Embryonic stem cell is a crucial factor in the developmental process of a multicellular organism. Embryonic stem cells (ESCs) are derived at early stages during development and eventually give rise to all type of tissues and organs in an animal. The ES cell lines have three properties consisting of self-renewal, primary chimera formation and pluripotency [1]. Pluripotency, a key feature of ESCs, refers to the capacity of a cell to differentiate into several different types of tissues [2]. An array of cellular factors play important role in maintaining pluripotency and determining cell fate in the developmental pathway. Negative effects on these factors may pose adverse consequences on the embryonic development. Over the past few decades, numerous studies have been conducted to identify these key factors and to understand their roles in pluripotency and cell differentiation. Also, a significant amount of research is being conducted to manipulate these cellular factors to make use of ESCs in veterinary science, bio-medical science, tissue transplantation, tissue recovery, wildlife conservation and many more real-life applications. Manipulation of embryonic stem cells deals with pre-implantation embryo and leads to killing of potential life-form. Therefore, studies are also being conducted to invent ways so that the pluripotent stem cells can be generated from any somatic cell and these induced stem cells then can be used in different bio-medical applications. The concept of pluripotency and cell differentiation, the roles of the major molecular factors involved in this process and how these factors are being manipulated to benefit mankind are further discussed in this essay.
Multicellular organisms evolve from a fertilized single cell that replicates and specializes into different tissues and eventually into different organs to form an individual organism. Fertilization refers to a process where two homozygous cells (sperm and egg) merge to form one heterozygous cell (the zygote). This heterozygous zygote is the starting point of the development of a multicellular organism comprised of tissues and organs with completely different structure and functionality. Immediately after fertilization the growth and replication of this single cell takes place. This replication process continues with the division and formation of cell organelles such as nucleus, mitochondria, and ribosomes and eventually of the whole cytoplasm to hold the integrity and functionality of the infrastructure of the resulting daughter cells [3]. Once cell replication is triggered after fertilization, cells in early embryo enter a stage known as morula that is characterized by multicellular sphere formation [3]. Subsequent stage is known as blastula characterized by the presence of a cavity (blastocoel) in the middle of the multicellular sphere structure. The inner cells of mammalian blastula are called blastocyst and embryonic stem cells are primarily derived from this cell mass [4].
Cell differentiation starts in a subsequent stage of development known as gastrula. This stage is characterized by the formation of rudimentary form of mouth known as blastopore and (in vertebrates) a precursor of backbone known as notochord [5]. In the case of a higher form of vertebrate organism (including human), the formation of heart, bones, muscles and nervous system take place after these basic organs develop. The development continues to form a fetus and eventually to form an individual.
This complex phenomenon of development of a whole organism from a single cell indicates that there is a universal template lies within this single cell that provides it with the potential to differentiate into all type of cells required to form an individual. Furthermore, there seem to be a decisive force that influences the cells decision to remain as a pluripotent cell or to commit on to a specific development pathway. This decisive force includes various intrinsic factors such as DNA configurations, transcription factors and gene expressions. Micro-environment contributes as an extrinsic factor in maintaining pluripotency and/or in the differentiation of pluripotent cell by providing signals and cues that affect intrinsic factors.
At early stage of developmental process, it is crucial for the embryonic cell to maintain its pluripotency and self-renewal capacity. Pluripotency of the embryonic stem cells is attributed to the pluripotent genome characterized by unique epigenetic features such as histone modifications. One of the epigenetic characteristics essential to pluripotent cell is the presence of sufficient level of histone modification namely H3K9 acetylation. In one of the studies, embryonic stem cell line E14 ESC was observed to be less pluripotent. Correlatively, the E14 ESC cells contained a significantly low amount of H3K9 acetylation. To find out that if this correlation between less pluripotency and low level of H3K9 actually suggests a causative relation or not, the less potent E14 ESC cells were treated with histone deacetylase inhibitors (HDACi). Microarray and CHIP-seq analysis performed on the treated cells demonstrated that HDACi increased the pluripotency in these cells by enabling them to produce ECM (extracellular matrix) [6]. This provides evidence that pluripotency of ES cells depends on the epigenetic feature H3K9 acetylation.
