The Significance of Stem Cell Regulation

Before delving into the molecular biology and therapeutic potentials of induced pluripotent stem cells, it is crucial to provide foundational definitions and descriptions. A stem cell commences as an undifferentiated cell that can either undergo self-renewal, whereby it generates daughter cells that remain as stem cells, or mature into a specific cell type via differentiation (Can/Hematol 2008). Specific stem cell types possess unique potencies, or abilities to self-renew. That is to say, one type of stem cell may be capable of differentiating into all adult cell types, while another may only be capable of maturing into one specific somatic cell type.

Overall, the presence of stem cells is essential for proper human development and function as they contribute to the growth, maintenance, and repair of numerous physiological systems (Kara et al.).

Potency

On a macro scale, the two classifications of stem cells are embryonic and adult stem cells. Embryonic stem cells have the unique ability to exist in an undifferentiated state indefinitely while also being pluripotent. This pluripotency enables these cells to produce daughter cells of all differentiated somatic cell types, germ cells, and cells of all three embryonic germ layers (Can/Hematol 2008). In contrast, adult stem cells can only give rise to differentiated somatic cells of the particular tissue from which these cells originated (Can/Hematol 2008). To delve further into the specifics, embryonic and adult stem cells fall into potency categories reflecting their ability to differentiate: totipotent, pluripotent, multipotent, and unipotent. Totipotent stem cells contain all the constituents necessary to produce a living being, given that these cells can supply all embryonic and extraembryonic tissues required for proper growth (Can/Hematol 2008). The zygote and the cells within the initial zygotic divisions are considered totipotent (Kara et al.). Pluripotency, which ranks below totipotency in this hierarchy, is the next category.

A pluripotent stem cell has the potential to form all cells of the embryo, but cannot form a placenta. Multipotent stem cells are capable of forming many, but not all, cell types of the body. "Until recently, it was believed that they were tissue-specific...however, this concept has been challenged... (multipotent cells) can differentiate in vitro and in vivo into various cell types not only from the tissue of origin" (Can/Hematol 2008). Hematopoietic stem cells, which can produce several different types of blood cells, are prime examples of multipotency. Oligopotent stem cells, though not often widely discussed, have the ability to differentiate into a few cells (Kara et al.). Lymphoid and myeloid stem cells are ideal examples of oligopotent stem cells. Finally, unipotent stem cells, which can only produce differentiated daughter cells of one type, are another category of stem cells. Muscle stem cells are the typical example of unipotency. Using these definitions, embryonic stem cells can be classified as totipotent or pluripotent at different developmental stages and locations, whereas adult stem cells can be categorized as pluripotent, multipotent, or unipotent (Figure 1).

History

More can be added about the history if needed, using Kara et al.'s paper. In the early 1980s, scientists began to exploit two major classes of stem cells: embryonic and non-embryonic, or adult stem cells. While promising, these cells were solely murine-derived, limiting their applicability to humans. It wasn't until over a decade later, in the late 1990s, that human-derived stem cells were propelled to the forefront of experimentation. Marking a vast leap in reproductive therapy, it became possible to isolate these human embryonic stem cells for reproductive purposes, such as in vitro fertilization. However, the crowning achievement arose in 2006 when researchers at Kyoto University discovered the necessary means to reprogram differentiated adult cells to possess embryonic stem cell-like functionality, as if reversing time itself. The methodology included inducing differentiated somatic cells with the primary genes responsible for embryonic stem cell potency. These reprogrammed cells were classified as induced pluripotent stem cells (iPSCs). Due to the ethical concerns that surround the usage of embryonic stem cells derived from human blastocysts for therapeutic potential, this exploitation of differentiated adult cells held extreme significance. These findings culminated in promising therapies and breakthroughs, such as induced pluripotent stem cells, that have life-altering and quite possibly, life-saving, clinical potentials.

Induced Pluripotent Stem Cells

Can add more content and sources: Why are iPSCs important? They don't cross ethical lines and largely eradicate potential immune rejection from the host from a foreign cell. What is the promise behind them? The benefit is that we can use the host's own cells to create tissues and even organs from these cells. Are they effective? How are they therapeutic or what makes them therapeutic?

Inducible Genetic Factors

In the case of induced pluripotent stem cells (iPSCs), how were the aforementioned genes utilized to produce pluripotent stem cells from differentiated adult somatic cells? These mature differentiated cells are not renewing themselves; they have reached a terminally differentiated state and do not retain stem cell-like capabilities. Therefore, to attain stem cell-like properties, there needs to be an induction of factors to completely alter the cell from one that has no potency to a pluripotent state. It was discovered that four transcription factors, Oct4, Sox2, c-Myc, and Klf4, when administered to mouse fibroblasts, were in fact able to reprogram them to an undifferentiated pluripotent stem cell state (Yu et al). This is what is classified as an induced pluripotent stem cell (iPSC). The main adversary of this process was the addition of c-Myc. This genetic factor can cause differentiation and apoptosis of the stem cell, which is counteractive to the purpose of the process of achieving an undifferentiated condition (Yu et al).

