why the assay for embryonic
surface antigens on them was positive? Clearly, the claim that no human
embryos are involved in such research is not true.
B. Creation of patient-specific "stem cells" for transplant therapies
As admitted by both researchers in their publications, these iPS cells
cannot be used in patient "therapies" -- not only because transcription
/and /viral foreign genes can cause tumors (as admitted in both
studies), but also because both sources of foreign genes (virus and
transcription factors) could cause foreign antigens on iPS cell surfaces
that would in turn cause immune rejection reactions in patients -
regardless if such genes were currently being expressed or not.
Likewise, foreign surface antigens (both genetic and molecular) could
also be caused in iPS cells by media used (e.g., mouse and primate
media), feeder cells used for cultivation (e.g., fetal cells),
inoculations of cell cultures, etc. (see details below). Therefore, such
cells would never be "patient-specific", and could cause serious immune
rejection reactions in the patients. These patients too are innocent
living human beings whose dignity and safety must also be sustained.
In fact, there are so many artifacts and antigens introduced into the
material and methods (as especially detailed by Yamanaka, but similarly
assumed for Thomson, and as listed more fully below) that one must
seriously question the accuracy and reliability of the data itself. /Did
the data presented in these studies ever pass a t-test? /This is not
clear at all in the details of the research studies.
C. Creation of models of specific diseases
Because of the incorporation of so many known foreign and even unknown
antigens into these iPS cells and the multitude of artifacts inherent in
the research, one wouldn't really know what disease one was studying -
even if original disease cells were derived from a patient with a
specific disease for the purpose of studying abnormal cell biochemistry.
That is, the actual diseased cells of the patient would not match or
function like the diseased iPS cells derived from them - like comparing
apples and oranges. Thus how could such studies seriously provide
"models of specific diseases"? Further, with so many unknowns, the data
obtained would probably not be accurate, reliable, or repeatable,
especially in other labs.
D. Use iPS cells to screen drugs, vaccines, chemical and
biological agents, etc.
Perhaps "some" legitimate information could come out of such studies,
e.g., how certain drugs, etc., function /in iPS cells/. However, as with
"studying models of specific diseases", there are so many artifacts and
foreign antigens involved in both studies that the data obtained in
screening drugs would seem to be quite useless if it were extrapolated
to "normal" cells, or to treat real diseased cells/patients.
E. The techniques are so simple to do
One look at Yamanaka's study, especially his "materials and methods"
section, and it is clear that the study was not "simple". At least he
was far more forthcoming with his details than was Thomson.
In sum, the various claims in the introductions of these iPS cell
studies hardly match the scientific details buried in the body of the
articles. Are they more fiction than fact?
IV. Admitted problems in the studies
However, credit must be given to the authors for admitting to some
important problems inherent in their studies. Among them are the following.
A. Foreign genes could end up in germ line cells
Theoretically, there is no reason why they couldn't deliberately make
ip-sperm and ip-oocytes, as well as ip-embryos. If ip-sperm and/or
ip-oocytes are used to reproduce ip-embryos, the foreign genes (all of
them) would become part of the genome of the germline cells of the
ip-embryo, and expressed as antigens on the cell surfaces. The same
applies if they created an ip-embryo outright by means of reprogramming.
There is no theoretical impossibility for continuing the reprogramming
of these cells back to the zygote (or earlier) stage of differentiation.
These foreign genes would then also become part of the germline of the
ip-embryo and be passed down through its generations.
Further, the fact that these iPS cells form chimeras when injected into
blastocysts demonstrates that their foreign genes are or can still
become active - even if not picked up in assays. One can only test for
what one already is looking for.
B. Tumor markers on iPS cells are picked up in assays
Not unexpectedly, both researchers state in their studies that these iPS
cells could not be used in patient therapies because of their ability to
form tumors. However, the fact that these iPS cells could cause tumors
in patients is /not the same /problem as the fact that these genes, and
the non-tumoric foreign genes, cause specific antigens on the surface of
iPS cells - regardless if such genes are being expressed at the time
intra-cellularly. Such foreign antigens could thus cause rejection
reactions in the patients.
