|Index to this page|
|Human Embryonic Stem Cells (ESCs)|
|Making transgenic animals using embryonic stem cells|
|Cloning mammals using somatic cell nuclei|
Stem cells are cells that divide by mitosis to form either
How the choice is made is still unknown. However, several genes have been found whose activity prevents a daughter cell from differentiating.
The only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage (as shown by the ability of mammals to produce identical twins, triplets, etc.).
In mammals, the expression totipotent stem cells is a misnomer — totipotent cells cannot make more of themselves.
Three types of pluripotent stem cells occur naturally:
All three of these types of pluripotent stem cells
In mice and rats, embryonic stem cells (ESCs) can also:
Induced pluripotent stem cells (iPSCs) Using genetic manipulation in the laboratory, pluripotent stem cells can now be generated from differentiated cells. These induced pluripotent stem cells (iPSCs) are described below.
Multipotent stem cells are found in adult animals; perhaps most organs in the body (e.g., brain, liver, lungs) contain them where they can replace dead or damaged cells. These adult stem cells may also be the cells that — when one accumulates sufficient mutations — produce a clone of cancer cells.
While progress has been slow, some procedures already show promise.
Using multipotent "adult" stem cells.
|Link to discussions of|
One way to avoid the problem of rejection is to use stem cells that are genetically identical to the host.
This is already possible in the rare situations when the patient has healthy stem cells in an undamaged part of the body (like the stem cells being used to replace damaged corneas or those used to treat junctional epidermolysis bullosa).But even where no "autologous" stems cells are available, there may be a solution: using somatic-cell nuclear transfer .
In this technique,
Using this procedure it possible to not only grow blastocysts but even have these go on to develop into adult animals — cloning — with a nuclear genome identical to that of the donor of the nucleus. The first successful cloning by SCNT was with amphibians [View procedure]. Later, mammals such as sheep (Dolly), cows, mice and others were successfully cloned. And in the 11 November 2007 issue of Science, researchers in Oregon reported success with steps 1–4 in rhesus monkeys (primates like us).
This should reassure people who view with alarm the report in May 2013 by the same workers that they have finally succeeded in producing embryonic stem cells (ESCs) using SCNT from differentiated human tissue. The workers assure us that they will not attempt to implant these blastocysts in a surrogate mother to produce a cloned human. And their failure with monkeys suggests that they would fail even if they did try.
While cloning humans still seems impossible, patient-specific ESCs
Whether they will be more efficient and more useful than induced pluripotent stem cells [below] remains to be seen.
Sperm and eggs each contain certain genes that carry an "imprint" identifying them later in the fertilized egg as being derived from the father or mother respectively.
|Link to discussion of gene imprinting.|
Creating an egg with a nucleus taken from an adult cell may not allow a proper pattern of imprinting to be established.
When the diploid adult nucleus is inserted into the enucleated egg (at least those of sheep and mice), the new nucleus becomes "reprogrammed". What reprogramming actually means still must be learned, but perhaps it involves the proper methylation and demethylation of imprinted genes. For example, the inactive X chromosome in adult female cells must be reactivated in the egg, and this actually seems to happen.
In primates (in contrast to sheep, cattle, and mice), the process of removing the resident nucleus causes molecules associated with the centrosome to be lost as well. Although injecting a donor nucleus allows mitosis to begin, spindle formation may be disrupted, and the resulting cells fail to get the correct complement of chromosomes (aneuploidy).
In other words, mutations that might be well-tolerated in a single somatic cell of the adult (used to provide the nucleus) might well turn out to be quite harmful when they become replicated in a clone of cells injected later into the patient.
Furthermore even if the initial stem cells are free of deleterious mutations, as they proliferate in culture, they can acquire mutations. Recently (2017) it has been shown that human embryonic stem cells grown in culture gradually acquire mutations in the TP53 gene — the most frequent "driver" gene in human cancers. The acquisition of a mutant TP53 seems to provide a growth advantage in the culture so that as time goes on, this subclone comes to dominate the population.
This phenomenon should provide a cautionary note in proposals to inject humans with cultures stem cells.
The goal of this procedure (which is often called therapeutic cloning even though no new individual is produced) is to culture a blastocyst that can serve as a source of ES cells.But that same blastocyst could theoretically be implanted in a human uterus and develop into a baby that was genetically identical to the donor of the nucleus. In this way, a human would be cloned.
And in fact, Dolly and other animals are now routinely cloned this way. Link to a description.
