Stem Cell Biotechnology 101: Self-Renewal and Maintenance

Foreword

The field of stem cells is brimming with promise and intrigue. As the understanding of these remarkable foundational components of life continues to evolve, so does the capacity to harness their extraordinary potential. Stem cells have enchanted the minds of scientists, medical professionals, and curious individuals alike, presenting unparalleled opportunities to revolutionise healthcare and regenerative medicine. This series of articles aims to unravel the mysteries of stem cell differentiation, explore the mechanisms governing their self-renewal, and examine the factors that shape their destiny. Within these articles, readers may discover the immense potential of induced pluripotent stem cells (iPSCs) in reshaping personalised medicine while uncovering their awe-inspiring regenerative capabilities. Moreover, this series sheds light on the critical challenges and limitations inherently intertwined with this field, from ensuring the safety and efficacy of stem cell therapies through navigating the complex terrain of regulatory frameworks and public perception to debating ethical uncertainties.

This 101 series is divided into eight articles:

1. Stem Cell Biotechnology 101: Exploring the Potential of Stem Cells

2. Stem Cell Biotechnology 101: Sources and Isolation Methods

3. Stem Cell Biotechnology 101: Differentiation and Lineage Commitment

4. Stem Cell Biotechnology 101: Self-Renewal and Maintenance

5. Stem Cell Biotechnology 101: Induced Pluripotent Stem Cells (iPSCs) and Genetic Engineering

6. Stem Cell Biotechnology 101: Stem Cells in Regenerative Medicine

7. Stem Cell Biotechnology 101: Stem Cells in Disease Modelling and Cancer Research

8. Stem Cell Biotechnology 101: Ethical Considerations and Future Perspectives

Stem Cell Biotechnology 101: Self-Renewal and Maintenance

Self-renewal is a fundamental characteristic of stem cells that allows them to generate daughter cells identical to themselves. This process ensures the maintenance of a population of undifferentiated stem cells while enabling the production of more specialised cell types. At the cellular level, self-renewal involves the division of a stem cell into two daughter cells. One retains the stem cell identity and the ability to self-renew, and the other becomes committed to differentiation. A network of signalling pathways, transcription factors, and epigenetic regulators orchestrates the molecular mechanisms underlying self-renewal. These factors collectively maintain the stem cell's ability to proliferate without losing potency (He et al., 2009).

Maintenance of stem cells refers to the mechanisms that sustain the unique attributes and properties of stem cells over time. It encompasses processes that prevent stem cells from prematurely differentiating into specialised cell types and ensure long-term survival. One critical aspect of maintenance is preserving the stem cells' self-renewal capacity, allowing them to continuously divide and generate both identical stem cells and cells committed to differentiation. Additionally, maintenance involves regulating various genetic and epigenetic factors that govern the stem cell's gene expression profile and ensure its identity and function remain stable (Xi Chen et al., 2015).

Basically, self-renewal and maintenance mechanisms work in concert to uphold the unique properties of stem cells, allowing them to last as a reservoir of undifferentiated cells while, at the same time, contributing to the generation of specialised cell lineages during development, tissue repair, and homeostasis.

Figure 1: The comparison of stem cell differentiation and self-renewal (Samanthi, 2020).

An especially important aspect of stem cells that links this article to the previous one is asymmetric and symmetric cell division. They represent fundamental processes in stem cell biology, each bearing unique implications for cellular fate and tissue dynamics. Asymmetric division yields one stem cell and one differentiated cell, ensuring cellular diversity and contributing to developmental processes (Zimdahl et al., 2014). Conversely, symmetric division generates two identical daughter cells, which is vital in stem cell maintenance and self-renewal. Aside from generating two stem cells, as depicted in Figure 2, symmetric division can also yield two differentiated cells. This outcome is contingent upon contextual factors and the needs of the given tissue or organism. A comprehensive exploration of both division modes is essential to grasp the convoluted mechanisms underlying stem cell dynamics (Venkei & Yamashita, 2018).

Figure 2: Symmetric and asymmetric cell division (Chen, 2015).

