Developmental Regression: Making Placental Cells from Embryonic Stem Cells

A research group from Copenhagen, Denmark has discovered a way to make placental cells from embryonic stem cells. In order to do this, the embryonic stem cells must be developmentally regressed so that they can become wither placenta-making cells rather than inner cell mass cells.

This study is significant for two reasons. First of all, it was thought to be impossible to make placental cells from embryonic stem cells because embryonic stem cells (ESCs) are derived from the inner cell mass cells of 4-5-day old human blastocysts. These early embryos begin as single-celled embryos that divide to form 12-16-cell embryos that undergo compaction. At this time, the cells on the outside become trophoblast cells, which will form the trophectoderm and form the placenta and the cells on the inside will form the inner cell mass, which will form the embryo proper and a few extraembryonic structures. Since ESCs are derived from inner cell mass cells that have been isolated and successfully cultured, they have already committed to a cell fate that is not placental. Therefore, to differentiate ESCs into placental cells would require that ESCs developmentally regress, which is very difficult to do in culture.

Secondly, if this could be achieved, several placental abnormalities could be more easily investigated, For example, pre-eclampsia is a very serious prenatal condition that is potentially fatal to the mother, and is linked to abnormalities of the placenta. Studying a condition such as pre-eclampsia in a culture system would definitely be a boon to gynecological research.

Because human ESCs can express genes that are characteristic of trophoblast cells if they are treated with a growth factor called Bone Morphogen Protein 4 (BMP4), it seems possible to make placental cells from them (see Xu R.H., Chen X., Li D.S., Li R., Addicks G.C., Glennon C., Zwaka T.P., Thomson J.A. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 2002;20:1261–1264, and Xu RH. Methods Mol Med. 2006;121:189-202). However, a study by Andreia S. Bernardo and others from the laboratory of Roger Pedersen at the Cambridge Stem Cell Institute strongly suggested that BMP4 treatment, even in the absence of FGF signaling (another growth factor that has to be absent for BMP4 to induce trophoblast-like gene expression from ESCs) the particular genes induced by BMP4 are not exclusive to trophoblast cells and more closely resemble mesodermal gene profiles (see AS Bernardo, et al., Cell Stem Cell. 2011 Aug 5;9(2):144-55).

Into the fray of this debate comes a paper by stem cells scientists at the Danish Stem Cell Center at the University of Copenhagen that shows that it is possible to rewind the developmental state of ESCs.

In this paper, Josh Brickman and his team discovered that if they maintained mouse ESCs under specific conditions, they could cause the cells to regress into very early pre-blastocyst embryonic cells that can form trophoblast cells or ICM cells.

“It was a very exciting moment when we tested the theory, said Brinkman. “We found that not only can we make adult cells but also placenta, in fact we got precursors of placenta, yolk sac as well as embryo from just one cell.”

“This new discovery is crucial for the basic understanding of the nature of embryonic stem cells and could provide a way to model the development of the organism as a whole, rather than just the embryonic portion,” said Sophie Morgani, graduate student and first author of this paper. “In this way we may gain greater insight into conditions where extraembryonic development is impaired, as in the case of miscarriages.”

To de-differentiate the ESCs, Brinkman and his colleagues grew them in a solution called “2i.”  This 2i culture medium contained inhibitors of MEK and GSK3.  MEK is a protein kinase that is a central participant in the “MAP kinase signaling pathway, which is a signaling pathway that is central to cell growth and survival.  This particular signaling pathway is the target of the anthrax toxin, which illustrates its importance,  GSK3 stands for “glycogen synthase kinase 3,” which is a signaling protein in the Wnt pathway.

When the mouse ESCs were grown in 2i medium they expressed genes normally found only in pre-blastocyst embryos (Hex, for example).  Therefore, the 2i medium directs mouse ESCs to de-differentiate.  When ESCs grown in 2i were implanted into mouse embryos, they divided and differentiated into cells that were found in placental and embryonic fates.  This strongly argues that the ESCs grown in 2i became pre-blastocyst embryonic cells.  When the ESCs grown in 2i were also grown with LIF, which stands for “leukemia inhibitory factor” (LIF is a protein required for the maintenance of mouse ESCs in culture), the 2i cells were maintained in culture and grew while maintaining their pre-blastocyst status.  These cells differentiated into placental cells, embryonic or fetal cells.  Essentially, the 2i-cultured cells when from being pluripotent to being “totipotent,” or able to form ALL cell types in the embryo, fetus, or the adult.

ESC de-differentiation in totipotence

“In our study we have been able to see the full picture unifying LIF’s functions: what LIF really does, is to support the very early embryo state, where the cells can make both embryonic cells and placenta. This fits with LIFs’ role in supporting implantation,” said Brinkman.

This study definitively shows that ESCs are NOT embryos.  ESCs can regress in their development but embryos develop forward, becoming more committed as they develop and more restricted in the cell fates they can form.  This should effectively put the nail in the coffin of Lee Silver’s argument against Robert P. George that embryonic stem cells are embryos.  They are definitely and unequivocally, since embryos do NOT develop in reverse, but ESCs can and do.

