Scope of Embryonic Stem Cells in research

Abhijit Ray*

HOD, Department of Biotechnology, Raipur Institute of Technology, Raipur (CG)

*Corresponding Author Email:abhijitray_2001@yahoo.com

ABSTRACT:

Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the embryoblast orinner cell mass (ICM) results in destruction of the fertilized human embryo. They can be propagated as homogeneous stem cell cultures and expanded without apparent limit. ES cells retain the character of embryo founder cells, even after prolonged culture and extensive manipulation. Thus, they are able to reintegrate fully into embryogenesis when returned to the early embryo. The ability to propagate pluripotent ES cells presents unique opportunities for experimental analysis of gene regulation and function during self-renewal, cell commitment, and differentiation. The combination of intrinsic and extrinsic factors that maintain developmental identity and potency is beginning to be defined. Progress is also being made toward understanding and controlling lineage- and/or cell-type-specific differentiation of ES cells in vitro.

KEYWORDS: Embryonic stem cell, embryo, pluripotency, fetus, cell culture.

INTRODUCTION:

Embryonic stem cells are distinguished by two distinctive properties:

§ Their pluripotency

, and

§ Their ability to replicate indefinitely (Ying et al., 2003).

ES cells are pluripotent, that is, they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, andmesoderm. These include each of the more than 220 cell types in the adult body. Pluripotency distinguishes embryonic stem cells from adult stem cells found in adults; while embryonic stem cells can generate all cell types in the body, adult stem cells are multipotent and can produce only a limited number of cell types. Human ES cells measure approximately 14μm while mouse ES cells are closer to 8μm (Thomson and Zwaka 2010).

Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely. This allows embryonic stem cells to be employed as useful tools for both research and regenerative medicine, because they can produce limitless numbers of themselves for continued research or clinical use.

Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. Diseases that could potentially be treated by pluripotent stem cells include a number of blood and immune-system related genetic diseases, cancers, and disorders; juvenile diabetes; Parkinson's; blindness and spinal cord injuries. Besides the ethical concerns of stem cell therapy, there is a technical problem of graft-versus-host disease associated with allogeneic stem cell transplantation.

However, these problems associated with histocompatibility may be solved using autologous donor adult stem cells, therapeutic cloning, stem cell banks or more recently by reprogramming of somatic cells with defined factors (e.g. induced pluripotent stem cells). Other potential uses of embryonic stem cells include investigation of early human development; study of genetic disease and as in vitro systems for toxicology testing.

Pluripotent Stem Cells in the Early Embryo

The mammalian fetus develops from a founder population of cells that are present before and shortly after implantation. These cells are pluripotent, meaning that they are individually capable of giving rise to derivatives of each of the three primary germ layers and to germ cells. Initially defined as the entire internal cell component of the blastocyst, the inner cell mass (ICM), pluripotent cells are segregated to a subcompartment, the epiblast, prior to implantation. After implantation the epiblast expands rapidly to generate the cellular substrate for gastrulation and formation of the embyro proper. Once gastrulation commences, the epiblast cells (often termed primitive or embryonic ectoderm at this stage) progressively differentiate into definitive mesoderm, endoderm, and ectoderm.

However, a range of experimental interventions in mouse embryos have revealed that epiblast cells are highly plastic and that their self-renewal and differentiation are regulated according to the embryonic context. Thus, the ICM and epiblast can adjust to either the removal or addition of significant numbers of cells and still give rise to a normal fetus. Indeed, this capacity to accommodate extra cells provides the foundation on which chimeric fetuses are produced. Most strikingly, if the early mouse embryo is removed from the uterus and the epiblast cells are grafted to a permissive ectopic site, such as the testis or kidney capsule of a syngeneic or immunocompromised mouse, they will generate large multidifferentiatedtumors known as teratocarcinomas. Teratocarcinomas contain differentiated cell types of all germ layers and, in addition, an undifferentiated, proliferative component that can be maintained on serial transplantation. Teratocarcinomas can be produced at a high frequency from single epiblasts, but not at all from postgastrulation embryos. The persistence and expansion of undifferentiated stem cells in embryo-derived teratocarcinomas indicates that mouse epiblast cells do in fact have an intrinsic potential for prolonged self-renewal.

