The Making of Pancreatic β Cells: Advances and Apprehensions

Diabetes is a dreadful disease, which in its acute stages, causes severe multiple organ failure. It is also one of the world’s oldest diseases. Type 1 Diabetes is characterized by the absence of insulin and exogenous insulin dependency. Stem cell therapy is one of the promises of this era, as there are numerous studies on Rodents, Frogs, Zebra fish, Dog and Chick, elucidating the wide array of genes, transcription factors, signaling pathways and compounds, which could promote β cell neogenesis, regeneration, differentiation and trans-differentiation. Even though, a recent PubMed search on the keyword ‘Pancreatic beta cell proliferation’ revealed around 3000 reports, this review focuses on the trends attempted in recent years and infers certain critical aspects in the observations.

Diabetes is a multifactorial disease sharing its origin and aggravations from various sources such as food habits, environment, microbial infections, and family genetics. Recently, Alicia R. Timme-Laragy et al. [15] reported that zebra fish embryos, when exposed to PCB-126, which is a polychlorinated biphenyl, commonly present in electrical equipments, rubber and plastics, could develop core molecular aberrations in the formation of pancreatic β cells. Diabetic cases were reported as ancient as 3000 BC, in the period of Hesyra (16), but not at such an explosive proportions, this 21 century experiences [17], as the total number of diabetic individuals in 2030 is calculated to be 366 million [18].
Various other reports lament the radical change in the food habits [19,20] and Chrono-Biological shifts [21] from the primitive hunter-gatherer groups to the present post-industrial societies to be the source of numerous complications. Circadian clocks and various clock genes are reported to be associated with various imbalances such as Lung Fibrosis [22], Oral Squamous Cell Carcinoma [23,24], Colorectal cancer [24,25], and also in Neurogenesis [26].
In Type-1 diabetes, the β cells are destroyed rapidly by autoimmunity, hence, hyperglycaemia arises, which leads to further complications. Repeated intake of exogenous insulin, on the other hand, increases the risk of recurrent hypoglycaemia.
Henceforth, one of the primary solutions for these problems could be the regeneration of pancreatic β cells. These artificially generated β cells must have the ability to withstand high endocrinal demands, despite the increased metabolic and immunological insults. evidences, collectively suggests the mechanism of autoimmunity as the cause of canine diabetes, open to further research. In addition to the canine model, pigs exhibit much less sensitivity to Insulin [28], chickens need STZ injection in ovo, for successful diabetic induction [29] and various classifications of diabetes occurs in dogs and cats [30]. In short, these reports indicate the radical modes operandi differences among diabetic subjects across the species.

Molecules and Compounds
Various naturally occurring or artificially designed molecules and compounds are being reported to be directly involved in the proliferation of pancreatic beta cells. They are mentioned in Table 1. Inhibition of dual specificity tyrosinephosphorylationregulated kinase 1A (DYRK1A) and glycogen synthase kinase-3 beta (GSK3B).

Signalling pathways
Many signaling pathways and their intermediates are being reported to be involved in the proliferation of beta cells. The whole organism high-content screening by Naoki Tsujiv et al. [65] suggested 20 hit-compounds involving Retinoic acid, Serotonin and Glucocorticoid signaling pathways.

Adenosine signaling
Many effective drugs and compounds were screened in the study by Olov Andersson et al. [66], to be activating the Adenosine signaling in their course towards the regeneration of the β cells in Zebra fish. Some of them are: 1. NECA (5'-N-Ethylcarboxamidoadenosine)-a nonspecific adenosine agonist that activates the adenosine G Protein Coupled Receptor (GPCR) signaling, a potent β cell-specific enhancer acting upon A2aa receptor. Its proliferative effects were confirmed also in the studies by Ersin Akinci et al. [67] 2. A-134974-An adenosine kinase inhibitor that blocks the degradation of adenosine, therefore, increasing the endogenous adenosine 3. Cilostamide-It could affect adenosine signaling by inhibiting phosophodiesterase (PDE3), therefore, decreasing the degradation of intracellular cAMP. 4. Zardaverine -It affects the adenosine signaling by inhibiting PDE3 and PDE4. 5. IB-MECA-An adenosine agonist 6. EHNA-An adenosine deaminase inhibitor

