What is an Embryo?

Addressing controversy over embryoids

Even a school child can answer the question after he or she is initiated into the secrets of sexual reproduction! An embryo forms after an egg unites with sperm at fertilization to make an early edition of a fetus and then a baby. Fertilization is a canonical process in biology, featured in chapter one of my undergraduate textbook, Waddington’s Principles of Embryology.

Wait! Not so fast. We must update some tenets of embryology we learned in classes and taught our students. Laboratories have crafted multicellular bodies to look like embryos without negotiating fertilization. They blaze news headlines, giving full employment for bioethicists to debate potential human life and when it begins.

In the past sixty years, revolutionary discoveries in reproductive biology and embryology roared like waves on the shores of social and political debate. The first to rustle pebbles was oral contraception, followed by abortifacient drugs, then IVF and its accessories, then prenatal genetic testing, then reproductive cloning, and now embryo artifacts. We call them embryoids (oid: to resemble).

While some people welcome more control of the vagaries of fertility others resist any disruption of the natural order. Endless arguments over a controversial innovation only pause when a more radical technology steals the news. And inevitably, futurology (a pseudoscience) speculates about the march of embryology toward ectogenesis that would make Aldous Huxley chuckle.

Most visionary scientists sixty years ago (nay, even 30) didn’t even dream of making look-alike embryos from somatic cells. The match improves every year. Nature is more plastic than we realized before the advent of mammalian cloning and DNA editing. Of course, Darwin and Wallace showed that species are not immutable and we can change their forms rapidly by selecting breeders. To radically alter the development of cells resembling our make-up presents unsettling questions, including whether we are wise enough to handle that power.

Embryoids are bodies conceived without fertilization. They inherit a diploid genome from an ancestral parent cell to appear and develop like early-stage embryos (to untrained eyes). Other chromosome makeups are possible. There are three basic types according to their origin. Two are known by other names (clones and partenotes) and a third, newer to science, is variously called an embryo “model” or a “synthetic” embryo. I discuss each type of embryoid in turn:

·         Cloning by somatic cell nuclear transfer

·         Parthenogenesis from uniparental development

·         Embryoid bodies from pluripotent stem cells 

If you skip the details you can find my impressions at the end.

1. Nuclear transfer to the cytoplasm of another cell is a familiar if technically tricky procedure in experimental biology but was absent from clinical medicine until 1992. It found a role in treating oligospermic men by injecting sperm to fertilize eggs (called intra-cytoplasmic sperm injection or ICSI). Four years later somatic cell nuclear transfer (SCNT) to empty (enucleated) eggs generated cloned embryos after activation. Dolly the sheep gestated in a surrogate uterus to become the most celebrated clone in history. Since the nucleus came from an adult animal the question arose that if a sheep can be cloned, why not a human? Dolly’s team at Roslin, outside Edinburgh, diplomatically scotched the speculation; they affirmed the natural process is best for a baby and more pleasurable for parents. Cloning is more risky than difficult and to be safe and fully successful epigenetic marks must be reprogrammed to prescribe the genes needed for timely expression.

It has succeeded in farm animals, laboratory rodents, domestic animals, and wild animals, but not in humans to date, except monozygotic twins formed naturally. Not all clones are equal. Some appear healthy but most are abnormal or fail to thrive from incomplete reprogramming. Dolly didn’t have a full lifespan and grew obese, for which visitors like me can be blamed for feeding her treats!

In my opinion, the cloning of animals is a milestone in developmental biology of limited practical value. To overcome its limitations we will need a clever epigenetic repair toolkit to express or repress multiple genes during development. A majority of people in polls say cloning is ethically dubious and I heard that only one company at Roslin remains open of those launched after Dolly. They hoped for a pharmaceutical bonanza from milking body fluids from genetically modified clones. To say anything here about human reproductive cloning will waste your time. 

2. A second type of embryoid happens naturally from conception without a father. This is the process of parthenogenesis, akin to vegetative propagation in plants. It is common in invertebrates, rare in vertebrate animals, and strictly absent in mammals. So why do we need genes from a father?

