β-cell proliferation is most prominent during embryogenesis and the subsequent postnatal growth. Following this initial burst, the proliferative capacity of β-Cells is maintained at a low ratio throughout adulthood (Dahiya et al., 2024; Granger and Kushner, 2009; Jacovetti and Regazzi, 2022; Spears et al., 2021).

Studying the mechanisms that control β-cell proliferation with the overall goal of generating new endogenous β-cells has been a main focus for diabetes therapy research. Although the “proliferation” approach could potentially compensate for the reduced number of functional β-cells in Type 2 Diabetes, the success of such a strategy for treating Type 1 Diabetes is rather obscured, as reoccurrence of autoimmunity against new β-cells remains a concern. β-Cell proliferation strategies may open a door to combination therapy in diabetes treatment (Eguchi et al., 2022b; Xie et al., 2023). Combination therapies of proliferation agents with immunosuppressant and anti-oxidative substances have been shown to improve overall long-term outcomes of diabetes treatment. These therapeutic approaches have been highlighted in numerous reports (Eguchi et al., 2022a; Ludvigsson, 2016).

Given that β-cell proliferation in both human and rodent islets has been extensively reviewed elsewhere (Bernal-Mizrachi et al., 2014; Stewart et al., 2015; Elghazi et al., 2007; Rafacho et al., 2009; Elena et al., 2021; Cui et al., 2024), here we will primarily focus on recent advances in β-cell neogenesis.

It has long been presumed that the longevity of adult β-cells primarily relies on their replication capacity rather than neogenesis. Nevertheless, ongoing research tries to explain whether this doctrine holds for humans as well (Dor et al., 2004). The generation of insulin-producing cells to compensate for their absolute or relative shortage in type 1 and type 2 diabetes is an obvious therapeutic strategy. The low proliferative rate of pancreatic β-cells has led to the search for other sources of β-cell generation such as embryonic or adult stem cells, progenitor cells, facultative stem cells or terminally differentiated cells residing within or outside the adult pancreas. In this section, we will highlight some of these efforts (Márquez-Aguirre et al., 2015; De Haro-Hernández et al., 2004; Ye et al., 2016; Granger and Kushner, 2009).

To address the β-cell defective characteristic in T1 and T2 diabetes, researchers have focused on generating pancreatic β-cells from stem cells. They have also concentrated on re-building the β-cells' normal cellular niche (Choi et al., 2004; Zhang et al., 2013; Santana et al., 2006; Roche et al., 2009). Zhang et al. studied the functions of pancreatic islets isolated from human fetal pancreatic progenitor cells in vitro and in vivo. In their study, human fetal pancreatic progenitor cells were expanded in a culture medium enriched with fibroblast growth factor (FGF) and leukemia inhibitor factor (LIF). In order to direct the cells towards pancreatic endocrine cell differentiation, glucagon-like peptide-1 (GLP-1) and Activin-A were added to the cell culture medium. α- and β-cell endocrine and exocrine functions were analyzed by immunofluorescent staining and ELISA technique. Islet-like structures were transplanted into renal capsules of diabetic nude mice to evaluate the functions of these islets in vivo. Immunohistochemistry staining for human C-peptide and human mitochondrion antigen was used to show the human origin of the cells and viability of grafted islets (Zhang et al., 2013). The differentiated cells expressed insulin, glucagon, glucose transporter-1 and -2 (GLUT1 and GLUT2) and voltage-dependent calcium channel (VDCC) (Zhang et al., 2013). These cells were also able to form islet-like structures containing α- and β-cells. These islet-like structures were glucose responsive, leading to normoglycemia in diabetic nude rodents post transplantation (Zhang et al., 2013).

ESCs have demonstrated promising potential to differentiate into various committed cell types, including pancreatic β-cells. The self-renewal capacity of ESCs makes them a competent source for insulin-producing β-like cells.

Other reports have indicated that pancreatic endocrine progenitor cells from ESCs can give rise to insulin-producing cells when transplanted into mouse models (Bruin et al., 2013; Liu and Lee, 2012).

Bruin et al. had previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells can be differentiated into insuline producing cells when transplanted under the kidney capsule in a mouse model of diabetes (Bruin et al., 2013). To overcome the undesired diffrentiation of hESC into bone and cartilage cells, they developped an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue post-transplantation. They also reported that the variation within the host tissue environment has an impact on the development of pancreatic progenitor cells in vivo (Bruin et al., 2013).