TGF β signaling
TGF-β signaling is probably the most frequently investigated pathway, in its relation to the proliferation of pancreatic beta cells. Inhibition of this pathway is a common therapeutic strategy for treating pancreatic ductal adeno carcinoma (PDAC) and hepatocellular carcinoma (HCC) [68][69]. Inhibition of TβR1 (ALK5) by SB431542, leads to the down-regulated nuclear expression of the cell cycle regulator p27, therefore, promotes β cell proliferation [70]. Nodal, a member of this pathway almost specifically promotes β cells without disrupting the cellular viability [71].
The TGF-β2 pathway also reportedly [72] promotes endothelial cell (EC) lineage, upon induced pluripotent stem cells (iPSCs), in a miR-21-dependent manner. Knockdown or disruption of this pathway could result in significant reduction of EC markers like VE-cadherin. Deletion of this pathway in CD4+ T cells could result in the progression of Autoimmune Diabetes [73] and In vitro redifferentiation of β cells [76]. Other reports [74] suggest that both TGFβ-R1 and TGFβ-R2 are not responsible for neogenesis, but for normal β cell proliferation. Yousef El-Gohary et al. [75] reports the involvement of the TGF-β signaling regulators smad 7,2 and 3 in the course of β-cell dedifferentiation.

Retinoic Acid (RA) Signaling
Another pathway which is studied in the context of pancreas development and pancreatic cancer (PC) is the Retinoic Acid Signalling (RA) pathway. Methylation of Cellular Retinoid Binding Protein 1 (CRBP1), an important regulator of this signaling, is strongly associated with exocrine acinar regeneration [77]. It should be noted that, in the work of Georgia Pennarossa et al. [78], in their attempt to generate pancreatic β cells from skin fibroblasts using demethylation procedures, the addition of RA along with Activin-A, resulted in increased differentiation towards pancreatic cell lineage and formation of clearly distinguished reticular patterned cell aggregates. From these two studies, it could be hypothesized that the demethylation step is the strong reason in the development of pancreatic β cells. In Chick and Mouse models, this pathway is positively associated with the olfactory neurogenesis [79].

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Vol. 5

Transcription factors
The study by Chutima Talchai et al. [80] suggests dedifferentiation of β cells to be the cause of diabetes. This report effectively pioneered further studies (Table 2) involving various transcription factors (TFs) in this field.

EFFECT UPON β CELLS OTHER FUNCTIONS REFERENCES
Pdx-1 Specific to beta cells.cdk4 dependent beta cell proliferation.
Maintains regional cellular identity in adult foregut in mice.
Mutation of this lead to decreased body size.
PAX balance between α cells and β /δ cells is maintained by the antoganistic actions of the two homeodomain transcription factors Aristaless-related homeobox (ARX) and PAX4, respectively [95][96][97]. Development of β and δ cells from the dorsal pancreatic bud in mouse, but not in zebra fish.
Protection from stress-induced apoptosis. In Drosophila, the Ey protein is the counterpart of PAX6.

miRNAs
miRNAs are small, non-coding RNA sequences which could affect the gene expression either post-transcriptionally or post-translationally. Overexpression and knockout studies with many invertebrates reveal that miRNAa are inevitable for many cellular and developmental regulations as many are stage-specific and tissue-specific [103]. Recently, Ksenia Tugay et al. [104] reported the association of miRNAs with age-related decline of β cell function. Other similar reports are mentioned in Table 3. It should be noted that gene therapy strategies using miRNA mimics could lead to non-specific gene expression changes, failed target gene suppression, interferon responses, accumulation of unnatural strands and other molecular changes, unless handled with caution [105]. -Adaptive β cell expansion.
-Mass andTurnover rates of both α and β cells.

