Stem Cells

Stem cells are cells that divide by mitosis to form either

• two stem cells, thus increasing the size of the stem cell "pool",
• or
  • one daughter that goes on to differentiate, and
  • one daughter that retains its stem-cell properties.

How the choice is made is still unknown. However, several genes have been found whose activity prevents a daughter cell from differentiating.

1. Totipotent cells. In mammals, totipotent cells have the potential to become
   • any type in the adult body;
   • any cell of the extraembryonic membranes (e.g., placenta).

   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.

2. Pluripotent stem cells. These are true stem cells, with the potential to make any differentiated cell in the body (but probably not those of the placenta which is derived from the trophoblast).

   Three types of pluripotent stem cells occur naturally:

   • Embryonic Stem (ES) Cells. These can be isolated from the inner cell mass (ICM) of the blastocyst — the stage of embryonic development when implantation occurs. For humans, excess embryos produced during in vitro fertilization (IVF) procedures are used. Harvesting ES cells from human blastocysts is controversial because it destroys the embryo, which could have been implanted to produce another baby (but often was simply going to be discarded).
   • Embryonic Germ (EG) Cells. These can be isolated from the precursor to the gonads in aborted fetuses.
   • Embryonic Carcinoma (EC) Cells. These can be isolated from teratocarcinomas, a tumor that occasionally occurs in a gonad of a fetus. Unlike the other two, they are usually aneuploid.

   All three of these types of pluripotent stem cells

   • can only be isolated from embryonic or fetal tissue;
   • can be grown in culture, but only with special methods to prevent them from differentiating.

   In mice and rats, embryonic stem cells can also:

   • contribute to the formation of a healthy chimeric adult when injected into a blastocyst which is then implanted in a surrogate mother;
   • enter the germline of these animals; that is, contribute to their pool of gametes;
   • develop into teratomas when injected into immundeficient (SCID) mice. These tumors produce a wide variety of cell types representing all three germ layers (ectoderm, mesoderm, and endoderm).

   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.

3. Multipotent stem cells. These are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. [Discussion]

   Multipotent stem cells are found in adult animals; perhaps most organs in the body (e.g., brain, liver) 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.

Using Stem Cells for Human Therapy

The Dream

Examples:

• Type 1 diabetes mellitus where the beta cells of the pancreas have been destroyed by an autoimmune attack;
• Parkinson's disease; where dopamine-secreting cells of the brain have been destroyed;
• spinal cord injuries leading to paralysis of the skeletal muscles; [View]
• ischemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvation;
• multiple sclerosis with its loss of myelin sheaths around axons.
• blindness caused by damage to the cornea.

While some success has been achieved with laboratory animals, not much has yet been achieved with humans.

Two exceptions:

• culturing human epithelial stem cells and using their differentiated progeny to replace a damaged cornea. This works best when the stem cells are from the patient (e.g. from the other eye). Corneal cells from another person (an allograft) are always at risk of rejection by the recipient's immune system.
• the successful repair of a damaged left bronchus using a section of a donated trachea that was first cleansed of all donor cells and then seeded with the recipient's epithelial cells and cartilage-forming cells grown from stem cells in her bone marrow. So far the patient is doing well and needs no drugs to suppress her immune system.

The Immunological Problems

A Solution?

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).

In this technique,

1. An egg has its own nucleus removed and replaced by
2. a nucleus taken from a somatic (e.g., skin) cell of the donor.
3. The now-diploid egg is allowed to develop in culture to the blastocyst stage when
4. embryonic stem cells can be harvested and grown up in culture.
5. When they have acquired the desired properties, they can be implanted in the donor with no fear of rejection.

Their procedure:

• removal of the spindle and thus all nuclear material from secondary oocytes at metaphase of meiosis II;
• fuse each enucleated egg with a skin cell taken from a male monkey;
• culture until the blastocyst stage is reached;
• extract embryonic stem cells from the inner cell mass;
• establish that they have the nuclear genome of the male (but mostly the mitochondrial genome of the female);
• culture with factors to encourage differentiation: they grew cardiac muscle cells (which contracted), and even neuron-like cells;
• inject into SCID mice and examine the tumors that formed. These contained cells of all three germ layers: ectoderm, mesoderm,and endoderm.
• However, all attempts to implant blastocysts in the uterus of a surrogate mother in the hope of producing a clone of adult animals genetically identical to the skin cell donor failed.

While their rate of success simply producing ES cells was low, for the first time there is now a probability of being able to apply the procedure to humans. However, the goal for humans should be limited to developing the formation of patient-specific cells that

• could be used in cell-replacement therapy or, failing that,
• provide the material for laboratory study of the basis of — and perhaps treatment of — genetic diseases.

