Genomic Imprinting Maintains A Reserve Pool of Blood-Forming Stem Cells


Hematopoietic stem cells or HSCs reside in the bone marrow and give rise to the wide variety of specialized blood cells that inhabit our bloodstreams. Within the bone marrow, HSCs come in two varieties: an active arm of HSCs that proliferate continually to replace our blood cells and a reserve arm that sits and quietly waits for their time to come.

New research from the Stowers Institute at Kansas City, Mo, in particular a research team led by Linheng Li, discovered a mechanism that helps maintain the balance between those HSCs kept in reserve and those on active duty.

According to Dr. Li, genomic imprinting, a process that specifically shuts off one of the two gene copies found in each mammalian cell , prevents the HSCs held in reserve from being switched to active duty prematurely.

Li explained: “Active HSCs form the daily supply line that continually replenishes worn-out blood and immune cells while the reserve pool serves as a backup system that replaces damaged active HSCs and steps in during times of increased need. In order to maintain a long-term strategic reserve of hematopoietic stem cells that lasts a lifetime it is very important to ensure that the back-up crew isn’t mobilized all at once. Genomic imprinting provides an additional layer of regulation that does just that.”

Sexual reproduction produces progeny that have once set of chromosomes from the mother and one set of chromosomes from the father. The vast majority of genes are expressed from both sets of chromosomes. However, in placental mammals and marsupial mammals a small subset of genes are imprinted, which means that they receive a mark during the development of eggs and sperm and these marks shut down expression of those genes in either the sperm pronucleus or the egg pronucleus. Therefore, after the fusion of the sperm and the egg and the eventual fusion of the egg and sperm pronuclei, these imprinted genes are only expressed from one copy of genes. Some are only expressed from the paternal chromosomes and others are only expressed from the maternal chromosome. Imprinting is essential for normal development in mammals.

The importance of genetic imprinting is shown if an egg loses its pronucleus and is then fertilized by two sperms. The resulting zygote has two copies of paternal chromosomes and no copies of the maternal chromosomes. Such an embryo is called an andogenote, and the embryo fails to form but the placenta overgrows. If this occurs during human development, it can lead to a so-called “molar pregnancy” or “hydatiform mole.” This fast growing placental tissue can become cancerous and lead to uterine cancer. For that reason, molar pregnancies are usually dealt with expeditiously.

However, if the sperm that fertilizes the egg is devoid of a pronucleus, and the egg pronucleus duplicates, then the resulting zygotes can two copies of the maternal chromosomes, and this entity is known as a gynogenote, and it develops with a poorly formed placenta that dies early in development.

In previous experiments in mice, Li and his colleagues indicated that the expression of several imprinted genes changes as HSCs transition from quiescent reserve cells to multi-lineage progenitor cells.

In their current study, Li and other Stowers Institute researchers examined a differentially imprinted control region, which drives the reciprocal expression of a gene called H19 from the maternal chromosome and IGF2 (insulin-like growth factor-2) from the paternal chromosome.

The first author of this study, Aparna Venkatraman developed a mouse model that allowed her to specifically delete the imprinted copy from the maternal chromosome. Thus, in these mice, H19, which restricts growth, was no longer active and Igf2,, which promotes cell division, was now active from the paternal and the maternal chromosome. To access the effect of this loss of imprinting on the maintenance of HSCs, Venkatraman examined the numbers of quiescent HSCs and active HSCs. in mouse bone marrow.

Venkatraman explained: “A large number of quiescent HSCs was activated simultaneously when the epigenetic control provided by genomic imprinting was removed. It created a wave of activated stem cells that moved through different maturation stages.”

She followed this experiment with a closer look at the Igf2 gene. Misregulation of Igf2 leads to overgrowth syndromes such as Beckwith-Wiedmann Syndrome. It exerts its growth promoting effects through the Igf1 receptor, which induces an intracellular signaling cascade that stimulates cell proliferation.

