Introduction
Understanding the mechanisms of beta cell differentiation and maturation is integral to studies of diabetes pathophysiology, regenerative therapies and stem-cell-derived models of beta cell dysfunction and replacement. While the developmental processes that lead to a stable beta cell identity are relatively well known, the mechanisms underlying the functional maturation of beta cells are less clear. In simple terms, beta cell ‘differentiation’ defines the acquisition of a terminally differentiated insulin-positive cell identity throughout in utero development or in vitro stem-cell-based protocols. Conversely, beta cell ‘maturation’ is a measurement of the phenotypic properties of beta cells and their ability to respond to, and control, blood glucose levels through glucose-sensitive insulin secretion (GSIS). Acquisition of this function occurs postnatally in rodents [
1‐
3] and humans [
4], and therefore is a property of beta cell development, but one that occurs beyond the attainment of fetal beta cell identity. This divide in cell identity and mature functionality is exemplified in early stem-cell-based differentiation studies that generated beta-like cells with high expression patterns of canonical beta cell markers but with limited functional activity [
5,
6]. The issue of defining what constitutes a mature beta cell can be therefore quite problematic if only certain aspects of beta cell biology are investigated, as has been recently reported [
7]. Some hallmark features of beta cell ‘identity’ and ‘maturity’ are outlined in the Text box and will be elaborated upon further throughout this review.
The current understanding of beta cell maturation is that such a process is a spectrum rather than a binary state [
8‐
10]. Mature functionality may itself also be a dynamic process, whereby beta cells flux from active to inactive states, or be dependent on the interplay of functionally heterogeneous beta cell pools [
11‐
16]. Functional maturation can be a reversible process, as beta cell dedifferentiation and senescence, with resultant functional deterioration, are known to be associated with the onset of diabetes [
17‐
19]. Therefore, the study of functional maturation of beta cells is necessary to understand the underlying mechanisms of dysfunction eventually leading to diabetes, as well as to improve the efficacy of therapeutic interventions and stem-cell-based islet replacement therapies.
This review will primarily focus on the reported multi-faceted mechanisms that drive and maintain beta cell functional maturation within in vivo and in vitro models.
Signalling pathways and gene markers of beta cell maturation
The extent of beta cell differentiation is generally evaluated, in vitro and in vivo, through the upregulation and maintenance of a set of known beta cell marker genes (including
INS,
PDX1 [
97],
NKX6.1 [
98],
NEUROD1 [
99],
MAFA [
100] and
UCN3) [
3]. However, the presence of these genes is not necessarily an indication of mature beta cell functionality [
10,
101]. Indeed, upregulation of urocortin 3 (UCN3) occurs during the postnatal maturation of beta cells [
3,
10] but UCN3 itself appears to be functionally redundant in driving this maturation process [
102]. It is therefore important to understand the difference between genes that are critical for maintaining beta cell identity and those that further determine the functional properties of beta cells, and of course, the intrinsic overlap between these two groups. The direct transcriptional regulation of beta cell-specific transcription factors and the influence on metabolic gene regulation is largely unknown. However, certain regulatory patterns have been discovered [
103]. For instance, MafA may help repress ‘disallowed’ metabolic genes while maintaining expression of specific glucose transporter genes (
GLUTs), glucokinase (
GCK) and
PGC1α (coding for a regulator of mitochondrial biogenesis and circadian oscillation) within beta cells [
104,
105]. A transcriptional network driven by oestrogen-related receptor γ (
ERRγ) has been shown to regulate multiple OxPhos-related genes, as well as regulating the pyruvate–citrate cycle-related enzyme encoded by
MDH1, during beta cell maturation [
106]. It has also been reported that transcriptional regulation through calcineurin–nuclear factor of activated T cell (NFAT) pathways tailor the expression of
GCK and
GLUT2 (GLUT2 is the predominant glucose transporter in murine beta cells) [
107,
108]. In contrast, the transcription factor activity of regulatory factor X6 (RFX6) is linked to regulation of
GCK but not
GLUT2 [
109]. These overlapping functions in metabolic gene regulation may be due to the web of beta cell-enriched transcription factors directly regulating each other, although there is evidence that the physical interaction of multiple transcription factors is necessary to maintain metabolically mature states, such as the co-binding of neuronal differentiation 1 (NEUROD1) and cAMP responsive element binding protein 1 (CREB1) in beta cell-specific enhancer regions [
110].
