An Insight into Vital Genes Responsible for ß-cell Formation.

The regulation of glucose homeostasis and insulin secretion by pancreatic ß-cells, when disturbed, will result in diabetes mellitus. Replacement of dysfunctional or lost ß-cells with fully functional ones can tackle the problem of ß-cell generation in diabetes mellitus. Various pancreatic-specific genes are expressed during different stages of development, which have essential roles in pancreatogenesis and ß-cell formation. These factors play a critical role in cellular-based studies like transdifferentiation or de-differentiation of somatic cells to multipotent or pluripotent stem cells and their differentiation into functional ß-cells. This work gives an overview of crucial transcription factors expressed during various stages of pancreas development and their role in ß-cell specification. In addition, it also provides a perspective on the underlying molecular mechanisms.

Production of Bioactive Human PAX4 Protein from E. coli.

Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for ß-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.

Biological activity of recombinant human PDX1 protein produced from Escherichia coli.

Pancreatic and duodenum homeobox 1 (PDX1) is considered as a pivotal transcription factor that acts as a "master regulator" in pancreatogenesis and maintenance of ß-cells. Earlier study has reported that PDX1 also functions as a tumor suppressor in human gastric cancer cells by inhibiting cell growth. Here, we report the bioactivity of the purified human PDX1 fusion protein using various assays like cell migration, proliferation, cell cycle analysis, and gene expression. In cancer cells, recombinant PDX1 protein reduced cell migration and proliferation, and arrested cell growth by inducing apoptosis in gastric cancer cells. In pancreatic ductal cancer cells, the application of the PDX1 protein resulted in the induction of insulin gene expression. The results of these experiments demonstrate the biological activity imparted by recombinant human PDX1 fusion protein on gastric and pancreatic cancer cells and its usefulness as a biological tool to elucidate its function in various cellular processes.

Direct Reprogramming of Somatic Cells into Induced ß-Cells: An Overview.

The persistent shortage of insulin-producing islet mass or ß-cells for transplantation in the ever-growing diabetic population worldwide is a matter of concern. To date, permanent cure to this medical complication is not available and soon after the establishment of lineage-specific reprogramming, direct ß-cell reprogramming became a viable alternative for ß-cell regeneration. Direct reprogramming is a straightforward and powerful technique that can provide an unlimited supply of cells by transdifferentiating terminally differentiated cells toward the desired cell type. This approach has been extensively used by multiple groups to reprogram non-ß-cells toward insulin-producing ß-cells. The ß-cell identity has been achieved by various studies via ectopic expression of one or more pancreatic-specific transcription factors in somatic cells, bypassing the pluripotent state. This work highlights the importance of the direct reprogramming approaches (both integrative and non-integrative) in generating autologous ß-cells for various applications. An in-depth understanding of the strategies and cell sources could prove beneficial for the efficient generation of integration-free functional insulin-producing ß-cells for diabetic patients lacking endogenous ß-cells.

Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli.

Neurogenin 3 (NGN3) transcription factor is vital for the development of endocrine cells of the intestine and pancreas. NGN3 is also critical for the neural precursor cell determination in the neuroectoderm. Additionally, it is one of the vital transcription factors for deriving human ß-cells from specialized somatic cells. In the current study, the production and purification of the human NGN3 protein from Escherichia coli (E. coli) is reported. First, the 642 bp protein-coding nucleotide sequence of the NGN3 gene was codon-optimized to enable enhanced protein expression in E. coli strain BL21(DE3). The codon-optimized NGN3 sequence was fused in-frame to three different fusion tags to enable cell penetration, nuclear translocation, and affinity purification. The gene insert with the fusion tags was subsequently cloned into an expression vector (pET28a( +)) for heterologous expression in BL21(DE3) cells. A suitable genetic construct and the ideal expression conditions were subsequently identified that produced a soluble form of the recombinant NGN3 fusion protein. This NGN3 fusion protein was purified to homogeneity (purity > 90%) under native conditions, and its secondary structure was retained post-purification. This purified protein, when applied to human cells, did not induce cytotoxicity. Further, the cellular uptake and nuclear translocation of the NGN3 fusion protein was demonstrated followed by its biological activity in PANC-1 cells. Prospectively, this recombinant protein can be utilized for various biological applications to investigate its functionality in cell reprogramming, biological processes, and diseases.

Recent Advances in the Generation of ß-Cells from Induced Pluripotent Stem Cells as a Potential Cure for Diabetes Mellitus.

Diabetes mellitus (DM) is a group of metabolic disorders characterized by high blood glucose levels due to insufficient insulin secretion, insulin action, or both. The present-day solution to diabetes mellitus includes regular administration of insulin, which brings about many medical complications in diabetic patients. Although islet transplantation from cadaveric subjects was proposed to be a permanent cure, the increased risk of infections, the need for immunosuppressive drugs, and their unavailability had restricted its use. To overcome this, the generation of renewable and transplantable ß-cells derived from autologous induced pluripotent stem cells (iPSCs) has gained enormous interest as a potential therapeutic strategy to treat diabetes mellitus permanently. To date, extensive research has been undertaken to derive transplantable insulin-producing ß-cells (iß-cells) from iPSCs in vitro by recapitulating the in vivo developmental process of the pancreas. This in vivo developmental process relies on transcription factors, signaling molecules, growth factors, and culture microenvironment. This review highlights the various factors facilitating the generation of mature ß-cells from iPSCs. Moreover, this review also describes the generation of pancreatic progenitors and ß-cells from diabetic patient-specific iPSCs, exploring the potential of the diabetes disease model and drug discovery. In addition, the applications of genome editing strategies have also been discussed to achieve patient-specific diabetes cell therapy. Last, we have discussed the current challenges and prospects of iPSC-derived ß-cells to improve the relative efficacy of the available treatment of diabetes mellitus.

Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells.

Over a decade ago, a landmark study that reported derivation of induced Pluripotent Stem Cells (iPSCs) by reprogramming fibroblasts has transformed stem cell research attracting the interest of the scientific community worldwide. These cells circumvent the ethical and immunological concerns associated with embryonic stem cells, and the limited self-renewal ability and restricted differentiation potential linked to adult stem cells. iPSCs hold great potential for understanding basic human biology, in vitro disease modeling, high-throughput drug testing and discovery, and personalized regenerative medicine. The conventional reprogramming methods involving retro- and lenti-viral vectors to deliver reprogramming factors in somatic cells to generate iPSCs nullify the clinical applicability of these cells. Although these gene delivery systems are efficient and robust, they carry an enormous risk of permanent genetic modifications and are potentially tumorigenic. To evade these safety concerns and derive iPSCs for human therapy, tremendous technological advancements have resulted in the development of non-integrating viral- and non-viral approaches. These gene delivery techniques curtail or eliminate the risk of any genomic alteration and enhance the prospects of iPSCs from bench-to-bedside. The present review provides a comprehensive overview of non-integrating viral (adenoviral vectors, adeno-associated viral vectors, and Sendai virus vectors) and DNA-based, non-viral (plasmid transfection, minicircle vectors, transposon vectors, episomal vectors, and liposomal magnetofection) approaches that have the potential to generate transgene-free iPSCs. The understanding of these techniques could pave the way for the use of iPSCs for various biomedical applications.