Epigenetic Instability in ES Cells and Cloned Mice

Science v.293 6jul01

[ Epigenetic Instability in ES Cells and Cloned Mice Science v.293 6jul01 below SF Chronicle article]

Huge Genetic Defects Discovered in Mice Cloned from Stem Cells

Carl T. Hall / SF Chronicle 6jul01

Cloned mice have profound genetic abnormalities not apparent at birth, researchers report today, a finding that bolsters the belief among many scientists that current cloning techniques are too risky to be used on humans.

The new study is sure to reverberate in the highly politicized debate over cloning and embryonic stem cells. The Bush administration is considering whether to allow federal funds for stem cell research, and Congress is considering whether to make human cloning a federal crime.

The findings show that a cloning method using embryonic stem cells appears to be inherently hazardous, producing genetically abnormal offspring that give no outward sign of problems. The study did not look at other methods that use mature cells taken from an adult donor.

"Even cloned animals which look normal may not be," said biologist Rudolf Jaenisch of the Whitehead Institute for Biomedical Research at the Massachusetts Institute of Technology. Jaenisch is the senior author of the study, which appears today in the journal Science.

Most experts say that reliable techniques for cloning even a nonhuman primate are nowhere close at hand, despite a lengthening parade of barnyard clones -- including cattle, goats and pigs -- that began with the 1997 birth announcement of Dolly the sheep in Scotland.

"All kinds of subtle problems crop up, and now it looks like the problems are even more subtle than a lot of people anticipated," said Gregory Stock, director of a program on medicine, technology and society at the University of California at Los Angeles School of Medicine.

"It's not a trivial process," he added. "But even if it's ludicrous to think someone will be cloning a human baby in a year doesn't mean it won't happen in five or 10 years."

Ethical Questions

Many argue that it's unethical to attempt human reproductive cloning given the risks, even if it's acceptable to use the same technology to produce human cell lines for medical purposes, which is known as therapeutic cloning.

Jaenisch and his colleagues set out to discover how genes turn on and off in a cloned animal, in this case mice produced from embryonic stem cells.

Stem cells are the modeling clay of mammalian development -- undifferentiated lumps of cells that can be molded into virtually any of the hundreds of specialized cell types that form a mature organism.

Although clones, in theory, can be made from virtually any kind of adult cell DNA, the process is said to be easier using embryonic stem cells as the starter material, because such cells don't have to be extensively reprogrammed when transferred into an egg.

In the study, researchers removed DNA from mouse stem cells and inserted it into a mouse egg that had been stripped of its DNA. The resulting embryos were then implanted in mother mice and allowed to grow to birth.

There was no evidence that the genes in the cloned animals were altered. However, the way in which the genes made proteins was flawed and unstable. In effect, the researchers found that even though the biological blueprint was intact in the cloned animals, the way that the blueprint was read and interpreted was flawed.

Past cloning efforts have already demonstrated that cloned embryos are difficult to bring to term, and many that are born carry visible defects, in particular growth abnormalities. Extreme obesity, for example, has developed in many cloned animals.

Defects Not Apparent at Birth

The most surprising result of the study was that the gene expression errors can be tolerated well enough to result in a successful live birth of normal- seeming clones.

"We see huge errors in gene expression in mice that make it to term," said David Humpherys, a graduate student at MIT and lead author of the Science study.

Moreover, there's no ready way to tell just how much of a clone's genome might harbor abnormalities, since errors can appear in different body parts at different times, and may or may not cause noticeable health problems.

Given today's gene-monitoring methods, "there would be no way to ensure that a cloned person would have normal attributes," Humpherys said.

Errors seem to spring from the stem cells, rather than the cloning process itself. This could further complicate efforts to use stem cells for other medical purposes, such as to produce genetically pure transplant material.

Jaenisch said this instability of gene expression in embryonic stem cells, combined with the lack of a normal sperm and egg maturation process, appears to be what gives rise to the type of errors observed in the new study.

The very idea of human reproductive cloning has raised profound controversies that go well beyond the safety issues. Even if a human could be cloned, some argue, the prospect challenges traditional notions of parenthood, identity and human rights.

California lawmakers have enacted a moratorium that imposes civil penalties on anyone who attempts to clone a human being.

