Breakthroughs in stem cell technology have contributed to disease modeling and drug screening via organoid technology. Organoid are defined as three-dimensional cellular aggregations derived from adult tissues or stem cells. They recapitulate the intricate pattern and functionality of the original tissue. Insulin is secreted mainly by the pancreatic β cells. Large-scale production of insulin-secreting β cells is crucial for diabetes therapy. Here, we provide a brief overview of organoids and focus on recent advances in protocols for the generation of pancreatic islet organoids from pancreatic tissue or pluripotent stem cells for insulin secretion. The feasibility and limitations of organoid cultures derived from stem cells for insulin production will be described. As the pancreas and gut share the same embryological origin and produce insulin, we will also discuss the possible application of gut organoids for diabetes therapy. Better understanding of the challenges associated with the current protocols for organoid culture facilitates development of scalable organoid cultures for applications in biomedicine.
Diabetes mellitus (DM) is a well-known disease increasing the risk of morbidity and mortality due to disease complications. Current therapies do not result in complete recovery. The lack of insulin production due to T cell-associated autoimmune damage of pancreas β cells is the main cause of type 1 DM. Therefore, efficient therapeutic strategies designed to restore insulin production are needed.
Transplantation of human islets into the portal vein to replace the damaged β cells resulted in regulation of blood glucose levels and significant improvement in the patient health condition (1, 2). However, this method is limited by the shortage of islet donors and the need to induce immunosuppression to prevent immune rejection (3). Moreover, intricate mechanisms such as redifferentiation and dedifferentiation are prerequisites for the expansion of mature β cells (4, 5). Accordingly, application of pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is one of the leading strategies for the generation of β cells with alternative function due to the high capacity of PSCs to differentiate into a wide range of lineages (6). Various protocols generate β cells from PSCs following treatment with signal-modulating compounds, hormones, and growth factors that induce multi-step pancreatic endoderm (PE) differentiation (7–9). The elevated expression of β cell-specific transcription factors and insulin secretion following glucose challenge guarantee authentic β cells (10). Ectopically transplanted PSCs-derived pancreatic β cells in immunodeficient diabetic mice triggered insulin secretion upon glucose challenge (11). However, generation of pancreatic β cells from PSCs is labor-intensive, time-consuming, expensive, and inefficient, and the clinical applications of PSCs are restricted by biosafety issues (12). Notably, the conversion of duct and islet cells into cells similar to mesenchymal cells is the main obstacle hindering the expansion of pancreatic cells in the monolayer platform (4, 13, 14). Therefore, the development of a new system based on three-dimensional (3D) organoid culture is essential for efficient differentiation and maturation of pancreatic β cells, as it simulates the inherent cellular environment (cell polarization, alignment, and niches) (15). Here, we will discuss methods of pancreatic organoid culture and their challenges along with its impact on the large-scale generation of PSCs-derived pancreatic β cells. Further, we discuss the possible application of gut organoids for regeneration of damaged pancreatic cells in diabetes therapy.
Organoid culture or organs-in-a-dish is the
In 2009, the Clevers research group demonstrated the organoid culture for the first time, using stem cells derived from the intestine, which laid the initial groundwork in the organoid era (19) followed by the development of optic cup from ESCs (20). Subsequently, a series of studies investigating organoid culture using various cells derived from liver, kidney, pancreas, brain, stomach, and prostate have emerged (21–26). Here, we focus on pancreatic and gut organoids and their application in diabetes therapy.
Pancreas is an organ manifesting both endocrine and exocrine functions and plays a crucial role in diseases such as diabetes, pancreatic cancer, and disease associated with pancreatic inflammation. The exocrine function is attributed to acinar function, which is associated with the secretion of digestive enzymes, whereas the endocrine function is related to epithelial clusters (islets of Langerhans) including α, β, δ, ɛ and pancreatic polypeptide (PP; formerly known as γ) cells, which secrete glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide, respectively (27).
