Porous Material Helps Deliver Molecules to Stem Cell-Derived Cells


A Swedish group has successfully tested a new porous material that allows for the efficient delivery of key molecules to transplanted cells that have been derived from stem cells. Such a material can dramatically improve the way stem cell-based treatments for neurodegenerative diseases.

This research project included a collaboration between Danish, Swedish and Japanese laboratories, and it tested a new type of porous material that efficiently delivers key molecules to transplanted cells derived from stem cells in an animal model.

Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p  .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.
Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p < .001. No statistically significant differences were observed between groups with direct or MesoMim administration of the factors (p > .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.

This potentially versatile and widely applicable strategy for the efficient differentiation and functional integration of stem cell derivatives upon transplantation, and it can serve as a foundation for improving stem cell-based neurodegenerative protocols, for example, Parkinson’s disease.

Alfonso Garcia-Bennett of Stockholm University, one of the lead authors of this study, said: “We are working to provide standard and reproducible methods for the differentiation and implementation of stem cell therapies using this type of approach, which coupled material science with regenerative medicine.”

Garcia-Bennett continued: “We demonstrated that delivering key molecules for the differentiation of stem cells in vivo with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also improved their long-term survival.”

This research group is already working together with two companies to speed up the commercialization of a standard differentiation kit that will allow other scientists and clinicians to reproduce their work in their own laboratories.

“Noncontroversial” Embryonic Stem Cells?


An article from Bioscience Technology, a working scientist’s rag, has argued that everyone can have their lifetime supply of embryonic stem cells. Below is a summary of the article, after which I will comment on it.

Susan Fisher is the director of the UCSF Human Embryonic Stem Cell program. Last week, her lab reported that they have efficiently created embryonic stem cell lines from the cells removed from early embryos for Preimplantation Genetic Diagnosis (PGD) clinics. PGD takes a single cell from an early embryo that was created by means of in vitro fertilization, and subjects that single cell to genetic analyses to determine if the embryo carries a genetic disease. Because early human embryos have the ability to “regulate,” the removal of a single simply spurs the cells of the embryo to undergo extra cell divisions. The embryos subjected to PGD are then either destroyed, if they harbor a genetic disease, or implanted into the mother’s womb and gestated.

However, these cells removed from embryos could also be used to make an embryonic stem cell culture, since they could be seeded in culture to make an embryonic stem (ES) cell line. Therefore, in theory, cells could now be routinely removed from in vitro fertilization (IVF) clinic embryos, to provide them with a lifetime supply of their own embryonic stem cells. Because these cells were made without destroying embryos, they would be uncontroversial.

“Back in the mid-2000’s, when California was trying to decide whether to fund ES cell research, thousands of interested people would come out to hear us speak about topics like this,” says Fisher, interviewed after her report to the New York Stem Cell Foundation conference last week. “It is possible this particular, refined approach will generate that kind of interest now.”

ES cells have the greatest potency of any human stem cells and they can potentially form every cell type in the adult human body. Because such cells were recently harvested, they would not possess any of the mutations that ES cultures can acquire when they are grown for long periods of time in culture.

Traditionally, ES cell lines have been derived from stored, spare embryos from IVF clinics that were donated by other patients. Therefore, they are not immunologically identical to patients who potentially need them. Patients who receive non-matching tissues must take harsh immunosuppressive drugs for years to avoid rejecting the cells, and even then, over time the immune eventually wins the fight in some cases.

In recent years, scientists have turned to induced Pluripotential Stem Cells (IPSCs). IPSCs are made by genetically engineering adult cells to express four genes that de-differentiate the cells so that they are embryonic-like cells. IPSCs have been a boon to research, since scientists hace used them to make “disease in a dish” models on which to try drugs. But IPSCs are often riddled with mutations, as they come from adults. They have not yet hit the clinic as a result (although trials are upcoming).

However, Fisher, following on the heels of very preliminary work published in the journal Nature by the biotechnology company ACT, has refined the ability to create possibly uncontroversial stem cells—that are immunological matches to patients. By removing one cell from a very young human embryo, Fisher thinks that scientist might be able to produce a veritably unlimited supply of ES cells that are immunologically identical to the embyros from which they came. And as the embryos aren’t destroyed, but implanted into the mothers’ uteruses, the derivation of these tailor-made ES cells should be uncontroversial. “We will see how this is received,” Fisher says.