The latest evidence points to what many consider to be an even more disturbing danger: Defective clones might survive to birth, and seem normal in early life, only to reveal potentially profound defects later on.

Jaenisch said there's no clear path to solve the problems, given the number of genes that might be prone to misfiring and the difficulty of finding them all in the vastness of the genome.

Limitation of Study

One limitation of the new study is that it has no direct bearing on cloning with mature, or "somatic," cells from an adult donor, such as the udder cell used to produce Dolly.

For this reason, some cloning experts cautioned against drawing too many conclusions about all types of cloning methods based on the new evidence.

"This is a very important study, but their conclusions seem too broad," said Jose Cibelli, vice president of research at Advanced Cell Technology, a company in Worcester, Mass., active in therapeutic cloning and stem cell research.

The politics of cloning

• The scientific implications
  The presence of undetected genetic glitches could help explain why so many clones do not survive to birth. The study found that it will be difficult to come up with a genetic test able to predict which developing clones will be healthy, a strike against some human cloning advocates.
  
  
• The political implications
  The research could influence the debate over a separate use of embryonic stem cells to create tissues for research on diseases and their treatments. The Bush administration is expected to decide soon whether researchers who take government money should be allowed to work on tissues derived from embryonic cells. The work is controversial because obtaining the stem cells requires the destruction of embryos.

Epigenetic Instability in ES Cells and Cloned Mice 

Science v.293 6jul01

David Humpherys, ^1,2 * Kevin Eagan, ^1,2 * Hidenori Akutsu,³* Konrad Hochedlinger,¹ William M. Rideout III, ¹ Detlev Biniszkiewicz,¹ Ryuzo Yanagimachi, ³ Rudolf Jaenisch ^{1,2 +}

Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed inmost cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

¹Whitehead Institute for Biomedical Research, ²Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge MA 02142, USA. ³Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, USA.

* These authors contributed equally to this work.
+ To whom correspondence should be addressed. Email: jaenisch@wi.mit.edu 

Nuclear transfer technology has been used to derive live clones in several species including sheep (1) cattle (2) goats (3) pigs (4, 5) and mice (6), but only a few percent of nuclear transfer embryos develop to term. Even those clones that survive to term frequently die of respiratory and circulatory problems and show increased placental (7- 9) and birth (10) weights often referred to as "large offspring syndrome" (11). Abnormal regulation of imprinted genes has been shown to affect fetal growth (12, 13), and it has been proposed that improper expression of these genes may contribute to the abnormalities observed in cloned offspring (14, 15).

With one notable exception, poor survival of NT embryos has so far been independent of the donor tissue; a significantly higher fraction of blastocysts cloned from ES cell nuclei than from any somatic cell type survive to adulthood (10, 16, 17). This result is consistent with the idea that the nucleus from an undifferentiated embryonic cell might be more amenable to or require less reprogramming than the nucleus from a differentiated somatic cell. In an effort to correlate changes in gene expression with the survival and fetal overgrowth of cloned animals, we have examined imprinted gene expression in mice cloned by nuclear transfer and in the donor ES cell populations from which they were derived.

Cloned embryos were produced by transfer of low passage (p8 to pl2) ES cell nuclei into enucleated oocytes, which were then transferred into surrogate mothers and delivered by caesarian section (c-section) at 19.5 days post coitum (dpc) as previously described (10). Total RNA was isolated from placentas and organs of newborn mice, and Northern hybridization analysis (18) was used to quantity expression of H19 and Igf2 in normal and cloned neonates. H19 and Igf2 RNA levels were similar in placentas of normal pups (Fig. l A, lanes l through 3) and in placentas derived from normal zygotes that had been cultured in vitro to the blastocyst stage before transfer to surrogate mothers (lanes 4 and 5). In contrast to controls, expression of both genes varied widely between the placentas of cloned embryos. Some clones expressed both genes at levels similar to control placentas (lanes 6, 13, 14, 26, 27, 32, 33), whereas most clones showed aberrant levels of H19 and/or Igf2 transcripts. In many clones, H19 was silenced and Igf2 was expressed at higher levels than normal (lanes 8, 9, 18 through 22, 24, 29), as one would predict from the usual reciprocal expression pattern of these two genes (19). However, the reciprocal expression of H19 and Igf2 was not observed in a few clones (lanes 15 through l7).