In stem cells, symmetric division takes centre stage. This division mode strategically generates identical stem cell progeny, effectively replenishing the stem cell pool and sustaining the population of undifferentiated cells within their specialised niches. The balance between asymmetric and symmetric divisions significantly influences tissue homeostasis and regenerative processes. An excessive occurrence of symmetric divisions can culminate in uncontrolled self-renewal and the potential for tumorigenesis. Conversely, insufficient symmetric divisions impede tissue repair and hinder the restoration of damaged areas (Morrison & Kimble, 2006).

Embryonic Stem Cells

The Maintenance of Stemness In Vitro

When provided with suitable conditions, embryonic stem cells (ESCs) exhibit an extraordinary capacity for continuous proliferation and self-renewal (Pal et al., 2011). The establishment of these conditions has evolved, initially involving mouse fibroblasts as trophoblast cells on feeder layers (Evans & Kaufman, 1981). Subsequent investigations unveiled the pivotal role of leukaemia inhibitory factor (LIF) in this process, as it maintains ESC growth without differentiation, substituting the need for trophoblast support (Sutherland et al., 2018). This role has been further complemented by BMP4, which fosters stem cell growth and replaces serum requirements (Ying et al., 2003). The interplay of key factors such as Oct3/4 and Nanog plays a central role in maintaining the ground state of ESCs (Chambers et al., 2003; Niwa et al., 2000). Figure 3 highlights the impact of Oct4, Sox2, and Nanog on the behaviour of stem cells.

Figure 3: Oct4/Sox2/Nanog form a core transcriptional network regulating the stem cell machinery (Bosnali, 2009).

Various transcription factors, such as Stat3 and others, have been identified in regulating murine ESC stemness (Mossahebi-Mohammadi et al., 2020). When it comes to human ESCs (hESCs), distinct conditions emerged for maintaining their growth and stemness. Serum-free culture mediums, bFGF, and Wnt signalling pathway activation have been explored to support hESCs in an undifferentiated state (Dahéron et al., 2004; Sato et al., 2004). Notably, the elaborate interplay of Oct4, Nanog, and long intergenic non-coding RNAs contributes significantly to hESCs stemness (Keller, 2005; X. Lu et al., 2014; Richards et al., 2004). Despite significant progress, the precise mechanisms underpinning the long-term maintenance of hESCs stemness and self-renewal remain as areas of ongoing investigation.

Transcription Factors

Transcription factors operate as molecular switches, influencing gene expression and orchestrating the developmental trajectory of cells. In the context of embryonic stem cells, the trio of Oct3/4, Sox2, and Nanog stands out as the core transcription factors, wielding significant influence over the maintenance of pluripotency and the preservation of the ground state of these cells. Among them, Oct3/4 appears particularly crucial, as its presence is encoded by the Pou5f1 gene and characterised by two POU domains (Chan et al., 2011). A POU domain is a specific type of DNA-binding domain found in transcription factors. The term "POU" is derived from the names of the three transcription factors in which this domain was initially identified. This transcription factor has a dichotomous role, as its conditional expression fosters both the retention of pluripotency and the engagement of multipotent differentiation pathways (Rodda et al., 2005).

Figure 4: The transcriptional regulatory network managing self-renewal and differentiation in stem cells (He, 2009).

The suppression of Oct3/4 leads to a decline in pluripotency and an impairment in the potential to embark on multipotent journeys (Abulaiti et al., 2017). Another influential player, Nanog, finds prominence in early embryonic development, expressing itself during the undifferentiated phases of both mouse and human stem cells. Its augmentation induces ESCs' exponential expansion and concurrently maintains Oct3/4 levels (Heurtier et al., 2019). The synergy between Sox2 and Oct3/4 activates Oct-Sox enhancers, resulting in a cascading effect that bolsters the expression of Nanog and Oct3/4, thereby reinforcing the pluripotency of ESCs (Masui et al., 2007). The regulatory network extends beyond this trio and encompasses multifarious entities, including BMP, LIF, Wnt signals, Smad1, STAT3, and Tcf3 (Soucie et al., 2016). These components form a complex organisation of transcriptional regulation, where Nanog-Oct3/4-Sox2 clusters intricately interact with downstream factors like Smad1 and STAT3, spurred by BMP and LIF pathways (J. Kim et al., 2008). This complicated interaction stabilises the transcription factor complexes, culminating in escalated transcriptional activity controlling ESC-specific genes. The partnership between Wnt signalling pathways and Oct3/4 and Nanog strengthens their roles in keeping cells pluripotent and maintaining the balanced state of embryonic stem cells (Cole et al., 2008).

