Insulin Precursor Research Articles

Construction of a full length α-factor secretory signal sequence for human insulin precursor expression in Pichia pastoris

Saccharomyces cerevisiae and Pichia pastoris are yeast known as a potential expression system to produce recombinant protein. The full-length α-factor (α-mating factor) secretory signal of S. cerevisiae plays an essential role in the secretion and processing of the mature protein of interest. Here, we attempted to construct the full-length α-factor signal sequence of S. cerevisiae in the pD902-IP (Insulin Precursor) expression vector for secreted expression of human insulin precursor in P. pastoris. We have isolated the full-length α-factor secretory signal sequence in a pTA2 cloning vector. The full-length α-factor was then inserted into the IP-cassette of pD902-IP and transformed into E. coli TOP10. The E. coli transformants, which were able to grow on the Zeocin selection medium, harbored the full-length α-factor for the IP expression in the pD902 vector validated by PCR and sequencing. Furthermore, the construct electroporation into P. pastoris X-33 was done and followed by IP protein expression confirmation visualized with SDS-PAGE.

Capture and intermediate purification of human insulin precursor from Pichia pastoris culture using cation exchange chromatography

Purification has an important role in obtaining protein with a high degree of purity, particularly for human therapeutic purposes. Purification of pharmaceutical proteins requires several unit operations, involving chromatographic separation techniques. To increase purification efficiency and shorten process development, it is necessary to examine the chromatography system in performing a capture and intermediate purification in a single step. We use one of the best Pichia pastoris clones obtained from previous studies to produce a human insulin precursor (HIP). To capture and purify HIP from the culture, we clarified the cells through centrifugation and filtration. The supernatant was then loaded into a cation exchange column. Purification was carried on by two-step elution and monitored based on UV absorbance. Effects of loading concentration, flow rate, and pH of samples were evaluated. Fractions of elution were collected and verified by SDS-PAGE. Concentrations of HIP protein were quantified by using ImageJ by incorporating lysozyme as standard and reversed phase HPLC. Loading concentration and pH of the sample have an impact on the recovery. In this study, the best HIP recovery at ∼ 47% resulted from purification with 10% volume of loading concentration and 500 ml loading volume.

Jazf1 acts as a regulator of insulin-producing β-cell differentiation in induced pluripotent stem cells and glucose homeostasis in mice.

Genetic susceptibility of type 2 diabetes and Juxtaposed with another zinc finger protein 1 (Jazf1) has been reported; however, the precise role of Jazf1 in metabolic processes remains elusive. In this study, using Jazf1-knockout (KO)-induced pluripotent stem cells (iPSC), pancreatic beta cell line MIN6 cells, and Jazf-1 heterozygous KO (Jazf1+/- ) mice, the effect of Jazf1 on gradual differentiation was investigated. We checked the alterations of the genes related with β-cell specification, maturation, and insulin release against glucose treatment by the gain and loss of the Jazf1 gene in the MIN6 cells. Because undifferentiated Jazf1-KO iPSC were not significantly different from wild-type (WT) iPSC, the size and endoderm marker expression after embryoid body (EB) and teratoma formation were investigated. Compared to EB and teratomas formed with WT iPSC, the EB and teratomas from with Jazf1-KO iPSC were smaller, and in teratomas, the expression of proliferation markers was reduced. Moreover, the expression of the gene sets for β-cell differentiation and the levels of insulin and C-peptide secreted by insulin precursor cells were notably reduced in β-cells differentiated from Jazf1-KO iPSC compared with those differentiated from WT iPSC. A comparison of Jazf1+/- and WT mice showed that Jazf1+/- mice had lower levels of serum insulin, pancreatic insulin expression, and decreased pancreatic β-cell size, which resulted in defects in the glucose homeostasis. These findings suggest that Jazf1 plays a pivotal role in the differentiation of β-cells and glucose homeostasis.

Stability analysis of a Pichia pastoris recombinant clone expressing human insulin precursor

Current recombinant human insulin production utilizes two major expression systems, Escherichia coli and Saccharomyces cerevisiae-based expression systems. Methylotropic yeast, Pichia pastoris, has appeared as a promising alternate yeast recombinant host for insulin precursor (IP) expression due to its ability to produce high titers, efficient secretion, and growth to very high cell densities. Similar to the S. cerevisiae system, P. pastoris secreted soluble IP into the culture supernatant. In the previous study, we have established P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. It is essential to verify that the expression cassettes of the IP encoding gene remain stably integrated with the genome during such prolonged methanol induction. Therefore, we aimed to analyze the stability of one recombinant clone (CL-4) expressing the human insulin precursor by verifying the stable integration of the IP expression cassette into the genome by PCR, and the IP protein expression after prolonged methanol induction over 70 generations. We found that the expression cassette was stably integrated into the genome of the CL-4 recombinant clone and the IP expression was sustained after 72 generations of cultivations in the culture and induction media without antibiotic selection.