Similarly, Northern analysis of the imprinted genes Peg1/Mest and Meg1/Grb10 revealed variability in the RNA levels amongst the clones, although the extent of variation was less than that observed for H19  (Fig. l A). Expression levels of these genes were noticeably lower than for controls in many clones (Peg1, lanes 13 through 16, 23, 28 and Meg1, lanes 23, 25, 28, 32, 33). When the expression of Peg3 and Snrpn was examined, there was less variation than for the other genes (data not shown). A comparison of expression levels among several genes in a given placenta revealed that the abnormal expression of one imprinted gene did not correlate with abnormal expression at other imprinted gene loci (Fig. 1A).

It has been established that the differentially methylated region (DMR) upstream of the H19 gene affects its expression with the expressed maternal allele being hypomethylated and the silent paternal allele being methylated (20). To determine whether the abnormalities in gene expression were associated with epigenetic alterations in the imprinted domain upstream of H l9, we analyzed the methylation pattern of the H19 DMR as previously described (21). As predicted, the DMR was highly methylated in those placentas that had silenced the H19 gene and was partially unmethylated in those that showed H19 expression (22). In contrast, we did not observe a difference in DNA methylation levels between control and cloned offspring at the Igf2r DMR 2 (data not shown), again indicating that epigenetic alterations at one imprinted locus did not necessarily predict changes at other loci.

To determine whether similar alterations in H19 and Igf2 expression were present in the organs of newborn clones, we examined RNA isolated from the kidney, heart, and liver of NT mice. H19 expression was silenced in the heart and kidney and reduced in the liver of all four clones examined (Fig. 1B). Igf2 expression was increased in kidney and heart but was comparable to controls in the liver (Fig. 1B). lanes 15 through l8). The less dramatic elevation in hepatic Igf2 expression compared with mesodermal expression changes is consistent with previous observations (19).

Increased placental and birth weights are common phenotypes of cloned animals (7, 10). We investigated, therefore, whether the altered expression of imprinted genes observed in cloned animals correlated with fetal overgrowth and neonatal mortality. The results summarized in Fig. 1Alaw show that the majority of NT pups, regardless of their postnatal survival, exhibited increased birth and placental weights and displayed stochastic changes in the expression of a number of imprinted genes. However, no significant correlation between any of these parameters was established.

The results described so far indicate that expression and methylation of imprinted genes varied widely in placentas and tissues of neonatal ES-cell NT mice. Because these clones were derived from ES cells that had been cultured in vitro, we investigated whether faulty reprogramming during nuclear cloning caused improper imprinted gene expression or whether these errors were the result of preexisting losses of imprinting in the donor cell population, as has been previously observed (23). The ES cell lines used in the nuclear transfer experiments were differentiated in the absence of feeders or LIT (Leukemia Inhibitory Factor) in media containing l0^-7M all-trans retinoic acid. H19 expression was then determined by Northern analysis. Wide variation in expression was observed in different ES cell lines; the J1 and V26.2 lines showed the lowest H19 expression levels (Fig. IC, lanes 3 and 6) and exhibited the highest degree of H19 DMR methylation, as determined by Southern analysis (data not shown).

Given the variability of H19 gene expression in the placentas of cloned mice even when derived from the same cell line, we assessed the extent of heterogeneity in H19 expression and methylation among subclones derived from individual colonies of the V6.5 ES cell line (24). As judged by Northern analysis, ES-cell subclones differed widely in H19 expression after differentiation in retinoic acid (Fig. 2A). Subclones that expressed

H19 (subclones 39 and 72) showed a methylation profile consistent with one hypomethylated H19 allele (Fig. 2B), whereas subclones that silenced H19 (subclones 10, 23, 43, 89) were hypermethylated in both alleles (Fig. 2, A and B). Methylation differences between subclones were often more dramatic than the differences observed between the various ES cell lines.

To assess the epigenetic heterogeneity among individual clonally related cells, we investigated whether gene expression would be similar in animals derived from cells of the same subclone. We attempted to generate mice from each of four subclones that expressed both Peg] and H19 (subclones 39 and 72), Peg] but not H19 (subclone 89), or neither Peg] nor H19 (subclone 23, Peg] expression not shown). Mice derived entirely from donor ES cells were generated by two different methods: (i) tetraploid embryo complementation and (ii) nuclear cloning.

