Embryonic Stem Cells
Proliferation of Embryonic Stem Cells under Defined Conditions
1.1 Stem Cells
Stem cells are defined functionally as cells capable of perpetuating through self-renewal, while retaining the ability to generate differentiated cells . Fundamentally stem cells are a clonal, self renewing entity that can be multipotnent or pluripotent and thus can generate different cell types. These exceptional characteristics offer hope of cell replacement and regenerative therapy for a litany of currently untreatable diseases.
However in order to exploit stem cells to their full theoretical potential it is essential to completely understand their biological properties, and achieve their differentiation into clinically relevant populations of specific, functional cells and tissues. Stem cells also present many ethical concerns which will need to be addressed before their clinical application can be fully realised. Classified by their origin, stem cells may be embryonic, germinal, somatic (adult) or induced pluripotent stem cells (iPSCs).
1.2 Embryonic Stem Cells (ES Cells)
Embryonic stem cells are derived from the inner cell mass of the pre-implantation embryo after formation of a cystic blastocyst . These cells would usually produce the epiblast and ultimately all adult tissues. ES cells are the in vitro counterparts to the in vivo epiblast [1, 3] . The basic characteristics of an ES cell include: the ability to self renew; pluripotency; clonogenicity; the ability to retain a normal karyotype; indefinite proliferation in vitro under cell defined culture conditions, and they can be frozen and thawed. A central characteristic of ES cells is their pluripotency. Pluripotent cells have the ability to differentiate into any into all cells and tissues of the three primary germ layers: the endoderm, mesoderm or ectoderm.
In 1981 Evans and Kufman  isolated the first EC cells from the blastocyst of mouse pre-implantation embryos. This breakthrough was a product of tireless research into germ cell tumors, also know as teratocarcinomas. A teratocarcinoma is a spontaneous tumor of the testis in mice and humans, consisting of a diverse population of cells. As early as 1957, Stevens and Hummel  observed that the cells and tissues of germ cell tumors are varied, lacking in organization and at different levels of maturation. Current evidence suggests that germ cell tumors develop as a result of defective germ cell development during embryogenesis and because the majority of germ cells are found in the gonads, teratocarcinomas are primarily located in the ovaries and testes . In the 1970's developmental biologists confirmed that teratocarcinomas contained undifferentiated stem cells and could be induced in mice when embryos are inserted into extra-uterine sites. These cells, dubbed embryonal carcinoma (EC) stem cells, could be extracted and, under appropriate culture conditions, grown while maintaining their ability to differentiate . Developmental biologist began to search for a method to directly isolate stem cells from mouse embryos, in an effort to eliminate the teratocarcinoma stage. This was achieved by Evans and Kuffman in 1981who established the first mouse embryonic stem lines. This discovery paved the way for development of human embryonic stem lines in 1998 by Thompson et al .
1.3 Adult (Somatic) Stem Cells
Adult stem cells are undifferentiated cells found in differentiated tissues. Their ability to differentiate and self-renew is restricted in comparison to ES cells. They are generally limited to cell types of their original tissue. Adult stem cells maintain the ability throughout adult life to proliferate; they are capable of self-renewal and are multipotent. Multipotent stem cells are capable of differentiating into multiple, yet limited number of cells lineages. These abilities are essential for tissue homeostasis. Adult stem cells continuously supply new cells to restore populations of highly differentiated yet short-lived cell types such as blood, skin and sperm. The origin of adult stem cells in some mature tissues is still under investigation.
Adult stem cells are most frequently isolated from the mesoderm-derived bone marrow. Both hematopoietic and stromal mesenchymal stem cells are found in bone marrow. Mysenchymal cells may be valuable for future clinical application as they are capable of differentiating, in vivo and in vitro, into several types of adult mesenchymal tissues, including muscle, bone, cartilage and adipose . The first bone marrow transplant took place in 1968 for the treatment of severe combined immunodeficiency, and since the early 70's, bone marrow transplants are common practice for the tackling malignancies and immunodeficiency syndromes.
1.4 Induced Pluripotent Stem Cells (iPSC)
Induced pluripotent stem cells (iPSCs) are differentiated somatic cells that have been reprogrammed to a pluripotent state, capable of producing a cell from any of the three primary germ layers. This is achieved by the introduction of a defined set of transcription factors and culturing the cells under ES cell conditions. IPSCs were first described in 2006, through pioneering work by Shinya Yamanaka  and colleagues. This seminal study demonstrated that simultaneous retroviral-mediated introduction of the four transcription factors Oct4, Sox2, Myc and Klf4, could reprogram mouse fibroblasts to a pluripotent state. Since its publication several groups have applied the methodology to other types of mouse cells  and even to human somatic cells . In 2008, Hanna et al  reprogrammed terminally differentiated B-lymphocytes to iPSCs. The well defined reprogramming criteria make the procedure straightforward and have been independently repeated by several groups.