Stem cells
Dilli Ram Bhandari et al. [116], had come up with a simple and time saving method for the effective In vitro production of the insulin producing cells (IPCs) from UCB-MSCs (Umbilical Cord Blood derived Mesenchymal Stem Cells), WJ-MSCs (Wharton's Jelly derived Mesenchymal Stem Cells) and AE-SCs (Adult Embryonic Stem Cells).
This method utilizes some of the previously reported methods [117][118][119][120] with slight modifications. Growth factors such as Fibroblast growth factor (FGF), Epidermal growth factor (EGF), Keratinocyte growth factor (KGF), Vascular endothelial growth factor(VEGF) and Insulin like growth factor-1(IGF-1) were added in the medium. Activin A along with sodium butyrate was used as key factor for the endodermal differentiation in this study. Sanna Toivonen et al. [121], reported the stimulation with Activin A and Wnt3a for the development of hepatic and pancreatic progenitors from human pluripotent stem cells (hPSCs). Georgia Pennarossa et al. [143] also used the protocol of Shi Y et al. [118] to generate pancreatic β cells from adult dermal fibroblasts. Expression of SOX17, PDX1 and positive response to glucose stimulation, production of Insulin and C-peptide from the differentiated cells was found to be significantly increased than the undifferentiated cells.
Rui Wei et al. [122] compared the two routes of production, the first involving nestinpositive progenitors and second involving the definitive endoderm (DE), for the successful differentiation of cells into insulin producing cells (IPCs) from human embryonic stem cells (hESCs). In spite of the similarities of the results such as islet-like cell aggregation, expression of transcription factors such as Pdx1, MafA and Nkx6.1, production of pancreatic hormones such as Insulin, C-peptide and PP, Glucose -stimulated insulin production and IPC morphology among the both methods, there were certain differences observed (Table 4). Altogether, this study prescribes the nestin protocol, as it involves the neuronal-trait of β cells and anticipates similar works with same cell lines to elucidate and modify the different obtained results.
In addition to the report by Rui Wei et al. [122], Mahmoud M.Gabr et al. [123] compared three IPS protocols involved in the human bone marrow derived mesenchymal stem cells (HBM-MSC) production. The first protocol was the one-step protocol. This method was utilized by Hisanaga et al. [124], which used Fetal bovine serum, Conophylline and Betacellulin. The second one was the two-step protocol. This protocol was utilized by Tayaramma et al. [125]. It utilized Trichostatin-A (TSA), fetal bovine serum and glucagon like peptide-1(GLP-1). The third was the three-step protocol used by M. M. Gabr et al. [126]. It utilized β-mercaptoethanol, basic fibroblast growth factor, epidermal growth factor, betacellulin, activin-A, B27 supplement and nicotinamide.
They report that the cells generated using two-step (TSA-based) protocol synthesized relatively higher Insulin and Glucagon. The relative expression of pancreatic endocrine genes was also higher in this group. Other reports suggest that factors such as culture medium density [127] could be very helpful in achieving optimal differentiation.

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Vol. 5 Dong-Sik Ham et al. [128] reports an efficient formation of Insulin-producing cells from Neonatal Porcine Liver-Derived Cells (NPLCs) by the addition of PDX1/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA). After transplantation in STZ-mice these cells cured hyperglycaemia and Insulin secretion was achieved in 6 weeks. The Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrinal cells also was reported [129]. They have performed adenoviral induction of MafA, Neurog3, Pdx1 and PAX6 upon the cultured ductal cells. Significant levels of Insulin expression, Insulin + cells, Proinsulin levels, and other endocrinal islet markers such as endocrine markers, including SST, GCK, PCSK1, KCNJ11, and ABCC8 were observed as results in this study.
Fazel Sahraneshin Samani et al. [130] accomplished in-vitro differentiated human umbilical cord blood CD133+ cells into insulin producing cells. Intriguingly, the insulin levels secreted by these cells in response to glucose challenge have varied. Exendine-4 (EX-4) [131] and Laminin 411 [132] could also be used to optimally differentiate the adipose-derived mesenchymal cells (ADMSCs) and umbilical cord MSCs, respectively, into insulin producing cells. The conversion of δ cells into β cells also is an age-dependent process [133].