Fig. 1. Perinatal silencing of H19 in placentas and tissues of mice cloned from ES cells. (A). Northern blot analysis of H79, Igf2, Peg1/Mest, Meg1/Grb10, and Gapdh in placentas after c-section at 19.5 dpc The ES cell line used as a nuclear donor is indicated above the lanes. Genetic backgrounds of the ES cell lines are given in (10). V18.6, J1, and V26.2 are inbred lines, whereas the remainder are Fr lines. Samples in lanes 1 through 3 were from normal embryos and those in lanes 4 and 5 were from embryos that were in vitro cultured to the blastocyst stage. The placentas are ordered by weight from smallest to largest among clones from a given cell line. Igf2 and Gapdh are shown for the same blot, whereas the blots shown for H79, Peg1/Mest, and Meg1/Grb10, are from equivalently loaded blots showing similar Gapdh expression. Placental and birth weights for the mice examined are indicated below the respective lanes [ solid bar indicates that the mouse initiated normal breathing, hatched bar indicates death soon after delivery; compare results with (10)). For controls, an average birth weight is indicated for normal mice (10). (B) Northern analysis of newborn kidney, heart, and liver from wild-type 129 mice (lanes 1, 2, 7, 8, 13, and 14) and  mice cloned from the J1 and V6.5 ES cell lines. The blot was sequentially probed, without stripping, for H19, Igf2, and Gapdh, Igf2 transcripts from multiple promoters are present, including one just smaller than the  prominent H79 transcript (see, for example, lanes 9, 10, 15, and 16). (C) Northern analysis of ES lines for H79 and Gapdh after 7 days differentiation in retinoic acid (RA). Undifferentiated F, _1,2-3 ES cell RNA (lane 1) is included for comparison.

Fig. 2. H19 methylation and expression in subclones of the V6.5 ES cell line and expression in mice cloned from these subclones. (A) Northern analysis of V6.5 subclones after 6 days RA treatment. The original ES line from which the subclones were derived after 7 days RA treatment is shown for comparison (lane 1). subclones have been given numerical designations. (B) DNA from indicated undifferentiated subclones digested with Sac I and Hha I. The blot was probed with an H79 DMR Sac I probe. The profile seen for subclone 72 most closely resembles the pattern observed in control placentas at term. The intense low molecular weight band prominently visible in subclones 39 and 72 is the primary restriction fragment seen in Dnmt1^-1- ES cells (21). (C) Comparison of H79 and Peg1 expression by Northern analysis in neonatal kidney and liver from mice generated via tetraploid embryo complementation and nuclear transfer from three (89, 23, 39) of the different V6.5 (B6/129) subclones characterized in (A) and (B). Neonatal RNA (lanes 1 and 2) from 129 inbred mice is shown for comparison. Peg1 expression was analyzed because subclone 23 had failed to express Peg1 upon RA differentiation. Placental and birth weights and postnatal survival are indicated as in Fig. 1.

Tetraploid embryo complementation is technically easier than nuclear transfer and involves production of composite embryos by injecting ES cells into a tetraploid blastocyst (10, 25, 26). Because the host tetraploid blastocyst cannot contribute to embryonic lineages, the composite embryos give rise to mice that are entirely derived from descendants of the injected ES cells. We successfully generated tetraploid ES-cell pups from three of the four subclones (Table l). Most pups had increased birth weights relative to normal but not in vitro cultured controls, and they initiated normal breathing after delivery by c-section. Fig. 2C shows the great variability observed among pups derived from both different and identical ES-cell subclones. For example, two pups derived from subclone 89, which had silenced H19 in the donor cells, did not express H19 in kidney and liver (lanes 3 and 5), but one did (lane 4). Consistent with the expression pattern found in the donor cell population, none of the pups derived from subclone 23 expressed H19. Two of the pups generated from subclone 39 showed high H19 expression levels (lanes 15 and l6), which is consistent with the donor cells; however, a third pup had silenced H19 (lane l4). Normal Peg 1 expression was seen in all pups derived from ES cell subclones 39 and 89 but not in pups derived from subclone 23.