PLURIPOTENCY OF EMBRYONIC STEM CELLS

Teratocarcinoma Formation

ES cells closely resemble EC cells in morphology, growth behavior, and marker expression. This relationship extends to the capacity to give rise to multidifferentiatedteratomas and teratocarcinomas. ES cells readily produce tumors containing well-differentiated mesodermal, ectodermal, and endodermal tissue and cell types. The representation of undifferentiated stem cells in the tumors tends to be less than in EC cell-generated teratocarcinomas, most likely reflecting the latter’s history of tumor selection. The ability clonally to give rise to teratocarcinomas is a defining feature of pluripotent embryo cells, shared by ES, EG, and EC cells.

Integration into the Developing Embryo

The most extraordinary feature of ES cells is that, even after extended propagation on tissue culture plastic in synthetic media, they remain capable of participating in normal embryogenesis. Several techniques can be used to introduce ES cells into the preimplantation mouse embryo, but regardless of method of delivery, the ES cells can colonize all fetal lineages plus yolk sac mesoderm. Consistent with their epiblast origin, ES cells contribute poorly to extraembryonic endoderm and rarely, if ever, to trophoblast. In contrast to EC cells, ES cells behave relatively consistently in their ability to integrate into the embryo and produce viable chimeras. ES cells produce functional differentiated progeny in all tissues and organs. Incorporation into embryogenesis not only confirms that ES cells are pluripotent, but also demonstrates that they can respond appropriately to developmental cues for proliferation, differentiation, migration, and patterning. ES cells thus retain in full the identity and capacity of resident epiblast cells.

Targeted Gene Modification

The major use of ES cell genetic modification to date, however, has been for the directed modification of nominated genes, known as gene targeting. Pioneering work in the mid-1980s established that transfected DNA could be integrated into designated loci in the ES cell genome via homologous recombination. In 1989 the first incidence of germ-line transmission of a targeted allele was reported, demonstrating that the manipulations and drug selections involved in isolating homologous recombinant clones did not in themselves compromise ES cell pluripotency. There are now well-established procedures for introducing a range of different types of modifications, such as deletion, point mutation, reporter insertion, or coding sequence replacement, into the mouse genome. Conditional mutations can be created by incorporation of site specific recombinase technology.

Chromosome Engineering

The use of site-specific recombination can be extended to the engineering of long-range modifications in the ES cell and thence the mouse genome. Deletions, inversions, duplications, or translocations can be generated according to the respective orientation and cisor translocalization of the recombinase recognition sequences. This is a powerful method for interrogating the genome, increasingly so with the amassing of sequence information and gene localization data. Autonomous chromosomal elements have also been introduced into ES cells via cell fusion. These minichromosomes can be maintained stably in ES cells and chimeras, and in some cases, can be transmitted through the germ line. This creates the foundations of a system for genetic dissection of centromere function in mammalian mitosis and meiosis. Minichromosome vectors may also find applications in biotechnology; for example, the creation of humanized antibodies.

HUMAN EC CELLS

While embryogenesis in the laboratory mouse became progressively more accessible to experimental study, analysis of human development remained, and indeed still remains, severely restricted, not only by ethical considerations but also by the logistical problems of working with human embryos. Nevertheless, although recent developments in biology indicate a strong conservation of regulatory mechanisms throughout phylogeny, stretching from the nematode worm all the way to mammalian development, there is no doubt that each species presents unique features and that human development differs in significant ways from that of other mammals. Human EC cell lines provide an opportunity to investigate mechanisms that regulate embryonic cell differentiation in a way that is pertinent to early human development, while also shedding light on a medically significant form of cancer. Cell lines were first derived from human germ cell tumors and maintained as xenografts in the 1950s. Later, several lines were established in culture, notably TERA1, TERA2, and SuSa, described during the 1970s. Initial studies of these cell lines highlighted similarities with the mouse EC cells. In particular, some of the human GCT-derived cell lines were reported to express the F9 antigen and, later, SSEA1. It was first assumed that this was consistent with human EC cells expressing the F9 antigen, like murine EC cells. However, a comparative study by Andrews et al. (1980) of a range of cell lines derived from GCT, and a more detailed analysis of one of these, 2102Ep (Andrews et al. 1982), led to the conclusion that human EC cells differ in a number of respects from their murine counterparts. In particular, SSEA1, which had become a hallmark of murine EC cells, appeared not to be expressed by human EC cells, in contradiction to the earlier studies, although it is expressed by some derivative cells following differentiation.