The process, she reported, is robust, if still not easy to pull off. This procedure, however, is labor-intensive and required a great deal of skill to pull off. In Fisher’s lab at UCSF, they derived ten human ES cell lines from four eight-cell embryos and one 12-cell embryo from a single couple.

When compared to standard ES cells, the UCSF lines were healthy and “formed derivatives of the three germ layers” like standard ES cells. Furthermore, these cells could form trophoblasts (placental cells), and Fisher’s team used them to create the first human trophoblast stem cell line. This is something that standard ES cells cannot do and this could make the UCSF cells useful in the clinic for diseases affecting the placenta.

Will patients begin turning to such cells? A few companies in the mid-2000s started offering designer ES cells like these, but that practice ended due to lack of interest or understanding, Fisher says. Additionally, some technical problems—later fully rectified—associated with the earlier Nature ACT paper may have cast a pall on enthusiasm for the approach, others in the field note.

“It remains to be seen if a place will be found for both iPS and ES cells,” Fisher concludes.

Now follows my comments:

Human embryos are very young human beings.  They do not have the right to vote, own property, or get a driver’s license, but they at least have the right not to be harmed.  By withdrawing cells from the embryo, you are potentially harming it.  “But wait,” proponents will tell you, “there are hundreds or even thousands of children who have been born who grew from embryos that were subjected to PGD and their rates of birth defects are no higher than everyone else’s.”  So their rates of birth defects are lower, but have we followed them for the rest of their lives to establish that removing a blastomere during early development does no harm?

“Oh come on,” you say.  But there are studies in mice that show that removing blastomere from early embryos does not cause higher rates of birth defects, but it does cause higher rates of neurological defects that manifest later in life.  Yu and others found that “mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy.”  In another paper, Yang and others showed that “blastomere biopsy, increases the rate of embryo death at 4.5-7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos.”  In human embryos, time-lapse photography of biopsied embryos by Kirkegaard K, Hindkjaer JJ and Ingerslev HJ showed that “blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.”  Finally, Sugawara and others showed that “The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.”

There is reason to believe that this procedure potentially hurts the embryo.  Also, not all blastomeres in the early embryo are equally competent to make ES lines (see Lorthongpanich et al., Reproduction. 2008 Jun;135(6):805-1).  Therefore, if more than one blastomere must be taken from the embryo, the risks to it definitely increases (see Groossens et al., Hum. Reprod. (2008) 23 (3): 481-492).  The embryo has a basic right not to be harmed, but PGD potentially harms it without its consent.  This is barbaric.  With any other procedure we would say so, but this seems to be alright because we are dealing with embryos and they are too small and young.  This is ageism and size discrimination.  These are not “uncontroversial stem cells.”  They are anything but.  

Microparticles and Local Control of Stem Cells


Using stem cells to grow three-dimensional structures, such as organs or damaged body parts, requires that scientists have the ability to control the growth and behavior of those cells. Also, adapting such a technology to an off-the-shelf kind of process so that it does not cost an arm and a leg is also important.

A research project by scientists from Atlanta, Georgia has used gelatin-based microparticles to deliver growth factors to specific areas of aggregates of stem cells that are differentiating. This localized delivery of growth factors provides spatial control of cell differentiation, which enables the creation of complex, three-dimensional tissues. The local delivery of growth factors also decreases the amount of growth factor used and, consequently, the cost of the procedure.

This particular microparticle technique was used on mouse embryonic stem cells and it proved to provide better control over the kinetics of cell differentiation since it delivered that promote cell differentiation or inhibit it.

Todd McDevitt, associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, said, “By trapping these growth factors within microparticle materials first, we are concentrating the signal they provide to the stem cells. We can then put the microparticle materials physically inside the multicellular aggregate system that we use for differentiation for the stem cells. We have good evidence that this technique can work, and that we can use it to provide advantages in several areas.”

The differentiation of stem cells is largely controlled by external cues, including protein growth factors that direct cell proliferation, and differentiation that are available in the three-dimensional environment in which the cells live. In most experiments, stem cells are grown in liquid culture and growth factor is equally accessible to the growth factors. This makes the cultures quite homogeneous. But delivering the growth factors via microparticles gives better control of the spatial and temporal presentation of these growth factors to the stem cells. This gives scientists the means to make heterogeneous structures from stem cell cultures.