IGF signaling pathway
IGF signaling pathway

The expression of the Igf1 receptor itself is regulated by H19, which encodes a regulatory microRNA (miR-675) that represses translation of the Igf1 receptor gene and therefore prevents production of Igf1 receptor protein. Venkatraman explained that once the “imprinting block is lifted, the Igf2-Igf1r signaling pathway is activated.” Venkatraman continued: “The resulting growth signal triggers the inappropriate activation and proliferation of quiescent HSCs, which eventually leads to the premature exhaustion of the reserve [HSC] pool.”

Interestingly, the roundworm, Caenorhabditis elegans, provided the first clues that diminished insulin/IGF signaling can increase lifespan and delay aging. Li again: “Here the IGF pathway is conserved by subject to imprinting, which inhibits its activation in quiescent reserve stem cells. This ensures the long-term maintenance of the blood system, which in turn supports the longevity of the host.”

Human Stem Cells From Cloned Embryos: What Horrors WIll Follow?


First the news, then the commentary. Here’s the news:

In the May 14th edition of the international journal Cell, Shoukhrat Miltalipov from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. This is the first time this has been successfully reported. In 2004, a South Korean researcher, Woo Suk Hwang, reported that his laboratory had succeeded in making patient-specific human embryonic stem cells from cloned embryos, but his papers were later shown to be completely fraudulent, and Hwang, in a word, walked. For more on this sad, sordid event, see my “Catastrophic Cloning Caper” here.

Many laboratories have tried and failed to get cloned human embryos to live long enough to get embryonic stem cells from them. The cloning procedure produces a very abnormal embryo that dies very early during development.

How did Mitalipov succeed when so many others before him had failed? Mitalipov honed his cloning protocol in work with early embryos from Rhesus macaques, and during this work, Mitalipov and his coworkers discovered that including caffeine with the mix of chemicals used during donor removal and transplantation into the host egg prevents the oocytes that have just had their nuclei removed from dividing prematurely, and if these oocytes are used in a cloning experiment, they survive longer than oocytes treated with standard cloning techniques.

“It was a huge battery of changes to the protocols over a number of different steps,” said Mitalipov. “I was worried that we might need a couple of thousand eggs to make all these optimizations, to find that winning combination.”

The procedure used in this paper, cloning, is more technically known as “somatic cell nuclear transfer” or SCNT. SCNT requires human eggs that are extracted from female volunteers of reproductive age who are given several drugs to hyperstimulate their ovaries, which then ovulate several eggs at a time. The eggs are harvested by means as aspiration, and used in SCNT.

For SCNT, the egg nucleus is removed by means of a micropipette. The egg is ever so gently squeezed until the nucleus, which is usually off to one side in the egg, protrude through the cell membrane, and the nucleus is sucked off with the micropipette. Then a body cell; in this paper, fibroblasts from the skin were used, is laid next to the nucleus-less egg, and an electric current is pulsed through the two cells, which causes them to fuse. This fusion converts the egg, which used to have one set of every chromosome, into a cell that now has two sets of every chromosome, and the egg cell, begins to divide and recapitulate the events of early development. This is also referred to as cloning.

Somatic_cell_nuclear_transfer-image

Sperm and eggs have chromosomes that have been modified in specific ways. When the sperm and egg fuse, the process of fertilization begins, and the modifications to the chromosomes serve their purpose during the early stages of development, but those modifications and gradually undone as development proceeds. This phenomenon is known as genetic imprinting and it is very common in mammals. For a good paper on genetic imprinting see Wood AJ, Oakey RJ (2006) Genomic Imprinting in Mammals: Emerging Themes and Established Theories. PLoS Genet 2(11): e147. doi:10.1371/journal.pgen.0020147.

Since cloned embryos have a genome that is not properly imprinted, its development is hamstrung to one degree or another. Most researchers were unable to get cloned human embryos to survive past the 8-cell stage. However, by including caffeine in the SCNT medium during egg nucleus removal and transplantation of the donor nucleus into the host egg, enough of the cloned embryos survived to the 150-cell blastocyst stage to allow for the derivation of embryonic stem cells. Even though SCNT is an exceedingly inefficient process, Mitalipov was able to derive six embryonic stem cells lines from 128 eggs, which is about a 4% success rate.