Robert P. George argues that early human embryos, like the kind used to make ESCs are very young  members of the human race and deserve, at the minimum, the right not to be harmed.  Silver counters that George’s argument is inconsistent because George would not extend the same right to an ESC cell line, which is the same as an embryo.  His reasoning is that mouse ESCs can be transplanted into other mouse embryos that have four copies of each chromosome.  The messed up mouse embryo will make the placenta and the ESCs will make the inner cell mass and the mouse will develop and even come to term.  This is called tetraploid rescue, and Silver thinks that this procedure is a minor manipulation, but that it shows that ESCs are functionally the same as embryos.

I find Silver’s argument wanting on just about all fronts.  This is not a minor manipulation.  The tetraploid embryo is bound for certain death, but the implanted ESCs use the developmental context of the tetraploid embryo to find their place in it and make the inner cell mass.  The ESCs do not do it all on their own, but instead work with the tetraploid embryo in a complex developmental give-and-take to make an embryo with the placenta from one animal and the embryo proper from another.

Thus Silver’s first argument does not demonstrate what he says it does.  All it demonstrates is that ESCs can contribute to an embryo, which is something we already knew and expected.  This new data completes blows Silver’s assertion out of the water, since ESCs can take developmental steps backward and embryos by their very nature and programming, do not.  Thus these two entities are distinct entities and are not identical.  The early embryo is a very young human person, full stop.  We should stop dismembering them in laboratories just to stem our scientific curiosity.

New Cell Type Derived from Embryonic Stem Cells for Possible Treatment of Brain Diseases

This story comes from my alma mater, UC Irvine. Go anteaters!! No really, UC Irvine’s mascot is the anteater, not to be confused with the aardvark.

Edwin Monuki at the Sue and Bill Gross Stem Cell Research Center with his graduate student Momoko Watanabe and other colleagues devised culture conditions to differentiate embryonic stem cells in “choroid plexus epithelial cells.” Monuki and he team were able to make choroid plexus epithelial cells (CPECs) from mouse and human embryonic stem cell (ESC) lines.

Now you are probably reading this and screaming, “what the heck are CPECs?” Calm down, we will explain:

The central nervous system is surrounded by a clear fluid known as cerebrospinal fluid or CSF. CSF flows all around the brain and spinal cord and also flows inside it. The CSF has several functions. These functions include buoyancy, protection, stability, and prevention of stroke (ischemia).

The CSF provides buoyancy to the brain, since the brain is large a potentially heavy. However, by filling the brain from within and around it with CSF decreases the density of the brain. CSF allows the brain to maintain a density that prevent it from collapsing under its own weight. If the brain were denser, then it would compact and cut off the blood supply of the cells in the lower part of the brain. This would kill off neurons.

Protection provided by the CSF comes during those times the head is struck. The CSF prevents the brain from coming into contact with the skull. The stability provided by the CSF

CSF flows throughout the inside of the brain through cavities known as “ventricles.” These ventricles provide reservoirs through which the CSF flows and is absorbed back into the bloodstream. This constant movement of the CSF through the CNS rinses it and removes metabolic wastes from the central nervous system. It also guarantees an even distribution of neural materials through the central nervous system.

CSF also helps prevent strokes, since the low pressure of the CSF in the skull translates into low intracranial pressure, which facilitates blood perfusion.

Now that have seen that CSF is very important in the life of the brain, where does it come from?  The answer is the CPECs make CSF as a fine filtrate from blood plasma. A minority of the CSF is also made by the walls of the brain ventricles. Nevertheless, CSF circulates from the lateral ventricles to the interventricular foramen, to the third ventricle, through the cerebral aqueduct, to the fourth ventricle, through the median aperture and lateral apertures to the subarachnoid space over the brain and spinal cord where arachnoid granulations return the CSF to the bloodstream.

Thus, CPECs make CSF, which helps remove metabolic wastes and other toxic compounds from the brain. In various neurodegenerative diseases, the choroid plexus and CPECs degenerate too and fail to efficiently remove debris and other rubbish from the central nervous system. Transplantation experiments in rodents have demonstrated that implanting a healthy set of CPECs can restore CSF function and slow down the damage done to the brain by neurodegenerative diseases (see Matsumoto et al., Neurosci Lett. 2010 Jan 29;469(3):283-8).  The problem is the lack of good cultures of CPECs.

Monuki and Watanabe and company seem to have fixed this problem. Monuki commented on his publication, “Our method is promising, because for the first time we can use stem cells to create large amounts of these epithelial cells (CPECs), which could be utilized in different ways to treat neurodegenerative diseases.” Monuki is an associate professor of pathology and laboratory medicine and cell and developmental biology at UC Irvine.

To make CPECs from ESCs, Monuki and his team differentiated cultured ESCs into neural stem cells. The neural stem cells were then differentiated into CPECs. This strategy makes a great deal of sense, since a neural stem cell population seems to exist in the CPECs (see Itokazu Y et al., Glia. 2006 Jan 1;53(1):32-42.

According to Monuki, there are three ways that these cells could be used to treat neurodegenerative diseases. First, the CPECs could be used to make more CSF and that would help flush out the proteins and other toxic compounds that kill off neurons. The down side of this is that it would also increase intracranial pressure, which is not optimal. Secondly,. the CPECs could be engineered into superpumps that transport high concentrations of therapeutic agents into the brain. Third, cultured CPECs could be used as a model system to screen drugs and other agents that shore up the endogenous CPECs in the patient’s brain.

Monuki’s next step is to develop an effective drug screening system in order conduct proof-of-concept studies to determine how CPECs afect the brain in mouse models of Huntington’s disease and other pediatric neurological diseases.