Continued research of both ES and iPSCs is vital. With our limited information of the molecular processes of reprogramming it is still premature to predict what theories will materialize regarding iPSC technology. The use of both ES cells and iPSCs allows the comparison of developmental processes, and the possible acceleration of our understanding of human development. Comparative analyses might also provide useful information as cell therapies are developed using human ES cell and iPSC sources.
1.5 Growth of Embryonic Stem Cells in Culture
Growth in Culture
Mouse ES cells can be maintained and grown rapidly in culture for indefinite passages under certain conditions that activate self-renewal pathways and thus retains their undifferentiated state [14-16].This has been shown conclusively by their complete integration into a developing embryo after being reintroduced into the blastocyst . It is established that ES cells in culture are destined for three fates: they can self-renew (divide into two identical daughter cells); differentiate (change their pattern of gene expression to become another cells type and continue to divide or proliferate as differentiated cells); or die, generally from a major (necrosis) or minor (apoptosis) insult .
Initial mouse and human embryonic stem lines were grown in the presence of a medium supplement, containing fetal calf serum (10%) and on a “feeder” layer of non-dividing cells that ensured the growth of the ESC in an undifferentiated state. The “feeder” cells were usually mouse embryonic fibroblasts (MEF) and were chemically treated to prevent cell division [4, 8, 19-20]. In 1988, the cytokine, leukemia inhibitory factor, was identified as the component secreted by the fibroblast feeder cells. It was shown that addition of LIF to the culture medium was a sufficient to sustain undifferentiated growth in the absence of the MEF [15-16].
Mechanism of LIF Action: STAT3 vs. MAPK/ERK
Leukemia inhibitory factor (LIF) belongs to the interleukin-6 (IL-6) cytokine family. Mice embryonic stem cells express the LIF receptor, which is a heterodimeric receptor complex consisting of the LIF-specific receptor subunit LIFRβ and the signal transducer gp130 (glycoprotein-130) . LIF exerts its effects by binding to LIFR, which results in activation of JAK (Janus-associate tyrosine kinase) . The activated JAK phophorylates several tyrosines of gp130 which then act as docking sites for proteins containing the Src homology 2 (SH2) domains, including the signal transducer and activator of transcription (STAT) family of transcription factors . In mice ECSs LIF predominantly activates STAT3.
The LIF/STAT3 pathway is essential for self-renewal and pluripotency in mESCs, knockout studies show that certain genes in the pathway are expendable
ICM development is not affected in mutant mouse embryos lacking these genes. Mice deficient in LIF develop normally , while mice deficient in LIF-receptors exhibit perinatal lethality [25-26]. Gp130-deficient embryos die after 12.5 dpc . Stat3-deficient embryos die around 6.5 dp .
This evidence would suggest that while contributing, the LIF/gp130/STAT3 pathway is not the sole protagonist in the complex molecular mechanism of embryonic stem cell self-renewal.
1.6 Transcription Factors: Regulators of Pluripotency
The transcription factors Oct4, Sox2 and Nanog comprise the core regulatory network that governs embryonic stem cell pluripotent identity . This trio of transcription factors appears to promote self-renewal by suppressing extra embryonic fate options through continual repression of trophoblast and hypoblast specifier genes, Cdx2/Eomes and Gata4/Gata6 respectively .
Oct 4 is a POU (Pit, Oct Unc) domain-containing transcription factor encoded by Pou5f1. Oct4 is a member of the Octamer group of transcription factors that recognize an 8-bp DNA site with the consensus ATGCAAAT [31-32]. This maternally inherited transcription factor is developmentally regulated in mice. It is present at low levels in blastomeres until activated at the four cell stage and is later restricted to the pluripotent cells. Oct4 is highly expressed in undifferentiated human and mouse ES cells and is diminished when cells differentiate and lose their pluripotency. A strict level of Oct 4 expression is essential for ES cells' pluripotent identity. It was found that a 150% increase above endogenous levels results in differentiation into ectoderm and mesoderm, while a 50% decrease causes differentiation into trophectoderm . However, ES cells expressing Oct3/4 constitutively from an exogenous promoter still required LIF for self-renewal .
As Oct4 expression must be maintained within certain parameters to ensure an undifferentiated state it is an excellent maker for undifferentiated ES cells. The IOUD2 ES cell line used in our study is genetically altered with a LacZ gene inserted directly after the Oct4 promoter, IOUD2 ES cells are therefore very valuable for assessing cellular division and self renewal.
