Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine

2.1 PDX1 is key in transcription factor reprogramming strategies

PDX1 plays a key role in early embryonic pancreas formation, directed differentiation of different endocrine lineages, and late maturation of β cell function.¹⁵ PDX1 expression was detected on the 8.5th day of mouse embryonic development (E8.5) and around the fourth week of human gestation.^{13, 16} As early as 2000, Ferber et al demonstrated the capacity of PDX1 to reprogram extrapancreatic tissue into a similar β cell phenotype.¹⁷ To study the effects of ectopic expression of PDX1 in the liver, the researchers delivered PDX1 recombinant adenovirus to male mice aged 11–14 weeks. The results showed that PDX1 was mainly expressed in the liver, whereas the expression of this gene induced the expression of endogenous mouse insulin 1 (mI-1) and mI-2 genes in the liver. A single PDX1 gene expression is sufficient to induce the expression and activation of endogenous insulin genes in tissues outside the pancreas. When PDX1 recombinant adenovirus was delivered to streptozocin (STZ)-induced diabetic mice for treatment, blood glucose levels gradually decreased. This suggests that the expression of PDX1 is sufficient to induce mature, biologically active insulin production in the liver. In islets, PDX1 works synergistically with other transcription factors to regulate the expression of insulin and other islet-specific genes.^{18, 19} Transcription factors that occur naturally in the liver are ubiquitous and tissue specific and may work synergistically with ectopic PDX1 to induce and regulate insulin gene expression in this organ. In 2006, Yamada et al introduced the adenovirus-mediated PDX1 gene into hepatocytes cultured in vitro and detected insulin expression at the mRNA and protein levels. Hepatocyte albumin expression is downregulated during transdifferentiation, and cytokeratin-19 (CK19) and alpha-fetoprotein mRNA are upregulated. These results suggest that hepatocytes have the potential to transdifferentiate into insulin-producing cells in vitro. Transcriptome analysis showed that fetal hepatocytes introduced into PDX1 expressed a variety of β cell-related genes as well as genes expressed in non-β islet cells, such as glucagon and pancreatic polypeptide (PP). Although cells respond to glucose stimulation, they secrete very low levels of insulin, about 2% of normal human islets.²⁰ These results suggest a large difference between hepatocytes transdifferentiated with PDX1 alone and islet β cells.

2.2 PDX1 and Ngn3 combine to improve the efficiency of the transcription factor reprogramming strategy

Ngn3 encodes an essential helix-ring-helix transcription factor that is also considered a major regulator of pancreatic development.^{21, 22} At the mouse embryonic stage of 8.5 days (E8.5), Ngn3 was first detected during the primary transition of islet development, and E11.5 expression decreased to near zero. Ngn3 is reexpressed during the secondary transition of islet development and peaks at E15.5.²³ Pancreatic endocrine cells disappear in mice with Ngn3 gene deletion and die shortly after birth.²² In the adult mouse pancreas, a small number of Ngn3 expression-positive cells can be detected at normal homeostasis. Based on lineage tracing, these Ngn3-positive cells are a population of islet progenitor cells.²⁴ In damaged pancreatic tissue, Ngn3 expression is upregulated, and Ngn3-positive cells contribute to adult β cell regeneration.^{25, 26} When expressed ectopically in non-endo epithelial cells, Ngn3 can confer endocrine cell fate on these cells.²² Ngn3 is necessary for pluripotent stem cell-derived maturation of human β cells.²⁷ Previous studies have shown that expression of PDX1 in the liver via adenovirus vectors can produce some insulin-expressing cells in the liver and alleviate symptoms of diabetes in animal models.^{17, 28} In a 2005 study, researchers' overexpression of PDX1, which carries a VP16 transcriptional activation domain modification, along with NeuroD or Ngn3, significantly increased insulin gene promoter activity in HepG2 cells and was more effective than wild-type PDX1. Furthermore, PDX1/VP16 overexpression along with NeuroD or Ngn3 significantly improves glucose tolerance in STZ-induced diabetic mice.²⁹

However, the study did not determine whether those cells that expressed insulin were β-like cells. In a 2007 study, Soonsang Yoon demonstrated that coexpression of PDX1 and Ngn3 produced a small number of β-like cells in the liver.³⁰ Coexpression of PDX1 and Ngn3 induces the expression of various islet cell-specific genes to initiate a series of events that induce a small number of hepatocytes into β-like cells. Chang et al showed that PDX1 and Ngn3 combined can reprogram hepatocytes into platelet-derived growth factor receptor A (PDGFRα)-positive β-like cells.³¹ The addition of PDGF-AA to glucagon-like peptide-1/exendin-4-containing medium promotes the expansion of β-like cells, and the cells can secrete insulin in response to glucose stimulation, enabling the treatment of diabetic mice after transplantation. Compared to previous work using PDX1 alone to reprogram hepatocytes into insulin-producing cells, the improved strategy developed based on the current work not only improves the efficiency of reprogramming hepatocytes into β-like cells but also enhances the ability of reprogrammed cells to secrete insulin.