George Daley of Boston Children’s Hospital and the Harvard Stem Cell Institute, who was not involved in the research, said of it: ““I think it is a beautiful piece of work.” He continued: “This group has become remarkably proficient at a very technically demanding procedure and [has] shown that SCNT-ESCs may in fact be a practical source of cells for regenerative medicine.”

Mitalipov and his group analyzed four of the cloned embryonic stem cell lines and found that their NT-hESCs could successfully differentiate into beating heart cells in culture dishes. Also, they could differentiate into a variety of cell types in teratoma tumors when transplanted into live, immunocompromised mice. These stem cells also had no chromosomal abnormalities, and displayed fewer problematic epigenetic leftovers from parental somatic cells than are typically seen in induced pluripotent stem cells (although, for the life of me, no one has shown that these epigenetic holdovers are a big problem for regenerative medicine). Mitalipov said more comparisons are required, however.

“We are now left to analyze the detailed molecular nature of SCNT-ES cells to determine how closely they resemble embryo-derived ES cells and whether they have any advantages over iPS cells,” added Daley. “iPS cells are easier to produce and have wide applications in research and regenerative medicine, and it remains to be shown whether SCNT-ES cells have any advantages.”

Mitalipov, however, did point out one fundamental difference between NT-ESCs and iPSCs: their nuclear genomes come from the donor cell, but NT-hESCs contain mitochondrial DNA (mtDNA) from the host egg cell. Therefore, SCNT reprograms the cell but also corrects any mtDNA mutations that the donor may carry. Thus, patient-specific NT-hESCs could be used to treat people with diseases caused by mitochondrial mutations. “That’s one of the clear advantages with SCNT,” Milatipov said.

The cells used for this cloning experiment came from infants.  It still remains for cloning to succeed with adult cells as the donor cells.

Now for the commentary:

Regular readers of this blog will already know that I am deeply opposed to human cloning in any form.  It is the equivalent of making people for spare parts.  This is immoral and barbaric.  I predicted some time ago (OK not so long ago, 4 years to be exact), that the technical problems with human cloning would be solved and scientists would one day clone a human embryo.  Now that it is here, I hope that people are as horrified by it as I am.

“Get over it.  It’s an embryo and a cloned one at that.” you might say.  But what if the malady that doctors want to cure is poorly served by embryonic stem cells made from cloned embryos and a cloned fetus is a better source of cells?  Do we allow gestation of the cloned embryo to the fetal stage so that we can dismember it and take its tissue?  Let’s bring this home.  What if the cells needed to come from a five-year old?  Can we justify that because the kid was cloned?

“But wait, that’s a five-year old and this is an embryo,” you say.  But you were once a blastocyst.  You did not pass through the blastocyst stage, you WERE a blastocyst.  The only difference between the blastocyst and you now is time, environment, degree of dependence, and size.  Are any of these differences morally significant when it comes to whether or not we can kill you?  Can we kill all the short people?  Can we kill all the younger people because they are not as well-developed?  Can we kill people who are dependent on others (that includes everyone mate, so put your hand down)?  Can we kill those in a different location (genocide anyone)?  None of these categories constitutes a good reason for terminating someone’s life.  Likewise, none of these changes renders you essentially different from who and what you are.  To kill someone at the earliest stages for their tissue is simple murder, and we use size, location, extent of development, location and degree of dependence to salve of consciences for doing it, but that won’t define what we are doing.

People will go on and on about the great advances that could lead to.  Sorry, I’m not buying that one.  Embryonic stem cells have been promising that one for the last 15 years with pert near little to nothing to show for it.  This discovery is a great technical advance, but it opens to door to reproductive cloning – an even bigger mistake, and fetus farming, in which we destroy our own children in the womb, not because they are in inconvenience to us, but because we want their tissues to save our lives.  Now children, rather than being a blessing, are merely tissues to be harvested.  We have become like the Greek gods from the stories of old who ate their own children.  May God forgive us.