The genetic factors Oct4, Sox2, Nanog, and Lin28 were transduced into IMR90 fetal fibroblasts and within the first twenty days, not only were there almost 200 iPS colonies that manifested embryonic stem (ES) cell morphology, but also the clones showed a normal karyotype, expressed telomerase activity, and prototypical human embryonic stem cell surface physiology (Yu et al). To further the excitement of this promising experiment, it was demonstrated that the clones could produce daughter cells of all three germ layers, and that the present variation in gene expression between these two cell lines, iPS and true ES cell, was less than that of the variation between different human embryonic stem cells themselves (Yu et al). This led to the discovery that Oct4, Sox2, Nanog, and Lin28 were the true transcription factors needed to rightfully reprogram differentiated somatic cells to achieve pluripotency and replicative functions similar to that of embryonic stem cells.

Stem Cell Regulation

Regulation Importance

The regulation of stem cells is pertinent because it offers a lens into the foundation of stem cell functionality at a molecular level, the issues that can arise when checks and balances fail or are surmounted, and the potential for these cells to be exploited for therapeutic benefits. The pathways that allow stem cells their fascinating ability to renew themselves and, simultaneously, form specialized cells are intricately regulated to mitigate their complexity. There are two systems in place to maintain the self-renewal condition of stem cells: intrinsic and extrinsic signals. The extrinsic signals, which impact intrinsic genetic factors of the stem cell, are provided by a unique environment where the stem cell resides called the niche (Zhang et al.). This region comprises cells that function to maintain the stem cell population by providing physical anchorage and by secreting proliferative, self-renewal, and specialization factors (Li and Neaves). However, the stem cells located within this niche contend with each other for these growth signals, resulting in a balance between those that attain the factors necessary for self-renewal and those that do not, subsequently leading them to differentiate (Nusse et al). Dysregulation of this balance can disrupt the equilibrium between the cell’s renewal and specialization properties, potentially resulting in tumor formation, premature cell differentiation, and other severe cellular defects (Zhang et al.).

Bone morphogenetic proteins (BMP) are a group of polypeptides that reside in various tissues and interact with a range of receptors to influence key stem cell properties such as cellular specialization, apoptosis, and growth (Zhang et al.). The functions of these proteins vary greatly, from promoting self-renewal in embryonic stem cells, encouraging osteoblastic specialization in mesenchymal stem cells, to managing the number of stem cells by manipulating the niche in hematopoietic stem cells. However, BMPs are not the sole drivers of these regulatory pathways; numerous other macromolecules are responsible for essential stem cell properties.

Specifically, the Wnt/Beta-catenin signaling pathway is one primary avenue utilized by the niche that is responsible for stem cell proliferation and self-renewal (Clevers et al.). This molecular cascade has exhibited its importance throughout many key elements of human growth that rely on stem cell support as a basis for survival, such as embryonic development and adult homeostasis (Nusse et al.). In particular, this cellular mechanism can be held accountable for the highly important and constant regeneration of cells and tissues in adult physiological systems such as the skin, stomach, and blood (Clevers et al.). If mutated or disrupted, this novel regeneration will be impacted, and unique tissue development will atrophy (Clevers et al.). Molecularly, Wnt binds its 7-transmembrane domain receptor, termed Frizzled, which is coupled to LRP, a low-density lipoprotein co-receptor (Nusse et al.). Following this, the protein messenger Dishevelled propagates through the cellular cascade to prevent the degradation of Beta-catenin, which can then insert into the nucleus and regulate transcription (Nusse et al.). This protein, beta-catenin, then stimulates DNA transcription and results in enhanced cell proliferation, a capacity very integral in the stem cell arsenal. The downside of this cascade? Changes, whether through mutations or extrinsic signals, for example, can then abuse the growth capability of beta-catenin, or the Wnt signaling cascade as a whole, and lead to excessive cell growth and tumor formation, or interfere with the Wnt factor Tcf4 to decrease the stem cell population (Zhang et al.).

Regulation of cell proliferation is only half of the battle. Without apoptosis, or programmed cell death, continued cell growth could not be equilibrated and cellular masses are more likely to manifest. Interestingly enough, apoptosis and its opponent, growth, harmonize when it comes to stem cell self-renewal through the integration of the aforementioned Wnt/Beta-catenin pathway. The Bcl-2 proto-oncogene is an inhibitor of apoptosis initiation that, when overexpressed, has shown to increase the cell cycle duration by avoidance of apoptosis induction and lengthening of the G1 phase (Domen et al.). Coupling this knowledge of Bcl-2 activity with Beta-catenin’s proclivity to stimulate cell proliferation, it appears evident that these factors would combine to maintain the self-renewal capacity of a stem cell. In fact, this was proved to be true. Experimentally, in a Bcl-2-transgenic mouse, it was the expression of Beta-catenin protein that maintained hematopoietic stem cells (HSC) in an undifferentiated state through not only the expansion of the hematopoietic stem cell population by means of proliferation, but also through preventing differentiation (Reya et al.).