When embryonic stem cells grow in culture, they tend to clump together. When the growth medium is withdrawn or if growth factors that induce differentiation are added, the cells form an “embryoid body” that is stuff with cells differentiating into all kinds of cell types. When McDevitt and his co-workers added microparticles with the growth factors BMP4 (bone morphogen protein 4) or Noggin (which inhibits BMP4 signaling), they centrifuged the cells and found that the microparticles found their way into the interior of the embryoid bodies.

When they examined the embryoid bodies, with confocal microscopy they found that BMP4 directed the cells to make mesodermal and endodermal derived cell types. However, because the microparticles were in direct contact with the cells, they needed 12 times less growth factor than was required by solution-based techniques.

“One of the major , in a practical sense, is that we are using much less growth factor,” said McDevitt. “From a bioprocessing standpoint, a lot of the cost involved in making stem cell products is related to the cost of the molecules that must be added to make the stem cells differentiate.”

Beyond more focuses signaling, the microparticles also provided localized control that was not available through other techniques. It allowed researchers to create spatial differences in the aggregates and this is an important possible first step toward forming more complex structures with different tissue types such as vascularization and stromal cells.

“To build tissues, we need to be able to take stem cells and use them to make many cell types which are grouped together in particular spatial patterns,” explained Andres M. Bratt-Leal, the paper’s first author and a former graduate student in McDevitt’s lab. “This spatial patterning is what gives the ability to perform higher order functions.”

Once the stem cell aggregates were made and treated with growth factor-endowed microparticles, McDevitt and his colleagues saw spheres of cells with differentiating cells.

“We can see the microparticles had effects on one population that were different from the population that didn’t have the particles,” said McDevitt. “This may allow us to emulate aspects of how development occurs. We can ask questions about how tissues are naturally patterned. With this material incorporation we have the ability to better control the environment in which these cells develop.”

The microparticles could provide better control over the kinetics of cell differentiation; slowing it down with molecules that antagonize differentiation or speed up with other molecules that promote stem cell differentiation.

Despite the fact that McDevitt and his colleagues used mouse embryonic stem cells in this paper, he and his co-workers are already testing this technology on human embryonic stem cells, and the results have been comparable.

“Our findings will provide a significant new tool for tissue engineering, bioprocessing of stem cells and for better studying early development processes such as axis formation in embryos,” said Bratt-Leal. “During development, particular tissues are formed by gradients of signaling molecules. We can now better mimic these signal gradients using our system.”

Both Copies of the Nanog Gene Are Expressed in Embryonic Stem Cells


Commonly held ideas are sometimes held because there is a great of evidence to substantiate them. However, other times, an idea is commonly held because simply because it has been repeated over and over and over even though the evidence for it is poor. Thus, when new evidence come to light showing the commonly held believe to be untrue, it becomes incumbent on us to readjust what we think.

When it comes to embryonic stem cells and the genes that keep them pluripotent, the transcription Nanog plays a very critical role in the self-renewal of embryonic stem cells and there is a great deal of evidence for this assertion. However, the expression of the gene that encodes Nanog was thought to follow the same mode of expression as some of the other pluripotency promoting genes. Namely, that only one of the copies of the Nanog gene were thought to be expressed in embryonic stem cells. This turns out to be probably false.

First a little background. In 2007, Ian Chambers and others published a paper in the journal Nature that examined the expression and function of Nanog in embryonic stem cells. Chambers and others found that Nanog expression levels in individual embryonic stem cells from a culture derived from a single cell varied wildly.  The figure from the Chambers et al paper is shown below.

Immunofluorescence of TNG cells for Oct4 and Nanog. Individual signals from 4,6-diamidino-2-phenylindole (DAPI), GFP, anti-Oct4 and anti-Nanog are shown on the left alongside a combined view of GFP with the stainings from anti-Oct4 and anti-Nanog.
Immunofluorescence of TNG cells for Oct4
and Nanog. Individual signals from 4,6-diamidino-2-phenylindole (DAPI),
GFP, anti-Oct4 and anti-Nanog are shown on the left alongside a combined
view of GFP with the stainings from anti-Oct4 and anti-Nanog.

The reason for this fluctuation in Nanog levels was uncertain, but Chambers and others showed that Nanog could be deleted from mouse embryonic stem cells without affecting their ability to contribute to various sundry embryonic tissues during mouse development, even though they do not make functional gametes (eggs and sperm).  In fact, mouse embryonic stem cells can self-renew under particular conditions without a functional copy of the Nanog gene even though they are prone to differentiation.  From this, Chambers and others concluded that Nanog stabilized rather than promoted pluripotency of embryonic stem cells by “resisting or reversing alternative gene expression states.”