PAK1
The p21-activated kinase 1 (PAK1) is a Serine/threonine kinase, important for the whole body glucose homeostasis and also for the insulin secretion and signaling. Survivin is a multifunctional protein involved in cell cycle regulations. In pancreas, it is selectively expressed in the β cells and its over-expression restores the proliferation of β cells. Survivin is strongly positively regulated by PAK1 [134]. Activation or over expression of PAK1 is associated with inflammation and colitis-associated carcinogenesis NF-κB pathway [135], Gastric cancer [136,137] and Lung cancer [138]. Therapeutic targeting of PAK1 in Acute myeloid leukemia (AML) and Myelodysplastic syndrome (MDS) is highly effective in achieving tumor-specific apoptosis and differentiation of AML cells [139].

CCN3
Studies on knock-out mice [140], cDNA microarrays [141] and recent works by Renée Paradis et al, in 2013 [142], show that the nephroblastoma overexpressed gene (Nov or Ccn3), is a novel, and, one of the transcriptional targets, up-regulated by FoxO1 in the β cells. The expression of Ccn3 is increased in patients with obesity. It negatively regulates β cell proliferation [142].

Impact of epigenetic alterations
Few evidences were reported on the impact of epigenetic modulations upon the β cell phenotype and proliferation. Both DNA methylation [143] and Histone methylations (H3K4me3 and H3K27me3) [144] are reported to be associated with the formation of pancreatic converted cells (PCCs) from adult dermal fibroblasts and α-β cell plasticity, respectively.

The effect of systemic microenvironment and ageing
The effect of ageing upon β cell survival and regeneration is contraversial [145][146][147]. Similar studies were conducted in various organs such as Muscle [148], Skin [149] and Neuronal cells [150]. Heterochronic and isochronic transplant studies [151] have shown the increased proliferation rates of old islet β cells which were transplanted to young mice and of young islet β cells which were transplanted to young mice. Conversely, decreased proliferation rates were observed in young islet β cells transplanted to old mice and old islet β cells transplanted to old mice.
The Ink4a (Cyclin dependent kinase inhibitor 2a) locus has certain impact upon β cell mass in the course of aging [152]. This locus encodes the protein p16 Ink4a , which is an inhibitor of CDK4 activity.CDK4 increases β cell proliferation by increasing β cell number [153].This locus is regulated by the Polycomb group (PcG) of protein complexes such as Polycomb repressive complex

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Volume 5 1(PRC1) and Polycomb repressive complex 2(PRC2) [154], which contain Bmi/ubiquitin ligase-Ring1B proteins and the histone methyltransferase, named as enhancer of zeste homolog 2 (EZH2), respectively. Repression of PcG complex and expression of EZH2 enhanced β cell proliferation in older mice. The conversion of δ cells into β cells is also an age-dependent process [133]. The inhibition of Insulin-IGF signaling (IIS) pathway by the selective ablation of median neurosecretory cells (mNSCs) could prevent ageing complications and obesity in Drosophila [155]. These evidences suggest the detrimental effects of excess sugar which has been forshadowed in earlier reports [19]. In Drosophila, the Insulin producing cells (IPCs) are regulated by the Drosophila insulin like peptides (DILPS), which control the metabolic and ageing processes. The receptor of Drosophila tachykinin-related peptides (DTKs), DTKR regulates the survival of IPCs as suggested by increased transcript levels of Dilp2 and Dilp3 [156]. In addition to DTKs, the drosophila Cbl family proteins (dCbI) also modulates the ageing processes by directly controlling DILP production via the EGFR-ERK pathway [157].