Also, Liu et al. presented an efficacy strategy for the differentiation of mouse embryonic stem cells into insulin-producing cells using a two-step differentiation protocol for the formation of endoderm in monolayer cellular culture by activin A, and the differentiation of this monolayer endoderm into insuline producing cells using substances such as nicotinamide, insulin, and laminin. They have demonstared that the differentiation process is successfully obtained with appx. 7 days and the newly formed insuline producing cells are capable of realising insulin in a dose dependent manner based on the amount of added glucose (Liu and Lee, 2012). Therefore, these cells demonstrated an appropriate response to changes in serum glucose levels (Liu and Lee, 2012).

Further, in attempts to use differentiation protocol for neuronal progenitor cells, a few numbers of nestin⁺ ESCs were shown to differentiate into β-like cells. However, a handful number of cells within the small cell population expressed specific insulin genes. These cells were also incapable of Pdx-1 expression (Memon and Abdelalim, 2020; Hori et al., 2005; Ben-Yehudah et al., 2009; Pan et al., 2019)^, inspite of possesing neuronal cell phenotype (Pan et al., 2019).

The study showed that the cells continued differentiation after transplantation in vivo, leading to insulin secretion in mice. This result has been also in accordance with other reports indicating that human ESCs can respond to environmental signals, and differentiate further according to external signals (Vazin and Freed, 2010; Yim and Sheetz, 2012; Mullen and Wrana, 2017; Mossahebi-Mohammadi et al., 2020; Lanner and Rossant, 2010; Huang et al., 2020). It is to conclude that in this case, the message for ESCs has been to further differentiate to endocrine pancreatic islet cells (Huang et al., 2020; Jacobson and Tzanakakis, 2017). Some studies also investigated and compared the murine and human-derived cells in vivo (Yu and Xu, 2020; Baeyens et al., 2018). They revealed that the islet development machinery in rodents (mouse) and human is conserved (Yu and Xu, 2020; Baeyens et al., 2018; Nair and Hebrok, 2015). In this regard, a supportive finding revealed that insulin-producing cells co-cultured with islet cells for approximately 1 month responds to glucose concentration under physiological condition (Oh et al., 2015; Campbell and Newgard, 2021). This study found a co-culture strategy that better promoted differentiation efficiency compared to chemical enforcement methods with soluble chemical factors (Pavathuparambil Abdul Manaph et al., 2019). Due to a limited knowledge around the mechanisms of cell maturation, this effective strategy for promoting islet maturity and regeneration has not yet been used in the clinical trials.

By now, scientists have been able to produce functional insulin-producing, glucose responsive β-like cells. However, the low efficiency of the presented methods and inability to regenerate highly competent β-like cells have been challenging. More recently, scientists have put a great effort in the improvement of the existing protocols for ESC differentiation into pancreatic progenitor cells (Ben-Yehudah et al., 2009; Mayhew and Wells, 2010; Shahjalal HM. et al., 2018; Memon et al., 2018; Wesolowska-Andersen et al., 2020). It should however be noted that the ethical and safety issues might diminish the efficacy of such treatment strategies. In this regard, the tumourigenicity of ESCs is one major concern and obstacle for the success of the therapeutic strategy. This appears as an important aspect for the patient’s health (Herberts et al., 2011). Advanced techniques in enhancing cell maturation and purification can ensure the safety procedure of implantation of fully differentiated, mature cells. These techniques involve fluorescence- and magnetic activated cell sorting, genetic selection, use of biomarkers, and embryonic cell lines. As a result, the risk of tumorigenesis can be reduced (Martin, 2017; Wuputra et al., 2020). The biggest problem, however, remains the ethical conflict about the isolation and harvest of human ESCs Therefore, the majority of studies have focused on animal models, which is not always translatable to human cell and organ machinery.

To date, a handful number of clinical trials for stem cell-based treatment of T1 diabets using human embryonic stem cells (hESC) have been registered (Park et al., 2024).