Fast forward to 2012 and another Nature paper by Yusuke Miyanari and Maria-Elena Torres-Padilla from the IGBMC in Strasbourg, France, which showed that before mouse embryos implanted into the uterus, only one copy of the Nanog gene was expressed, but after implantation, both copies of the Nanog gene was expressed.  Miyanari and Torres-Padilla also made mouse embryonic stem cells that had copies of the Nanog gene labeled with different glowing proteins.  This ingenious experiment showed confirmed that Nanog levels were variable, but also showed that only one copy of the Nanog gene was expressed in growing embryonic stem cells in culture.

a, Schematic of the Nanog knock-in reporter NGR. A PEST motif in the carboxy terminus of the fluorescent proteins allows monitoring of dynamic Nanog expression. iHyg, internal ribosome entry site (IRES) hygromycin; iNeo, IRES neomycin; mChe, mCherry; NLS, nuclear localization signal; tGFP, TurboGFP. b, Representative image of NGR ES cells cultured with LIF or 2i/LIF. Scale bar, 10 µm. c, The incidence of allelic switching of Nanog expression in ES cells. Cells were classified into four groups: monoallelic (TurboGFP-positive, green), monoallelic (mCherry-positive, red), biallelic (TurboGFP- and mCherry-positive, yellow) and no expression (black). The proportion of cells undergoing a transition between these four groups during a single cell cycle is indicated. Overall, 47% of cells showed a colour change in this period. n, number of cells analysed. d, The asymmetric replication of Nanog in ES cells cultured with LIF changes to symmetric replication upon treatment with 2i. The cell nuclei were classified as single/double (SD), single/single (SS) and double/double (DD) according to DNA-FISH signals5. n, number of nuclei analysed. *, P < 4 × 10−7; **, P < 1.4 × 10−3 (Fisher’s exact test). e, Representative image of DNA-FISH for Nanog (arrowheads) and Oct4 in ES cells cultured with LIF or 2i/LIF. Scale bar, 2 µm. f, Nanog allelic expression is unaffected in the absence of DNA methyltransferase activity. Quantification of RNA-FISH for Nanog in wild-type (WT) ES cells and ES cells lacking all three DNA methyltransferases (TKO) cultured with LIF or 2i/LIF. g, ChIP for H3K4me3, MED12 or NIPBL along the Nanog locus (black line, top) in ES cells cultured with LIF or 2i/LIF. The position of the ChIP amplicons is depicted by the thick boxes below the line, the TSS by an arrow, the first exon by the black box on the line, and the distal enhancer by the blue box on the line. The Oct4 promoter region (Oct4 Pro) and distal enhancer (Oct4 DE) were positive controls (right)20. The mean ± s.d. of three independent biological replicates is shown.
a, Schematic of the Nanog knock-in reporter NGR. A PEST motif in the carboxy terminus of the fluorescent proteins allows monitoring of dynamic Nanog expression. iHyg, internal ribosome entry site (IRES) hygromycin; iNeo, IRES neomycin; mChe, mCherry; NLS, nuclear localization signal; tGFP, TurboGFP. b, Representative image of NGR ES cells cultured with LIF or 2i/LIF. Scale bar, 10 µm. c, The incidence of allelic switching of Nanog expression in ES cells. Cells were classified into four groups: monoallelic (TurboGFP-positive, green), monoallelic (mCherry-positive, red), biallelic (TurboGFP- and mCherry-positive, yellow) and no expression (black). The proportion of cells undergoing a transition between these four groups during a single cell cycle is indicated. Overall, 47% of cells showed a colour change in this period. n, number of cells analysed. d, The asymmetric replication of Nanog in ES cells cultured with LIF changes to symmetric replication upon treatment with 2i. The cell nuclei were classified as single/double (SD), single/single (SS) and double/double (DD) according to DNA-FISH signals5. n, number of nuclei analysed. *, P < 4 × 10−7; **, P < 1.4 × 10−3 (Fisher’s exact test). e, Representative image of DNA-FISH for Nanog (arrowheads) and Oct4 in ES cells cultured with LIF or 2i/LIF. Scale bar, 2 µm. f, Nanog allelic expression is unaffected in the absence of DNA methyltransferase activity. Quantification of RNA-FISH for Nanog in wild-type (WT) ES cells and ES cells lacking all three DNA methyltransferases (TKO) cultured with LIF or 2i/LIF. g, ChIP for H3K4me3, MED12 or NIPBL along the Nanog locus (black line, top) in ES cells cultured with LIF or 2i/LIF. The position of the ChIP amplicons is depicted by the thick boxes below the line, the TSS by an arrow, the first exon by the black box on the line, and the distal enhancer by the blue box on the line. The Oct4 promoter region (Oct4 Pro) and distal enhancer (Oct4 DE) were positive controls (right)20. The mean ± s.d. of three independent biological replicates is shown.

