Reprogramming human oocytes to a pluripotent stage – using triploid embryos

In the October 6, 2011 edition of the journal Nature, Scott Noggle from the New York Stem Cell Laboratory, and his collaborators from the University of San Diego and Columbia University have made a remarkable observation of cloned human embryos. When human embryos are cloned, an egg has its nucleus removed and replaced with a nucleus from a body cell. After stimulation, the egg divides and begins to recapitulate the stages of early embryonic development. This technique is often called “somatic cell nuclear transfer” or SCNT. Typically, cloned human embryos fail to develop for very long. They tend to die before they develop to the blastocyst stage, and they have massive abnormalities in gene expression. Noggle and colleagues found that if the egg nucleus is not removed, and the nucleus from the body cell is added to it, the cloned embryo develops to the blastocyst stage much more easily. Apparently, in human eggs, the removal of the nucleus conveys tremendous abnormalities upon the newly formed embryo, and prevent it from regularly developing to the blastocyst stage.  Noggle and his coworkers provide reasons for the development failure of cloned human embryos.

In this paper, Noggle and others made cloned human embryos through SCNT, but all of them stop dividing at the 6-10 cell stage.  They tried a few other experiments to determine if they could activate the egg to divide without causing developmental arrest.  First they fertilized 21 eggs with frozen sperm and 16 of these 21 eggs formed embryos that developed all the way to the spherical blastocyst stage after six days.  Because 76% of the eggs formed viable embryos, whatever problem afflicts the cloned embryos, it is not due to the quality of the eggs.  Next, the artificially activated unfertilized eggs with a molecule that let Calcium ions into the egg (called calcium ionophore).  7/52 (13.5%) of these artificially activated eggs divided and formed spherical blastocyst embryos.  Such embryos are called “parthenotes: because they were made without the benefit of fertilization and only have one copy of each chromosome (a condition that is called “haploid”).  This again confirms that the problem is not with the eggs.  To determine if developmental arrest was due to the removal of the egg’s nucleus, they removed nuclei from the eggs, and transferred egg nuclei from other eggs, and then artificially activated those eggs.  Once again, 1/7 (14.3%) of these eggs divided and formed spherical blastocysts.  In another control experiment, a nucleus from a body cell was fused with the egg, and the egg was then artificially activated, after which the body-cell nucleus was removed.  4/7  (57%) embryos developed to blastocysts after 6 days.  These control experiments suggest that the experimental manipulation the eggs are experiencing is not the cause of their developmental arrest.  Instead it is the absence of the egg nucleus that causes the developmental arrest of cloned human embryos.

To overcome the developmental arrest, they fused unmanipulated eggs with body cells and artificially activated them.  These, according to the authors, generated cells with three copies of each chromosome ( a triploid).  The problem is that they report using “MII stage oocytes.”  This is a fancy way of saying that the eggs are arrested in the last stage of meiosis.  This creates a problem:  the egg completes meiosis after fertilization and becomes a haploid cell.  The other nucleus is extruded in the form or a second polar body, which is a tiny bled of cell material attached to the egg.  Without completing meiosis, the egg has two copies of each chromosome and therefore the embryo should have four copies of each chromosome (tetraploid).  Nevertheless, they authors claim that they have generated triploid embryos, and their data support their conclusion.  They should explain more completely how they generated these cells, since the procedure they have detailed does not make complete biological sense.

Of these triploid embryos, 13/63 (20.6%) formed spherical blastocyst-stage embryos and these were used to make embryonic stem cells cultures.  From these 13 blastocysts, two embryonic stem cells lines were made.  These two lines (soPS1 and soPS2) were examined for their ability to form tumors in mice with sick immune systems.  Now only did they form the right kinds of tumors, but they expressed all the types of genes embryonic stem cell lines express.  Thus, triploid embryos can form blastocysts, and embryonic stem cell lines can be made from them.

Their gene expression studies showed that the cloned embryos failed to initiate the program of gene expression that is common observed in 4-8 cell stage human embryos.  This gene expression program, which is called “zygotic gene expression” is essential for further development, and the cloned embryos fail to properly initiate zygotic expression.