Sox2 is transcription factor and belongs to the Sox (Sry-related HMG box containing) family of proteins that bind to DNA through the High mobility group (HMG) box DNA-binding domain. Oct4 and Sox2 are co-expressed in ES cells. The expression of Sox2 and Oct4 are regulated by Sox2 and Oct4, suggesting a positive feedback system may be employed. Abolition of Sox2 in ES cells, like Oct4, was found to cause differentiation towards the trophectoderm. Current belief is that Sox2 functions to maintain Oct4 expression. In 2007 Masui et al revealed that enforced expression of Oct4 can prevents ES cells differentiation induced by Sox2 loss .
In 2003, Chambers and Mataui [35-36] reported on the identification of Nanog a unique divergent homeodomain protein; it shares a maximum of 50% amino acid identity with the NK2 family.
Over expression of Nanog is sufficient for self-renewal in ES cells, even in the absence of LIF or other cytokines . However, Nanog cannot replace Oct4 and Sox2 function and is not regulated by STAT3 as Nanog and STAT3 exert their functions differently. Nanog and Oct4 work together to support ES cell pluripotency and self-renewal. This statement is supported by the discoveries that Nanog is expressed in Oct4 deficient embryos and that Nanog over expression cannot stop differentiation caused in ES cells by down regulation of Oct4 expression.
Nanog-null embryos die shortly after implantation. In vivo, Nanog is essential fro ICM determination and germ cell development. In culture, it prevents progression to differentiation and protects pluripotency.
1.7 Embryonic Stem Cells Potential
Embryonic stem cells (ESCs) can potentially give rise to any differentiated cell in the body. This unique ability coupled with their capacity to self-renew indefinitely in culture makes them ideal candidates for biomedical research, regenerative medicine, and tissue engineering and cell replacement therapies.
Human ESCs are potentially a valuable tool in disease modeling and the study of genetic abnormalities. There are many processes which we are incapable of examining in vivo in humans, in the past, mice and other animals, have provided good replacement models but the opportunity to examine these abnormalities in human cells is invaluable. ESCs could help clarify the genetic basis of diseases and lead to the identification of new targets for drug development.
However, it is the use of ESCs in regenerative medicine that has really caught the imagination of the scientific community. Many significant human diseases troubling society today are caused by loss or dysfunction of specific cell types in the body. Heart failure, diabetes, stroke, Parkinson's, neurodegenerative disorders, spinal cord injury, osteoarthritis, and kidney failure all result from absent or damaged cell populations that the body is unable to replace. It is predicted that these diseases may be treated by replacing damaged tissues with healthy cells derived from pluripotent sources.
This theory has been driven primarily by experiments in mice on experimentally induced or genetic lesions. Cardiomyocytes differentiated from mouse ESCs have been transplanted to the heart and survived , after receiving mouse ESC derived embryoid bodies rats with spinal chord lesion could again bear their own weight . Insertion of ES-derived neural cells in mice with Parkinson-like lesions caused a functional improvement . These studies are indeed encouraging but the question is now to what extent studies in mice can translate to humans?
1.8 Challenges before Clinical Application
Before allogeneic transplantation of ESCs becomes a reality, host immune mechanisms must be controlled. Several strategies have been formulated to address the problem, including lifelong immunosuppresssion, creating an ESC bank with a catalog of tissue types or even development of a universal donor cell.
Transplanted cell death is a major problem for regenerative therapy. Recent studies suggest that cell-death pathways are driven by stresses caused by transplantation, including ischemia, loss of matrix attachments and inflammation .
ESCs and iPSCs are pluripotent cells and raise serious safety concerns because they can potentially form teratomas upon transplantation. Results have shown that normal or injured adult tissues lack the cues required to induce ESCs to form appropriate differentiated cell types . To prevent teratoma formation, ESCs must be at least partially differentiated in advance, enriched for the desired cell type and screened for undifferentiated cells.
Before regenerative therapy can be considered it will be necessary to generate a sufficient number of desired cell types in a homogenous population. Since the isolation of both mouse and human ESC lines, there has been sizeable progress made in directing the differentiation of ESCs. Traditionally, ESCs are differentiated in suspension culture as embryoid bodies (EBs). EBs are small aggregates of ESCs enclosed by an outer layer of visceral ectoderm. EBs are so called because of their size, differentiation capacity and their likeness to the early post-implantation embryo. Differentiation of ESCs has been achieved by exposing cells of simple EBs to soluble factors or by introduction of transcription factors for near 20 years. Conditions that direct both human and mouse ESCs are well documented. Currently purification of desired differentiated cell types is required for most applications.
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