Other factors that play a role in maintaining pluripotency, like Dax1, Nac1, Zfp281, Nr5a2, and Klf4, work in coordination with the central control system involving Oct3/4. These factors attach to regions of the genome located upstream from the transcription initiation site. This strategic attachment allows them to influence the activity of the Oct3/4 gene. The interchange between Oct3/4 and these additional factors is a crucial part of maintaining pluripotency (Dejosez et al., 2010; Heng et al., 2010).

At the same time, other key players are involved, such as c-Myc, n-Myc, E2f1, Zfx, Rex1, and Ronin. Said control switches are situated closer to the gene's starting point. They can affect the activities of the proteins that come after the gene is turned on. By attaching to specific areas, these factors can carefully guide the sequence of events that support pluripotency. These interactions create a precise regulatory network that helps maintain the delicate balance needed to keep stem cells in a pluripotent state. (Young, 2011).

Figure 5: Transcriptional regulators implicated in the control of ESC state (Young, 2011).

The Leukaemia Inhibitory Factor

As previously mentioned, the involvement of trophoblast cells is required during the early stages of establishing and sustaining embryonic stem cells. Then the role of LIF, a member of the IL-6 cytokine family, was unveiled (J. J. Kim et al., 2010). To keep the stem cells in their unique state, LIF activates a pathway called JAK/STAT3. LIF also triggers the PI3K pathway in both mouse and human ESCs through the glycoprotein 130 (gp130) receptor. This activation is vital in keeping these stem cells in an undifferentiated state. PI3K then sets off a cascade, leading to a chemical modification (phosphorylation) of a protein called Akt. Once the phosphorylated Akt protein reaches the cell's nucleus, it influences specific genes responsible for promoting stem cell renewal and preventing differentiation into specific cell types (Nicola & Babon, 2015).

Consequently, when LIF activates the PI3K/Akt pathway (Figure 5), it encourages ESCs to continually renew themselves while inhibiting their transformation into various cell types. When PI3K is deactivated through gene mutations or specific inhibitors, a different pathway called ERK becomes more active, leading to ESC differentiation. It is also worth noting that PI3K also influences the proliferation of ESCs. The inactivation of a protein called PTEN, which usually inhibits PI3K/AKT signals, accelerates ESC proliferation and reduces cell death by boosting Akt phosphorylation. LIF also plays a role in activating the SHP2-Ras-ERK pathway through gp130 (G. Chen et al., 2022).

Figure 6: LIF regulates self-renewal of the stem cell by influencing the PI3K/Akt pathway (Ke, 2018).

To sum up, LIF manages a network of signals that keeps ESCs pluripotent in a dual manner. When LIF interacts with ESCs, it triggers the JAK-STAT3 pathway through gp130, which promotes ESCs renewal. Additionally, it activates the PI3K pathway via gp130, supporting their growth. Furthermore, it can activate the SHP2-Ras-ERK pathway, possibly pushing them towards differentiation. The gist of maintaining ESC self-renewal is maintaining a precise balance among these three pathways.

Small Molecules

By diving into the detailed network of signals that guide stem cells' behaviour, researchers have found that introducing small molecules during ESCs culture can influence these signals and, significantly, keep ESCs in their youthful state. For instance, a certain molecule, which can be thought of as a "stop" sign for a specific pathway named GSK3β, helps prevent ESCs from turning into different cell types. Another molecule can be likened to a "roadblock" on a MEK pathway, ensuring that ESCs keep renewing themselves and do not lose their identity. Similarly, a different molecule works as a "blocker" for a pathway called FGFR, stopping ESCs from following a path that leads to their transformation. These molecules help ESCs stay young and undifferentiated when grown in a dish outside the body (G. Chen et al., 2022).

One well-known combination of these molecules, often referred to as "2i," involves using two small compounds together to maintain ESCs in their particular state. However, it is essential to use relatively high amounts of these molecules, which can sometimes slow down cell growth and make research challenging (Ying et al., 2008). Additionally, Chen et al. (2014) have found that a molecule called Sunitinib can help ESCs maintain their youthful form during extended periods of lab culturing. By blocking a signal known as VEGFR, Sunitinib prevents ESCs from ageing prematurely and turning into different cell types, as presented in Figure 6. This discovery sheds light on how to keep ESCs in their youthful condition for more extended periods, and it could have implications for potential therapies in the future.