The evolution of pregnancy in viviparous species presented a conflict between the interests of the mother and father (Haig hypothesis). She should invest in a few, healthy offspring instead of cramming more into the limited accommodation of a uterus. He, on the other hand, has no such constraints on fertility and can father as many babies as he pleases to maximize his genetic fitness (i.e. more of his genes in the next generation).

This hypothesis is alien to us as parents embracing mutual love and cooperation as the best safeguards for healthy children, but evolution by natural selection has no such feelings. The agency of clashing sexes is epigenetic and, in particular, the imprinted genes that are turned on or off according to whether the allele is inherited from the mother or father. The genes H19 and Igf2, to take well-studied examples, have counteracting effects early in development, being expressed either in the fetus or placenta, but not in both at the same time. Picture a genetic see-saw. Japanese researchers provided proof by making a parthenogenetic mouse (though only one) after fixing its imprints.

No more needs to be written here about embryoids from a single parent. The epigenetic limitations are reminiscent of those in reproductive cloning, but the third type of embryoid faces no such problem, though plenty of other issues.

3. Of the three types of embryoids, those constructed from pluripotent stem cells command the most attention now. They develop from a scrambled ball of cells to form the equivalent stage to Day 9 in mouse pregnancy or Day 14 in humans after implantation. I imagine today’s stem cell conferences that I no longer attend are packed with audiences while newspaper reporters rush out after a lecture to publish a scoop. I scratch my head wondering what to make of it, like many other colleagues. If you allow me another page or two, this is what I see and think.

The pace of discovery is being set by big research teams, though still small in number. Some of their published papers have dozens of authors, something more familiar in astronomy but never seen before in embryology. The teams are international and interdisciplinary to combine rare skills and expertise in a race to push technology ahead.

This story began in 1981 with the genesis of mouse embryonic stem cells (ES cells). They are derived in culture from a tiny founder population in the blastocyst from which they would have grown a whole body from three germ layers (ectoderm, mesoderm, and endoderm). We waited seventeen years until human ES cells arrived. It is generally safe to say that what can be done with mice can be replicated in humans given enough effort and patience.

Pluripotent stem cells have remarkable properties. Under stable conditions, they can multiply almost forever, but altering their environment turns them toward a variety of types, up to 200 in theory. Years ago, Alan Trounson mesmerized me with a video showing rhythmically beating heart muscle cells after differentiation from ES cells. Sad to say, the hasty character of science makes past dramas seem banal. Stem cells herald a revolution in regenerative medicine using grafts to treat heart disease diabetes, and so much more but mostly unrealized as yet. Like Australia’s Foster Lager which loses its fizz from waiting too long for a drinker, a society with a short attention span turns to the countertop for the latest mouthwatering recipe—embryoids from stem cells.

A ball of ES cells placed in the uterus cannot recreate an embryo and may become a tumor (teratocarcinoma). They represent ancestral stem cells for all organs and tissues but are not totipotent like a zygote or early embryo. Their destiny is already restricted to the epiblast that makes the germ layers. The blastocyst’s outer sphere of trophectoderm cells is discarded when ES cells are isolated in culture. They are a missing ingredient of an embryo needed for the hypoblast to make a yolk sac and primary endoderm before a placenta can be formed. An epiblast without a hypoblast is blasted! For ES cells to be eased into the uterus they must be carried in a Trojan horse of extra-embryonic cells. What does that mean?

Janet Rossant’s team in Canada overcame the block when they combined tetraploid embryos with ES cells. The abnormal cells with four sets of chromosomes provided what was missing. The aggregates grew into fetuses and readied for delivery. The pups were exclusively diploid from ES cells whereas tetraploid cells were discarded at birth as the placenta.

The experiment provided more proof that ES cells are pluripotent and, more significantly, a direct way of making transgenic mice compared to the standard method of injecting ES cells into blastocysts. Their achievement back in 1990 also helped to inspire a current spate of embryoid engineering which had to wait for sophisticated genetic tools to become available.

The Nobelist Shinya Yamanaka made the first great stride by constructing pluripotent stem cells from fibroblasts and other somatic cells. This step overcame ethical and juridical restrictions on human embryo research because induced pluripotent stem cells (IPS cells) are almost identical to ES cells. To make them required crossing cell membranes with “gene transfer” of specific transcription factors (“gene switches”) for reprogramming the somatic genome. Transfection didn’t alter the DNA sequence. Recapitulation of the developmental clock mimics the process that made Dolly, but instead of one egg at a time it yields thousands more cells for selection.