Notably, Schulz et al. created a product consisting of pancreatic endoderm cells from hESCs and a drug delivery system, (Schulz, 2015). This product went through phase 1/2 clinical trial. However, the study was terminated due to the lack of immunosuppressing strategy resulting in a host reaction against the implant (Pullen, 2018). On a second attempt, a Phase 1/2 clinical study involving 17 T1D patients was carried out using the modified product. This study showed successful engraftment and insulin secteration in significant number of the cases. Half a year post-implantation, significant number of the participants showed positive levels of C-peptide, demonstrating the potential of the product as a promissing method for T1DM treatment (Shapiro et al., 2021). The results of this clinical study underscores innovative approach that utilizes pancreatic endodermal cells derived from hESC for treating diabetes. The hESCs have been also genetically modified by the CRISPR/Cas9 technology. This modification also resulted in enhancement of cellular survival rate against the patient’s immune system, deminishing the risk of graft versus host disease (Ellis et al., 2023). In addition, another product consisting of differentiated pancreatic islet cells derived from human for treating T1D has been desigened for clinical investigations (Reichman et al., 2023). As of yet, the data analysis from this study has revealed promissing results, demonstrating restored insulin production in the first couple of participants in the study.

With all being discussed about the impact of embryonic stem cells in generating insuline producing cells, however, the ethical and safety concerns exist remain, hampering the potential of using these cells in clinical trials. Besides, the cell differentiation process itself may cause partially undesired complications in the cell phenotype that might lead to immunogenicity and force an unforeseen immune reaction in the body. Here, therapies that protect new β-cells from being destructed are critical. Hence, the β-cell protection side approach for applying cell therapy strategies pro β-like cell regeneration comes in handy. The immunomodulatory therapy in which mesenchymal stem cells can be also involved promotes the development of efficient protocols for the production of ESC-derived pancreatic β-cells. several protocols have been developed for differentiation of human ESCs towards insulin-producing cells (Naujok et al., 2011; Jiang et al., 2007; Trounson, 2007; Lee and Chung, 2011; Nagaya et al., 2019; White et al., 2008; Mastracci and Sussel, 2012).

Adult stem cells are the most viable source for cell therapy in several disease models (Bai, 2020; Editorial; Mousaei Ghasroldasht et al., 2022; Sng and Thomas, 2012). They do not demonstrate ethical issues in most countries and seem to provide an unlimited resource for advanced regenerative therapies. Many reports indicated that β-like insulin-producing cells can be generated from stem and progenitor cells residing in bone marrow, adipose tissue, liver, spleen, neuronal tissues, and umbilical cord blood. Precise and detailed studies are required to identify the most effective tissue-specific adult stem cells for generation of insulin-producing, glucose-responsive fully committed β-like cells (Naujok et al., 2011). It is also much needed to develop more efficient differentiation protocols to allow for testing these strategies in clinical studies.

Despite an obvious need for further assessments, optimizations, and clinical trials, the initial success of the (re)generation of glucose-responsive insulin-secreting β-like cells gives promise for a therapeutic strategy for diabetes using stem cell-based cell therapy (Pokrywczynska et al., 2013).

Stem and progenitor cells can be derived from a variety of tissues such as cord, blood, bone marrow, adipose tissue, oral cavity tissues such as pulpa, periost and spongiosa, and epithelium. These cells have been characterized, expanded and applied for transplantation procedures in which adult stem cells give rise to committed cells such as osteocytes, adipocytes, and chondrocytes as well as β-like insulin-producing cells (Choi et al., 2004; Rohban and Pieber, 2017).

Several scientific reports have indicated the role of tissue-specific adult mesenchymal stem and progenitor cells (MSPCs) in the regeneration of insulin-producing β-like cells. In most of the studies, MSPCs (Rendra et al., 2020) isolated from bone marrow, adipose tissue, and umbilical cord blood have been shown to contribute more effectively in the regeneration of β-like cells compared to cells from other tissue of origins (Rohban and Pieber, 2017; Jun HS. and Park EY., 2009; Liu D. et al., 2020).

Figure 2 depicts an overview on cellular playmakers and strategies toward β-cell regeneration.

Using α-cells as a source for generating new β-cells has been the focus of many investigators in the past decade (Habener and Stanojevic, 2012; Lu et al., 2014; Wei et al., 2022; Dahiya et al., 2024).

Near total ablation of α-cells using diphtheria toxin has been shown to promote spontaneous α-to β-cell conversion in mice (Thorel et al., 2010). In mouse models, the expression of β-cell-specific markers, e.g., PDX1 and PAX4 and Maf-A, as well as impaired expression of Arx and Dnmt1 in α-cells lead to the trans-differentiation of α-cells to β cells (Zhong and Jiang, 2019; Hunter and Stein, 2017; Nishimura et al., 2015).