Figure 7: Schematic representation of the autocrine VEGF signaling (Chen, 2014).

Non-Coding RNA and Stemness Maintenance

Many research groups are revealing the multifaceted potential of RNA beyond its conventional roles as a messenger or a component of the ribosome (Dinger et al., 2008; Mello & Mattos, 2009). Emerging evidence indicates that non-coding RNAs (ncRNAs) are versatile molecules involved in various cellular functions, from orchestrating gene expression to shaping protein translation and post-translation modifications, making them integral players in essential life processes. An array of ncRNAs has been scrutinised in stemness maintenance, mainly focusing on long non-coding RNAs (lncRNAs) and small RNAs like microRNAs (miRNAs) (Jia et al., 2013; Song & Tuan, 2006). The former are an intriguing class of RNAs situated between protein-coding genes and are characterised by a length of at least 200 bases. Conversely, miRNAs are diminutive non-coding RNAs, spanning about 22 nucleotides, and are recognised for their considerable conservation across species and their spatiotemporal specificity. They are indispensable for the self-renewal and differentiation of stem cells (Marson et al., 2008).

These diverse non-coding RNAs fine-tune the delicate balance between these two crucial processes, exerting control over genes and their expression levels. For instance, long non-coding RNAs interact with DNA, RNA, and proteins to influence gene expression and epigenetic states (W. Lu et al., 2019). They actively participate in chromatin remodelling, transcriptional regulation, and establishing higher-order chromatin structures. Meanwhile, small RNAs, such as microRNAs, play a distinct regulatory role by binding to target messenger RNAs, leading to their degradation or translational repression. Through these intricate mechanisms, non-coding RNAs contribute significantly to stem cell populations' overall stability and robustness (Naeli et al., 2023).

Role of Metabolic Processes

Environmental factors and cellular metabolic processes play a pivotal role in regulating the metabolic homeostasis of embryonic stem cells, which serves their primary objective of furnishing essential building blocks for cellular growth and function. Mounting evidence underscores the active involvement of cellular metabolic reactions in decisively shaping the fate of stem cells. Notably, the fate decisions of ESCs are intimately linked to several key metabolic pathways, including carbohydrate, lipid, and glutamine metabolism, and the orchestrated interplay of these pathways profoundly influences the preservation of ESC identity (Mans & Haramis, 2014). For instance, the mTOR pathway, attuned to cellular energy status, ensures stem cell viability even under energy-deficient conditions. Another essential facet involves reactive oxygen species (ROS), metabolic by-products generated during aerobic metabolism. They have emerged as influential secondary messengers managing the complicated signalling cascades that govern stem cell self-renewal and differentiation (Ezashi et al., 2005). Hypoxia-inducible factors (HIFs), instrumental in regulating ROS homeostasis, intertwine glucose metabolism and redox reactions. As such, they wield critical influence over the self-renewal of ESCs (Lee et al., 2012). Figure 7 illustrates the correlation between the stem cell's energy demands and stemness maintenance/differentiation.

Figure 8: Stem cell metabolism is dynamically modulated to control stemness, proliferation, and cell commitment (Rigaud, 2020).

Further insights stem from forkhead-box transcription factors, essential regulators of glucose metabolism, which control cellular responses tied to ROS. Sirtuins, acting as modulators of glucose and lipid metabolism through NAD+-dependent deacetylation of specific proteins and histones (proteins that package DNA), also influence the self-renewal of stem cells through ROS mitigation (Matsui et al., 2012). In this complex metabolic landscape, the AMP-activated kinase, a master regulator of cellular energy homeostasis, governs downstream signalling cascades, therefore dictating stem cells' equilibrium and fate (Young et al., 2016).