The News and Views commentary on this article by George Q. Daley suggests that triploid embryonic stem cell lines might be a potential patient-specific cell line for use in regenerative medicine.  I find this unlikely for several reasons.  Triploid embryos constitute 2-3% of all human conceptions (See D E McFadden and W P Robinson, “Phenotype of Triploid Embryos,” J Med Genet 43 (2006): 609-12), and is one of the major causes of spontaneous abortion (MR Creasy, JA Crolla, and ED Alberman, Hum Genet 1976; 31: 177–196; Kajii T, Niikawa N, Cytogenet Cell
1977; 18: 109–125; Brajenovic-Milic B, et al., Fetal Diagn Ther 1998; 13: 187–191).  Triploid result from either “digyny” (extra haploid set from mother) or “diandry” (extra haploid set from father).  Diandry tends to result in fetuses whose developmental growth is either mostly normal is or shows slow growth on one side of the fetus, but the placenta is abnormally large and filled with fluid-filled cysts.  This condition is given the formidable name of “partial hydatidiform mole,” (PHM) and it is potentially deleterious for the mother, since the huge placenta can become cancerous.  The digyny fetuses showed marked asymmetric growth in the uterus, and poor development of the adrenal hypoplasia.  The placenta is also small and underdeveloped.  The poorly developed placenta can, in some cases, or can cause preeclampsia in the mother, which is a life-threatening condition for a pregnant mother in which her blood pressure becomes dangerously high (see See Clasien van der Houwen, Tineke Schukken, and Mariëlle van Pampus, Journal of Medical Case Reports 2009, 3:7311).  Additionally, triploid fetuses may have a many other congenital anomalies that include fusion (syndactyly) of the third and fourth fingers and fusion f the toes, abnormal genitals, and cardiac, urinary tract, and brain anomalies.  These abnormalities  appear in both digynic and diandric triploids.  Given the poor developmental potential of triploid fetuses, it seems quite dangerous to use triploid embryonic stem cells for regenerative medicine, since they might cause more problems than healing.

My other problem with tis paper is that they paid women of reproductive age for their eggs.  In the first place this violates the guidelines of the National Academy of Science, which state: “Women who undergo hormonal induction to generate oocytes specifically for research purposes (such as for NT) should be reimbursed only for direct expenses incurred as a result of the procedure, as determined by an IRB. Direct expenses may include costs associated with travel, housing, child care, medical care, health insurance, and actual lost wages. No payments beyond reimbursements, cash or in-kind, should be provided for donating oocytes for research purposes. Similarly, no payments beyond reimbursements should be made for donations of sperm for research purposes or of somatic cells for use in NT.” (Final Report of the National Academies’ Human Embryonic Stem Cell Research Advisory Committee and 2010 Amendments to the National Academies’ Guidelines for Human Embryonic Stem Cell Research , Appendix C, Page 27 3.4(b) Payment and Reimbursement. See  Yet Jan Helge Solbakk, faculty member at the Centre for Medical Ethics at the University of Oslo say that the authors “deserve praise rather than criticism, because their approach helps to draw attention to a possible way out of th regulatory quagmire resulting from reduction of oocyte providers to ‘donors’ or ‘gift givers’ deserving mere;y compensation for their gifts.” so, let’s praise the authors even though they broke the rules.  Folks, those rules are in place for a reason.  Luring young women to donate their eggs with money will tend to attract those who need the money; that is poor college students, or poor women.  These procedures have real risks, and women will close their eyes to the risks, because the egg donation will help them make payments.  This is exploitation of women, and Jennifer Lahl made a movie about it called “Eggsploitation” that documents what happens to when women are paid for their eggs.  Who speaks for them?  This is a very disturbing trend in this paper, and Nature should have had the backbone to reject it our of hand for that alone.  Having said, that, the paper does make some very original observations, but it is doubtful that these cell lines will plays a significant in regenerative medicine.  Also, the developmental arrest problem in cloned embryos is real, and this underscores why cloned mouse embryos are not that good a model system for cloned human embryos.