Pluripotent embryonic stem cell is also characterized by highly capable DNA repair mechanism that provides the pluripotent stem cell with extreme self-renewing capacity [10]. The role of DNA repair in maintaining pluripotency and self-renewal capacity of the embryonic stem cells has been proven in many studies. Damages and failures in the DNA structure can yield devastating consequences in the integrity of the embryonic stem cells and/or the subsequent differentiated cells. Double strand breaks in DNA can accumulate and this damage can eventually cause exhaustion of the pluripotency of the embryonic stem cells and may also lead to the cell’s death. This process of DNA damage accumulation is believed to be the primary cause of aging [7].
Another feature of pluripotent embryonic stem cell is the short growth (G-1) phase in cell division cycle. The length of this cellular growth phase is regulated by LIF pathway. Coronado and colleagues showed that LIF withdrawal results in longer growth phase and cells with longer G1 phase starts showing lineage specific marker. Supplementation of LIF returns the cell into self-renewing naïve ES stage with shorter G1 phase. In their experiment, FUCCI reporter system was used to track down cell cycle progression and distinguish cell’s in the G1 phase. The experiment also demonstrated the role of Cyclin E in the duration of G1 phase and consequently self-renewing capability of ESCs. Cyclin E is a key factor that initiates the transition from G1 phase to S phage. As a result, overexpression of Cyclin E1 increases the self-renewing capability of stem cells by shortening the G1 phase. On the other hand, when Cyclin E is knocked down, cells exhibit longer G1 phase and starts the differentiation process [8].
Wnt signalling pathway, originally identified as cell signalling mechanism in carcinogenesis, plays role in maintaining pluripotency. In an experiment, the role of wnt receptor FZD7 was explored in maintaining undifferentiated pluripotent state. Using RNA-seq data set and qRT PCR, it was shown that FZD7 gene was the most highly expressed FZD gene in undifferentitated cells. On the other hand, FZD7 was significantly downregulated in already differentiated cells. Also, when the FZD7 function was disrupted by short hairpin RNA-mediated knockdown and/or FZD7 specific Fab (fragment antigen binding molecule), the consequent adverse effect on Wnt signaling pathway led to the decrease in pluripotency in hESCs. This finding suggests that cell surface molecule such as FZD7 in addition to other receptors FGF, insulin like growth factor, epidermal growth factor receptors, E-cadherin and L1 adhesion molecule play crucial role in maintaining pluripotent state [9]. In addition to this surface receptors, pluripotency in embryonic stem cell is also maintained by a suite of nuclear transcription factors consisting of Oct4,Sox2, Klf4, Myc, and Nanog [10].
LIF pathway activates STAT3 that plays role in “stemness” state in mouse embryonic cell. Unlike mouse ESCs, human ESCs (hESC) does not maintains their undifferentiated state through LIF (leukemia inhibitory factor)/ gp130 pathway. Human embryonic stem cells require other extrinsic factors secreted from feeder layers in order to maintain their pluripotent state. In vitro, mouse feeder layer can keep human embryonic cells in undifferentiated pluripotent state by secreting extrinsic factor known as activin A. As a result, the pluripotent state in hESCs can be maintained for a long term without the help of any animal feeder layer when cultured in a media containing activin A, nicotinamide and keratinocyte growth factor. Human ESCs maintained in such way exhibit the expression of regular pluripotent markers such as Oct-4, nanog, TRA-1-60 [11]. This demonstrates that actin A can play an important role as a soluble extrinsic factor in maintaining pluripotency in hESCs.
A Recent study identifies the factors that lead to exit from a pluripotent state in order to pave the way for lineage commitment at later developmental stages. The pluripotent embryonic stem cells occur in two different state namely the naïve and the primed states; where the former indicates a stable state and the latter indicates preparation of differentiation of the cell. This finding improves the knowledge of pluripotency and its pre-differentiation mechanism. The shift from naïve to primed pluripotent cell is facilitated by the dissolution of the core pluripotency transcription-factor circuit. This involves expression of tumor suppressors Tsc1/2,Folliculin (Flcn), and Flcn’s interaction factors (Fnip1 and Fnip2). In the experiment, shRNA knock down of Flcn kept cells uncommitted after withdrawing other inhibitors and even after exposing to diffferentiation facotrs actin A, fibroblast growth factor (FGF4) and BMP4 or bovine serum. This demonstrates the importance of Flcn to exit naïve pluripotent states. Activities of Flcn in committing the cell to primed pluripotent state mainly involve inhibition of bHLH associated Tfe3 transcription factors that consequently reduces the balance in pluripotency [12].