2.3 Specific combinations of Ngn3, PDX1, and MafA are central to the transcription factor reprogramming strategy

MafA is a transcription factor with a leucine zipper structure that belongs to the macrophage activating factor (MAF) family.³² MafA is the only specific islet β cell insulin gene-activating transcription factor found so far, which can bind to the conserved cis-regulatory element RIPE3b in the insulin gene promoter region as a strong transaction factor to regulate insulin expression. Islet β cell maturation and functional maintenance depends on the normal expression of the MafA protein.^33-35

In 2008, Zhou et al identified 20 transcription factors specifically expressed in embryonic pancreatic cells out of 1100 by in situ hybridization.²² Of these, nine genes were highly correlated with pancreatic development and were selected for the initial reprogramming experiment. A specific combination of three transcription factors, Ngn3, PDX1, and MafA, was identified, which reprogram pancreatic exocrine cells into cells that closely resemble islet β-cells. Induced islet β-like cells are indistinguishable from endogenous islet β cells in size, shape, and ultrastructure. They express genes necessary for β-cell function and can improve hyperglycemia by remodeling the local vascular system and secreting insulin.

Banga et al showed that the combination of PDX1, Ngn3, and MafA transcription factors was also able to reprogram cells in the liver into insulin-positive cells.³⁶ These cells exhibit duct-like structures expressing epithelial-specific marker proteins E-Cadherin and CK19 while being positive for insulin and C-peptide. The content of insulin released after the corresponding glucose stimulation reached 20% of the islets of normal mice. However, these cells also secrete other pancreatic hormones. The study also showed that the combination of PDX1, Ngn3, and MafA was more reliable and effective than previously used combinations, with liver cells maintaining insulin expression for longer.

4.1 Islet organoids based on pancreatic cell origin

By day 11.5 of the mouse embryonic stage, the pancreas is essentially made up of pluripotent pancreatic progenitor cells that can differentiate into all types of pancreatic cells. These cells express Sox9, PDX1, Hnf1b, and Ptf1a.^{24, 56} In 2013, Greggio et al isolated embryonic pancreatic dorsal from mouse E10.5 to prepare pancreatic progenitor cell suspension and established a pancreatic progenitor cell culture medium with DMEM/F12-based medium and the addition of serum-free substitutes and β-mercaptoethanol, phorbol myristate acetate, Y-27632, epidermal growth factor (EGF), mouse R-spondin1, FGF10, FGF1, heparin, and other small molecule progenitor cell culture media.⁵⁷ The cell suspension was mixed with growth factor-removed Matrigel and cultured in a 96-well plate. In this 3D culture system, the dissociated mouse embryonic pancreatic progenitor cells were efficiently expanded. By adjusting the composition of the medium, hollow spheres consisting mainly of pancreatic progenitor cells can be generated, as well as complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. Experiments revealed the community effect of pancreatic development, through which small cell populations maintain the characteristics of progenitor cells better than isolated cells, as well as the requirement for 3D culture. Sox9⁺ pancreatic epithelial cells are considered pancreatic progenitor cells and are capable of developing into exocrine duct or acinar cells in vivo, as well as endocrine progenitor cells that produce all islet cells.^{56, 58, 59}

Sugiyama et al isolated and purified single Sox9⁺ pancreatic epithelial cells from mouse embryo E11.5, similarly mixed early Sox9⁺ pancreatic epithelial cells with growth factor-removed Matrigel and cultured under the condition of supplementation with insulin, transferrin, and IGF1 in PrEBM-based medium. To achieve cell expansion and growth spheroids, cells were cultured at 21% oxygen concentration and supplemented with 150 ng/mL human FGF10, 2% (vol/vol) B27, and 0.33 μM all-trans retinoic acid in basal medium. To induce spheroid differentiation, spheroids were cultured at 5% oxygen concentration and supplemented with basal medium with 50 ng/mL FGF10, 10 mM nicotinamide. The resulting islet organoids were cultured at 21% oxygen. Basal medium was supplemented with 150 ng/mL FGF10, 10 mM nicotinamide, and 0.5 mg/mL mouse R-spondin1-Fc or 200 ng/mL purified Wnt3a. By establishing an in vitro culture system, islet organoids with self-proliferation and multidirectional differentiation potential were successfully obtained. Mouse embryonic pancreatic progenitor cells cultured under 3D conditions reflect in vitro differentiation of exocrine and endocrine cells but do not support the long-term expansion of pancreatic cells. Tissue culture strategies using adult ducts, acinar, and low insulin cells have only limited function for expanding and differentiating presumed progenitor cells as β cells.⁶⁰

