Do No Harm
GENE EXPRESSION OF EMBRYONIC STEM CELLS EXTREMELY UNSTABLE
A report from the Whitehead Institute explains why there are so many problems with cloning and foreshadows serious problems with use of embryonic stem cells to treat human disease. The researchers cloned mice starting with mouse embryonic stem cells as the donor of the clone's genetic material. They found wide variations and significant instabilities in how different genes were turned on and off in the mice. The researchers also analyzed the gene expression in the starting embryonic stem cells. The answer was startling—the embryonic stem cells themselves were also unstable. The authors note that the gene expression "of the embryonic stem cell genome was found to be extremely unstable." This has far-reaching implications. Proponents of embryonic stem cell research in humans make extravagant promises of cures for a multitude of diseases, based on the assumption that they will be able to form any of the 210 tissues of the human body from a dish of human embryonic stem cells. In reality, there have been very few successes along those lines, in the culture dish or in mice. Instead, the cells tend to just grow, or form tumors when injected into mice, or form a mixed collection of partially-formed tissue. The results of the study again may point to the reason for this—the extremely unstable state of the embryonic stem cell genome.
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…"
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.
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…"
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."
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 (EB's) from embryonic germ (EG) cells, isolated and cultured the cells from EB's. 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 EB's 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."
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.
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 therapies—much 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.
Further excerpt from article
"But ES cells have plenty of limitations, too. For one, murine ES cells have a disturbing ability to form tumors, and researchers aren't yet sure how to counteract that. And so far reports of pure cell populations derived from either human or mouse ES cells are few and far between--fewer than those from adult cells."
ARTICLES NOTING TOTIPOTENT ABILITY OF ES CELLS, including forming trophoplast
Nagy A et al.; "Derivation of completely cell culture-derived mice from early-passage embryonic stem cells"; Proceedings of the National Academy of Sciences USA 90, 8424-8428; Sept 1993
Wang ZQ et al.; "Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection"; Mechanisms of Development 62, 137-145; Mar 1997
Iwasaki S et al.; "Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos"; Biology of Reproduction 62, 470-475; 2000
Thomson JA et al.; "Embryonic stem cell
lines derived from human blastocysts"; Science 282, 1145-1147;
Nov 6, 1998
Eggan K et al.; "Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation"; PNAS 98, 6209-6214, May 22, 2001.