Now if we fast forward one more year and a paper from the journal Cell Stem Cell and a letter to the same edition of this journal, we have an article by Dina Faddah and others from the laboratory of Rudolf Jaenisch at the Whitehead Institute (MIT, Cambridge, MA), and a supporting letter from Adam Filipczyk and others from Germany and Switzerland.  In this article, the authors also double-labeled mouse embryonic stem cells and examined multiple cells and showed that BOTH copies of Nanog were expressed, and that the range of variability of Nanog expression was approximately the same as other pluripotency genes.

Filipczyk and others used a similar approach to examine the expression of Nanog in mouse embryonic stem cells and they came to the same conclusions as those of Faddah and others.

What is the reason for the differences in findings?  Faddah and others did an important experiment to answer this question.  Some of the cells that with labeled copies of the Nanog gene disrupted the production of a functional Nanog protein.  The constructs used in the papers by Faddah and others and by Filipczyk and others did not disrupt Nanog protein production.  When Faddah and others tested these other constructs that disrupted Nanog protein production to determine is the amount of glowing protein tracked with the amount of Nanog protein produced, it was clear that the amount of Nanog protein made by the cells did not reflect the amount of glowing protein produced.  According to Dina Faddah, “The way the reported was inserted into the DNA seems to disrupt the regulation of the alleles so that when the reported said Nanog isn’t being expressed, it actually is.”

Jaenisch sees this as an instructional tale for all stem cell scientists.  He noted: “Clearly, the conclusions for this particular gene need to be reconsidered.  And it raises the question for other genes.  For some genes, there might be similar issues.  For other genes, they might be more resistant to this type of disturbances caused by a reporter.”

Bottom line – read the materials and methods part of the paper carefully because the way these experiments are done can determine if the results are trustworthy.

Lab-Made Eggs Raise New Fertility Options


Katsuhiko Hayashi of Kyoto University is the lead author of a landmark paper that reports an achievement that has eluded scientists for decades.

In their most recent publication in Science magazine, Hayashi and his colleagues made mouse eggs from induced pluripotent stem cells in a culture dish, and then fertilized them with mouse sperm to create healthy, fertile mice.

This work is a continuation of reports published by the same core group of scientists at Kyoto University who made healthy mouse sperm in the lab from induced pluripotent stem cells and embryonic stem cells (K. Hayashi, H. Ohta, K. Kurimoto, S. Aramaki, M. Saitou, Cell 146, 519 (2011)). If this work can be applied to humans, it will revolutionize fertility treatments.

During the development of mammals, primordial germ cells (PGCs) become one of two cell types depending on the sex of the embryo. For example, if the embryo has an X and a Y chromosome, the PGCs differentiate into spermatozoa, but if the embryo is female and has two X chromosomes, they form oocytes. Sperm and eggs combine during sexual reproduction to form a single-celled embryo known as a zygote, and zygotes have the full developmental potential to grow into the adult animal.

In this paper, Hayashi used mouse embryonic stem cells and surrounded them with cells from the embryonic ovary. This creates a kind of “reconstituted ovary” which is then transplanted into a living mouse to develop. After being cultured in a mouse body for four weeks and four days, this culture system induced the embryonic stem cells to form PCG-like cells that went through all the stages of oocyte development. Fertilization of these oocytes produced by this reconstituted ovary system produced fertile, viable offspring. They also repeated this experiment with induced pluripotent stem cells and they successfully converted these stem cells into PGC-like cells that also underwent successful fertilization.

This experiment has already provided lots of fodder for bioethics bloggers all over the globe. Wesley Smith at his Human Exceptionalism blog at National Review has written the following:

“That mind-exploding point aside, the primary purpose for using this technique in humans would probably be to create mass egg quantities for use in cloning experiments. Each cloning attempt (using SCNT, the technique resulting in Dolly) requires a human egg. At present, human cloning has not been reported–primarily because of the “egg dearth” that inhibits researchers from the kind of repeated trial and error experiments necessary to perfect technique in humans.