In 2022, Wang et al identified a population of protein C receptor positive (Procr⁺) cells from the adult mouse pancreas by single-cell RNA sequencing (scRNA-seq).⁶¹ These cells are located in islets, do not express differentiation markers, and are characterized by epithelial-mesenchymal transformation. Through genetic lineage tracing, Procr⁺ islet cells undergo clonal expansion and produce all four endocrine cell types during adult homeostasis. Sorted Procr⁺ cells make up approximately 1% of islet cells, and when cultured at clonal density, islet-like organoids can be formed. β cells dominate in differentiated islet organoids, whereas α, δ, and PP cells occur less frequently. Organoids respond to glucose and secrete insulin. After transplantation in diabetic mice, these organoids can reverse STZ-induced diabetes in mice.⁶²

4.2 Islet organoids based on ESCs and iPSCs sources

In the past few decades, the in-depth study of ESCs and iPSCs to islet β cell differentiation has provided a solid foundation for ESCs and iPSCs to islet organoid differentiation. In 2016, Kim et al constructed islet-like clusters derived from hESCs and secreted glucose-responsive insulin, which lowered blood sugar after the transplantation of STZ-induced diabetic mice.⁶³ In 2017, Chaimov et al developed a novel artificial pancreas encapsulation platform based on solubilized whole porcine pancreatic ECM. The ECM-microcapsule platform provides a natural fibrous 3D niche that differentiates captured hepatocytes into insulin-producing cells through ectopic expression of PDX1, Pax4, and MafA. In vivo, ECM-encapsulated cells have been shown to be nonimmunogenic and, most important, can significantly improve glycemic control in preclinical models of diabetic mice.⁶⁴ In 2018, Candiello et al developed a novel class of hydrogel systems, Amikagel, that generate regenerative islet organoids with precise size and cellular heterogeneity on this gel. Amikagel-induced hESC-PP spheroid formation enhances the expression of the β cell-specific INS1 gene and C-peptide protein, as well as functional insulin production in response to glucose stimulation in vitro.⁶⁵ In 2019, Tao et al used an organ-on-a-chip platform to induce the differentiation of iPSCs into islet organoids. The organ microarray platform contains a multilayer microfluidic device that enhances the expression of pancreatic β-cell-specific genes PDX1 and Nkx6.1 under perfusion 3D culture conditions compared with static culture, at the same time promoting the increase of insulin secretion.⁶⁶ Hepatocytes present themselves as favorable candidates for reprogramming into differentiated insulin-producing cells (DIPCs) due to their shared developmental lineage with pancreatic cells, alongside their superior safety profile in comparison to stem cells (Table 1). In 2020, Lee et al fabricated DIPC spheroids derived from human hepatocytes using concave microwells. Higher levels of islet-related gene expression and insulin secretion were observed in spheroids compared to single-cell DIPC.⁶⁷ This suggests that the attainment of a uniform size for the spheroids could potentially enhance the functionality of DIPC.

4.3 Islet organoids based on coculture of islet cells with cells from other sources

With the maturation of in vitro induction of stem cell differentiation into islet-like organs, how to avoid immune rejection and promote angiogenesis after islet-like organ transplantation has become a problem that needs to be solved. As early as 2011, Jung et al cocultured bone marrow mesenchymal stem cells (BM-MSCs) with isolated pancreatic islets and found that BM-MSCs had a protective effect on cocultured and isolated pancreatic islet cells. After transplantation, pancreatic islets cocultured with BM-MSCs showed stronger activity and glucose-induced insulin secretion function. The levels of tissue inhibitor of metalloproteinase-1 and vascular EGF increased at 4 weeks.⁷³ In 2013, Jun et al used the concave micropores of polydimethylsiloxane to carry out 3D cocultures of primary islet cells and hepatocytes. They found that compared with the single cultured spheres, the islet spheres mixed with hepatocytes have higher stability and closer cell–cell junction, which improves the survival time of islet cell transplantation in vivo.⁶⁸ In 2012, Kang et al transplanted endothelial progenitor cells from umbilical veins and porcine islets into nude mice with diabetes. The cotransplanted cells accelerated the vascular reconstruction of the graft, thus improving the functional state of the graft vascular system and greatly improving the transplantation β cell quality.⁷⁴ In 2019, Lebreton et al successfully cocultured human amniotic epithelial cells (hAECs) with mouse pancreatic islet cells to produce a dynamic and functional pancreatic organ composed of hAECs and dissociated islet cells.⁷⁵