In 2009, Collombat et al. reported the trans-differentiation of glucagon-producing cells into functional β-cells upon overexpression of PAX4 (Collombat et al., 2009a; Collombat et al., 2009b). In a different set of experiments, they blocked the expression of aristaless related-homeobox (known as ARX) in α-cells (Miura et al., 1997; Marsh and Golden, 2013). Cumulatively, both studies demonstrated that the overexpression of PAX4 and/or blockade of ARX led to the generation of functional β-cells. The regenerated β-cells were also shown to be able to reverse diabetes in STZ-treated transgenic mouse models (Yin et al., 2006).

Further, studies reported that the absence of glucagon-producing cells ran an endocrine-specification setting in the duct epithelium progenitor cells upon re-expression of NGN3⁺ in these uncommitted cells (Prasadan et al., 2010; Magenheim et al., 2011). This alteration in the endocrine phenotype was also detected upon diphtheria toxin-mediated α-cell death (Habener and Stanojevic, 2012; Thorel et al., 2010). There are also several reports indicating the trans-differentiation from α-to β-cell under certain culture medium supplements, drugs and conditions in vitro (Yu and Xu, 2020; Xing et al., 2023; Sarnobat et al., 2023; Nihad et al., 2021). The question, however, still remains as to whether these regenerative aspects can take place in humans since most of the studies have been carried out in rodents.

Another important question is regarding the translation of these findings into potential pharmacological protocols and/or cell based-therapy strategies. In 2009, a study revealed that the expression of Pax4 was enough for α-cells to trans-differentiate into β-cells (Gittes, 2009). Also, lack of Pax4 expression leads to loss of β-cells identity while boosting α-cells quantity (Collombat et al., 2009b). The data clearly indicates the developmental relation of these two pancreatic hormone-releasing cell types and their close gene expression profile. One year later in 2010, Herrera and colleagues showed that reprogramming of α-cells towards β-cells can be initiated by β-cell ablation (Thorel et al., 2010). They conducted their experiments by developing a transgenic mouse and used lineage tracing method for α-cells while ablated β-cell thoroughly using diphtheria toxin receptor machinery. The findings indicated that a strategy to completely destroy β-cells promoted regeneration of β-cells that are mainly developed from residual α-cells. Most interestingly, not only did the cells start to express β-cell specific biomarkers such as Pdx1 and Nkx6.1, but they also started to secrete insulin. Here again, the similarity in the function and origin of these 2 cell types might be the reason for α-to β-cell trans-differentiation. What pushes the α-cells to convert into insulin-producing cells is still a matter of debate. One theory is that extreme, abnormal conditions such as lack of insulin signaling in the microenvironment is responsible for initiating a series of changes that ultimately activates the conversion system. There are reports indicating that α-cells possess the Pdx1 and Mafa genes in their DNA content, which are known to present actively in β-cells (Thorel et al., 2010). In a study aimed at investigating the potential of transcriptional reprogramming, pancreatic islets were exposed to histone methyltransferase inhibitor that eventually led to the expression of Pdx1 biomarker and insulin production in glucagon producing α-cells (Bramswig et al., 2013; Spaeth et al., 2016).

A study published by Furuyama et al. reported that they have isolated islet non-β-cells admix with α-cells and pancreatic polypeptide (PPY)-producing γ-cell population from non-diabetic or diabetic human donors (Furuyama et al., 2019). The cells then undergo lineage-tracing and reprogramming using the transcription factors PDX1 and MAFA to produce insulin in response to glucose. Upon cellular transplantation into diabetic mice, converted human α-cells reverse diabetes by producing insulin up to 6 months post-transplantation (Furuyama et al., 2019). This study indicated that insulin-producing α-cells could still express α-cell markers detected by deep transcriptomic and proteomic profiling characterization. These findings also provide insight into molecular mechanisms for the treatment of Diabetes (Furuyama et al., 2019).