Epigenetics and Self-Renewal

The balance between the renewal and differentiation of embryonic stem cells is finely orchestrated through epigenetics. As described in the previous article, epigenetic modifications encompass diverse regulatory processes like DNA methylation changes, histone modifications, and regulatory tweaks guided by non-coding RNAs (Papatsenko et al., 2018). These subtle but influential changes regulate the destiny of stem cells, dictating whether they retain their self-renewing potency or enter paths of specialisation (Wu & Yi, 2006). A relevant phenomenon, DNA demethylation, occurs in the promoter regions of critical transcription factors within ESCs, boosting their expression. The contrary holds for differentiated cells, where this methylation curtails expression. For example, essential factors that control gene activity, like Oct4 and Nanog, have lower levels of expression in specialised ESCs due to a process called methylation carried out by specific molecules (Wu et al., 2021).

Additionally, modifications of histones differ between ESCs and their more mature counterparts. In ESCs, histone H3K4 and H3K27 trimethylations are limited, unlike in differentiated cells, where they are abundant (Oleksiewicz et al., 2017). This distinction aligns with activating specific genetic signals and silencing others in ESCs (Bernstein et al., 2006). Epigenetic overseers like TRIM28 guide the renewal of human ESCs by controlling the addition of specific molecular tags and DNA methylation. Crucial contributors like histone modifiers Jmjd1a and Jmjd2c prevent excessive tagging in the key genes of ESCs, maintaining their activity and promoting self-renewal (Loh et al., 2007). When the genes responsible for encoding these proteins become impaired, resulting in the absence or incorrect production of the corresponding proteins, the normal biological functions of TRIM28, Jmjd1a, and Jmjd2c are disrupted. In consequence, the proper regulation of ESCs is obstructed, leading to their inclination towards differentiation. The activities of histone modifiers like Jarid2 and Mtf2, which are parts of a more extensive regulatory system, are tied to modifying histone H3K27. This action suppresses Oct4 levels and speeds up the process of ESC differentiation (Landeira et al., 2010). Oct4 boosts the activity of a molecule called H3K9 demethylase and the components of PRC2. Oct4 and Sox2 team up to regulate Utf1, a protein related to the packaging of DNA, ensuring that ESCs maintain their ability to self-renew and multiply by influencing specific modifications—H3K27 trimethylation— and controlling PRC2's involvement (Mansour et al., 2012). Beyond merely renewing themselves, epigenetic factors also play a role in preserving stem cells' unique properties and developing cancer stem cells (Chen et al., 2019).

Figure 9: Epigenetic response to extrinsic signals occurs through the transcriptional factors network (n.d.).

Adult Stem Cells

Adult stem cells are a vital component of tissue homeostasis and regeneration, and a complex interplay of intrinsic and extrinsic factors orchestrates their ability to maintain stemness and govern self-renewal. Since adult stem cells are secluded within specialised niches residing within mature tissues, they are attuned to their microenvironment's cues. Local signalling cues, including growth factors, cytokines, and extracellular matrix components, play a central role in instructing adult stem cells to either self-renew or differentiate. The niche provides mechanical and biochemical support, dictating stem cell behaviour by regulating the activation of specific signalling pathways, such as Wnt, Notch, and TGF-β. These pathways modulate the balance between self-renewal and differentiation through intricate molecular networks (Donnelly et al., 2018).

Transcription factors, distinct to each tissue type, govern adult stem cell maintenance by regulating the expression of genes associated with stemness and differentiation. These transcriptional regulators include Oct4, Sox2, and Nanog, analogous to their roles in embryonic stem cells, although context-specific. Epigenetic modifications, such as DNA methylation and histone modifications, imprint stem cell identity by maintaining chromatin configurations that enable genes required for self-renewal to remain accessible while repressing those linked to differentiation. DNA methylation patterns and histone marks establish a "memory" that guides adult stem cells' response to environmental cues (Lin et al., 2022). Cell-cell interactions within the niche further contribute to adult stem cell maintenance. Adhesive connections forged with neighbouring cells and stromal partners contribute to essential survival cues, preserving a quiescent state that forestalls premature differentiation (Brown et al., 2019).

Figure 10: Parameters of the stem cells and their niches (Donnelly, 2018).