Pancreatic development occurs concomitantly in the ventral as well as the dorsal anterior foregut endoderm (on embryonic day 9.5 (E9.5)), followed by expansion into the surrounding mesenchyme and proliferation, differentiation, and branching resulting in mature organ formation (28–31). The early-stage multipotent pancreatic progenitor cells, which express Sry (sex-determining region Y)-like box 9 (Sox9), hepatocyte nuclear factor 1 homeobox B (Hnf1B), pancreas transcription factor 1 subunit alpha (Ptf1A), and pancreatic and duodenal homeobox 1 (Pdx1), are developed before E11.5, and generate all the pancreatic cells including endocrine lineages, exocrine cells (acinai), and ductal cells subsequently (32, 33). The two progenitor cells generated by E14.5 yield a limited range of pancreatic cells; at this stage, Sox9-positive cells generate acini (34).
Fibroblast growth factor (FGF) and Notch (named after the appearance of a notch in the wings of mutant
In 2013, Greggio
In 2018, Takahashi
The reprogramming of acinar cells into pancreatic endocrine β cells with the transcription factors regulating endocrine formation, namely neurogenin-3 (Neurog3 or Ngn3; a basic helix-loop-helix (bHLH) transcription factor), Pdx1, and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) represents an important alternative source to regenerate the damaged β cells (42). In this regard, the overexpression of Ngn3, Pdx1, and MafA in mouse ductal pancreatic organoids using lentiviral vectors resulted in the generation of pancreatic β-like cells that showed a robust resemblance to β cells in insulin secretion and transcriptome-wide analysis (43). Interestingly, the phospho-mutant form of Ngn3 together with Pdx1 and MafA markedly promoted the expression of β cell-associated genes and showed a four-fold increase in reprogramming toward insulin-positive endocrine cells compared with the wild-type Ngn3 (43). The phospho-mutant form of Ngn3 was prepared via replacement of serine with alanine and therefore, cannot be phosphorylated with cyclin-dependent kinases (CDKs). Therefore, the Ngn3 mutation via mutations associated with the phosphorylation site resulted in enhanced endocrine cell reprogramming for ductal organoids.
In 2018, Loomans
Further details of the organoid cultures derived from adult and fetal pancreatic tissues are discussed and summarized elsewhere (47).
In 2015, Shim
In 2016, Kim
In 2009, Wang
In 2017, Wang
In 2018, Candiello
In 2019, Tao
Scientific efforts were targeted at the regeneration of damaged islets and the development of an alternative islet. However, gastrointestinal tract (GIT) represents an alternative and renewable source of pancreatic β cells for diabetes therapy. From a developmental perspective, pancreas and GIT originate in the same gut tube. In particular, stomach- and intestine-derived stem cells yielded a continuous supply of hormone-producing endocrine cells (58, 59). The endocrine cells are located only in the islet of Langerhans in the pancreas, but distributed in all the gastric epithelium (60).
In 2014, Chen
In 2016, the study by Ariyachet
The deletion of the transcription factor Foxo1 is another approach for the generation of insulin-secreting cells in gut organoids (63, 64). However, other studies demonstrated the key protective role of Foxo1 in the sustained survival of β cells (60, 65).
Taken together, these facts and scientific findings pave the way for further studies for practical application of gut organoids generated from patient tissues or PSCs based on their insulin secretion efficacy, and further therapeutic application in diabetes as an alternative source of pancreatic β cells.
Recently, various studies reported the generation of functional pancreatic β cells from PSCs (ESCS or iPSCs). However, these methods are laborious, time-consuming, expensive, and lack reproducibility (Fig. 3A). Furthermore, many protocols generated immature β cells and showed short survival upon
This work was supported by the National Research Foundation of Korea (NRF) grant provided by the Korean government (MSIT) (No. 2017M3A9C6032056) and by the Konkuk University Researcher Fund in 2018.
The authors have no conflicting interests.