Fibroblast Growth Factor (FGF) and Copper Transport protein (Ctr-1) have been identified as another two key differentiation factors. FGF contributes in mesoderm development through Ras-MAPK pathway. Ctr1 is a key regulatory factor in this FGF induced pathway that ensures normal embryonic development. Mouse embryonic stem cell that is homozygous and deficient for Ctr1 (Ctr1 -/-) remains undifferentiated and pluripotent even in the presence of differentiation-favorable condition. In Xenopus, Ctr1 is associated with tyrosin kinase Laloo and helps FGF in mesoderm differentiation. Disruption of Xctr1 also effects adversely ectoderm formation in frog embryo [13].
In addition to the cellular factors mentioned above, epigenetic diversity created by histone and nucleic acid modifiers such as DNA methyl tansferase play important roles in differentiation process. This epigenetic diversity acts as a suite of potentials at molecular level. In this process, The enzyme DNA methyltransferase moves methyl groups on the DNA strand (methylation process) and this process consequently increases the epigenetic diversity of pluripotent cells based on the location of methyl groups along the DNA strands. Downregulation of DNA methyl transferase 3a nd 3b (Dnmt3a/3b) by demethylase enzymes can increase self-renewal capacity in ESCs. This inhibits cell differentiation [14].
In addition, an experiment performed on Xenopus embryo showed that activin and FGF also play a crucial role in cell differentiation. In a dose-dependent manner, Activin can induce 5 different cell types including epidermis, postlateral mesoderm, muscle, notochord and neural inducing organizer cells. It causes muscle differentiation when presented in relatively narrow range of concentration. On the other hand, higher dose of acitvin is required to induce notochord differentiation. FGF induce expression of postereolateral cell type and it mainly modules the activity of activin [15].
The generic pattern of the differentiation process from the progenitor ESCs into specialized cells is still under investigation [16]. However, several studies show that specific inductive signals and transcription factors direct the differentiation process of the progenitor cell towards specific tissue and organs. For example, heart development in vertebrates is regulated by Nkx2-5 homeodomain protein, HLH protein, Mef-2 family protein, zinc finger protein GATA-4, and cardiac-restricted ancyrin repeat protein (CARP) [16]. Muscle cell differentiation takes place under the direction of MyoD, Myf5, Myogenin, and MRF4 regulatory factors [17]. Inductive signal derived in the presence of signalling agent such as the retinoic acid (RA) drives the differentiation of ESCs into motor neuron progenitor cells. Thus, recovery of parts of the nervous system such as spinal cord and axon can be achieved, as these induced motor neuron cells can populate the designated parts of the neural system [18].
Any disruption to these molecular regulatory facotrs can have serious detrimental effect on developmental process. Disruption to the transcription regulation may occur in form of overexpression, downregulation, or even a complete absence of transciption regulatory factors. The deviation of transcriptional regulation activity from normal level may lead to developmental disorder such as the neural tube development syndrome [19]. Also, disruption to the pluripotent ESCs by toxin such as ethanol can affect the transcription process of Oct4 and Sox2 and consequently diverts early neuronal differentiation. This can lead to neurodevelopmental deficit such as the Fetal Alcohol Syndrome Disorders [20].
An example of embryonic stem cell application in regenerative medicine is the utilization of ESCs for regenerating skin tissues after severe burn or severe allergic reaction. Under normal condition, skin lesions are replaced by connective tissue resulting in the formation of scar. Using the pluripotency of the embryonic stem cells, recovery of skin lesion can be done by introducing the stem cells into the lesion along with the appropriate trigger factor(s) to induce stem cell differentiation into skin tissue [21]. This will avoid the formation of scar tissue and will ensure smooth recovery of the skin. Recently, functional pancreas and kidneys have been generated from porcine embryonic stem cells indicating that missing organs can be regenerated from exogenic pluripotent cells in vivo [22].