Transcriptomics studies comparing healthy and diabetic beta cell pools offer many clues to the subsets of genes that may be necessary for maintaining beta cell function, either through candidate transcription factors or through direct regulation of beta cell metabolism. One example of the latter is the higher expression of glucose-6-phosphatase catalytic subunit 2 (
G6PC2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (
PFKFB2) within healthy beta cell samples [
111‐
116] and the upregulation of these genes during maturation of SC-islet beta cells [
35,
61]. Both genes encode glycolytically linked enzymes that have been shown to have direct regulatory control over the glucokinase-mediated step of glycolysis [
117‐
120]. The functional maturation of beta cells therefore correlates with heightened control over this initial step of glycolysis, which may regulate the pattern of downstream metabolism and glucose trafficking. The governing mechanisms of glycolytic flux within mature beta cells may also encompass the regulatory effect of cytosolic citrate and PEP (as products of the TCA metabolite cycles outlined previously) on phosphofructokinase 1 (PFK1) activity [
112], with the generation of fructose-1,6-bisphosphate as a possible direct modulator of glucose-sensitive AMPK/mTOR activity [
95] (Fig.
2). Even the oligomerisation state of the glycolytic enzyme GAPDH (rather than expression level) has been associated with beta cell functional maturation [
34]. Some recent findings have also identified the SIX homeobox 2 (SIX2) and SIX homeobox 3 (SIX3) transcription factors as regulators of beta cell functional maturation [
121,
122]; this has been demonstrated in SC-islet knockdown models of SIX2, wherein GSIS function was strongly impaired [
123]. Interestingly, although SIX2 is necessary for SC-islet functional acquisition in vitro, SIX3 expression appears to be important for advanced maturation events and is not detected in SC-islets in vitro or after extended murine engraftment [
35,
123].
The regulation and temporal sequence of genes within this context must at some level be run through transcription factor networks that are responsive to cell lineage signalling and systemic nutritional cues [
101]. The ‘holy grail’ within the field of SC-islet generation is an optimised cocktail of signalling and patterning factors that would trigger in vitro beta cell maturation to the same level that is seen post-engraftment. Therefore, SC-islet generation protocols represent fertile ground to test candidate maturation signalling molecules, while simultaneously providing information on processes occurring during postnatal maturation [
9,
124,
125].
A recent study found that non-canonical Wnt signalling, through Wnt4, may be one such signalling pathway that triggers maturation events within SC-islet beta cells [
126]. It has long been established that Wnt signalling has a variety of important functions throughout islet organogenesis that are spatially and temporally controlled [
127]. Furthermore, in SC-islets at earlier stages of differentiation, Wnt signalling affects the balance and penetrance of pancreatic progenitor formation [
128,
129]. The exogenous application of Wnt4 to SC-islets increases an assortment of beta cell marker genes as well as mitochondrial OxPhos responsiveness to glucose [
126], a pattern that is also seen when Wnt4 is added to human islet and beta cell lines [
13].
WNT4 expression has also been observed in neonatal rat islets, suggesting a role in postnatal functional maturation [
85]. However, another SC-islet study was unable to detect any discernible improvement in beta cell maturation following Wnt4 treatment, and conversely found that canonical Wnt signalling inhibition improved SC-islet maturation [
130]. Regardless, the interplay of Wnt signalling in different aspects of beta cell differentiation and maturation is well founded. Tantalising evidence of Wnt signalling-derived AMPK/mTOR pathway changes in the regulation of the
Tcf7l2 gene in beta cell proliferation in mice is a clear demonstration of the holistic interactivity of cell signalling, energy-sensing machinery and mature beta cell functionality [
131].
Many members of the TGF-β family have also been connected with beta cell maturation, although again with some inconsistent findings between research groups. Inhibition of the transforming growth factor β receptor 1 (TGFBR1, also known as ALK5) during SC-islet maturation has been shown to increase many beta cell marker genes, including
MAFA [
5]. However, more recent studies have shown either a marked improvement of SC-islet beta maturation in the absence of ALK5 inhibitors [
132] or, in contrast, an increase in insulin expression in the presence of ALK5 inhibition [
31]. Another member of the superfamily, bone morphogenetic protein 4 (BMP4), may also aid in the postnatal maturation of beta cell function following temporally controlled release from islet pericytes [
133]. However, one study found that BMP4 treatment inhibited GSIS through the reduction of calcium currents [
134], again indicating that particular cellular milieus and developmental timings elicit strong control over specific signalling outcomes. The thyroid hormone triiodothyronine (T3) has also been shown to accelerate the postnatal maturation of beta cells and boost
MAFA expression in SC-islet models [
105,
135], demonstrating that beta cell maturation is affected by systemic hormonal exposure.