Control of cell cycle molecules upon β cell proliferation
Based on the previous works [158,159] on β cell proliferation, Nathalie M. Fiaschi-Taesch et al. [160], reported the roles of key G1/S phase regulators during the course of proliferation based on adenoviral integration with both cdk6 (cyclin dependent kinase-6) and cyclin D3 (Ad.C6+D3) and its transduction into human cadaveric and rat insulinoma cells (Ins1 832/13).
Ki67 was used in this study, instead of BrdU, as it reflects the cell cycle progress right at the time of labeling. The results of labeling of cells post-proliferation with the transduction combination are shown in Table 5. These results were confirmed by live cell imaging by GFP tagged cdk6.The expression levels were verified with Rat islets, human colon cancer cells (HCT116) and human embryonic kidney cells (HEK293).
These observations suggest optimal concentrations of p21 and 27 in promoting cell cycle and a selective trafficking of molecules across nucleus during proliferation. Although this study explains some important regulations, it anticipates more details on other important molecules in different β cell types. The same research group [161] reported the combinatorial actions of early and late phase Cyclins and Cdks upon the proliferation of β cells and their In Vitro expansion. They also suggest that human β cells are more resistant to proliferation than the β cells of Rats. Recently, High-Throughput Screening methods (HTS) suggested p18 and p21 to be the prime candidates for the proliferation of beta cells [162].

Impact of nervous system
The synthesis of insulin, in response to stimulation of the vagus nerve, is very common in normal glucose homeostasis. Blocking the parasympathetic nervous system negatively affects β cell proliferation [163]. γ-aminobutyric acid (GABA), is an important neurotransmitter of the central nervous system. In the β cells, it is synthesized by the two isoforms of Glutamicacid decarboxylase (GAD), GAD 65 and GAD67 This signaling has significant role in the late-fetal and early post-International Journal of Pharmacology, Phytochemistry and Ethnomedicine Vol. 5 natal development of pancreas in mice, as GABA signaling precede Insulin expression [164]. Seratonin, another neurotransmitter and its related signaling molecules also are playing vital role in the proliferation of β cells [65].

Interactions among inter and trans-dermal derivatives
The endocrinal insulin ameliorates schizophrenia; the vagus nerve and the neurotransmitter GABA stimulate β cell proliferation. Also 1. Andrew V. Biankin et al [165], reported with Exome sequencing and Copy number variation(CNV) methods that mutations in the genes such as ROBO1, ROBO2, SLIT2, SEMA3A, SEMA3E, SEMA5A, EPHA5 and EPHA7, which are involved in pancreatic ductal adeno carcinoma (PDAC) are also involved in axon guidance. GWAS studies suggest that ROBO2 is associated with expressive vocabulary in infants [166]. Robos are also involved in the midline crossing of axons [167] and cellular senescence [168].
3. Hussein A.N. Al-Wadei et al [169], reports the therapeutic ability of GABA along with Celecoxib, a COX-3 inhibitor, in preventing pancreatic cancer by inhibiting β-adrenergic effectors. Athymic nude mouse and the pancreatic lines BXPC-3 and Panc-1 were used in this study.15nM of Epinephrin was used as stress-trigger. The inhibitory effects of Celecoxib were significantly increased by the addition of GABA.
4. By expressing the key Pdx1, MafA and Ngn3 (PMN) factors in intestinal cells, Y.J.Chen et al. [170] reports a De Novo formation 'Neo-β Cell Islets'. They used double transgenic (DTG) mice generated from R26Tetβ (Rosa26 locus) and R26rtTA*M2 mice. These neo-islets cells responded well to glucose challenge in STZ-treated mice. Islet cells could also be generated from gall bladder using the induction of NEUROG3, Pdx-1 and Maf-A [171].

Conclusion
Altogether, these evidences suggest that there is an appreciable integration among the dermal derivatives and they could assist each other in various ways. Yet it should be noted that this 'integration' could also sabotage any therapeutic interventions upon pancreatic β cells, as it could affect other dermal networks. We believe that more elaborate studies on morphogenesis and predermal determinants are required to confront this dilemma.