Problems with Stem Cells
Being Obtained From Embryos
Reference:
Humpherys D et al.; Epigenetic instability in
ES cells and cloned mice; Science 293, 95-97;
July 6, 2001
Culture of embryonic stem (ES) cells affects
their totipotency and may give rise to fetal abnormalities.
Altered allelic methylation patterns were detected in
all 4 genes examined. All the methylation changes
that had arisen in the ES cells persisted on in vivo
differentiation to fetal stages. ES fetuses
derived from two of the four ES lines appeared developmentally
compromised
Reference:
Dean W et al.; Altered imprinted gene methylation
and expression in completely ES cell-derived mouse fetuses:
association with aberrant phenotypes; Development
125 2273-2282; May 19 1998
Culture of preimplantation mammalian embryos and cells
can influence their subsequent growth and differentiation.
Culture of embryonic stem cells is associated with deregulation
of genomic imprinting and affects the potential for
these cells to develop into normal fetuses. Preimplantation
culture in presence of serum can influence the regulation
of multiple growth-related imprinted genes, thus leading
to aberrant fetal growth and development.
Reference:
Khosla S et al.; Culture of preimplantation mouse
embryos affects fetal development and the expression
of imprinted genes; Biology of Reproduction 64,
918-926; 2001
Problems Achieving Specific Cells, Tumor Formation With Embryonic Stem Cells
Rarely have specific growth factors or culture
conditions led to establishment of cultures containing
a single cell type.
Furthermore, there is significant culture-to-culture
variability in the developments of a particular phenotype
under identical growth factor conditions.
[T]he possibility arises that transplantation
of differentiated human ES cell derivatives into human
recipients may result in the formation of ES cell-derived
tumors.
[T]he poor availability of human oocytes, the
low efficiency of the nuclear transfer procedure, and
the long population-doubling time of human ES cells
make it difficult to envision this [generation of human
embryos by nuclear reprogramming] becoming a routine
clinical procedure
Reference:
Odorico JS, Kaufman DS, Thomson JA, Multilineage
differentiation from human embryonic stem cell lines,
Stem Cells 19, 193-204; 2001
In this study researchers used human ES cells, and
added mixes of growth factors in an attempt to get specialized
cell types formed in culture. While partially differentiated
cells formed, no specific tissues were derived. The
authors note, The work presented here shows that
none of the eight growth factors tested directs a completely
uniform and singular differentiation of cells.
Reference:
Schuldiner M et al.; Effects of eight growth factors
on the differentiation of cells derived from human embryonic
stem cells; Proc. Natl. Acad. Sci. USA 97, 11307-11312;
Oct. 10, 2000
In this study the authors formed aggregated embryoid
bodies (EBs) from embryonic germ (EG) cells, isolated
and cultured the cells from EBs. Cells show long-term
population doubling (PD), normal karyotypes (checked
at 20 PD, but not in the long-term cultures), can be
stably transfected with extra genes for gene therapy.
The cells are relatively uncommitted precursor or progenitor
cells.
EB-derived cells may be suited to studies
of human cell differentiation and may play a role in
future transplantation therapies.
Although
a compelling demonstration of the potential of human
EG cells, the limited growth characteristics of differentiated
cells within EBs and difficulties associated with
their isolation would make extensive experimental manipulation
difficult and limit their use in future cellular transplantation
therapies.
For PSCs [pluripotent stem cells]
to be of practical use, methods to generate large numbers
of homogeneous cell types must be developed.
Reference:
Shamblott MJ, Axelman J, Littlefield JW, Blumenthal
PD, Huggins GR, Cui Y, Cheng L, Gearhart JD; Human
embryonic germ cell derivatives express a broad range
of developmentally distinct markers and proliferate
extensively in vitro; Proc Natl Acad Sci USA 98,
113-118; Jan 2 2001
Report on the study from UniSci News Report, Jan 7
2001
EBDs reproduce readily and are easily maintained, Gearhart
said, and thus eliminate the need to use fetal tissues
each time as a source a step that should quell
many of the political and ethical concerns that swirl
around stem cell studies. "We thought from the
first that problems would arise using hPSCs [human pluripotent
stem cells] to make replacement tissues," says
molecular biologist Michael Shamblott, Ph.D.
The early-stage
stem cells are both difficult and slow to grow. "More
important," says Shamblott, "there's a risk
of tumors. If you're not very careful when coaxing these
early cells to differentiate to form nerve cells
and the like -- you risk contaminating the newly differentiated
cells with the stem cells.
"Injected into the body,
stem cells can produce tumors. The EBDs bypass all this."
EBDs readily divide for up to 70 generations, producing
millions of cells without any apparent chromosomal abnormalities
typical of tumor cells.
Problems with Embryonic Stem Cell Differentiation
The following quotes are from an article in Science
describing first exciting new results with adult stem
cells, transforming bone marrow stem cells in brain
and liver. The article then goes on to contrast the
successes of adult stem cell research with the following
description of human embryonic stem cell research.
Reference:
Vogel G; Stem cells: New excitement, persistent
questions; Science 290, 1672-1674; Dec 1 2000
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, whose
use has been restricted by legal and commercial hurdles.
Last month, a group led by Nissim Benvenisty of The
Hebrew University in Jerusalem, in collaboration with
Douglas Melton of Harvard University, reported in the
Proceedings of the National Academy of Sciences that
they could nudge human embryonic stem cells toward a
number of different cell fates. But the results did
not produce easy answers; some cells expressed markers
from several kinds of lineages.