Intracellular pathways such as PI3K/Akt, MAPK, and mTOR integrate niche-derived signals, nutrient availability, and energy status to regulate adult stem cell behaviour. These pathways transduce extracellular cues into cellular responses, ensuring that self-renewal is activated only when necessary for tissue repair. More precisely, the PI3K/Akt pathway is closely linked to ASCs' maintenance of pluripotency and self-renewal. Its activation in response to growth factors and nutrient signals sets in motion a cascade of events that promote cell survival, inhibit apoptosis, and enhance cell cycle progression. Similarly, the MAPK pathway, particularly the ERK branch, translates environmental cues into cellular decisions, influencing ASC proliferation, differentiation, and survival. The mTOR pathway is an integral regulator of cell growth and metabolism, adjusting cellular responses based on nutrient availability and energy status. (Wamaitha et al., 2020). Additionally, metabolic processes wield significant influence over ASC behaviour. The ratio between oxidative phosphorylation and glycolysis, the two main modes of cellular energy production, becomes finely tuned to modulate the equilibrium between self-renewal and differentiation in response to energy demands. This metabolic flexibility allows ASCs to adapt their energy utilisation strategy based on the varying requirements for self-renewal or differentiation in tissue repair and homeostasis (Wani et al., 2022).

The roles of these intracellular pathways and metabolic processes in embryonic stem cells diverge due to the unique nature of pluripotent cells. The intracellular pathways, while potentially sharing some common components with those in ASCs, predominantly serve to amplify and fine-tune the pluripotency-associated signalling cascade in ESCs. PI3K/Akt and MAPK pathways might play roles in coordinating ESC proliferation and survival, but their dynamics are intertwined with the pluripotency circuitry directed by the core transcription factors. Similarly, mTOR's functions in ESCs are likely to be geared towards supporting rapid cell proliferation and biomass accumulation during the early stages of embryonic development. Metabolically, ESCs exhibit different energy demands and metabolic profiles compared to ASCs. The highly proliferative nature of ESCs is characterised by robust glycolytic activity, promoting the generation of building blocks necessary for cellular growth and rapid DNA synthesis. The metabolic landscape of ESCs, influenced by the high demands of pluripotency maintenance and early embryonic development, contrasts with the more quiescent and differentiated state of adult tissues housing ASCs (Cheng, 2008).

Figure 11: Overview of PI3K/AKT/mTOR signalling pathway (Rinne, 2021).

Principally, there is a degree of overlap in intracellular pathways and metabolic mechanisms between ESCs and ASCs, but their functions are predetermined by the contrasting requirements dependent on the nature of the stem cell.

Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs), akin to embryonic stem cells, uphold stemness and self-renewal potential. An elaborate mixture of internal and external factors governs these processes (Ma et al., 2018). Manipulating culture conditions eliminates differentiation-inducing pathways, while consolidated biosynthesis and suppressed differentiation pathways sustain iPSCs. Extrinsic cues, like cytokines, regulate signalling pathways, with growth factors like fibroblast growth factor and BMP4 nurturing iPSCs self-renewal. Epigenetic changes and transcriptional regulators guide iPSCs maintenance, mirroring patterns seen in ESCs and ASCs (Zakrzewski et al., 2019). Human iPSCs have unique aspects, yet the core principle remains the same: iPSCs retain pluripotency through gene networks and signals, proving pivotal in regenerative medicine and disease modelling (Mossahebi-Mohammadi et al., 2020). The next article in the 101 series will delve deeper into iPSC intricacies, offering comprehensive insights into their transformative potential.

The Surprising Influence of Vitamin C

An emerging player in the field of stem cell self-renewal and maintenance is vitamin C (ascorbic acid), a vital nutrient with epigenetic implications. Vitamin C is an essential dietary requirement for humans, serving as an antioxidant and a cofactor for a group of enzymes called Fe2+/α-KGDDs. These include collagen prolyl hydroxylases and epigenetic regulators involved in histone and DNA methylation processes (Kuiper & Vissers, 2014). The profound influence of vitamin C extends across multiple types of stem cells, illustrating its versatile impact on varied aspects of stem cell functionality. In the context of ESCs, vitamin C's modulatory effects come to the fore, exerting influence to enhance pluripotency and amplify self-renewal capacities, ultimately finetuning the delicate balance that defines the stem cell state (Chung et al., 2010). This dualistic role grants vitamin C a pivotal role in shaping the fundamental attributes of ESCs, accentuating their malleability and regenerative aptitude.

