Friday, January 26, 2007

Stem Cells Part IV: Human embryonic stem cells.

(A comment on illustrations: illustrations are helpful in working through this material. Unfortunately, I lack the time to produce them myself, and copying them gets into copyright issues. I recommend the illustrations found in the 2001 and 2006 NIH stem cell documents referenced at the end of this essay. They are available in downloadable format. Meanwhile, we shall persevere with words...)

We've seen that in the first week after conception, the pre-implantation human embryo goes through three distinct stages: single cell zygote, the morula, and the blastocyst. The zygote is the product of conception; that is, the union of the mother's oocyte and the father's sperm. The zygote is the earliest stage of a new human being, but it soon gives rise to the multi-celled morula, whose handful of cells all look more or less the same. By around day 5 or so, the morula has begun to differentiate into a blastocyst, an embryo of some 250 cells.[1] Most of these cells make up the outer shell, known as the trophoectoderm, which will become the placenta. The inside of the blastocyst is mostly hollow, with thirty or so cells stuck on the inside of the trophoectoderm shell as the inner cell mass. All this happens up in the fallopian tube, and by 5-7 days the blastocyst has made it's way through the tube, and implants in the uterus. Once implanted, the blastocyst begins to develop further, but it is the pre-implantation embryo which concerns us here.

The cells of the inner cell mass are not totipotent stem cells, because they do not normally give rise to the placenta[2]. That job is done by the cells of the trophoectoderm. However, the inner cell mass cells are pluripotent, being able to give rise to cells of any of the three germ layers, which gives them they value to medical researchers. In vivo, these cells are evanescent, existing only for a couple of days[3] between when the blastocyst forms, (day 4-5) and implantation, when the three primary germ layers appear (day 7-9). If human blastocysts could be formed outside of the body, in vitro, they could be mined for their inner cell masses, and the inner cell masses could be harvested, grown in petri dishes, and manipulated from there. What a great idea!

Human blastocysts have been being made in vitro for decades. Louise Brown, the first “test tube baby” (as they were then known), was born in 1984,[4] and in the quarter century since then the IVF - in vitro fertilization - industry has become a billion dollar business. It has also generated 400,000 leftover embryos[5], unwanted by their parents, residing in the freezers of IVF clinics across our land. In the late 1990’s, thirty six of these orphaned embryos were rescued from their indeterminate fate to become unknowing participants in a landmark study by Dr. James Thompson and his colleagues of the Wisconsin Regional Primate Research Center at the University of Wisconsin in Madison[6]. Actually, many of the 36 embryos were obtained frozen from the IVF Clinic at Rambam Medical Center in Haifa, Israel, though at least one was obtained fresh from the University of Wisconsin IVF clinic.[7] Based on his work with nonhuman primate embryos, Dr. Thomson had identified three essential characteristics for embryonic (ES) cells: “(i) derivation from the preimplantation or periimplantation embryo, (ii)prolonged undifferentiated proliferation, and (iii) stable developmental potential to form derivatives of all three embryonic germ layers...”.[8] In order for his experiments to be considered a success, the resultant cells would have to meet those criteria. Of the 36 blastocysts, 14 proved to be viable, and these were subjected to “immunosurgery” to isolate the inner cell mass. With immunosurgery, antibodies are made which are directed against markers found on the surface of cells in the trophoectoderm shell, but not the cells of the inner cell mass. When the blastocysts are incubated with the antibodies, the trophoectoderm shell is dissolved, leaving only the inner cell mass behind. In the process, of course, the 5 day old human embryo is killed, just as surely as if, were your skin to be dissolved, you would die, although individual cells of your organs would continue to live for a short while, and could be harvested and kept alive in culture.

Once the embryos were destroyed and their inner cell masses harvested, the cells were plated out in petri dishes containing nutrient serum and a layer of mouse embryo “feeder cells”. These feeder cells are necessary for proper growth of the human ES cells in culture.[9] After being placed on the feeder cells, the ES cells were allowed to grow and multiply for a week or two, then they were removed, dissociated into smaller clumps, and replated on fresh plates with fresh feeder cells. This procedure is known as a “passage”. Repeated passaging is necessary to keep the ES cells in an undifferentiated state: if passaging is not performed, the cells begin to differentiate spontaneously and uncontrollably into mature cell types. The other problem with cultured cells is that they can develop chromosomal mutations: the useful cell culture is one whose DNA remains unsullied. Of the 14 inner cell masses they began with, five developed into “cell lines” which could be successfully passaged repeatedly, and while doing so, kept their chromosomes clean. The five original cell lines are named H1, H7, H9, H13 and H14. Three of the lines have a normal XY karyotype ("boy", H1, H13, and H14) and two have a normal XX karyotype ("girl", H7 and H9). Thus Dr. Thomson was successful in meeting the first two criteria: (i) cell lines derived from a pre-implantation embryo, and (ii) ability to replicate in culture while maintaining genetic stability and lack of differentiation. What about the third criterion, "stable developmental potential to form derivatives of all three embryonic germ layers..."?

There's several ways that a putative stem cell, can be shown to have the capability to differentiate into mature tissue types. Explaining them segues into the topics of chimeras and cloning both of which are necessary to understand in order to see where human embryonic stem cell research must go if it is to ever be used in human medical therapy. So, next week, chimeras, and the week after that, cloning.

[1] "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
[2] However, embryoid bodies, which we will discuss later in this essay, have given rise to mature placental cells known as trophoblasts. See "Regenerative Medicine 2006", National Institutes of Health, Department of Health and Human Services pg. 8. Entire report downloadable in pdf format at In particular, see reference 53 of Chapter 1.
[3] "Stem Cells and the Future of regenerative Medicine" National Research Council/Institute of Medicine, National Academy Press, Washington, D.C. 2002. Pg.31.
[4] Of nominal interest, Louise Brown recently gave birth to her own child, and apparently without the use of IVF.,2933,243705,00.html
[5] Hoffman, DI, Zellman, GL, et al. Cryopreserved embryos in the United States and their availability for research. Fertility and Sterility 79(5):1063-1069, May, 2003
[6] Thomson, JA et al Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282:1145-1147, November 6, 1998.
[7] Stem Cells 2001, ibid. Pg. C-1, Appendix C.
[8] Thomson et al, ibid.
[9] The need for mouse embryonic fibroblasts as feeder cells represents one of the major technological problems in the development of human ES cell cultures for medical use. The risk is transmission of mouse pathogens into the ES cells and then, potentially, into the human recipient of whatever therapy the ES cell is used in. This problem has been solved only partially as of this writing. The other big technical problem, of course, is that no one can control what sort of differentiated cell the ES cell will grow up into.

No comments: