Cellular Differentiation: Comparison
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Please note this is a comparison between Version 2 by Catherine Yang and Version 1 by Sandeep Raha.

Across embryonic development to late adulthood, highly regulated cellular differentiation is imperative for proper development and growth, as well as for the maintenance of specialized tissues throughout life. In general, this crucial cellular process underlies organogenesis and tissue regeneration, and its dysregulation or pathological dysfunction may accelerate aging and/or the onset of disease. Furthermore, the effects of cannabinoids on cellular differentiation are seen across a broad variety of tissues, including many peripheral tissues such as muscle, bone, and blood.

  • stem cells
  • cannabinoids
  • mitochondria
  • cellular differentiation

1. Stem Cell Characteristics

Cells that have the ability to develop into more specialized cell types by undergoing differentiation are collectively referred to as "stem cells." The dynamic range and ability of stem cells to differentiate into more mature cell types allow them to be further categorized in terms of cell potency [14][1]. For instance, stem cells that can potentially differentiate into all cell types are totipotent, whereas stem cells that can differentiate into most but not all cell types are pluripotent. Embryonic stem cells (ESCs) are defined as totipotent and pluripotent, as they give rise to multipotent stem cells. Additionally, other stem cells have the ability to differentiate into a specifically related family of cell types and are thus multipotent. For example, NSCs are among the most well-appreciated multipotent stem cells, with a large body of literature dedicated to adult neurogenesis that takes place within the SVZ and hippocampal regions [15][2]. Multipotent mesenchymal stem cells (MSCs) can differentiate into bone, cartilage, muscle, and skin tissues. Immune cell populations originate from a common multipotent hematopoietic stem cell [16][3].
All stem cells share two fundamental characteristics: their inherent ability to both (1) self-renew and (2) differentiate into specialized cell types [17][4]. To elaborate, undifferentiated or partially differentiated stem cells receive metabolic signals that induce them to either self-renew, thereby replenishing the existing stem cell population, or to commit and differentiate into specialized tissues through asymmetric division [18][5]. The tendency of stem cells to favor one fate decision over another is dependent on several intracellular and extracellular signaling cues. Stem cells are not constitutively active; rather, a quality control mechanism exists wherein a subset of stem cells enter quiescence, a state of metabolic depression, to maintain a functional pool of stem cells throughout adulthood [19][6]. Within this subset of metabolically inactive stem cells, there exist naïve and primed pluripotency states, which possess inherent epigenetic distinctions [20][7]. In humans, the former closely resemble premature embryonic epiblast stem cells, and the latter resemble post-implantation epiblast cells [20,21][7][8]. The dynamic ability of stem cells to maintain or exit pluripotency is reliant on the coordination of pro-differentiation or pro-self-renewal genes at the transcriptional level.