Figure 12: Vitamin C enhances stem cell therapeutic potential (Chong, 2019).

Equally noteworthy is vitamin C's engagement with adult stem cells, known for their ability to replenish and repair tissue in mature organisms. Vitamin C is a catalyst for DNA and histone demethylase activity, underscoring its involvement in moulding epigenetic and gene expression profiles that dictate ASC behaviour. The detailed interplay between vitamin C and epigenetic orchestration suggests that vitamin C's leverage exceeds far beyond just upkeep. That would mean that its effects are not just about maintaining the status quo but involve more sophisticated adjustments that adapt stem cell behaviour to different conditions (Gasiūnienė et al., 2020).

Furthermore, vitamin C's involvement extends into cellular reprogramming, a transformative process that converts differentiated cells into pluripotent stem cells, i.e. iPSCs. That implies that vitamin C not only maintains the essence of stemness but also facilitates transformative reprogramming (Esteban et al., 2010). Recent investigations underscore the role of vitamin C in augmenting the efficiency of generating iPSCs from somatic cells like fibroblasts, thereby offering a promising avenue for propelling advancements in regenerative medicine and disease modelling (Figure 12).

Additionally, studies by Agathocleous unveiled vitamin C's complex function in controlling the dynamics of hematopoietic stem cells (HSCs), unveiling yet another layer of sophistication to ascorbic acid's role. They disclosed that vitamin C curbs HSC frequency and dampens the progression of leukaemia via a series of Tet2-dependent and -independent mechanisms (Figure 13). This work forges a connection between vitamin C and the orchestration of HSC dynamics, extending its involvement in stem cell biology beyond the confines of pluripotency (Agathocleous et al., 2017).

Figure 13: Vitamin C influences the rate of self-renewal, preventing cancer development (Schönberger, 2017).

The significance of vitamin C extends beyond its role in regular dietary intake, as pharmacological administration can attain therapeutic levels, showing potential with minimal patient toxicity. Its multifaceted impact is particularly notable in cancer prevention and treatment, where maintaining a vitamin C-rich diet could act as a deterrent against cancer progression, and pharmacological doses might synergise with existing therapies to enhance outcomes (Fu et al., 2020).

Despite its potential, the exact mechanism through which high-dose vitamin C exerts its anti-cancer effects has yet to be entirely elucidated. The prevailing hypothesis surrounding its pro-oxidant nature remains open to exploration, especially concerning the interactions with enhanced dioxygenase activity and its implications for conventional therapies upon vitamin C intervention. Unravelling these complexities becomes imperative to refine treatment strategies, possibly by identifying specific biomarkers of sensitivity (Cimmino et al., 2018).

In a more targeted approach, efforts to enhance the bioavailability of vitamin C and understand its structural role as a specific cofactor for α-KGDDs offer a glimpse of potential breakthroughs. This avenue holds significance for epigenetic regulators, including the TET proteins, which often become dysfunctional in cancer.

Conclusion

The world of stem cells reveals a captivating narrative of cellular plasticity, self-renewal, and the guided fate direction. Embryonic stem cells channel their pluripotent potential through a network of core transcription factors and epigenetic regulators that maintain their undifferentiated state. Adult stem cells, nestled within mature tissues, depend on precisely tuned interactions with their microenvironment to strike a balance between self-renewal and differentiation, thereby ensuring tissue homeostasis and repair. The emergence of induced pluripotent stem cells, an innovative feat of reprogramming, bridges the gap between ESCs and ASCs, offering an unmatched potential for personalised therapies and profound exploration of cellular behaviour. Each stem cell type's journey to uphold stemness and regulate self-renewal mirrors its origin, niche, and inherent genetic and epigenetic milieu. Comprehending these nuanced mechanisms is essential for advancing regenerative medicine and grasping the fundamental principles controlling cellular identity and destiny.

