Stem Cells Part V: Chimeras.

We left off last week with the dawn of the human embryonic stem cell era, Dr. Thomson's landmark Science paper published in November of 1998[1]. There are a number of "defining properties" of a human embryonic stem cell (hES cell),[2] but they are generally boiled down to three: first, it must come from a human pre-implantation embryo. Second, it must be able to replicate itself indefinitely in the laboratory, while maintaining genetic stability and remaining undifferentiated. Finally, it must possess "stable developmental potential to form derivatives of all three embryonic germ layers...". This last criterion is crucial, it is, in fact, the heart of "stem cell-ness". If a cell growing in culture isn't pluripotent, it's just a cell growing in culture, and will not have the miraculous ability to cure every disease known to man and keep us all age 28 forever. So, Dr. Thomson demonstrated that his five cell lines met the first two criteria: they came from pre-implantation human embryos, and they could be grown indefinitely in culture, without differentiating. This left only the third criterion: demonstrating pluripotentiality. Demonstrating this last criterion in a putative embryonic stem cell line is crucial. How is it done?

There are three primary ways a stem cell can be shown to be pluripotent[3]. The first way is in vitro differentiation. When hES cells (or, for that matter, mouse embryonic stem cells, which have been being researched since the early 1980's) are grown in culture, they must be carefully maintained on a layer of feeder cells, generally a specific cell known as mouse embryonic fibroblasts. As mentioned last week, this need for feeder cells is a major technological hurdle which has only been partially solved, but that doesn't concern us. If the hES cells aren't properly maintained on a feeder layer, they will spontaneously round up into embryoid bodies,[4]and it was this characteristic that Dr. Thomson used to demonstrate pluripotentiality in his cell lines. What are embryoid bodies? They are "clumps of cellular structures that arise when embryonic stem cells are cultured. Embryoid bodies contain tissue from all three germ layers: endoderm, mesoderm, and ectoderm. Embryoid bodied are not part of normal development and occur only in vitro."[5] We hear much from the opposition describing the early human embryo as a blob of tissue. This is not true, of course; the early human embryo is an early human being. However, embryoid bodies are, in fact, blobs of tissue. Human ES cell lines do not, in and of themselves, have the ability to self organize into a human embryo; that ability was lost when the true embryo, the blastocyst, was ripped apart for its inner cell mass. However, they may well develop into a meaningless aggregation of random tissues, not unlike the box in my garage filled with old engine parts. When Dr. Thomson's hES cells were removed from their feeder cells and placed in liquid suspension, they formed embryoid bodies. If the embryoid bodies are allowed to attach to a tissue culture plate, they will form mature tissue types, similar to a teratoma[6].

What is a teratoma? A teratoma is a naturally occurring tumour which consists of fully mature, random tissues. They are quite common in the ovary, and less commonly found in the testis. They are usually, but not always, benign, and the most common types are called "dermoid cysts" because they are big cystic things growing off the ovary, filled with hair and goo. The prognosis is generally excellent. They are dramatic, though, being filled with hair, skin, and other mature tissue types. Occasionally fully formed teeth are present, and, vary rarely, there may be a structure called a fetiform teratoma (also known as a homunculus)[7]. As we saw, the first method of demonstrating putative hES cell pluripotentiality is to let them form embryoid bodies, then put the bodies in a dish. They will form things similar to teratomas. The second method, though, of demonstrating hES cell pluripotentiality is to inject them into a mouse. If truly pluripotent, they will form teratomas. This was the second method Dr. Thomson used in his paper: he injected his hES cells into mice and, sho'nuff they developed teratomas of mature, human tissue types. This brings us to the last, "gold standard" method of demonstrating pluripotentiality: chimeras.

Chimera: An organism composed of cells derived from at least two genetically different cell types. The cells could be from the same or separate species.[8] As with teratomas, there are naturally occurring chimeras, but they don't concern us. In mouse ES cell research, the "gold standard" for demonstrating pluripotentiality is to produce a chimera. The mouse ES cells are injected directly into the cavity of a mouse blastocyst. The blastocyst is then implanted in the uterus of a female mouse, and the resulting babies will be chimeras, with organs and tissues derived from both the original blastocyst and the injected mES cells[9]. The reason that the production of chimeras is considered the "most rigorous test" of pluripotentiality is because the researcher can assess the functionality of the tissue thus produced, something one cannot do with teratomas or embryoid bodies. Thomson's original article did not produce human/mouse chimeras. However, today researchers want to. Why? Quoting from the National Academies of Science's 2005 publication, Guidelines for Human Embryonic Stem Cell Research directly,

"Those types of differentiation assays (embryoid bodies and teratomas) do not provide conclusive evidence that the resulting cell types are functioning normally, nor whether hES cells have the capacity to participate in normal development in the context of the three-dimensional embryo in the reproductive tract. Such conclusive evidence requires testing in blastocyst chimeras as is routinely done with mES cells.[10]

Make no mistake, human/animal chimeras are part and parcel of hES cell research. So is cloning, which we will take up next week.

Endnotes
[1] Thomson, JA et al Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282:1145-1147, November 6, 1998.
[2] "Stem Cells: Scientific Progress and Future Directions June 2001" National Institutes of Health, Department of Health and Human Services pg. 13 Entire report downloadable in pdf format at http://stemcells.nih.gov/info/scireport/2001report
[3] "Guidelines for Human Embryonic Stem Cell Research." National Research Council and Institute of Medicine of the National Academies National Academy Press, Washington, D.C. 2005. See Ch. 2, "Scientific background", pg. 30 ff.
[4] In fact, if the cells are kept in the dish on a lawn of feeder cells, and are simply allowed to grow up to the sides of their dish, becoming a bit crowded, they will begin to spontaneously differentiate. See Thompson et al, ibid.
[5] Guidelines, ibid, Glossary.
[6] Ibid, pg. 31.
[7] Crum, CP & Lee, KR Diagnostic Gynecologic and Obstetric Pathology Elsevier Saunders, Philadelphia. 2006. Pp 915-917.
[8] Guidelines, ibid, Glossary. To flesh this out a little, a chimera consists, essentially, of distinct populations of cells with two (or more) separate genetic make ups, and as such should not be confused with hybrids, where the genomes of two species, say a horse and a donkey, are combined at conception to produce a mule.
[9] Paraphrased from ibid, pp31ff.
[10] Ibid, page 33. My emphasis.