|Index to this page|
|Human Embryonic Stem (ES) Cells|
|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 can also:
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).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.
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).
"Reprogram" adult cells so that they develop into another type of differentiated cell without having to pass through a stem-cell-like stage ("transdifferentiation"). Workers in the Melton lab at Harvard University reported in the 2 October 2008 issue of Nature that they had succeeded in converting the exocrine cells in the mouse pancreas (that secrete digestive enzymes like amylase) into insulin-secreting beta cells. They did this by injecting the pancreas of living mice with an adenovirus vector carrying 3 genes for transcription factors. When the experiments were done with mice deliberately made diabetic (by destroying their pre-existing β-cells), the newly-created β-cells secreted enough insulin to bring blood sugar levels closer to normal.
In the Melton study, one type of endodermal cell (exocrine) was transdifferentiated directly into another type of endodermal cell (endocrine). More recently, another group has succeeded — using a different set of 3 genes encoding transcription factors — in transdifferentiating mouse fibroblasts (mesoderm) into neurons (ectoderm). In culture, these neurons generated action potentials and could form synapses. Their report is: Vierbuchen, T., et al., Nature, 463:1035, 25 February 2010.
Perhaps the most 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 (ES) cells.
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 ES cells. 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.These achievements point to another way that
Examples: researchers have succeeded in deriving iPSCs from
The result: all the signs of sickle-cell disease (e.g., anemia) in the treated animals showed marked improvement.
Let us hope that what works in mice can someday be developed into a safe therapy that will work in humans.
|Update. The Jaenisch lab reported in the 6 March 2009 issue of Cell that they have succeeded in making iPSCs (they call them hiPSCs) from fibroblasts taken from patients with Parkinson's disease. The cells were then differentiated into dopamine-releasing cells — the cells lacking in this disease. What is particularly exciting is that they accomplished this after using the Cre-lox system to remove all the genes (e.g., SOX2, OCT4, KLF4) needed for reprogramming the fibroblasts to an embryonic-stem-cell-like condition.|
|Since that report, other laboratories — using other methods — have also created iPSCs from which all foreign DNA (vector and transgenes) has been removed. Not only should such cells be safer to use in therapy, but these results show that the stimulus to reprogram a differentiated cell into a pluripotent state need only be transitory.|
Despite these successes, iPSCs may not be able to completely replace the need for embryonic stem cells and may even be dangerous to use in 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.