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Figure 1: The comparison of stem cell differentiation and self-renewal. (Samanthi, 2020). [Infographic]. DifferenceBetween.com. https://www.differencebetween.com/difference-between-stem-cell-differentiation-and-self-renewal/

Figure 2: Symmetric and asymmetric cell division. Chen, Xi, Ye, S., & Ying, Q. L. (2015). [Illustration]. Stem cell maintenance by manipulating signaling pathways: Past, current and future. BMB Reports, 48(12), 668–676. https://doi.org/10.5483/BMBRep.2015.48.12.215

Figure 3: Oct4/Sox2/Nanog form a core transcriptional network regulating the stem cell machinery. Bosnali, M., Münst, B., Thier, M., & Edenhofer, F. (2009). [Illustration]. Deciphering the stem cell machinery as a basis for understanding the molecular mechanism underlying reprogramming. Cellular and Molecular Life Sciences, 66(21), 3403–3420. https://doi.org/10.1007/s00018-009-0095-2

Figure 4: The transcriptional regulatory network managing self-renewal and differentiation in stem cells. He, S., Nakada, D., & Morrison, S. J. (2009). [Illustration]. Mechanisms of stem cell self-renewal. Annual Review of Cell and Developmental Biology, 25, 377–406. https://doi.org/10.1146/annurev.cellbio.042308.113248

Figure 5: Transcriptional regulators implicated in the control of ESC state. Young, R. A. (2011). [Illustration]. Control of the embryonic stem cell state. Cell, 144(6), 940–954. https://doi.org/10.1016/j.cell.2011.01.032

Figure 6: LIF influences the PI3K/Akt pathway, regulating self-renewal of the stem cell. Ke, M., He, Q., Hong, D., Li, O., Zhu, M., Ou, W. ... Wu, Y. (2018). [Illustration]. Leukemia inhibitory factor regulates marmoset induced pluripotent stem cell proliferation via a PI3K/‌Akt‑dependent Tbx‑3 activation pathway. International Journal of Molecular Medicine, 42, 131-140. https://doi.org/10.3892/ijmm.2018.3610

Figure 7: Schematic representation of the autocrine VEGF signaling. Chen, G., Xu, X., Zhang, L., Fu, Y., Wang, M., Gu, H., & Xie, X. (2014). [Illustration]. Blocking autocrine VEGF signaling by sunitinib, an anti-cancer drug, promotes embryonic stem cell self-renewal and somatic cell reprogramming. Cell Research, 24(9), 1121–1136. https://doi.org/10.1038/cr.2014.112

Figure 8: Stem cell metabolism is dynamically modulated to control stemness, proliferation, and cell commitment. Rigaud, V. O. C., Hoy, R., Mohsin, S., & Khan, M. (2020). [Illustration]. Stem Cell Metabolism: Powering Cell-Based Therapeutics. Cells, 9(11). https://doi.org/10.3390/cells9112490

Figure 9: Epigenetic response to extrinsic signals occurs through the transcriptional factors network. (n.d.). [Illustration]. Creative Diagnostics. https://www.creative-diagnostics.com/stem-cell-epigenetics.htm

Figure 10: Parameters of the stem cells and their niches. Donnelly, H., Salmeron-Sanchez, M., & Dalby, M. J. (2018). [Illustration]. Designing stem cell niches for differentiation and self-renewal. Journal of the Royal Society Interface, 15(145). https://doi.org/10.1098/rsif.2018.0388

Figure 11: Overview of PI3K/AKT/mTOR signalling pathway. Rinne, N.; Christie EL.; Ardasheva A.; Kwok CH.; Demchenko N.; Low C.; Tralau-Stewart C.; Fotopoulou C.; Cunnea P. (2021). [Illustration]. Targeting the PI3K/AKT/mTOR pathway in epithelial ovarian cancer, therapeutic treatment options for platinum-resistant ovarian cancer. Cancer Drug Resistance. 2021; 4(3): 573-95. http://dx.doi.org/10.20517/cdr.2021.05

Figure 12: Vitamin C enhances stem cell therapeutic potential. Lee Chong, T., Ahearn, E. L., & Cimmino, L. (2019). [Illustration]. Reprogramming the Epigenome With Vitamin C. Frontiers in cell and developmental biology, 7, 128. https://doi.org/10.3389/fcell.2019.00128

Figure 13: Vitamin C influences the rate of self-renewal, preventing cancer development. Schönberger, K., & Cabezas-Wallscheid, N. (2017). [Illustration]. Vitamin C: C-ing a New Way to Fight Leukemia. Cell Stem Cell, 21(5), 561–563. https://doi.org/10.1016/j.stem.2017.09.015