Embryonic Stem Cells From Cloned Embryos Vs Induced Pluripotent Stem Cells: Let the Debate Begin


In May of 2013, Shoukhrat Mitalipov and his coworkers from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. Other stem cell scientists have confirmed that Mitalipov’s protocol works as well as he says it does.

Mitalipov and others have also examined the genetic integrity of embryonic stem cells made from cloned human embryos and induced pluripotent stem cells made from mature adult cells through genetic engineering and cell culture techniques. This paper was published in Nature in June 2014 and used genetically matched sets of human Embryonic Stem cells made from embryos donated from in vitro fertilization clinics, induced Pluripotent Stem cells and nuclear transfer ES cells (NT-ES cells) derived by somatic cell nuclear transfer (SCNT). All three of these sets of stem cells were subjected to genome-wide analyses. These analyses sowed that both NT-ES cells and iPS cells derived from the same somatic cells contained comparable numbers of genetic variations. However, DNA methylation, a form of DNA modification for regulatory purposes and gene expression profiles of NT-ES cells corresponded closely to those of IVF ES cells. However, the gene expression provide of iPS cells differed from these other two cell types and iPS cells also retained residual DNA methylation patterns typical of the parental somatic cells. From this study, Mitalipov stated that “human somatic cells can be faithfully reprogrammed to pluripotency by SCNT (that means cloning) and are therefore ideal for cell replacement therapies.”

Now a new study by Dieter Egli of the New York Stem Cell Foundation (NYSCF) in New York City, which included Mitalipov as a collaborator, has failed to demonstrate significant genetic differences between iPS cells and NT-ES cells. This is significant because Eglin has long been a rather vigorous proponent of cloning to make patient-specific stem cells. Egli gave an oral preview of his forthcoming paper on October 22nd, at the NYSCF annual conference. Egli told his audience, “This means that all of you who are working on iPS cells are probably working with cells that are actually very good. So I have good news for you,” he told them, eliciting murmurs and chuckles. “What this exactly means for the SCNT program, I don’t know yet.”

Egli and colleagues used skin cells from two people—a newborn and an adult—to create both stem cells from cloned embryos (using donor eggs) and iPS cells. Then they compared the genomes of these two types of cell lines with the genomes of the original skin cells in terms of genetic mutations, changes in gene expression, and differences in DNA methylation. Both methods resulted in about 10 mutations compared with the average genome of the mature source cells. These changes didn’t necessarily happen during reprogramming, however, Egli says, since many of these mutations were likely present in the original skin cells, and some could have arisen during the handling of cells before they were reprogrammed.

Both types of stem cells also carried a similar amount of methylation changes. Overall, the method didn’t seem to matter, Egli and his team concluded. Because he is a longtime proponent of SCNT, Egli says it would have been “more attractive” to reveal significant differences between the two kinds of stem cells. “This is simply not what we found.”

Now it would be premature to conclude that iPS cells are as good as NT-ES cells for regenerative purposes, but this certainly seems to throw a monkey wrench in the cloning bandwagon. Cloning would be quite complicated and expensive and also requires young, fertile women to donate their eggs. These egg donors must undergo potentially risky procedures to donate their eggs. Jennifer Lahl’s documentary Eggsploitation provides just a few of some of the horror stories that some women experienced donating their eggs. The long-term effects of this procedure is simply not known and asking young women to do this and potentially compromise their health or future fertility seems beyond the pale to me.

Alternatively, iPS technology keeps improving and may come to the clinic sooner than we think. Also, is a cloned embryo essentially different from one made through IVF or “the old-fashioned way.?” This whole things seems to me to involved the creation of very young human beings just so that we can dismember them and use them as spare parts. Such a practice is barbaric in the extreme.

For those who are interested, please see chapters 18 and 19 of my book The Stem Cell Epistles to read more about this important topic.

New Pluripotent Stem Cell Production Protein Identified


Large scale production of stem cells requires an intimate knowledge of the genetic networks that convert adult cells into induced pluripotent stem cells (iPSCs). The original protocol established by Shinya Yamanaka and his colleagues used four genes all clustered on a retrovirus vector, but there are safer, more technically subtle ways to make iPSCs.

Because iPSCs are made from a patient’s own cells, they are less likely to be rejected by the patient’s immune system. They also show tremendous developmental flexibility, they can potentially be differentiated into any adult cell type in the body.  The problem with iPSCs comes from the difficulty of making large quantities of them in a reasonable amount of time.  However, a new research publication from scientists at the University of Toronto, the University for Sick Children and Mount Sinai Hospital, in collaboration with colleagues from the United States and Portugal, identifies specific proteins that play central roles in controlling pluripotency that may mean a potential breakthrough in producing iPSCs.

Researchers discovered these proteins by using something called the “splicing code.”  Benjamin Blencowe discovered the splicing code a few years ago.  “The mechanisms that control embryonic stem cell pluripotency have remained a mystery for some time.  However, what Dr. Blencowe and the research team found is that the proteins identified by our splicing code can activate or deactivate stem cell pluripotency,” said Brendan J. Frey, from the University of Toronto Departments of Electrical Engineering and Medicine, who published with Benjamin Blencowe the paper that deciphered this splicing code (see Nature 2010 465: 53-59).  While a complete recipe for producing iPSCs may not be available yet, it is beginning to look more likely, according to Frey.

In this paper, Blencowe and his collaborators identified two proteins known as muscleblind-like RNA binding proteins, or MBNL1 and MBNL2.  These proteins are conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between embryonic stem cells and other cell types.

RNA splicing occurs in plant, animal, fungal, and protist cells (only very, very rarely in bacteria), and involves the removal of segments of primary RNA transcripts.  When RNA molecules are transcribed in eukaryotic cells, they are engaged by cellular machinery called the RNA spliceosome.  The RNA spliceosome removes segments known as “introns” and the excised introns are degraded and the remaining RNA segments, which are known as “exons, are ligated together to form a mature messenger RNA.

mRNA splicing

Some introns are removed from primary RNA transcripts by all cells, but others are removed in some cells but not others.  This phenomenon is known “alternative splicing” and it is responsible for the differential regulation of particular genes.

alternative_splicing

Alternative splicing is mediated by sequences called splicing enhancers and splicing silencers that are six to either nucleotides long and bind proteins that either induce or repress alternative splicing in those cells that express the proteins that bind these splicing enhancers or silencers.

Alternative RNA splicing mechanism

MBNL is one of these proteins that bind to RNA splicing silencers.  If the quantity of MBNL proteins in differentiated cells is decreased, then these cells switch to an embryonic stem cell-like alternative splicing pattern for approximately half of their genes.  Conversely, overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns.  Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in a protein-coding gene called the forkhead family transcription factor FOXP1.  FOXP1 controls pluripotency, and consistent with a central and negative regulatory role for MBNL proteins in pluripotency, knockdown of MBNL significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

Thus MBNL proteins should be one of the main targets for the mass production of iPSCs.