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae.

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast-famine conditions to characterize the long-term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long-term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast-famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor

BackgroundTrypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate.ResultsThe tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL−1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg−1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL−1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris.ConclusionsSGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

Selecting Pichia pastoris recombinant clones for higher secretion of human insulin precursor into the culture supernatant

The methylotrophic yeast, Pichia pastoris, is one of the preferred yeast hosts for recombinant protein expression. It has been developed as a potential host to express a high level of recombinant proteins, and to achieve efficient secretion as well as growth to very high cell densities. Previously, we have obtained 19 P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. To select P. pastoris recombinant clones which exhibit high levels of protein expression, we conducted secreted expressions of IP protein in shake flasks. The secretion of IP into the culture supernatants was verified by SDS-PAGE. IP protein concentrations were estimated using ImageJ by applying lysozyme as standard. All of the 19 P. pastoris recombinant clones were confirmed to secrete the IP protein into their culture supernatants, and a single protein band with a molecular size of approximately 7 kDa was found in the SDS-PAGE gel. The six highest IP-expressing clones were selected for second screening in shake flasks. We selected three recombinant clones (CL-3, CL-4, and CL-18), which secreted the highest levels of IP proteins compared to the other clones. The secreted IP concentrations estimated by ImageJ for clones CL-3, CL-4, and CL-18 were 1230, 1143, and 1010 mg/L, respectively.

Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris

Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris

Expression, purification, and characterization of the Degludec precursor DesB30

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed.

The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

A spacer peptide (Glu-Ala repeats) connects N-terminal of a polypeptide precursor to the signal peptide of α-mating factor (α-MF) from Saccharomyces cerevisiae. During the polypeptide precursor secretion, the signal peptide will be first cleaved by signal peptidase in the endoplasmic reticulum, followed by Kex2 endopeptidase and finally the spacer peptide is removed by dipeptidyl aminopeptidase in the Golgi apparatus. In the case of insulin precursor, unfortunately, mixture forms of the insulin precursor containing a spacer peptide and the correct insulin precursor is observed in Pichia pastoris. To overcome this problem, a gene encoding insulin Aspart precursor without the spacer peptide has been constructed and its expression has been investigated in this study. The gene of insulin Aspart precursor has been successfully expressed extracellularly in P. pastoris KM71 with molecular mass of 6306.8 Da as determined by LC-ESI-MS. The expression level of insulin Aspart precursor is affected by aeration, cell density and methanol as an inducer. In shake flask production, the insulin Aspart precursor was optimally produced by 3% methanol induction every 24 h for two days, at initial cell density (OD600) of 60 and aeration (ratio of culture volume to flask volume) of 1:25. Based on Western blot analysis, there is no intracellular insulin Aspart precursor detected. Therefore, the spacer peptide is not essential for insulin Aspart precursor secretion in P. pastoris and removal of spacer resulted in insulin Aspart precursor homogeneity.

Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.

The Prohormone Proinsulin as a Neuroprotective Factor: Past History and Future Prospects.

Proinsulin was first identified as the primary translation product of the insulin gene in Donald Steiner’s laboratory in 1967, and was the first prohormone to be isolated and sequenced. While its role as an insulin precursor has been extensively studied in the field of endocrinology, the bioactivity of the proinsulin molecule itself has received much less attention. Insulin binds to isoforms A and B of the insulin receptor (IR) with high affinity. Proinsulin, in contrast, binds with high affinity only to IR-A, which is present in the nervous system, among other tissues and elicits antiapoptotic and neuroprotective effects in the developing and postnatal nervous system. Proinsulin specifically exerts neuroprotection in the degenerating retina in mouse and rat models of retinitis pigmentosa (RP), delaying photoreceptor and vision loss after local administration in the eye or systemic (intramuscular) administration of an adeno-associated viral (AAV) vector that induces constitutive proinsulin release. AAV-mediated proinsulin expression also decreases the expression of neuroinflammation markers in the hippocampus and sustains cognitive performance in a mouse model of precocious brain senescence. We have therefore proposed that proinsulin should be considered a functionally distinct member of the insulin superfamily. Here, we briefly review the legacy of Steiner’s research, the neural expression of proinsulin, and the tissue expression patterns and functional characteristics of IR-A. We discuss the neuroprotective activity of proinsulin and its potential as a therapeutic tool in neurodegenerative conditions of the central nervous system, particularly in retinal dystrophies.