Roughly 10 donor ES cells were injected into each blastocyst for tetraploid embryo complementation. To analyze gene expression in animals derived from a single nucleus, we used nuclear transfer to generate animals from different ES-cell subclones. A total of 613 enucleated oocytes were reconstituted with nuclei from the four ES-cell subclones, and five viable pups were obtained: two from subclone 23 and three from subclone 89. We were unable to obtain mice from subclones 39 and 72 (Table l). Low but variable levels of H19 were present in the kidney and liver of all five clones (Fig. 2C, lanes 6 through 8, 12, and l3), whereas Peg1 expression levels were similar to expression levels observed in the donor subclones. Peg1 was not expressed in the kidney and was barely detectable in the liver of two NT pups derived from subclone 23 (lanes 12 and 13) but was present at levels comparable to controls in NT pups derived from subclone 89 (lanes 6 through 8).

Table 1. Summary of pups derived by nuclear transfer (NT) and tetraploid (Tetra.) embryo complementation for V6.5 subclones. Percentage of live embryos at each stage are indicated. No., number; surv., surviving manipulation; activ., activated with psuedo pronuclei; trans., transferred to recipient.

Unlike NT placentas, which are derived exclusively from oocytes reconstituted with ES cell nuclei, the embryonic component of placentas generated by tetraploid complementation was largely derived from the tetraploid host blastocyst and to a lesser extent from the injected ES cells (27). Consistently, H19 expression was normal in the placentas of pups generated by tetraploid complementation but frequently abnormal in those from nuclear transfer animals (22)

We conclude that expression of H19, Peg1 and presumably other imprinted genes varies widely between individual ES-cell subclones. Surprisingly, expression was significantly different among mice derived from cells of the same ES-cell subclone, whether originating from several cells after tetraploid complementation or from a single cell after nuclear transfer. The variability of gene expression in these mice likely reflects epigenetic changes that occurred during in vitro culture among sister cells derived from a single cell, consistent with the notion that the epigenetic state of ES cells is extremely unstable. The level of H19 expression was not a useful criterion to predict whether given donor cell populations could be used to generate viable mice by either of the two methods. Because ES cells are a potential in vitro source of many cell types for transplantation medicine, it will be important to assess whether the epigenetic state of human ES cells is as unstable as that of murine ES cells. It should be emphasized, however, that epigenetic instability of murine ES cells does not impair the routine generation of normal chimeric mice.

No significant correlation between the anomalous fetal growth of cloned mice and abnormal expression of any single imprinted gene was seen (see weights in Fig. 1A and Fig. 2C). It is possible that the disturbance of placental and fetal growth is due to the cumulative action of many abnormally expressed genes which may have opposing influences on fetal growth (12, 13) and that the effect of a single imprinted gene is insufficient to produce a significant correlation with the overgrowth.

The widespread dysregulation of genes in cloned animals suggests that, contrary to previous conclusions (23), mammalian development may be rather tolerant to epigenetic abnormalities and that lethality may only result from the cumulative effects of a stochastic loss of normal gene regulation at multiple loci. Our results indicate that even apparently healthy cloned animals can have gene expression abnormalities that are not severe enough to impede develop ment to birth but that may cause subtle physiological abnormalities which could be difficult to detect

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18. Total RNA was isolated using TriReagent (Molecular Research Center, Cincinnati, OH). Five to ten micrograms were resolved in 1% agarose-formaldehyde gels and transferred to Zetabind (Cuno, Meridian, CT) or Genescreen (NEN, Boston, MA) filters, which were then hybridized with probe in Church buffer overnight. The probes were generated by random hexamer labeling of the following sources: H79 generated from a Sam Hl- I 754-base pair (bp) genomic fragment, Peg1/Mest was amplified from cDNA with primers 5'-CCAAAAGCTCCTCAAAGACG-3' and 5'-TCTGTTAGAAGCAGCCGATG-3', followed by nested primers 5'-CTTTCCCCCATCCTCACC-3' and 5'-CAAGCTCCTCCCTCACTCTG-3'; Meg1/Grb10 was generated from IMAGE clone #746564, and Igf2 was generated from a 1.2-kb cDNA fragment.

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28. We thank A. Wutz for providing subclones of the V6.5 cell line. Supported by the Victoria and Bradley Geist Foundation, the Kosasa Family Foundation, and the Harold Castle Foundation (R.Y.) and by NIH grants 5-R35-CA44339 and R01-CA84198 (R.J.).

6 April 2001; accepted 4 June 2001