4.4 Optimization of pancreatic islet organoid 3D culture protocol

Traditional cell differentiation research is mainly based on two-dimensional (2D) cell culture, but 2D cell culture cannot simulate the complex spatial organization structure and complex physiological processes of cells in vivo.^{48, 76, 77} This article has reviewed the transformation of cells into pancreatic islet β-like cells under a 2D culture system through various in vitro pathways, but the induced cells require specific tissue forms to perform normal physiological functions. Differentiated cells for regenerative applications, when using the established protocol, insulin-expressing β-like cells have low differentiation efficiency and final yield, lack functional characteristics, and show a lower ability to reverse disease conditions. In recent years, with the development of 3D culture technology, new ideas have been brought to the research and disease treatment of islet regenerative medicine. The natural ECM extracted from mouse sarcomas by Orkin et al provides a 3D environment for organoid culture. This ECM gel-like protein derived from tumor cell lines has been shown to support the self-renewal potential of stem cells and maintain their undifferentiated state.⁷⁸ However, the heterogeneous cancer cell line origin of Matrigel hinders its medical use, as well as batch-to-batch differences, hindering the reproducibility of the study, resulting in the inability of islet organoids to be produced under Good Manufacturing Practice (GMP) conditions for clinical treatment. The use of chemically synthesized matrices for organoid culture has been well studied.

Currently, polyethylene glycol (PEG), polylactic acid, poly ε-caprolactone, polyvinyl alcohol, and polylactide-co-glycolide artificial matrix gums synthesized from various materials are used to culture organoids.^{79, 80} Due to the lack of growth factors in synthetic Matrigels, the organoid field is pushed toward GMP by adding growth factors to specific media.⁸¹ Greggio et al crosslinked PEG hydrogels with laminin and thrombin activator XIIIa instead of Matrigel as a medium for pancreatic organoids. However, this hydrogel is less effective at maintaining the morphology and function of pancreatic embryonic organoids.⁵⁷ Youngblood et al synthesized a hydrogel with micropores of 250 ~ 400 μm to make the organoids cultured in it have a controllable cell density and volume/area ratio. However, it has been suggested that support mediators affect the maturation status of pancreatic organoids.⁸² Candiello et al synthesized a novel hydrogel Amikagel and cultured pancreatic progenitor cells induced from hESCs on Amikagel and found that the cells could spontaneously aggregate into circular colonies and gradually transform into pancreatic organoids with the ECM. Compared with Matrigel, the pancreatic organoids obtained from Amikagel culture have a higher maturity and can respond better to sugar stimuli.⁶⁵

The medium for pancreatic organoids was developed from the basis of intestinal organoids and consists of DMEM/F12 medium and EGF, Noggin, and R-Spondin1.⁸³ EGF promotes pancreatic cells while inhibiting the differentiation of pancreatic endocrine progenitor cells.⁸⁴ Noggin, as a member of the transforming growth factor superfamily, is the basic cytokine of pancreatic development and the basis for the development of solid organs, including the pancreas.⁸⁵ R-Spondin1 is an agonist of the LGR5 receptor of the Wnt/β-catenin pathway, which in turn is essential for the development and self-renewal of several types of adult stem cells.⁸⁶ At the same time, FGF10, nicotinamide, N-acetylcysteine, and B27 are also widely used as components of pancreatic islet organoid media.⁸⁷ FGF10 promotes PDX1⁺ pancreatic progenitor cell expansion and aids pancreatic progenitor cell growth and differentiation.^{88, 89} Nicotinamide induces fetal pancreatic endocrine cell differentiation and maturation and is commonly used for islet organoid culture.^90-92 Pancreatic organoids are expected to be a new diabetes treatment, but further studies are needed to evaluate their safety and efficacy.

4.5 CRISPR/Cas9 gene editing in organoid technology

In only a decade, CRISPR-associated nucleases were discovered to be capable of genome editing.^{93, 94} Traditional and emerging therapeutic modalities have been reshaped by this revolution. The ability of Cas9 to (a) identify nucleic acid sequences with high affinity and (b) precisely cleave target genes to trigger repair mechanisms to insert desired sequences has led to the development of several biological tools and translation applications.^{95, 96} Cho et al designed a nanocarrier consisting mainly of lecithin and self-assembling Cas9 complexes with single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) for injection into type 2 diabetes mellitus (T2DM) db/db mice.⁹⁷ Cas9-RNP complexes are efficiently delivered to hepatocytes through accumulation in the liver, disrupting the expression of the dipeptidyl peptidase 4 gene in T2DM mice while normalizing blood glucose levels and reducing liver and kidney damage. Studies have shown that liver-specific transcriptional enhancers regulated by FOXA2 are present at the intron T2DM sites represented by rs780094, rs780095, and rs780096SNP, and the expression of glucokinase regulatory protein gene can be increased by using the CRISPR-dCas9 transcriptional activator system.⁹⁸ Future CRISPR-Cas applications could design ways for hepatocytes to become β cells to circumvent the inherent limitations of β cells, including insufficient response to glucose-stimulated insulin and susceptibility to immune attacks after transplantation.