Totipotency

TOTIPOTENCY

Totipotency is defined as the ability of a single cell to divide and produce all the differentiated cells in an organism, including extraembryonic tissues. Totipotent cells formed during sexual and asexual reproduction include spores and zygotes. In some organisms, cells can dedifferentiate and regain totipotency. For example, a plant cutting or callus can be used to grow an entire plant. Mammalian development commences when an oocyte is fertilized by a sperm forming a single celled embryo, the zygote.

The zygote is totipotent, meaning that this single cell has the potential to develop into an embryo with all the specialized cells that make up a living being, as well as into the placental support structure necessary for fetal development. Thus, each totipotent cell is a self-contained entity that can give rise to the whole organism. This is said to be true for the zygote and for early embryonic blastomeres up to at least the four-cell stage embryo.

Experimentally, totipotency can be demonstrated by the isolation of a single blastomere from a preimplantation embryo and subsequently monitoring its ability to support a term birth following transfer into a suitable recipient. This approach was pioneered in rats and has been realized in several mammalian species including nonhuman primates. The ability of isolated blastomeres from two- and four-cell stage, IVF produced embryos of the rhesus monkey to support term pregnancies and to produce live animals. As embryo development progresses to the eight-cell stage and beyond depending on the species, the individual blastomeres that comprise the embryo gradually lose their totipotency. It is generally believed that this restriction in developmental potential indicates irreversible differentiation and specialization of early embryonic cells into the first two lineages, the inner cell mass that includes cells that will give rise to the fetus and the trophectoderm, and an outer layer of cells that is destined to an extraembryonic fate.

A complication in assessing the state of potency of blastomeres isolated from more advanced stages of development is insufficient cytoplasmic volume. Thus, although the blastomeres may in fact be totipotent, embryonic development of relatively small isolated blastomeres arrests at or near the time of blastulation. The zygote and early blastomeres undergo several unusual mitotic or cleavage divisions that are not accompanied by a corresponding growth of cytoplasm, that is, there is no change in embryo size despite the presence of more cells or blastomeres and each individual blastomere becomes smaller. The embryonic genome at these early stages is transcriptionally quiescent and development is regulated by maternally inherited factors present at the time of fertilization in the oocyte. The transition in developmental regulation with activation of the embryonic genome and a complete loss of dependence on oocyte factors occurs before the blastocyst stage in a species-specific manner. Additionally, by the late morula or early blastocyst stage the embryo ceases cleavage divisions and resumes normal mitotic divisions with concomitant increases in cell volume during the S-phase.

The likelihood that early blastomeres retain totipotency for a major part of preimplantation development but experimentally we cannot prove it is directly supported by the fact that the addition of oocyte cytoplasm to a blastomere of the eight- to sixteen-cell stage embryo can restore, or perhaps more appropriately allow expression of, its full developmental potential. This approach, embryonic cell nuclear transfer, has been employed in the monkey to demonstrate the totipotency of eight- to sixteen-cell stage blastomeres whereby reconstructed embryos when transferred to a recipient resulted in a term birth.

It is also known that conglomerates of embryonic cells at a later stage of development can develop into an organism. An experimental manipulation that supports this concept involves blastocyst splitting. Cutting the embryo into halves with an approximately equal distribution of trophectoderm and inner cell mass cells can lead to the production of viable infants. Obviously, embryo splitting that creates demi embryos with highly distorted ratios of inner cell mass to trophectoderm cells is inconsistent with the production of live births.

Pluripotency

It is "having more than one potential outcome." In cell biology, the definition of pluripotency has come to refer to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm, mesoderm or ectoderm. Pluripotent stem cells can give rise to any fetal or adult cell type. However, a single cell or a conglomerate of pluripotent cells cannot develop into a fetal or adult animal because they lack the potential to organize into an embryo. In contrast, many progenitor cells that are capable of differentiating into a limited number of cell fates are described as multipotent. Somatic stem cells such as neural, bone marrow-derived, or hematopoietic cells would fit into this latter category.

At least some of the embryo's inner cell mass cells are pluripotent, meaning that they can form virtually every somatic and germ cell type in the body. These inner cell mass cells are self sustained and their pluripotency is maintained by endogenously expressed factors. In vivo, pluripotent cells within the inner cell mass exist transiently; as the developmental program unfolds they differentiate into cells of the next embryonic or fetal stage. However, they can be isolated, adapted and propagated in vitro in an undifferentiated state as embryonic stem cells. Embryonic stem cells were first derived in nineteen eighty-one from the inner cell mass of the inbred mouse by Martin and Evans and Kaufman. In nineteen ninety-eight, embryonic stem cells were successfully isolated from surplus, IVF-produced human embryos.

Embryonic stem cells express specific markers or characteristics similar but not identical to the transient pluripotent cells of an embryo. This includes stage specific embryonic antigens, enzymatic activities such as alkaline phosphatase and telomerase, and "stemness" genes that are rapidly down-regulated upon differentiation, including OCT4 and NANOG. Under specific conditions, embryonic stem cells can proliferate indefinitely in an undifferentiated state, suggesting that the transcriptional activity and epigenetic regulators capable of supporting pluripotency can be maintained in vitro in embryonic stem cells. However, when released from the influence of these culture conditions or following their introduction back into a host embryo, embryonic stem cells retain their ability to differentiate into any cell-type, just like inner cell mass cells. Alternatively, they can differentiate in vivo in teratomas into cells representing the three major germ layers: endoderm, mesoderm and ectoderm or they can be directed to differentiate in vitro into any of the two hundred plus cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human embryonic stem cells may become an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating diseases.

Parenthetically, one of the challenges before clinical transplantation studies involving human embryonic stem cells can begin concerns the immune response anticipated after transplantation. Human embryonic stem cells are routinely derived from IVF embryos and transplantation of such cells into genetically unrelated patients will incite an immune response and result in rejection. Histocompatibility is one of major unsolved problems in transplant medicine. Rejection of unmatched transplanted tissues is provoked by alloantigens present on graft tissues by the recipient's immune system.

The alloantigens or antigenic proteins on the surface of transplant tissues that mostly cause immune rejection are the blood group antigens and the major histocompatibility complex proteins, also designated in humans as human leukocyte antigens. Matching donor and recipient HLA types is important to reduce a cytotoxic T-cell response in the recipient, and subsequently improve the chances of survival of the transplant. However, tissue or organ transplantation from one individual to another is a daunting task due to the existence of two classes of HLA molecules (Class One, and Two), each encoded by multiple genes and most importantly, each of these genes represented by multiple alleles. For example, there are twenty-two different alleles identified so far for the class one HLA-A gene and forty-two alleles for HLA-B. Thus, due to HLA polymorphism, the chances of finding a donor-recipient match based on just a few HLA genes (HLA-A, -B, and -DR) could be one in several million. Therefore, the need for developing approaches for deriving histocompatible pluripotent cells is commonly recognized.