In an attempt to investigate conversion strategies to regenerate insulin-producing cells, Li et al. published a paper with an approach to trans-differentiate glucagon-producing α-cells to insulin-producing β-like-cells (Li et al., 2017). It was already clear that the loss of regulatory transcription factor Arx is adequate to induce trans-differentiation of α-cells to functional insulin-producing-β-like cells (Li et al., 2017). Based on this knowledge, Li et al. identified the small molecule Artemisinin, an antimalarial medication, as a playmaker capable of repressing Arx. They showed that the mechanism of action of these molecules depends positively on GABAA receptor signaling (Li et al., 2017).

A head-to-head study published by Ben-Othman et al. (Ben-Othman et al., 2017) reported the identification of GABA as an inducer of α-to-β-like cell trans-differentiation in vivo. This trans-differentiation process induced mechanisms of α-cell replacement through mobilizing duct-lining precursor cells representing α-cell characteristics before conversion into β-like cells. This process was carried out solely through GABA exposure (Ben-Othman et al., 2017). The study also indicated that the newly generated β-like cells could reverse STZ-induced diabetes in vivo (Ben-Othman et al., 2017). This study underscored the treatment of transplanted human islets with GABA leading to a loss of α-cells and an enhancement in β-like cell counts and α-to-β-like cell trans-differentiation process also in humans. The last two studies both published in 2017 made a stir at the time. They indicated that α-cells can convert into functional insulin-producing β-like-cells via progenitor cells using the drug GABA.

However, ever since, there have been reports of a lack of reproducibility of these results. Several researchers, including Ackermann et al. (Ackermann et al., 2018), and Rohban, the author of this review, tried to reproduce the results indicated in these two studies and failed. As a result, these publications are currently considered to be dubious papers.

It should be again noted, however, that most of these experiments and related findings have been carried out in rodents, particularly mouse models. To date, the ‘extreme β-cell loss’ leading to α-to β-cell conversion cannot be recapitulated in T1D human patients due to the lack of suitable immunosuppressive and immunoregulatory strategies. The immunosuppressive and immunoregulatory role of adult stem cells in particular mesenchymal stromal cells have been revealed that it might be worth trying to see if these stromal cells can play a dual role in differentiating into insulin-producing cells while suppressing the immune system in T1D patients.

PDCs form around 35% of the human pancreatic cell population (Lemper et al., 2015). These cells can be isolated in vitro, and they bear trypsinization and other enzymatic dissociation and cell isolation procedures (Gmyr et al., 2001; Dorrell et al., 2008). These properties make PDCs good candidates for expansion experiments aiming to increase the cell mass required for transplantation. PDCs maintain their proliferation capacity in vitro including in 3D cell culture systems using collagen, matrigel, or agarose as a matrix. However, without manipulation, these cells undergo cell-cycle arrest rapidly (Jurczyk et al., 2014). In 2014, Jin and colleagues reported that PDCs develop cystic bodies when cultured in vitro that accelerate their proliferation capacity. They also acquire insulin expression after approximately 7 days in vitro (Jin et al., 2014).

The use of growth factors, e.g., EGF, hepatocyte growth factor (HGF), KGF, or nicotinamide to stimulate human PDC proliferation has been tested in several studies (Corritore et al., 2016; Bonner-Weir et al., 2000; Rescan et al., 2005; Hoesli et al., 2012). However, most of them failed to bypass the incidence of rapid senescence and restricted proliferation rate and growth. Mouse tissue, however, represented a different growth pattern, as reported by Oshima et al. (Oshima et al., 2007). They used purified CD133⁺/c-Met⁺ PDCs with HGF, EGF, nicotinamide, and dexamethasone substances as culture medium additives.

Most of the protocols that worked successfully for mouse PDC expansion did not show reproducibility in human cells. This could be due to telomere shortening in human cells, which limits the replication capacity and trigggers senescence. Contrary to human cells, telomere length faces minimal alteration during subculture in rodents (Bonner-Weir et al., 2010). In addition, PDCs alter their phenotypic characterization soon after enzymatic treatment in vitro. The loss of E-cadherin followed by mesenchymal biomarkers such as N-cadherin and Snail1 known as master regulators of epithelial-mesenchymal transition (EMT) occurs (Corritore et al., 2016; Bonner-Weir et al., 2010).