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 therapiesmuch
less the hoped-for replacement organs, says stem cell
researcher Oliver Brüstle of the University of
Bonn in Germany.
Brüstle was one of the first to
show that mouse embryonic stem cells could help treat
an animal disease model, in which neurons lack their
insulating coat of myelin. Even so, he is cautious about
the near-term prospects in humans. Says Brüstle:
"At present, it looks like it is really difficult
to differentiate these [human] cells into more advanced
cell types." Melton agrees. "It's unlikely
anyone will ever find a single growth factor to make
a dopaminergic neuron," as some might have hoped,
but the work provides "a starting place,"
he says.
Simply keeping human embryonic stem cells alive can
be a challenge, says Peter Andrews of the University
of Sheffield in England. For more than a year, he and
his colleagues have been experimenting with embryonic
stem cell lines that James Thomson derived at the University
of Wisconsin, Madison.
"They're tricky," Andrews
says. It took several false starts--and a trip to Wisconsin
--before the researchers learned how to keep the cells
thriving, he says. Melton uses almost the same words:
Human embryonic stem cells "are trickier than mouse,"
he says. "They're more tedious to grow."
Researchers from Geron Corp. in Menlo Park, California,
are having some luck. Company researchers have been
working with human embryonic stem cells as long as any
team has, because Geron funded the derivation of the
cells and has an exclusive license for their commercial
use.
They reported in the 15 November issue of Developmental
Biology that cell lines derived from a single embryonic
stem cell continue to replicate in culture for 250 generations.
This is important, says Geron researcher Melissa Carpenter,
because it means that a single human embryonic stem
cell, which might be modified in the lab, could produce
an essentially unlimited supply of cells for therapy.
That was known for mouse embryonic stem cells but had
not been shown in humans before. Even so, Geron researchers
seem no closer than other groups to devising therapeutic
uses for stem cells. Geron researchers reported last
month at the annual meeting of the Society of Neuroscience
that they had attempted to transplant human embryonic
stem cells into rats.
When they injected undifferentiated
cells into the brain, they did not readily differentiate
into brain cells, the researchers found. Instead, they
stayed in a disorganized cluster, and brain cells near
them began to die. Even partially differentiated cells,
the team reported, tended to clump together; again,
nearby brain cells died.
Excerpt from article in Science
Can Adult Stem Cells Suffice? by Gretchen
Vogel
Science vol. 292, pp. 1820-1822, 8 Jun 2001
In one tissue, at least, scientists agree that the
results are encouraging. In the past few months, a series
of papers has strengthened the idea that cells in the
bone marrow can respond to cues from damaged tissue
and help repair it. Until recently, doctors had only
attempted to use bone marrow stem cells to reconstitute
the blood or immune system.
But late last year, two teams reported that mouse cells
derived from bone marrow could become neuronlike cells
(Science, 1 December 2000, pp. 1775 and 1779). In April,
another two groups reported that bone marrow-derived
cells could help repair damaged heart muscle.
In one
study, Piero Anversa of New York Medical College in
Valhalla and Donald Orlic of the National Human Genome
Research Institute in Bethesda, Maryland, induced heart
attack-like damage in 30 mice. They then injected the
bone marrow cells into surviving heart tissue. Nine
days after the injection, the transplanted cells were
forming new heart tissue--muscle cells as well as blood
vessels--in 12 of the 30 mice, the team reported in
the 5 April issue of Nature.
In the other study, Silviu Itescu of Columbia University
in New York City and his colleagues isolated cells from
the bone marrow of human volunteers, then injected the
cells into the bloodstream of rats in which the team
had induced heart attacks.
Signals from the damaged
heart evidently attracted the transplanted cells, the
team reported in the April issue of Nature Medicine;
2 weeks after the injection, capillaries made of human
cells accounted for up to a quarter of the capillaries
in the heart. Four months after the operation, rats
that received the blood vessel precursors had significantly
less scar tissue--and better heart function--than control
rats.
Perhaps most impressive, in the 4 May issue of Cell,
scientists reported that a single cell from the bone
marrow of an adult mouse can multiply and contribute
to the lung tissue, liver, intestine, and skin of experimental
mice. Researchers knew that a tiny subset of cells purified
from bone marrow had the potential to multiply and give
rise to all the blood cell types, but isolating those
cells has been very difficult.
To increase their chances
of capturing the elusive cells, Diane Krause of Yale
University School of Medicine and Neil Theise of New
York University Medical School and their colleagues
performed a double bone marrow transplant. They first
injected bone marrow cells from a male mouse, tagged
with green fluorescent protein, into the bloodstream
of female mice that had received a lethal dose of radiation.
Two days later, they killed the recipient mice and isolated
a handful of green-tagged cells that had taken up residence
in the bone marrow. (Previous studies had suggested
that the most primitive transplanted cells lodge in
bone marrow.) They then injected irradiated mice with
just one of the green-tagged cells accompanied by untagged,
female-derived bone marrow cells that survive about
a month.
When the scientists killed the surviving mice
11 months after the second transplant, they found progeny
from the cells in lung, skin, intestine, and liver as well as bone and blood. "Bone marrow stem cells
can probably form any cell type," says Harvard's
Melton.
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