In parallel with the signalling pathways outlined above, beta cell maturation may also be self-regulated via the modulation of extracellular ATP release and purinergic receptor-based signalling, through the activity of ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) [
136]. This has been identified in numerous beta cell transcriptomic studies [
114,
137] and has also been shown to be a marker of beta cell maturation within SC-islets [
31]. An intriguing overlap between these findings and the model of glucose-sensitive purine synthesis within mature beta cells may imply yet another nexus point of cellular energy state (through AMPK/mTOR modulation), metabolic trafficking (ATP production and release) and the regulation of GSIS in mature beta cells [
138] (Fig.
1).
Another intriguing feature of beta cell functional maturation is the regulatory influence of microRNAs. Shifting patterns of microRNA expression have been shown to elicit robust regulatory effects on metabolic gene expression and beta cell functionality in a nutrient-sensitive manner, as well as throughout postnatal maturation [
139‐
141]. The upregulation of the miR-129 family in beta cells during postnatal weaning in mice correlated with enhanced glucose-responsive insulin release [
139]. This mirrors the postnatal increase in the expression of the miR-29 family, which has also been shown to repress the ‘disallowed’ genes
REST [
139] and
SLC16A1 [
142]. In contrast, downregulation of the miR-181 and miR-17 families during postnatal maturation leads to the upregulation of
GPD2,
MDH1 and
PFKP metabolic genes [
139]. Other microRNAs such as the miR-223 family (which ostensibly maintains
PDX1 and
NKX6.1 expression through suppression of forkhead box O1 [FOXO1] and SRY-box transcription factor 6 [SOX6] pathways [
143]) and the miR-7 family (which reportedly boosts GSIS and
PDX1 levels in SC-islets [
144] while suppressing mTOR signalling and proliferation [
145]) are all enriched in mature beta cells. However, conclusions about the presence or absence of a particular microRNA family should be assessed in relative terms. For example, the miR-375 family is upregulated during SC-islet maturation [
144], yet the forced overexpression of miR-375 in primary islets was reported to blunt GSIS responses and reduce glucose-responsive OxPhos [
146]. This drop in functional activity could be explained by the increased expression of
PDK4 and reduced
PC and
MDH1 expression within the primary islets. The shifting patterns and balance of microRNA family expression is therefore another key component of the onset and maintenance of beta cell maturity.
Finally, epigenetic signatures may help explain particular functional features of mature and immature beta cells. Both DNA methylation and histone modification are mechanisms by which beta cell identity and function are maintained, through tailoring the expression pattern of beta cell-enriched transcription factors, as well as being regulated by the transcription factors themselves [
147,
148]. Some relevant examples include DNA methylation through the activity of DNA methyltransferase 3 α (DNMT3A), which has been linked to the repression of beta cell ‘disallowed’ genes, regulated through the mTORC1 component raptor [
26] and through the inhibition of Wnt signalling during SC-islet maturation [
130]. Histone methylation involving the activity of the polycomb repressor complex (PRC2) may act in juvenile islets to maintain an immature transcriptomic state together with trithorax group (TrxG) proteins [
121,
149]. Evidence for extensive epigenetic shifts throughout beta cell maturation has also been seen in SC-islets [
50]. All of these aspects of regulation are intricately tied to the metabolic state of the beta cell, as each form of epigenetic modification is fuelled by specific metabolic inputs [
150].
In summary, the concept of tracking beta cell maturation through panels of marker genes is one that should be approached cautiously. While core beta cell identity genes are no doubt important for many facets of beta cell maturation, the upregulation of one particular gene is not necessarily a strong argument for predicting functional maturation. Indeed, much more needs to be uncovered about how signalling pathways fully trigger beta cell maturity and through which mechanisms they operate. Furthermore, interpreting expression levels of particular genes, especially those within core metabolic pathways, should be done carefully so as not to misconstrue what is necessary for specific beta cell function and what is key for basal cellular metabolism. Additionally, overexpression of particular genes within a pathway may not necessarily trigger systemic maturation events. Full understanding of beta cell maturity clearly needs to go beyond simplistic models of gene and protein expression, and the regulation and maintenance of epigenetic signatures in beta cell function and disease must be considered.
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