The expression of mesenchymal characteristics by PDCs has been initially detected in human exocrine cells in vitro (Fanjul et al., 2010). However, in several studies, this phenomenon was also observed in human primary islet cultures (Fanjul et al., 2010; Gershengorn et al., 2004; Ouziel-Yahalom et al., 2006). These human islet cells also showed the capability of redifferentiating into insulin-producing cells after a higher proliferation rate. The properties of these cells were further studied by other groups through experiments such as lineage tracing (Baeyens et al., 2018; Chen et al., 2019). In terms of these investigations, they were also able to produce β-like-cells with the potency to reduce blood glucose levels in NOD mice (Nir et al., 2007; Verhoeff et al., 2021). The EMT process can occur in human PDCs to stop the early senescence of cells within primary cultures in vitro (Corritore et al., 2016).

Using FACS-sorted CA19-9⁺ PDCs, it was revealed that the purified cells within the culture system were proliferating in the presence of endothelial growth media (Corritore et al., 2016). The cells were characterized and identified as human duct-derived cells (HDDCs) with a massive expansion ability (Corritore et al., 2016). Moreover, an E−to N-cadherin switch and low levels of CK19 and SOX9 expression was detected in HDDCs (Corritore et al., 2014). Another set of experiments revealed that incubation of CA19-9+ cells with TGFβ inhibitor A-83–01 blocked the normal characteristics of HDDCs, including the inability to clonally expand. in vitro (Corritore et al., 2014; Corritore et al., 2015). Culture conditions were designed in a way that mimicked pancreatic embryonic development (Guo T. and Hebrok M., 2009). In this regard, small molecules and growth factors were used to recapitulate the desired condition (Pavathuparambil Abdul Manaph et al., 2019; Kondo et al., 2017). After 2-week incubation time, HDDCs were finally prone to express typical β-cell features, including insulin production capability, albeit without glucose sensitivity properties (Cantley and Ashcroft, 2015). These findings promoted further works around the evaluation of HDDC diabetes-reversal potential in relevant animal models (Kumar et al., 2012; Adigbli et al., 2020).

In 2008, Inada and colleagues hypothesized that progenitor cells are duct epithelial cells that undergo regression to a less differentiated state post-replication and can form new endocrine and exocrine pancreas (Inada et al., 2008). To test their hypothesis on whether ductal cells can act as pancreatic progenitors postnatally and give rise to new islets, transgenic mice expressing human carbonic anhydrase II (CAII), a duct marker, were generated. The study demonstrated that CAII-expressing cells within the pancreas function as progenitor cells capable of forming new islets and acini after birth and upon injury (Inada et al., 2008). Despite some limitations in the report (CreER was not knocked into the CAII gene), the paper illustrates the existence of a “duct to β-cell” path by doing lineage tracing using CAII, (the duct marker). These cells can serve as sources for new islet regeneration for diabetes therapy (Inada et al., 2008).

In 2023, Doke et al. demonstrated the pioneering data on scRNA-seq analysis of human pancreas. Their study confirms a dynamic plasticity in the pancreatic tissue through an intermediate ducto-acinar stage leading to endocrine cell neogenesis (Doke et al., 2023). This dynamic plasticity also promotes trans-differentiation of non-β-cells into mature β-cells in the pancreas (Honzawa and Fujimoto, 2021).

The isolation of pancreatic islets gives rise to a large population of acinar cells that can be distinguished from all other pancreatic cells (Puri and Hebrok, 2010). Soon after the isolation of these cells, scientists explored their ability to convert into β‐cells both in vitro and in vivo (Lysy et al., 2013; Donga and Wu, 2018). They also found out that in an intermediate phase during acinar-to β‐like-cell conversion, a population of duct cells was generated (Basile et al., 2019). It was first in 1992 that Lemoine and colleagues revealed the ability of human acinar cells to convert into CK-19⁺ duct cells (Hall and Lemoine, 1992). In 2005 and later 2007, other studies conducted by Lardon et al. and Miyawaky et al. showed that rodent pancreatic exocrine cells can be converted into insulin‐expressing cells when treated with LIF and EGF (Okuno et al., 2007; Baeyens et al., 2005). The co-transplantation of these cells together with acinar cells into immune-deficient mouse models resulted in the trans-differentiation of acinar cells into endocrine cells. After isolation and upon purification, these cells were referred to as “non‐endocrine pancreatic epithelial cells” (Hao et al., 2006). They were able to go through endocrine differentiation and promote the survival of pancreatic progenitor population in the epithelium layer of the pancreas (Hao et al., 2006).