Scientists probably need thousands of eggs to figure out human cloning, but they are in extremely short supply because the only sources currently are women of child-bearing age. Efforts are ongoing to remedy that problem–such as using eggs taken from the ovaries of aborted female fetuses or removed from women surgically. If the iPSC approach can be made to work in humans, there would be an infinite supply of eggs, meaning that human cloning would just be a matter of time.”

Smith is right on this one. Human cloning is being held back by its ridiculously low efficiency and the paucity of eggs for such research. Human cloning would be done for research purposes, but its main purpose would be to replace people who have died, or to make embryos or babies to are organ donors for sick adults.

A few years ago, there was a Michael Bay movie entitled “The Island” with Scarlett Johansson and Ewan McGregor. In this movie, McGregor and Johansson are part of a society that lives in a highly controlled environment in which they are told what to wear, what to eat, when to sleep, where to go, and what to do. The only hope they have is to win a supposed lottery that lets them go to “The Island.” Winners are announced on a daily basis, and when they are announced, they are never seen again. McGregor serendipitously discovers that they lottery winners do not go to the Island, but rather go to a surgical room where they are put to sleep and robbed of their vital organs.

McGregor returns to inform Johanssonof the elaborate ruse under which they are living just as Johansson is announced to be the recent winner of the lottery. They escape from the compound and are relentlessly pursued be those company that runs the facility where McGregor and Johansson were housed.

It turns out that McGregor and Johansson are clones of wealthy people who can afford to have a replica of themselves as “insurance policies.” The clones are known as “products” by the scientists who produce them, and the medical staff hardly thinks twice about dispatching each clone for their organs or do deliver a baby for the super-rich who do not want to go through the pain of childbirth.

In one scene, the CEO of the company that produces the clones makes a sales pitch to potential customers in which he speaks of an entity called an “agnate” that contains organs and stem cells for treatments, but has no consciousness or human structure. These patrons think that they are buying the rights to a blog that has their organs, when in fact, they are buying a clone that is a human person that was made by a manufacturing process and is genetically identical to them.

While I do not know the bioethical views of Michael Bay, his movie makes a remarkably telling case against human cloning. Cloning produces a human embryo. While it might have some developmental abnormalities, it is a human person. Farming cloned embryos for tissues is exactly the same as farming cloning human adults for body parts. The only differences are the size, age, and developmental stage of the human persons. Neither size, nor age, nor stage of development are adequate criteria for disqualifying someone from the human race. If this was the case, then six graders would be more human than fifth graders, tall people would be more human than short people, and two-year olds would be more human than one-year olds all of which are patently absurd.

If you are going to argue that the developmental abnormalities of cloned embryos should disqualify them, then you are saying that the less well endowed among us do not have the right to live, which puts you in the same ethical category as Adolph Hitler. People are people, and their identity is the same regardless of their deformities.

This research should give us pause. Human cloning should be banned regardless of whether it is called therapeutic or reproductive cloning. Both manipulate human beings and that is wrong.

Embryonic Stem Cell Cultures Fluctuate Between Pluripotence and Totipotence


In the journal Nature, a fascinating paper appeared from the laboratory of Samuel Pfaff at the Howard Hughes Medical Institute at the Salk Institute for Biological Studies in La Jolla, California near San Diego. In this paper, Todd Macfarlan and his colleagues show that embryonic stem cells cycle between a very primitive developmental stage and a later stage. This cycling is also due to gene expression that is linked to transposable DNA elements.

First, we need some background. The term “totipotent” means that a cell can form any structure in the embryonic or adult body. For example, when the egg undergoes fertilization, it becomes a zygote, which has the capacity to grow into the embryo and all the extraembryonic membranes (amnion, chorion, allantois, placenta, and so on). Another example is a sponge. When a small piece of the sponge is cut from it, the cells in that small piece can de-differentiate and grow into an entire new sponge. Sponge cells are, therefore, totipotent.

Secondly, there is a term “pluripotent,” which means that the cells can form all the adult cell types. Embryonic stem cells are generally thought to be pluripotent and not totipotent. Once the embryo forms the two-cell stage, these two blastomeres are totipotent. However, when the blastocyst stage forms, the inner cell mass cells become pluripotent and lose the ability to form the placenta.