However, there are still questions with the method that must be answered.

Questions that Remain to be Answered

• Imprinted Genes.

  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.

• Aneuploidy.

  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).

• Somatic Mutations. This procedure also raises the spectre of amplifying the effect(s) of somatic mutations. [Link to discussion]

  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.

• Political Controversy.

  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).

Possible Solutions to the Ethical Controversy

"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.

Induced pluripotent stem cells (iPSCs)

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.

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.

• patient-specific cell transplants — avoiding the threat of immunological rejection — might be created for human therapy. And even if that is still a distant dream,
• these cells will provide the material for laboratory study of the basis of genetic diseases. Last year, for example, researchers succeeded in deriving iPSCs from patients with amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), and then causing them to differentiate into motor neurons (the cells affected in the disease) for study of their properties.

• harvesting some fully-differentiated fibroblasts from a sickle-cell mouse;
• reprogramming these to become iPSCs by infecting them with Oct4, Sox2, Klf4, and c-Myc;
• then removing (using the Cre-lox system) the c-Myc to avoid the danger of this oncogene later causing cancer in the recipient mice;
• replacing the β^S genes in the iPSCs with normal human β^A genes;
• coaxing, with a cocktail of cytokines, these iPSCs to differentiate in vitro into hematopoietic (blood cell) precursors;
• injecting these into sickle-cell mice that had been irradiated to destroy their own bone marrow (as is done with human bone marrow transplants). (Although the recipient mice were different animals from the fibroblast donor, they were of the same inbred strain and thus genetically the same — like identical human twins. So the procedure fully qualifies as "patient-specific", i.e., with no danger of the injected cells being rejected by the recipient's immune system.)

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.

Despite these successes, iPSCs may not be able to completely replace the need for embryonic stem cells. Several groups have found that iPSCs produced from adult mouse cells still retain some of the epigenetic marks (e.g., methylation of their DNA) of their tissue of origin and are biased toward differentiation into similar cell types.

Other approaches being explored

• ES cells can be derived from a single cell removed from an 8-cell morula. The success of preimplantation genetic diagnosis (PGD) in humans shows that removing a single cell from the morula does not destroy it — the remaining cells can develop into a blastocyst, implant, and develop into a healthy baby. Furthermore, the single cell removed for PGD can first be allowed to divide with one daughter used for PGD and the other a potential source of an ES cell line.
  
  
• In altered nuclear transfer (ANT) — a modified version of SCNT (somatic-cell nuclear transfer) — a gene necessary for later implantation (Cdx2 — encoding a homeobox transcription factor) is turned off (by RNA interference) in the donor nucleus before the nucleus is inserted into the egg. The blastocyst that develops
  • has a defective trophoblast that cannot implant in a uterus;
  • but the cells of the inner cell mass are still capable of developing into cultures of ES cells. (The gene encoding the interfering RNA can then be removed using the Cre/loxP technique.)
• Jose Cibelli and his team at Advanced Cell Technology report in the 1 February 2002 issue of Science that they have succeeded in
  • stimulating monkey oocytes to begin dividing without completing meiosis II (therefore still 2n)
  • growing these until the blastocyst stage, from which they were able to harvest
  • ES cells.
  If this form of cloning by parthenogenesis works in humans [It does! — success with unfertilized human eggs was reported in June 2007.], it would have
  • the advantage that no babies could be produced if the blastocyst should be implanted (two identical genomes cannot produce a viable mammal — probably because of incorrect imprinting);
  • the disadvantage that it will only help females (because only they can provide an oocyte!) (But men may have a procedure that works for them — below.)
• On 24 March 2006, Nature published an online report that a group of German scientists had been able to derive pluripotent stem cells from the stem cells that make spermatogonia in the mouse. Both in vitro and when injected into mouse blastocysts, these cells differentiated in a variety of ways including representatives of all three germ layers. If this could work in humans, it would
  • provide a source of stem cells whose descendants would be "patient-specific"; that is, could be transplanted back into the donor (men only!) without fear of immune rejection.
  • avoid the controversy surrounding the need to destroy human blastocysts to provide embryonic stem cells.
• The 7 January 2007 issue of Nature Biotechnology reports the successful production of amniotic fluid-derived stem cells ("AFS"). These are present in the amniotic fluid removed during amniocentesis. With the proper culture conditions, they have been shown to be able to differentiate into a variety of cell types including
  • ectoderm (neural tissue)
  • mesoderm (e.g., bone, muscle) and
  • endoderm (e.g., liver).
  So these cells are pluripotent. Although perhaps not as versatile as embryonic stem cells, they are more versatile than adult stem cells.

Applied to humans, none of the above procedures would involve the destruction of a potential human life.