In 2008, a group of scientists used adenoviruses for transporting Ngn3‐, Pdx‐1‐, and MafA genes for reprogramming processes within acinar cells (Zhou et al., 2008). The outcome gave rise to reprogrammed acinar cells that were able to produce insulin and decrease blood glucose levels in diabetic animals (Okuno et al., 2007). In contrast, Aldibbiat and colleagues showed in the same year that the converted acinar cells lacked the ability to process pro-insulin in trans-differentiated pancreatic acinar cells. This is due to deregulated insulin secretion and aberrant secretory pathway (Alidibbiat et al., 2008).

In a study in 2014, scientists induced the expression of Ngn3, Pdx‐1, and MafA through elastase 2A which is known to be a specific inducer of acinar cells. These genes were then introduced into exocrine cells (Li et al., 2014; Kim and Lee, 2016). The results showed that these genes play a distinct role in the process of conversion within the exocrine cells. It was further reported that Ngn3 and MafA expression mainly lead to trans-differentiation: Ngn3 gives rise to the pancreatic three islet endocrine cell types known as α‐, β‐ and δ‐cells. On the other hand, MafA showed to suppress the acinar and δ‐cell phenotypic properties while leading them to express more of α‐ and β‐cell properties. Pdx-1 has been also shown to play a role in the suppression of δ‐cell differentiation while directing them through β‐cell differentiation (Gannon et al., 2008). This study reported that the three main cell types in the pancreatic islets have been originated from the conversion of the acinar cells (Li et al., 2014). In another work, scientists were able to induce differentiation in murine acinar cells using substances such as nicotinamide and EGF (Baeyens et al., 2014; Wedeken et al., 2017). Using the lectin-labeling method for the acinar cells, they demonstrated that the regenerated β‐like-cells were of acinar cell-origin (Kim and Lee, 2016). Others such as Baeyens and colleagues in 2006 also reported that during β‐like-cell generation, kinase-associated signaling pathways such as JAK/STAT liaise with Ngn3 expression to promote β‐cell neogenesis (Baeyens et al., 2006). They highlighted that blocking Ngn3 expression by impairing EGF and LIF signaling pathways, as well as inhibiting EGFR, JAK2, and STAT3, led to a significant reduction in the formation of new insulin producing β‐like-cells in the pancreas. Normally, EGF and LIF signaling, along with molecular factors, promote the re-expression of the Ngn3 molecule in adult pancreatic cells through the JAK/STAT pathway. Conversely, inhibiting JAK/STAT led to a decrease in Ngn3 expression. This process is similar to neurogenesis, where EGF enhances the responsiveness of LIF signaling via the STAT3 cascade. Overall, the study demonstrated that EGF and LIF signaling through the JAK/STAT pathway promotes the re-expression of Ngn3 in pancreatic cells (Baeyens et al., 2006).

The researchers also determined that acinar-to β‐like-cell conversion takes place in the acinar cells upon MAPK overexpression (Lemper et al., 2015; Kim and Lee, 2016). Here gene therapy comes in handy for delivering the genes to the acinar cells through lentiviruses and/or microRNA manipulation, which resulted in overexpression of β‐cell-specific biomarkers, e.g., insulin and Pdx‐1 in the cells (Corritore et al., 2016; Williams et al., 2020). It is interesting to know that the acinar-to β‐cell conversion worked more effectively in vitro, e.g., in 3D culture methods (Yu and Xu, 2020; Baeyens et al., 2018; Hebrok, 2012). A lineage-tracing method using adenovirus recombinase elastase 2A promoter confirmed the origin of Ngn3‐ and insulin‐producing cells to be human acinar cells (Kim and Lee, 2016). The finding indicated the ability of human exocrine cells to convert into β‐like insulin‐expressing cells. Not only was it a groundbreaking scientific finding, but it also leads to a novel therapeutic and regenerative strategy in which plenty of exocrine cells can be obtained and used for regenerative therapy purposes. All in all, it is now known that acinar cells have the potential to trans-differentiate into β‐like insulin‐producing cells both in vitro and in vivo. Hence they serve as a promising source of cells that can be used in the treatment and even cure of diabetes.

Very recently in 2024, a study conducted by Esni and collegues demonstrated that preventing focal adhesion kinase activity leads acinar cells to convert into funtional, glucose responsive β-like cells (Dahiya et al., 2024), a significant step forward through diabetes cure.