The spectre of this is so abhorrent to many that they would like to see the procedure banned despite its promise for helping humans.
In fact, many are so strongly opposed to using human blastocysts — even when produced by nuclear transfer — that they would like to limit stem cell research to adult stem cells (even though these are only multipotent).
A promising alternative to the use of embryonic stem cells in human therapy are recently-developed methods of genetically reprogramming the nuclei of differentiated adult cells so that they regain the pluripotency of embryonic stem cells (ESCs).
In June 2007, three laboratories reported that introducing extra copies of only 4 genes into adult mouse skin cells (fibroblasts) enables them to regain the properties of ESCs. When these cells, named induced pluripotent stem cells (iPSCs for short), were placed in mouse blastocysts, they participated in building all the tissues of the chimeric mice that resulted. (When placed in tetraploid (4n) blastocysts — unable by themselves to develop normally — embryos were formed that thus were clones of the skin cell donor.) The four genes: c-Myc, Sox2, Oct3/4, Klf4.
|By 2009, several labs had succeeded in producing fertile adult mice from iPSCs derived from mouse embryonic fibroblasts. This shows that iPSCs are just a capable of driving complete development (pluripotency) as embryonic stem cells.|
Reprogramming works in humans, too! Using the same four genes, the Yamanaka lab in Japan reported on 20 November 2007, that they now had reprogrammed human skin cells to become induced pluripotent stem cells (iPSCs). And the Thomson lab in Wisconsin accomplished the same thing using SOX2, OCT4, NANOG, and LIN28.
|Further evidence of the remarkable role played by these few genes is the finding that during normal embryonic development of the zebrafish, the same or similar genes (SoxB1, Oct4, Nanog) are responsible for turning on the genes of the zygote. Earlier in development of the blastula, all the genes being expressed (including these) are the mother's — mRNAs and proteins that the mother deposited in the unfertilized egg [More]. It makes sense that the same proteins that can reprogram a differentiated cell into a pluripotent state (iPSCs) are those that produce the pluripotent cells of the early embryo.|
These achievements open the possibility of
Therapy with iPSCs has already been demonstrated in mice. Three examples:1. The Jaenisch lab in Cambridge, MA reported (in Science, 21 December 2007) that they had successfully treated knock-in mice that make sickle-cell hemoglobin with the human βS genes (and show many of the signs of sickle-cell disease in humans) by
The result: all the signs of sickle-cell disease (e.g., anemia) in the treated animals showed marked improvement.2. In the 25 July 2013 issue of Nature, a team of Japanese scientists report that they were able to manufacture three-dimensional buds of human liver cells. Their process:
The result: the implanted buds developed a blood supply and the mice began to secrete human albumin, human alpha-1-antitrypsin, and to to detoxify injected chemicals just as human livers do.3. Workers in the Melton lab at Harvard University reported in the 9 October 2014 issue of Cell that they had succeeded in differentiating large numbers of human beta cells from human iPSCs (as well as from human ESCs). When transplanted into diabetic mice, these cells brought their elevated blood sugar levels back down. (In the case of humans with Type 1 diabetes mellitus, even patient-derived beta cells will still be at risk of the same autoimmune rejection that caused the disease in the first place. Tests are underway to see if this can be avoided by putting the insulin-secreting (beta) cells in a semi-permeable capsule that allows insulin to diffuse out but blocks autoimmune T cells from getting in.)
Let us hope that what works in mice can someday be developed into a safe therapy that will work in humans.
|An Update. The same Japanese team that first succeeded in producing iPSCs reported in the 16 March 2017 issue of The New England Journal of Medicine that they had safely injected retinal cells into the eye of a patient with macular degeneration. The cells had differentiated from iPSCs that were, themselves, derived from her own skin cells. While there was little improvement in her vision, the surgery did halt further deterioration, and there were no bad side effects. The preparation of cells from each patient is a cumbersome and expensive process. However, the same lab found evidence that cells grown from a different donor, and at least partially-matched for MHC antigens, might work as well. Such cells could be grown in large numbers and banked for future use. Because the interior of the eye is an immunologically-privileged site, the same may not hold true for other organs.|
Despite these advances, iPSCs will have to be used cautiously for human therapy. Several groups have found that human iPSCs contain mutations as well as epigenetic patterns (e.g., methylation of their DNA) that are not found in embryonic stem cells. Some of the mutations are also commonly found in cancer cells.
Applied to humans, none of the above procedures would involve the destruction of a potential human life.