Infertility Treatment with Stem Cells is Unlikely

Because several laboratories have managed to differentiate embryonic stem cells into cells that look very much like human eggs and sperm, many have predicted that infertility will be treated with stem cell treatments (see Volarevic V, et al., Biomed Res Int. 2014;2014:507234). However, new work from the University of Gothenburg and Karolinska Institute has cast doubt on this hope.

At about 24 days of life, large, spherical sex cells are recognizable among the endodermal cells of the umbilical vesicle close to the allantois. These cells are the primordial germ cells and they are the progenitor cells of the sperm in men and eggs in women. As the embryo folds during about the late 4th week, the dorsal portion of the umbilical vesicle is incorporated into the embryo. This incorporation of the umbilical vesicle occur concurrently with the migration of these primordial germ cells along the dorsal mesentery of the hindgut to the gonadal ridges. During the 6th week of life, the primordial germ cells enter the underlying mesenchyme and are incorporated into the gonadal cords. Primordial germ cell migration is mainly regulated by three genes: Stella, Fragilis, and BMP-4.

PGC migration

These primordial germ cells divide as they migrate, and by five months of gestation, embryonic ovaries contain about six to seven million oogonia. Most of these oogonia experience cell death before birth, but the remaining oogonia begin meiosis toward the end of gestation. At this time, the oogonia are called primary oocytes. Meiosis is arrested in prophase of the first meiotic division, and this is the same stage at which spermatogenesis in the male is blocked. Primary oocytes decrease in number throughout a woman’s life. The ovaries of a newborn girl contain about two million primary oocytes and these are all the gametes she will ever have. Each primary oocyte is contained within a hollow ball of cells called the ovarian follicle. By the time a woman reaches puberty, that number of primary oocytes has been reduced to 400,000. Only about 400 of these cells will ovulate during a woman’s reproductive years. The rest will die by means of programmed cell death. Once all the primary oocytes are gone, ovulation stops and the woman undergoes menopause.

Kui Liu from the University of Gothenburg said: “Ever since 2004, the studies on stem cell research and infertility have been surrounded by hype. There has been a great amount of media interest in this, and the message has been that the treatment of infertility with stem cells is about to happen. However, many researchers, including my research group, have tried to replicate these studies and not succeeded. This creates uncertainty about whether it is all possible to create new eggs with the help of stem cells.”

In collaboration with Outi-Hovatta’s laboratory at the Karolinska Institute and Jan-Åke Gustafsson’ research team at the university of Houston in the US, Lui’s research team carried out experiments on mice that failed to demonstrate that functional gametes could be formed from pluripotent stem cells. Essentially, the only gametes that could that the female mice had were the ones they were born with.

In Liu’s opinion, fertility clinics should place their attention on using the eggs that women still have in order to treat infertility.

Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells are Epigenetically Rejuvenated

Earlier this year, Miltalipov and his research group published a paper in Nature that compared the genetic integrity of embryonic stem cells made from embryos, to induced pluripotent stem cells and embryonic stem cells made from cloned embryos.  All three sets of stem cells seemed to have comparable numbers of mutations, but the induced pluripotent stem cells had “epigenetic changes” that were not found in either stem cell line from cloned or non-cloned embryos.

Genetic characteristics have to do with the sequence of the DNA molecules that make up the genome of an organism.  Epigenetic characteristics have nothing to do with the sequence of DNA, but instead are the result of small chemicals that are attached to the DNA molecule.  These small chemical tags affect gene expression patterns.  Every cell has a specific epigenetic signature.

During development, the cells that will form our eggs and sperm in our bodies, the “primordial germ cells,” begin their lives in the outer layer of the embryo.  During the third week of life, these primordial germ cells or PGCs move like amoebas and wander into the yolk sac wall and collect near the exit of a sac called the “allantois.”  The PGCs are outside the embryo at this time or extraembryonal.  Incidentallyyolk sac is a terrible name for this structure, since it does not produce yolk proteins.  Therefore other textbooks have renamed it the “primary umbilical vesicle,” which is a bit of a mouthful, but it probably better than “yolk sac.”