2. Key Intracellular Signaling Pathways Involved in Stem Cell Function and Differentiation

In response to chemical signals in their microenvironment, stem cells undergo transcriptional changes at the nuclear level to facilitate developmental and regenerative processes. Extrinsic pressures due to oxygen tension and the presence of inflammatory cytokines have been demonstrated to mediate stem cell differentiation by altering cellular transcriptional programs [22][9]. Stem cells are maintained through the actions of the Wnt and Notch signaling pathways, both of which have been implicated in promoting “stemness” [23,24,25,26,27,28][10][11][12][13][14][15]. In general, the canonical Wnt/β-catenin signaling pathway leads to the stabilization of β-catenin, followed by the accumulation and localization of β-catenin in the nucleus, where it crucially regulates genes related to pluripotency [29,30][16][17]. In naïve human embryonic stem cells (hESCs), Xu et al. demonstrated that the inhibition of Wnt signaling decreases the proliferative capacity of the naïve stem cell population and further promotes a primed stem cell phenotype [31][18]. The addition of recombinant Wnt3 partially rescued the proliferative potential of hESCs, demonstrating that Wnt signaling is critical in maintaining self-renewal [31][18]. During placentation in early pregnancy, hypoxic conditions (2.5% O2) dominate the trophoblast microenvironment before the reestablishment of normoxic levels (20% O2) following placentation [32][19].
In some cases, stem cells respond to oxygen deprivation by recruiting stable hypoxia-inducible factors (HIFs), which promote vascularization and preserve cellular homeostasis through the regulation of bioenergetic pathways. Activated HIF transcription factor subunits localize to the nucleus and bind to the hypoxia response element (HRE), which promotes the transcription of cell proliferation and survival genes. However, the transcription factor HIF2α has also been shown to activate Wnt and Notch signaling pathways. To illustrate the key role of HIFs during trophoblast stem cell differentiation, an early study found that HIF1α- and HIF2α- null embryos fail to undergo placental morphogenesis as a result of dysfunctional stem cell fate determination [33][20]. Interestingly, Caniggia et al. observed substantial levels of HIF1α and TGFβ3, an inhibitor of extravillous trophoblast (EVT) differentiation, during early pregnancy [34][21], and further uncovered that HIF1α is located upstream of TGFβ3 gene expression, thus mediating cell fate genes [35][22].
The exact role of inflammation in mediating stem cell homeostasis is still unclear [36][23]. Some studies have reported a negative regulatory role for inflammation, wherein stem cell proliferation is diminished [37][24]. To emphasize the inhibitory role of inflammation on neurogenesis, Monje and colleagues found that treatment with indomethacin, an anti-inflammatory drug, rescued neural stem cell function following endotoxin-induced inflammation [38][25]. Likewise, in a mouse model of cortical development, it was observed that induced, systemic maternal inflammation resulted in less ventricular proliferation in the fetus, indicating negative regulation of stem cell function [39][26]. Finally, in mice lacking the tumor necrosis factor receptor 1 (TNFR1), it was shown that cell proliferation was considerably elevated in the dentate gyrus, suggesting that negative regulation might be the result of pro-inflammatory responses mediated by TNFR1 [37][24]. In contrast, Wolf et al. demonstrated that neural stem/progenitor cells (NSPCs) in the mouse hippocampus display enhanced proliferative capacity following bacterial endotoxin-induced inflammation [40][27]. Previous in vitro studies have identified key inflammatory cytokines, TNFα and IL-1β, as promoters of NSPC proliferation and differentiation by activating either NFKβ or JNK signaling pathways [41,42][28][29]. Taken together, it is apparent that inflammation plays a complex and multifaceted role in mediating stem cell function.
In addition to oxygen-sensing mechanisms, there exist various nutrient-sensing systems designed to coordinate cellular homeostasis with nutrient availability. Among these, the mammalian target of rapamycin (mTOR) protein kinase and AMP-activated kinase (AMPK) emerge as fundamental intracellular nutrient sensors with opposing downstream effects [43][30]. To elaborate, the activation of mTOR enhances anabolic processes, including cell growth, protein translation, and mitochondrial metabolism, whereas, under low nutrient conditions, AMPK activation promotes catabolic activities, such as glucose metabolism and autophagy. The cross-regulation of autophagy by mTOR and AMPK points to a crucial role in autophagic processes in stem cell dynamics. In HSCs and satellite cells, the deletion of an autophagy-related gene, Atg7, resulted in the loss of both HSC and satellite cell pools, phenotypes that were associated with increased ROS production and the accumulation of damaged mitochondria [44,45][31][32]. Likewise, Atg12-deficient HSCs exhibited similar deficits in HSC self-renewal [46][33], suggesting that autophagy might play a role in removing mitochondria to regulate stem cell bioenergetics. mTORC1 is complexed with several proteins, including the regulatory-associated protein of mTOR (Raptor) and the DEP-domain-containing mTOR interacting protein (Deptor), which have a range of functions from substrate recognition to the regulation of mTOR activity, respectively. The major downstream targets of mTOR, a serine/threonine kinase, include the ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein (4EBP1), which function in amino acid and ATP sensing. Specifically, being fundamental components for protein synthesis, amino acids indeed promote mTORC1 complex assembly, while ATP availability supports the energy requirement for downstream anabolic processes [47][34]. Upon its phosphorylation and activation by mTORC1, pS6K1 further phosphorylates eIF4B, a cofactor required for mRNA translation initiation due to its RNA helicase activity, and PDCD4, an inhibitor of eIF4B, thus promoting its degradation [48][35]. Alternatively, the native function of 4EBP in preventing the formation of the eIF4E complex required for translation by binding to and inhibiting eIF4E is hindered by mTORC1 phosphorylation, which ultimately dissociates 4EBP from eIF4E [49][36]. In addition, these mTORC1 effectors may possess multifaceted roles in cellular growth. For instance, de novo lipogenesis can be induced directly through an mTORC1-mediated pathway, wherein S6K1 activates the sterol-responsive element binding protein (SREBP), or indirectly, in which the absence of mTORC1 signaling leads to the association of Lipin1 and SREBP, thus preventing its activation [50][37]. Taken together, a complex interplay of feedback loops guiding mTORC1 signaling is responsible for promoting anabolic processes during conditions of substrate abundance.
In the context of aging, it is becoming increasingly apparent that mTOR contributes to tissue homeostasis by modulating stem cell maintenance, differentiation, and proliferation [51][38]. In a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, mTORC1 has been shown to promote the differentiation or proliferation of NSCs, HSCs, and mammary and germline stem cells upon stimulation by growth factors such as IGF [52,53,54][39][40][41]. IGF-mediated stimulation has been implicated in neuronal differentiation, wherein it induces an intracellular signaling cascade that results in the phosphorylation of Akt at Ser473 and Thr308 residues in differentiating olfactory bulb stem cells (OBSCs) [55][42]. Likewise, exit from pluripotency and initiation of differentiation have been linked to mTOR in hESCs, where these signaling cascades are tightly regulated. Easley et al. knocked down the rapamycin-insensitive companion of the mammalian target of rapamycin (Rictor), an associated protein of the mTORC2 complex, and tuberous sclerosis complex 2 (TSC2), an inhibitor of mTORC1 [56][43], using siRNA-mediated technology, and found increased activation of p70 S6K coupled with greater differentiation in hESCs [57][44]. In contrast, a recent study by Lee et al. found that inhibiting the well-established negative regulator of PI3K signaling, phosphatase, and tension homolog (PTEN), led to increased human NSC proliferation [58][45]. Furthermore, Schaub et al. demonstrated that mTORC1 and mTORC2 are involved in coordinating osteoblastic differentiation in MSCs [59][46]. In brief, MSC differentiation is achieved in vitro by incubating cells in an osteoblast induction medium for three weeks, after which the expression of key osteoblast marker proteins, such as osteopontin, collagen I and III, and Cbfa1, is highly expressed. However, it was found that osteoblast marker proteins were markedly reduced in MSCs treated with rapamycin, an mTOR inhibitor [59][46]. More specifically, their findings revealed that rapamycin exposure led to decreased phosphorylation of p70-S6K, a downstream effector of mTORC1, whereas mTORC2 activity was increased under the same conditions. Taken together, these observations suggest the existence of an intricate feedback loop between intracellular signaling targets, mTORC1, and mTORC2, in their co-regulation of cellular function.

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