One other stem cell habitat that necessitates stringent regulation is that of the reproductive system. Spermatogonial stem cells (SSCs) are not only outnumbered by the vast amount of spermatocytes, spermatids, and spermatogonia, but they also carry the immense proliferative burden of producing millions of sperm cells daily, making their self-renewal of utmost physiological importance. (Phillips et al.). As previously discussed, the niche is the extrinsic microenvironment that influences intrinsic functions of stem cells, such as self-renewal and differentiation. The spermatogonial stem cell (SSC) niche is located within the mammalian testes in the basal layer of the seminiferous tubules (Dadoune et al.). The components of the seminiferous tubules, Sertoli and pertibular myeloid cells, are responsible for facilitating the binding of SSCs to the niche (Tung et al.). How does the niche now accomplish this maintenance of SSC self-renewal and differentiation? Sertoli cells release GDNF which binds a GFR-alpha-1 co-receptor, attached to a Ret tyrosine kinase, ultimately stimulating downstream cellular cascades in undifferentiated spermatogonia (Meng et al.; Braydich-Stolle et al.). To further support its role as a key regulator of SSC self-renewal and differentiation, transgenic mice with an allelic GDNF knockout displayed a reduced population of spermatogonial stem cells (SSCs), while those experiencing an overexpression of GDNF manifested an increased accrual of undifferentiated spermatogonia (Meng et al.). Finally, mutations or protuberances of the Ets variant gene 5, which is a transcription factor in Sertoli cells, cause dysregulation in the SSC population and therefore, a lack of spermatogonia self-renewal support (Chen et al.).

Stem Cell Dysregulation Overview

The significance of stem cell regulation is not only crucial for positive developments such as therapies; it is also essential to fortify our knowledge of dysregulation and its consequences. The primary concern is cancer derived from uncontrolled cell proliferation, a potentially lethal outcome that mirrors self-renewal and regeneration characteristics found in life-supporting stem cells. It has been found that cancers may manipulate stem cells, deemed "cancer stem cells," to employ these self-renewal properties for their benefit (Li and Neaves). Along with this abuse of wild-type stem cells, cancers can also capitalize on the alteration and/or deregulation of the stem cell niche, feeding off pro-proliferative signals and utilizing cell-mobilizing signals for metastasis (Li and Neaves).

Dysregulation Within the Niche

In a wild-type physiological state, the niche functions to sustain stem cells in a quiescent state, whereby rapid proliferation is halted (Li and Neaves). It is only after receiving proliferative signals that stem cells are encouraged to begin the growth and division process (Li and Neaves). This aforementioned system of checks and balances ensures a physiological equilibrium between growth and quiescence. Cellular mutations or alterations that cause stem cells to become impartial to these growth signals or impervious to quiescent indicators can fuel uncontrolled growth, neoplasia, and/or tumorigenesis (Li and Neaves). With respect to manipulation of the niche itself, expansion or reduction of its size can have vast implications on stem cell numbers and tissue quantity. For instance, increasing the population of niche-specific cadherin cells that attach to hematopoietic stem cells boasted a marked increase in these stem cell numbers (Hematopoietic niche), while depleting the osteoblast count, a populace synonymous with the hematopoietic stem cell (HSC) niche (Askmyr et al.), led to a reduction in hematopoietic stem cells and, consequently, hematopoietic tissue in its entirety (Visnjic et al.). To further prove the implications of niche destruction, it was also found that mutating or destabilizing cellular components of the Drosophila ovary niche resulted in a loss of germline stem cells (Xie et al.). It was discussed previously that cellular mutations that hijack stem cells can lead to tumorigenesis, but research has shown that deregulation of the niche itself can also lead to uncontrolled stem cell proliferation and the possibility of cancer. Support of this notion was given when researchers discovered that molecular disturbance of the stem cell niche within mammary epithelium caused the promotion of TGF-alpha, a tumor-promoting growth factor, and led to the formation of breast cancer (Chepko et al.).

Dysregulation in Genetic Expression

The strikingly similar macro-physiological effects of self-renewal and differentiation exhibited by wild-type and cancer stem cells may give the impression that these cells are indistinguishable. However, after in-depth analysis of their cellular differences, it is clear that gene expression and micro-signaling are vastly variant. It was uncovered that the principal differing pathways amongst wild-type human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) were those incorporated in adherens junction, MAPK signaling, and Wnt signaling (Majeti et al.). To conclude that the aforesaid pathways were in fact the cancerous culprits when perturbed, the researchers mapped the relative expression levels of all genes in each pathway with respect to their signal transduction and communicative processes (Mateji et al.). In regards to leukemic stem cells (LSC), genes that were down-regulated in the adherens junction pathway were alpha-catenin, Afadin, and PAR3, whereas Axin and APC were up-regulated in the Wnt pathway.

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