Constructing embryoids from IPS or ES cells requires an abundance of skill and techniques. Transfection and gene-editing steer cells along lineages toward specific fates, even fabricating cells like the missing extra-embryonic types. Morphogens and adhesion molecules can be directed to promote growth, differentiation, and cell migration for shaping a body of up to 3,000 cells. It needs support in a 3-D matrix and never forms from cells left to lie at the bottom of a dish. At the end of an experiment when embryoids are fixed for microscopic study, fluorescent tags on molecules identify the range of cells in the body.

I am struck by their self-organization from a jumble of naïve stem cells with cells genetically modified to fill another role. Molecular signaling and cell-cell contact integrate the structure, the same as in embryos. Equally impressive is the unfolding process of gastrulation to form an axial primitive streak for the germ layers. A lacuna resembles the true proamnion. Some cells even contain human chorionic gonadotropin. The pregnancy hormone implies the presence of chorion cells and portends a placenta. I don’t underrate these achievements although the published images are not precise facsimiles of normal embryos on Day 14. Nevertheless, this research began only recently. I don’t doubt that after a lot of running, it will reach the goalmouth on Day 28 with a beating heart and the foundations of all major organs.

So what does this progress mean? And does it matter or worry you?

National watchdogs of human embryology will decide if breaking past Day 14 in development trespasses on the limit set by countries with laws permitting embryo research. The stem cell science organization, ISSCR, has banned members from trying to establish pregnancies with embryoids. Fat chance! Embryoids are too abnormal and too late a stage for implanting in the uterus. However, the brisk announcement may have helped to forestall anxiety from the memory of the Chinese scientist who used gene editing on human embryos. I don’t know if a child was damaged, but he temporarily dented public trust in science.

On the question of whether embryoids are useful for (not in) fertility treatment the answer is positive. They are embryo “models” (no more) for shedding light on the darkest chapters of human development, between the free-floating blastocyst and the earliest time to visualize embryos and fetuses with scanners. Patients with fertility problems will surely benefit. Early miscarriage is common. Embryos from IVF that may look healthy in a dish often fail after transfer.

Moreover, we may get more traction for research on birth defects occurring at stages of rapid cell growth and migration. A critical period for the effects of the infamous drug Thalidomide is late in the first month of pregnancy, soon to be in reach of embryoid cultures. The opportunity to screen new drugs can’t come soon enough. And since animals are poor substitutes for studying chromosome defects, human embryoids will occupy another research niche. “The proper study of mankind is man” (Alexander Pope).

I close by returning to the question of what is an embryo. A recent paper in EMBO Reports states that “what matters is potentiality, the origin should be irrelevant.” The authors imply a grey line between embryos and embryoids but don’t mention an ethical test that proves the potential to make a baby. The only way I know is by implantation in a uterus. We don’t call an object with a cockpit and wings an airplane unless it can fly!

It strains credibility to imagine babies made other than by nature’s wild way. And yet, it is not inconceivable that embryoids will closely match authentic embryos in the distant future. I already mused about nature’s permission to tinker. As one thing leads to another in science, viable embryoids would open a door to genetic manipulation or germ-line therapy, often regarded as a forbidden fruit. That scenario seems to tumble out of pages of science fiction, although it may be regarded quite differently one day by a different generation, either good or bad, or perhaps good and bad.

There are far too many other pressing matters today to add insurgent embryology to my worry list. Let embryoid research flourish to see where it leads. I think about Marie Curie who pioneered radioactivity. She didn’t live to see the atomic bomb yet gave us radium as a fruit for cancer treatment, saying “Nothing in life is to be feared, only understood.”

As I wrote at the beginning, some school children know the standard definition of an embryo as well as any scientist, though few will consider its moral value. That is a topic for a grownup essay which I will release in May.  Expect an essay pregnant with potential as the theme.

[Photo credit: Stufford on Pixabay. Below: the author with Dolly the sheep and one of her creators, the embryologist Bill Ritchie]