1 - Primordial germ cells 2 - Allantois 3 - Rectum 4 - Ectoderm 5 - Foregut 6 - Primordial Heart 7 - Secondary yolk sac 8 - Endoderm 9 - Mesoderm 10 - Amniotic cavity
1 – Primordial germ cells
2 – Allantois
3 – Rectum
4 – Ectoderm
5 – Foregut
6 – Primordial Heart
7 – Secondary yolk sac
8 – Endoderm
9 – Mesoderm
10 – Amniotic cavity

The embryo around this time undergoes a bending process as a result of its growth and the head bends toward the tail (known as the cranio-caudal curvature) and then the sides of the embryo fold downwards and eventually fuse (lateral folding).  This bending of the embryo allows the PGCs to wander back into the embryo again between the fourth and sixth week.  The PGCs move along the yolk sac wall to the vitelline and into the wall of the rectum.  After crossing the dorsal mesentery (which holds the developing intestines in place) they colonize the gonadal or genital ridge (which is the developing gonad). During their journey, and while in the gonadal ridge, the PGCs divide many times.

1 - Rectum 2 - Vitelline 3 - Allantois 4 - Nephrogenic cord (pink) 5 - Gonadal ridge (green) 6 - Primordial germ cells (red dots) 7 - Heart prominence
1 – Rectum
2 – Vitelline
3 – Allantois
4 – Nephrogenic cord (pink)
5 – Gonadal ridge (green)
6 – Primordial germ cells (red dots)
7 – Heart prominence

When the PGCs move into the developing gonad, the chemical tags on their DNA are completely removed (rather famous paper – Lee, et al., Development 129, 1807–1817 (2002).  This epigenetic erasure proceeds in order for the PGCs to develop into gametes and then received a gamete-specific set of epigenetic modifications.  These epigenetic modifications also extend to the proteins that package the DNA into chromosomes – proteins called histones.  Specific modifications of histone proteins and DNA lead to gamete-specific expression of genes.  Once fertilization occurs, and the embryological program is initiated, tissue-specific epigenetic modifications are conveyed onto the DNA and histones of particular cell populations.

This is a long-winded explanation, but because many cancer cells have abnormal epigenetic modifications, these epigenetic abnormalities in induced pluripotent stem cells (iPSCs) have been taken with some degree of seriousness.  Although, there is little evidence to date that links the cancer-causing capabilities of iPSCs with specific epigenetic modifications, although it certainly affects the ability of these cells to differentiate into various cell types.

A paper has just come from the laboratory of Wolfgang Wagner from the Aachen University Medical School, in Aachen, Germany that derived iPSCs from mesenchymal stem cells from human bone marrow, and then in a cool one-step procedure, differentiated these cells into mesenchymal stem cells (MSCs).  These  iPS-MSCs looked the same, and acted the same in cell culture as the parent MSCs, and had the same gene expression profiles as primary MSCs.  However, all age-related and tissue-specific epigenetic patterns had been erased by the reprogramming process.  This means that all the tissue-specific, senescence-associated, and age-related epigenetic patterns were erased during reprogramming.  Another feature of these iPS-MSCs is that they lacked but the ability to down-regulate the immune response, which is a major feature of MSCs.

Thus, this paper by the Wagner lab shows that MSCs derived from iPSCs are rejuvenated by the reprogramming process.  Also, the donor-specific epigenetic features are maintained, which was also discovered by Shao and others last year.  This suggests that epigenetic abnormalities are not an inherent property of the derivation of iPSCs, and that this feature is not an intractable characteristic of iPSCs derivation and may not prevent these cells from being successfully and safely used in the clinic.  However, this might be a cell type-specific phenomenon.  Also, the loss of the immune system regulatory capabilities of these iPS-MSCs is troubling and this requires further work.


Adult Mammals Lack the Stem Cell Activity to Make New Eggs

Recent research in mice and humans have discovered a stem cell population in ovaries that can form eggs. However, this discovery begs a question: namely, why do adult female mammals run out of eggs in their lifetime if they have a stem cell population that can produce eggs?