Many years ago, Beddington and Robertson, (1989) implanted mouse embryonic stem cells into the outer layer of cells (trophoblast) to determine if the inner cell mass cells could form the placenta (Development 105, 733–737). The embryonic stem cells were incorporated into the placenta at a very low rate. These data led to an intriguing question: Was the low incorporation due to contamination with trophoblast cells, or could a small proportion of the embryonic stem cells actually become placenta? When gene expression studies examined embryonic stem cells, gene expression was stable in the majority of the cells, but unstable in a small minority of cells (a condition called metastable). It was not surprising that embryonic stem cells were a mixed bag of different cells, but some cells expressed genes that were normally found at earlier developmental stages or were normally expressed in cells that make placenta:
1. Niakan, K. K. et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 24, 312–326 (2010).
2. Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391–401 (2008).
3. Singh, A. M., Hamazaki, T., Hankowski, K. E. & Terada, N. A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25, 2534–2542 (2007).
4. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)
5. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

Weird, huh?

Into the fray comes Macfarlan and company to save (or explain) the day. It turns out that our genomes are loaded with DNA from transposable elements. These DNA elements either have or had at one time, the ability to jump from one location in the genome to another. There are large numbers of these transposable elements in our genomes and almost 50% of the human genome is composed of the remains of these elements.  Current transposable elements include Long INterspersed Elements (LINEs), Short INterspersed Elements (SINEs) and SVA (SINE/VNTR/Alu) elements.  Others include elements such as Mariner, MIR, HERV-K, and others.  The significance of all this is that during development, when the embryo gets to the two-cell stages, in the mouse, particular transposable elements are expressed at very high levels (they produce 3% of the transcribed messenger RNAs, see Peaston, A. E. et al., Dev. Cell 7, 597–606 (2004); Evsikov, A. V. et al., Cytogenet. Genome Res. 105, 240–250 (2004); Kigami, D., et al., Biol. Reprod. 68, 651–654 (2003)), and after two-cell stage, the expression of these transposable elements is silenced (Svoboda, P. et al. Dev. Biol. 269, 276–285 (2004); Ribet, D. et al. J. Virol. 82, 1622–1625 (2008)).

Since these transposons are characteristic of gene expression at the two-cell stage, they can be used as a marker for embryonic stem cells that have reverted back to the two-cell stage.  MacFarlan and his co-workers made a reporter gene and placed it into embryonic stem cells that was controlled by the same sequences as the transposons that are activated at the two-cell stage.  After growing these engineered embryonic stem cells in culture, they discovered that a small minority of cells expressed this reporter gene.

Did these reporter-expressing cells have characteristics like unto those of the two-cell stage embryos?  Yes they did.  When Macfarlan and his buddies examined the genes expressed in the cells that expressed the reporter, they found that the traditional genes that are so characteristic of inner cell mass cells (Oct4, Nanog, Sox2, etc.) were not expressed and other genes normally expressed in two-cell stage embryos, such as Zscan4, were expressed.  Other features that are found in two-cell-stage embryos were also found in these cells that expressed the reporter gene. (methylation of histone 3 lysine 4 (H3K4) and acetylation of H3 and H4 for those who are interested).

Finally, the reporter-expressing cells were able to contribute to the formation of the placenta when transplanted into a mouse embryo.  This shows that these cells not only express the genes of the totipotent stage of development, but they also are totipotent.

These experiments show that most, maybe all embryonic stem cells pass through a short-lived state during which they display features that are characteristic of the totipotent two-cell stage: unlike the vast majority of the ES cells in the culture.  During this transition, they lack expression of the pluripotency-promoting proteins Oct4, Sox2 and Nanog, and have the ability to form cells of both the placenta and the fetus.

There is also a moral implication of these experiments.  In his book Challenging Nature and on the book’s web site, Lee Silver argues that embryonic stem cells are essentially embryos, and if we don’t object to using and discarding embryonic stem cells, then we should not have any problem with using and discarding embryos.  His reasons for asserting that embryonic stem cells are embryos is that in the mouse, embryonic stem cells can be inserted into the inner cell mass of an embryo that has four copies of each chromosome.  The tetraploid embryos can form the placenta, but they cannot form the embryo that is attached to the embryo.  Inserting embryonic stem cells into the inner cell mass of these embryos will rescue them from dying because the embryonic stem cells with make the embryo and the tetraploid embryos will form the placenta.  This experiment is called “tetraploid rescue” and Silver uses it to argue that embryonic cells are essentially embryos.