C. Unknown genetic errors and mutations in iPS cells render
"therapies" problematic
Even aside from the obvious and known induced antigens on these iPS
cells, it is also acknowledged that other genetic mutations could have
been caused in these cells but were just not picked up or tested for:
*Yamanaka: *"Thus, each clone had more than 20 retroviral integration
sites in total, which may increase the risk of tumorigenesis. ... This
issue must be overcome to use iPS cells in human therapies. ... minor
genetic alterations, which could not be detected by karyotype analyses,
or epigenetic alterations are required for iPS cell induction. These
issues need to be elucidated in future studies. ... /Human iPS cells,
however, are not identical to hES cells: DNA microarray analyses
detected differences between the two pluripotent stem cell lines.
Further studies are essential to determine whether human iPS cells can
replace hES in medical applications/" (emphases added).
Thomson: "For transplantation therapies based on these cells, with the
exception of autoimmune diseases, patient-specific iPS cell lines should
largely eliminate the concern of immune rejection. It is important to
understand, however, that before the cells can be used in the clinic,
additional work is required to avoid vectors that integrate into the
genome, potentially introducing mutations at the insertion site. ...
/However, further work is needed to determine if human iPS cells differ
in clinically significant ways from ES cells/" (emphases added).
As has already been suggested above, if there are so many "unknown"
genetic mutations, antigens, or artifacts, there are not only obvious
concerns about the use of these "patient-specific" cells in patients,
but also about any validity of the data derived from "studies of disease
mechanisms" and from screening drugs, vaccines, chemical and biological
agents, etc.
V. Other possible sources of artifacts and foreign antigens
Although not specifically addressed in these studies, there are a host
of other possible sources of artifacts and foreign antigens that could
render the data presented as useless, as well as call into serious
question the use of iPS cells in sick patients as "therapies". Although
some of the following examples may be too "scientific" for many readers,
at least it is worth registering and considering some of them.
In general, there are too many known and unknown variables throughout
the experiments; in fact, there is, as noted in the studies, genetic
variability even within the same iPS clone batches. It is also
critically relevant to determine from the studies if the assay machines,
gels, tests, etc., were reliably and properly calibrated before use, so
that the data would be reliable and reproducible. Otherwise, the data
presented in these studies are essentially useless. Again, the data
derived from just one or two studies could hardly pass a t-test. It is
not clear if the data presented in these studies were determined to be
statistically significant or not.
Other possible sources of data artifacts and problematic antigens in
these studies involves the /viral /genes used as vectors, as well as the
transmission genes transferred. For example, Thomson used the following
transmission genes, all of which would definitely show as problematic
antigens on the surfaces of iPS cells produced with them: OCT4, SOX2,
NANOG, and LIN28; Yamanaka used the following transmission genes:
Oct3/4, Sox2, Klf4, and c-Myc. And the questions arises as to whether
all of the transmission genes used in the studies were derived from
genetically /different /human embryos or fetuses? That too could result
in artifacts in the data presented, as well as the presence of more
foreign antigens on the surfaces of the iPS cells produced.
Also to be considered are foreign genes or antigenic molecules that
could be inadvertently derived from the media, inoculations and feeder
cell layers used, as well as from impurities in the several manufactured
biological products, laboratory contamination, etc.
The above are simply lists of standard good quality research controls
used throughout the various research fields. It would be important,
then, to turn again to the specific research studies to determine any
specific sources of artifacts and antigens.
A. Yamanaka et al
As noted, Yamanaka et al used the following transmission genes: Oct3/4,
Sox2, Klf4, and c-Myc. All of these genes, if incorporated into the iPS
cells, would cause foreign genetic antigens on the iPS cell surfaces. As
already noted, one also wonders if all of these "normal" transmission
genes were derived from different human embryos or fetuses. One cannot
assume, e.g., that the Oct3/4 transmission gene from one human embryo is
genetically identical to that of other human embryos. If such genetic
differences exist, then they could cause additional foreign antigens on
the surface of iPS cells.
Viral genes from the vectors used could also be incorporated into the
genome of iPS cells, also causing foreign antigens on the cell surfaces.
For Yamanaka, all four retroviruses used to transmit the "normal" human
embryonic genes could cause foreign antigens, not to mention
specifically the amphotropic retrovirus, the lentivirus, and the
ecotropic retrovirus used. Viruses have genes, too!