İnsan İnsülin Hormonu Öncülerinin Pichia pastoris AOX1 Promotoru Altında Klonlanması, Ekspresyonu ve Biyoreaktörde Üretimi

İnsülin
 pankreasın beta hücrelerinden üretilen ve vücutta glukoz dengesini sağlayan
 önemli bir peptit hormondur. Pankreasın yeterli miktarda insülin üretememesi ya
 da hücrelerin üretilen insüline cevap verememesi sonucu kan glukoz düzeyinin
 yükselmesiyle diyabet adı verilen metabolik bir hastalık meydana gelir.
 Günümüzde bu hastalığın tedavisinde insülin hormonu kullanılmaktadır. İnsülin
 hormonu genetik mühendisliği teknikleri kullanılarak rekombinant olarak
 üretilen ilk proteindir. İlk olarak Escherichia
 coli ve Saccharomyces cerevisiae’da üretilmeye başlanmış, ancak son zamanlarda rekombinant insülin üretiminde Pichia pastoris (Komagataella phaffi) ’in  kullanımı
 yaygınlaşmıştır. Bu çalışmada, insan insülin hormonu öncülerinin (IP) P. pastoris’in metanol ile
 indüklenebilir AOX1 promotoru altında
 üretimini sağlamak amacıyla; bu proteini kodlayan DNA fragmenti transformasyon
 ve ligasyon gibi moleküler biyoloji teknikleri kullanılarak plazmide aktarılarak
 istenen proteini kodlayan bir ekspresyon vektörü elde edilmiştir. Ekspresyon
 vektörünün lityum asetat yöntemiyle yetenekli hale getirilen P. pastoris X33 suşuna elektroporasyonla
 transferi sağlanmıştır. 5L ölçekli biyoreaktörde yapılan protein ekspresyonu
 çalışması sonrasında alınan örnekler SDS-PAGE ve ELISA yöntemleriyle analiz
 edilmiş ve 7.5 mg/L IP proteini üretildiği tespit edilmiştir.

Biosynthesis, structure, and folding of the insulin precursor protein.

Insulin synthesis in pancreatic β-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.

Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris

BackgroundSecretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation.ResultsAnalysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa).ConclusionsRapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ± 0.96 mg L−1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.

Reduced methanol input induces increased protein output by AOX1 promoter in a trans-acting elements engineered Pichia pastoris.

High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69g/(L supernatant) at the methanol feeding rate of 6.67mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12mL/(h·L broth). Correspondingly, the insulin precursor expression level in fermentation broth and maximum specific insulin precursor production rate was 137 and 77% higher than the WT-IP, respectively. However, oxygen consumption and heat evolution were reduced, and the highest oxygen consumption rate and heat evolution rate of the MF1-IP were 18.0 and 37.7% lower than the WT-IP, respectively.

Time-Resolved Fluorescence Assays for Quantification of Insulin Precursors in Plasma and Serum.

In metabolic diseases such as obesity and type 2 diabetes mellitus, the conversion of proinsulin to mature insulin can be impaired. This could mean that insulin molecules with lower activity toward the insulin receptor can be released under conditions of high metabolic demand, resulting in an inadequate glucoregulatory response. The chapter describes a fluorescent monoclonal antibody-based protocol for measurement of human proinsulin and the proinsulin conversion intermediates (split proinsulins). An example assay is presented using serum from non-diabetic, normal body mass index individuals.

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.

Mesenchymal cells are required for epithelial duct cell-to-beta cell maturation and function in an injured adult pancreas in the rat

The islet, the endocrine portion of the pancreas − develops from an invagination of the pancreatic duct epithelial cells (PDECs) into the surrounding tissue. The contact of the PDECs with mesenchymal cells (MSCs) may be an essential drive for endocrine cell fate. During pancreatic development, cells that express Neurogenin-3 (Ngn3) biomarker are precursors of insulin- producing beta cells. These precursors have been reported in the neogenesis of islets from adult tissues following the surgical ligation of the main pancreatic duct (PDL). But the capacity of these precursors to induce the appropriate signals to complete the entire neogenesis program has been questioned. We studied the fate of co-culture of PDECs and MSCs from the ligated adult pancreas and established the exact location of adult stem- or progenitor-like cells that give rise to beta cells. PDECs were cultured in direct contact with or without MSCs in serum-containing culture media. The cytomorphology of the cells in co-cultures was determined and the immunocytochemical study of the cells was carried out using anti-Ngn3, anti-insulin and anti-cytokeratin-7 (CK7) antibodies. Both the PDEC/MSC- and PDEC/MSC+ cultures showed out- pocketing from duct epithelium by the end of the second week, which are distinct as cell clusters only in PDEC/MSC+ cells later in week four, exhibiting numerous branching ducts. Co-expression of Ngn3 with insulin was observed in both cultures from the second week. However, characterizations of these Ngn3+ cells in the PDEC/MSC+ culture revealed that these cells also co-expressed a CK7 biomarker. This study provides new evidence of the ductal epithelial nature of beta cells in injured adult pancreata; and that the mesenchymal stromal cells are required to sustain Ngn3 expression for beta cell maturation and function.