I find this argument unconvincing for several reasons.  First of all, these embryonic stem cells are being manipulated by being inserted into an embryo.  Granted this embryo is abnormal, but it is an embryo all the same and it provides a vital function that the embryonic stem cells cannot supply – the making of the placenta.  This manipulation helps the embryonic stem cells make the embryo, but not everything else.  In this case the embryonic stem cells are only doing part of the job and they are also receiving the structure and inductive signals from the tetraploid embryo to form the embryo proper.  This is something that embryonic stem cells do not do in culture.

Secondly, embryos undergo development, a process that we understand rather well.  This process of development has a goal toward which the embryo proceeds during development.   Embryonic stem cells are not in the process of development.  They can be induced to form particular cell types or even tissues, but this is part of the embryo or fetus and forming part of the fetus does not constitute embryonic development but only a small part of it.  Embryos do not go backwards during development.  Cells that do go backwards are usually cancer cells that grow uncontrollably and cannot move to a more differentiated state that puts the brakes on cell division.  The fact that embryonic stem cells do move developmentally backward is another indication that they are not embryos.  They do something that embryos do not do and this disqualifies them from being embryos.

Thus another argument against the humanity of the early embryo falls into the pit of very bad arguments.

X (Chromosome) Marks the Plot


In female mammalian embryos, the X chromosome represents a problem. Since mammalian females have two X chromosomes, the embryo contains twice as much of the gene products of the X chromosome as opposed to male mammalian embryos, which only have one copy of the X chromosome. How is this problem solved? X chromosome inactivation (XCI). XCI occurs very early during female mammalian development, and it occurs on a cell-by-cell basis, and occurs randomly. The embryo has some cells that have one copy of the X chromosome inactivated and all the other cells have the other copy of the X chromosome inactivated. This is the reason the bodies of mammalian females are mosaics in which some cells have one copy of the X chromosome inactivated and yet other cells in which the other copy of the X chromosome is inactivated. Thus genetic diseases that map to the X chromosome will affect the entire body of the mammalian male but only a portion of the mammalian female’s body.

What does this mean for stem cells? Quite a bit. Embryonic stem cells are derived from the inner cell mass of the blastocyst-stage embryo. This is precisely the time when the cells of the embryo begin to randomly select a copy of the X chromosome to inactivate. The timing of XCI differs slightly from one species to another. In mice, for example, both copies of the X chromosome are active in mouse embryonic stem cells (ESCs) (Fan and Tran, Hum Genet 130 (2011):217-22; Chaumeil, et al., Cytogenet Genome Res 99 (2002):75-84), and XCI occurs when the cells differentiate (Murakami, et al., Development 138 (2011):197-202). Human ESCs, however, vary tremendously (Dvash and Fan, Epigenetics 4 (2009):19-22), with a few hESC lines showing activation of both copies of the X chromosome and many others showing inactivation of one or the other copy of the X chromosome. Human induced pluripotent stem cells (iPSCs) are derived from adult cells that already have one copy of the X chromosome inactivated. Therefore, de-differentiation of adult cells into iPSCs undoes XCI and activates both copies of the X chromosome (Maherali, et al., Cell Stem Cell 1 (2007):55-70 & Hanna, et al., PNAS 107 (2010):9222-7).

XCI is a process that is linked to pluripotency. The genes necessary for the maintenance of pluripotency (OCT4, Sox2, Nanog) all repress genes necessary for XCI (Xist) and activate genes that repress XCI (Tsix). Therefore, XCI seems to be a factor in the down-regulation of pluripotency in early embryonic cells.

There is a new study that underscores this link between XCI and pluripotency. Researchers at the Gladstone Institutes at the University of California, San Francisco have expanded upon the so-called Kyoto method for making iPSCs. The Kyoto method uses an animal cell line that grows in the culture dish and makes a protein called LIF (leukemia inhibitory factor). LIF activates the growth of cultured iPSCs and allows them to grow and establish an iPSC line.

According to Kiichiro Tomoda from the Gladstone Institute, iPSC derivation on LIF-making feeder cells always produces IPSCs that have two active copies of the X chromosome. However, if iPSCs are derived on feeder cells that do not make LIF, the result is very poor iPSCs derivation and the resultant iPSCs only have one active copy of the X chromosome. Furthermore, by passaging iPSCs that were derived from non-LIF-making feeder cells on LIF-making feeder cells, the inactivated X chromosome became active. This shows that iPSC derivation is highly sensitive to the environment in which the cells are derived. If also shows how to make iPSCs that more closely resemble early embryonic cells.