PRIMATE ES CELL LINES

Although human EC cells resemble mouse EC cells in some respects, they differ from them in a number of important areas. Thus, the relationship of human EC cells to human embryonic stem (ES) cells was, to some extent, in question not least because of the extensive chromosomal changes seen in EC cells. However, recently, primate ES cell lines have been isolated from rhesus monkeys, common marmosets, and human, and they have proved to share many of the characteristics previously defined for human EC cells. The primate ES cells were derived by plating an isolated ICM on fibroblasts in the presence of serum, but in the absence of other exogenous growth factors. These conditions are similar to those used for the derivation of some human EC cell lines, and for the derivation of mouse ES cells prior to the identification of leukemia inhibitory factor (LIF) as a critical mediator of mouse ES cell self-renewal. Human embryonic germ (EG) cell lines have also been isolated from fetal germ cells plated on fibroblasts in the presence of serum, but their derivation required supplementation of the medium with LIF, basic fibroblast growth factor, and forskolin. The factors produced by fibroblasts that are required for human ES and EG cell self-renewal are unknown. Mouse ES cells remain undifferentiated and proliferate in the absence of fibroblasts if LIF, or other LIF cytokine family members such as ciliary neurotropic factor (CNF) or oncostatin M, are present.

CONCLUSION:

The past 20 years have been marked by a dramatic increase in our understanding of mammalian development, most notably in the laboratory mouse. In part, this has been a consequence of the revolution in molecular genetics, and the finding that many regulatory mechanisms have been highly conserved throughout phylogeny. It has also had its roots in the study of teratocarcinomas and their EC stem cells. Those studies provided a stimulus to thought and approaches to embryonic development at a time when other tools were limited. They provided access to key molecules and genes that play a role in development, and they continue to provide a convenient experimental system for addressing some questions about the mechanisms that regulate embryonic cell differentiation. The culmination of that work was the development of techniques for culturing ES cells, which now provide the key route to genetic manipulation of the laboratory mouse. Although human embryogenesis undoubtedly resembles that of other mammalian species, there are certainly differences at the morphological as well as at the cellular and molecular levels. Given the logistical as well as ethical and legal difficulties surrounding work with human embryos, teratocarcinomas and their EC stem cells derived from human germ cell tumors provide a useful tool for helping to translate findings in species more tractable to experimental study to the human situation. For example, we have investigated the pattern of expression of various glycolipid antigens, and there are certainly aspects of their expression that are distinct between human and mouse EC cells and, evidently, embryos. The recent characterization of human and monkey ES cells has validated the presumption that human EC cells do relate to pluripotent cells of the very early embryo, as in the mouse model, so that results from EC cell lines will continue to provide important pointers for future study. In some ways, the capacity of EC cell differentiation is limited, and the availability of human ES cell lines will extend the range of questions that can be addressed in vitro. On the other hand, the limitations of EC cells can sometimes be put to advantage, as EC cells provide a simpler and more robust experimental system. Human EC and ES lines are likely to remain complementary tools. Apart from their value as experimental tools, pluripotent stem cells are likely sources of specific differentiated cell types for tissue replacement therapies for a whole host of diseases. Some have already made a start in this direction using EC-cell-derived neurons. Eventually, ES cells may provide the source of choice for such treatments because of their evident “normality” and the lesser likelihood of reversion to a malignant phenotype. Nevertheless, our current ability to culture human ES cells, and our understanding of their biology, rests on many decades of work with their tumor-derived counterparts, the EC cells of murine and human teratocarcinomas.

REFERENCES:

1. Thomson; Itskovitz-Eldor, J; Shapiro, SS; Waknitz, MA; Swiergiel, JJ; Marshall, VS; Jones, JM (November 1998). "Blastocysts Embryonic Stem Cell Lines Derived from Human". Science 282(5391): 1145–1147. 

2. Ying; Nichols, J; Chambers, I; Smith, A (October 2003). "BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3". Cell 115 (3): 281–292. 

3. Thomson, J A.; Zwaka (2010). "Homologous recombination in human embryonic stem cells". nature biotechnology 21 (3): 319–321

Received on 20.03.2012 Modified on 14.04.2012

Accepted on 25.04.2012 © A&V Publication all right reserved

Asian J. Nur. Edu. & Research 2(2): April-June 2012; Page 51-54