Recent studies indicate that pluripotency is not exclusive to embryonic stem cell derived from the blastocyst, as it can also be induced in already differentiated somatic cells using certain laboratory procedures. The induction of pluripotency is a process that goes in opposite direction to the differentiation process of embryonic stem cells. This reversal of cell differentiation process does not contribute directly to the development of the organism, but it offers enormous possibility for application in bio-medical science. The use of induced stem cells eliminates the needs to harvest embryonic stem cell directly from animals and human being. This circumvents ethical issues that may arise from the use of conventional harvest and use of animals and humans embryonic stem cells [23].
This revolutionary invention of inducing pluripotency in already differentiated somatic cell or iPSC technique was demonstrated in the experiment conducted by Takashaki and Yamanaka in 2006. In this experiment, four major factors (Oct3/4, Klf4, c-Myc and Sox 2) normally responsible for pluripotency in embryonic stem cell were used to induce pluripotency in mice fibroblast cells. They originally injected 24 genes usually acitve in embryonic stem cells to mice skin fibroblast cells using retrovirus and were able to narrow down the search to four major essential factors mentioned before. A stem cell specific gene Fbx15 was tagged with βgeo cassette (β-galactosidase and neomycine resistant gene) reporter gene, so the fibroblast cells that were transformed into pluripotent stem cells could be selected in a neomycine containing media. The study, however, ended leaving the question unanswered that if iPSC can be applied to induce pluripotency in human embryo or not [4].
In 2007, a major stride in regenerative medicine took place when pluripotency could be induced successfully in human fibroblast cell using the same method and factors used in the experiment with mice. The only difference is a different media had to be used to carry out the transformation. It demonstrates that the four major factors (Oct3/4, Klf4, c-Myc and Sox 2) are common in the process of inducing pluripotency in human and mouse; but they require two different microenvironment containing different extrinsic factor and signals. As using the retrovirus to induce pluripotency gave rise to cancer in 20% mice, non retrovirus methods such as adenovirus, cell permeable proteins or extrisnic factors to induce pluripotency is being developed. Another shortcoming of the experiment is that the induced stem cells were found to be different from human embryonic stem cells when a DNA microarray analysis was performed. Once these obstacles are overcome, iEPS can be used in patient-specific drug screening, organ transplant etc. [23].
Recently iPSC technology has been successful in restoring eysight in several patients. In an experiment, induced stem cells were successfully used to regenerate retinal pigment epithelia or retinal photoreceptor cells. The induced stem cells differentiated in vitro expressed several eye cell specific markers such as transcriptor factor genes Rf , Mitf, homeobox gene Crx and mature eye cell specific recoverin and rhodopsin in the presence of Wnt and Nodal antagonist. The iPSC cells that were initially obtained using Yamanaka factors at first were grown in culture free of bFGF (basic fibroblast grow factor) to induce differentitation. Over a period of 25 days to 8 months, all retina specific genes were observed to be expressed in these cells. Comparison made through PCR, immunocytochemistry, Immunoblot analysis, and ROS phagocytosis illustrated that the iPSC-RPE cells shared a great similarity with fRPE cells.The RPE (retinal pigment epithelia) cells obtained in this method can now effectively be used to treat age-related macular degeneration (AMD) [24].
So, in conclusion, pluripotency is the power that gives embryonic stem cells the ability to develop into any tissues and organs in the course of embryonic development. Without pluripotency a single cell can never differentiate into specific tissues and a multicellular organism can never form. The combined effects of different extrinsic and intrinsic factors play a very crucial role in this development process. Any disruption to these factors may give rise to various developmental disorders. Also, due to its vast potential, pluripotent embryonic stem cell also offers a wide range of bio-medical applications that can benefit mankind. Enormous studies on various factors that affect stem cell pluripotency and differentiation had been conducted over the past few decades. A lot more researches yet to be done in the coming years to elaborate our understanding and to improve technique to further manipulate these factors. This will allow us to genetically engineer pluripotent cells into tissue, organs, and may be a whole organism without any detrimental side effects. Ethical issues may arise from the possibility of such manipulation, but it all comes back to the responsibility and integrity of mankind to utilize this enormous capacity of a pluripotent embryonic stem cell to benefit nature in general.
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