New research from the Carnegie Institute for Science demonstrates that adult mice do not use stem to produce new eggs, thus answering this apparent conundrum.

Before birth, mouse and human ovaries contain an abundant supply of germ cells that originate from primordial germ cells that form from the inner layer of the primary umbilical vesicle (otherwise known as the yolk sac).  Between the time when the embryo is four to six weeks old, the primordial germ cells (PGCs) migrate from the wall of the primary umbilical vesicle to the gut tube.  From the gut tube, the PGCs migrate to the dorsal body wall by means of the mesentery that suspends the gut from the body wall.  Once in the body wall, the PGCs come to rest on either side of the midline in the loose mesenchymal tissue just inside the membranous lining of the body cavity (known more technically as the coelomic cavity).

PGC Migration Pathway2

Most of the PGCs populate the region of the body wall at the level that will form the gonads.  During their migration, PGCs continue to multiply by means of mitosis, which increase their numbers substantially.  Some PGCs may become stranded during their migration, coming to rest at extragonadal sites.  Occasionally, stray germ cells of this type may give rise to a type of tumor called a teratoma.


Once in their final location, the PGCs will stimulate the formation of the genital or gonadal ridge.

In females, PGCs (which are now called gonocytes) undergo a few more mitotic divisions after they are surrounded by the somatic support cells and become intimately associated with them.  The gonocytes differentiate into oogonia, and by the 5th month of fetal development all oogonia initiate meiosis.  After they initiate meiosis, the oogonia are called primary oocytes.  However, during an early phase of meiosis all sex cells enter a state of dormancy, and they remain in meiotic arrest as primary oocytes until sexual maturity.  Beginning at puberty, each month a few ovarian follicles resume development in response to the monthly surge of pituitary gonadotropic  hormones, but usually only one primary oocyte matures into a secondary oocyte and is ovulated. This oocyte enters a second phase of meiotic arrest and does not actually complete meiosis unless it is fertilized. These monthly cycles continue until the onset of menopause at approximately 50 years of age.

Near the time of birth, the ovaries of mice and humans contain an abundant supply of eggs that will be released from follicles during ovulation each menstrual cycle.  At the birth of the baby, she will possess a large reserve of primordial follicles that contain a single egg surrounded by supporting follicle cells.  Evidence of new follicle production is absent after birth.  Therefore, it has long been thought that the supply of follicles is fixed at birth and eventually is exhausted at menopause.

During the last decade, researchers have found primordial follicles in adult mouse ovaries that turn over and claimed that adult germ-line stem cells constantly resupply the follicle pool and sustain ovulation.  These claims were based on observations of ovarian tissue and one the behavior of extremely rare ovarian cells after these cells were cultured for some time in the laboratory.  Such criteria are subjective, especially in light of the fact that culturing cells for long periods of time in the laboratory can effectively reprogram them.

At Carnegie, Lei Lei and Allan Spradling used a technique that tracks individual cells and their progeny within living tissue over a specific time course.  The cells are marked with a gene, and this gene is inherited by the progeny of that cell, thus allowing the careful tracking of all the progeny of that cell or those cells.  This technique is called “lineage tracking” and it is a very popular technique in developmental and cell biology.

By subjecting primordial follicles to lineage tracking, Lei and Spradling showed that germ-line stem cell activity cannot be detected in mice.  Furthermore, primordial follicles are stable, and even if half the existing follicles die off, no germ-line stem cell activity is detectable.  This research does not prove that there are no germ-line stem cell divisions within the ovary of the mouse, but it does place an upper limit on the divisions of the germ-line stem cell population of one division every two weeks at the most, which is biologically insignificant.

What then can be said about the germ-line stem cell cultures isolated in the laboratory?  According to Alan Spradling, the cells “likely arise by dedifferentiation in culture,” and “the same safety and reliability concerns would apply as to any laboratory-generated cell type that lacks a normal counterpart” in the body.

This should be a warning to those conclusions that are solely derived from experiments conducted in culture alone and not in a living creature as well.

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.