And for brevity, the following is a further listing of sources of data
artifacts, foreign antigens in the iPS cells produced, and human
embryonic/fetal cell sources (at least as much as can be determined
solely from the studies):
* the mouse receptor for retroviruses, Slc7a1, was injected into the
original HDF cells
* firefly luciferase gene was injected as well
* one target cell used, HDF, was derived from the facial dermis of a
36-year-old Caucasian female, each detail of which would produce
specific antigens on the iPS cell surface
* another target cell used was a primary human fibroblast-like
synoviocytes (HFLS) from the synovial tissue of a 69-year-old
Caucasian male, each detail of which would produce specific
antigens on the iPS cell surface
* another cell used, BJ cells, has its own specific cell antigens,
as well as being a cell line established from neonate fibroblasts
* green fluorescent protein (GFP) used
* mitomycin C-treated SNL feeder cells used
* medium DMEM containing 10% FBS (fetal source)
* medium for primate ES cell culture used
* medium for primate ES cell culture containing bFGF used
* fibroblast growth factor (bFGF) used
* MEF-conditioned primate ES cell medium used
* PA6 feeder layer used
* coculture with PA6 cells
* activin A and bone morphogenetic protein (BMP) used
* 0.5% penicillin and streptomycin in media used
* DMEM containing 10% FBS (fetal source) used
* L-glutamine (Invitrogen), 1 3 10_4 M nonessential amino acids
(Invitrogen), 1 mM sodium pyruvate (Sigma) used
* PA6 stroma cells used
* Primate ES medium used
* recombinant human basic fibroblast growth factor (bFGF) used
* DMEM/F12 containing 1 mg/ml collagenase IV used
* 0.3 mg/ml Matrigel (growth-factor reduced) used
* MEFs derived from embryonic day 13.5 embryo pool of ICR mice used
* EcoRI fragment of pCR2.1-hOCT3/4 introduced into the EcoRI site of
pMXs retroviral vector used
* "20 bp random sequence, designated N20 barcode, into the NotI/SalI
site of Oct3/4 expression vector used; unique barcode sequence in
each experiment"
* KpnI/BglII digestion
* AatII (blunted)/NheI fragment of pQBI-polII inserted into the KpnI
(blunted)/NheI site of pGV-BM2
* Lentivirus production: 293FT cells (fetal cells), transfected with
3 mg of pLenti6/UbC-Slc7a1 along with 9 mg of Virapower packaging mix
* PLAT-E packaging cells; transfected with pMXs vectors with Fugene
6 transfection reagent
* 20% knockout serum replacement used
* PA6-feeder layer in Glasgow minimum essential medium used
* RPMI1640 (Invitrogen) plus B27 supplement (Invitrogen) medium
(RPMI/B27), supplemented with 100 mg/ml human recombinant activin
A used
* human recombinant bone morphologenic protein 4 used
B. Thomson
Whereas Yamanak's study provided many (but not all) details of their
materials and methods, the same is not true for Thomson's study. In
fact, there was a dirth of "methods and procedures" details. Therefore,
it is not possible to identify the same kind of list as above. However,
it is still obvious that foreign genetic cell surface antigens would be
caused in their experimental iPS cells because the cells were infected
and transformed with foreign transmission genes (OCT4, SOX2, NANOG, and
LIN28), using four different viral vectors whose own genes would be
foreign.
*VI. Conclusion *
Considering the enormous stakes involved, it would seem that it is
incumbent upon scientists involved in especially ethically sensitive
research to be as open and detailed in their publications as possible.
The trend, however, seems to be to camouflage and dilute the scientific
details as much as possible -- not only in order to evade professionally
appropriate questions from their scientific peers, but also to evade the
very questions that society and ethics have traditionally required of
all scientists. Rather than use and report the accurate scientific
facts, it would seem that scientists prefer to secure their successes by
using false and misleading manufactured "scientific" terms, verbal hype,
and empty promises.
Even the most basic requirements of research ethics appear to have been
abandoned, including the required use of the most accurate and reliable
scientific facts, as well as the "denial" of the age-old dictum that it
is simply wrong to purposefully kill innocent human beings - regardless
of their stage of development, ethnicity, culture, degree of illness,
etc., and regardless of whether or not their destruction could be of
benefit to other human beings.
Needless to say, even the most desperate of sick patients should not be
exposed to "therapeutic" research or clinical trials when such
participation unwittingly puts them into serious danger of harm and even
death. One even wonders how such patients could give legally valid
"informed consent".
We would all obviously welcome a viable scientific and ethical
resolution to the divisive politics of human embryo and fetal research
that has consumed us for so many years. These iPS studies, however, do
not appear to be that solution.
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