This protocol involves differentiating stem cells into pancreatic progenitor cells, which can further mature into β‐ cells that produce insulin. While this method has shown promising results in preclinical studies, it requires precise control of developmental signaling pathways to ensure the efficient generation of functional β‐cells (Hogrebe et al., 2021; Shim et al., 2007; Shi, 2010).

In this approach, stem cells are exposed to a defined series of growth factors and signaling molecules to mimic the natural developmental process of the pancreas. This method has the advantage of producing insulin-producing cells that closely resemble native β‐cells in both structure and function. However, the protocol can be complex and time-consuming, and requiring optimization for each cell line used (Shim et al., 2007; Zhang et al., 2009; Trounson, 2006).

Trans-differentiation involves directly converting non- β‐cells, such as liver cells, into insulin-producing cells. This method offers the advantage of bypassing the pluripotent stem cell stage, and reducing the risk of tumour formation. However, the efficiency of trans-differentiation can vary among cell types and may require genetic manipulation to enhance the conversion process (Yang et al., 2002; Okura et al., 2009; Oh et al., 2004).

Utilizing 3D culture systems can enhance the differentiation efficiency of stem cells into insulin-producing cells by providing a more physiologically relevant microenvironment. The spatial organization within 3D cultures can promote cell-cell interactions and signaling pathways crucial for beta cell maturation. However, optimizing 3D culture conditions for scalability and reproducibility remains a challenge (Khorsandi et al., 2015; Mendoza et al., 2018; Liang et al., 2023).

While each insulin-producing cell differentiation protocol posesses a certain level of effectiveness and has its strengths and limitations, a combination of these approaches may hold the key to maximizing efficiency and functionality. Future research should focus on refining existing protocols, identifying novel molecular targets, and developing standardized methodologies for generating reliable sources of insulin-producing cells for diabetes therapy (Gabr et al., 2017; Boyd et al., 2008; Enderami et al., 2017; Nemati et al., 2021; Shi, 2010).

The choice of cell source for generating insulin-producing β‐cells signifacantly influences their long-term stability and functionality post-transplantation (Lock and Tzanakakis, 2007; Jun Hee-Sook and Park Eun-Young, 2009)^,. Differentiation protocols, stem cell sources, and genetic manipulation and editing techniques all play crucial roles in determining the quality and maturity of β‐cells (Guo Tingxia and Hebrok Matthias, 2009; Melton, 2021). Recent advances in generating functional β‐cells from pluripotent stem cells and other sources provide optimism for enhancing long-term cell survival (Lock and Tzanakakis, 2007; Jun Hee-Sook and Park Eun-Young, 2009; Guo Tingxia and Hebrok Matthias, 2009; Melton, 2021).

Optimal transplantation techniques are essential for promoting the engraftment and survival of generated β‐cells in vivo. Site of transplantation, encapsulation technologies, and vascularization strategies all impact the long-term functionality of transplanted β‐cells (Rohban Rokhsareh et al., 2016; Farina et al., 2019; Pellegrini et al., 2016; Shahjalal HussainMd et al., 2018; Köllmer et al., 2016). Novel approaches such as bio-engineered scaffolds offer innovative solutions to improve the integration and longevity of β‐cell transplants (Gazia et al., 2019; Malik and Dhasmana).

Immune responses against transplanted β‐cells possess a significant challenge to their long-term stability and functionality (Tahbaz and Yoshihara, 2021; Caldara et al., 2023; Shi et al., 2022). Strategies to modulate the immune system, including immunosuppressive drugs, immune-privileged sites, and cell encapsulation, must be carefully considered to prevent rejection and ensure sustained cell survival (Tahbaz and Yoshihara, 2021; Caldara et al., 2023; Köllmer et al., 2016; Johannesson et al., 2015).

Promoting the long-term survival and functionality of transplanted β‐cells requires a multi-faceted approach. Factors such as oxygen tension, nutrient supply, and inflammatory environment all impact the viability and insulin-secreting capacity of β‐cells (Yitayew et al., 2024; Shi et al., 2022; Lazard et al., 2012; Ghasemi et al., 2021). Novel insights into enhancing cell survival through tissue engineering, gene editing, and co-culture systems offer exciting possibilities for improving the long-term outcomes of β‐cell therapy (Abadpour et al., 2021; Ghezelayagh et al., 2021; Gamble et al., 2018; Orlando et al., 2014).