Diabetes mellitus is a chronic disease with many debilitating complications. Treatment of diabetes mellitus mainly revolves around conventional oral hypoglycaemic agents and insulin replacement therapy. Recently, scientists have turned their attention to the generation of insulin-producing cells (IPCs) from stem cells of various sources. To date, many types of stem cells of human and animal origins have been successfully turned into IPCs
Diabetes mellitus is a common disease in many parts of the world with serious complications and no cure. Since the discovery of insulin more than 80 years ago, the treatment of diabetes mellitus has not changed much. Although cadaveric pancreatic transplantations have been explored, they are not without disadvantages and complications such as difficulty in finding a suitable donor and immune rejection of the transplanted pancreas or islet cells [
The pancreas is a gland organ that plays an important role in the digestive and endocrine systems. It consists of two major types of cells: (1) exocrine cells, which are organized in acini and secrete digestive enzymes and (2) endocrine cells, which are organized in the islets of Langerhans and secrete hormones into the bloodstream. In an adult pancreas, the islets contribute to 1% to 2% of the total pancreatic mass. There are approximately one million islets with three main types of cells, namely, alpha, beta, and delta cells. The insulin-secreting beta cells, and glucagon-secreting alpha cells contribute to 75% and 25% of the islets cells, respectively, while the remaining 5% of islet cells is made up of the somatostatin-secreting delta cells. Another type of cells, called the F cells (or PP cells), secretes pancreatic polypeptide. These cells are present mainly in the islets situated in the posterior portion of the head of pancreas, and the physiological function of pancreatic polypeptide is largely unknown [
The mature pancreas is a single organ, which is initially derived from two separate and distinct rudiments, that is a dorsal and a ventral bud arising from the primitive gut epithelium. The dorsal pancreatic bud grows into the dorsal mesentery while the ventral bud, the ventral mesentery. The differential rotation and fusion of the dorsal and ventral pancreatic buds, which take place late in the sixth week of fetal development, result in the formation of the definitive organ and are followed by the interconnection of their ductal systems [
The expression of the pancreatic and duodenum homeobox-1 (Pdx1) gene, also known as insulin promoter factor-1 (Ipf1), has been shown to be important in pancreatic development, and beta-cell maturation and inactivity leads to total absence of the organ [
Although many hormones are capable of increasing blood sugar levels, insulin is the only hormone that has a direct glucose-lowering effect. It is one of the key regulators of blood glucose concentration, and it plays an important role in the pathophysiology of diabetes mellitus. Insulin’s main actions on carbohydrate metabolism include (1) increasing glucose uptake in target cells, hence promoting storage in the form of glycogen, (2) stimulation of glycogenesis, (3) inhibition of gluconeogenesis, and (4) decreasing hepatic glucose output by inhibiting gluconeogenesis. Together, these actions contribute to insulin’s glucose-lowering effect. In addition, insulin also exerts multiple effects in fat and protein metabolisms [
Actions of insulin and glucagon and factors that affect their secretion.
Hormone | Action | Factors affecting secretion |
---|---|---|
Insulin | ||
Increases glucose uptake into target cells (e.g., skeletal and | Increased blood glucose concentration | |
adipose tissue cells) | ||
Stimulates glycogenesis in skeletal muscle and liver | Parasympathetic stimulation following food intake | |
Inhibits gluconeogenesis | Increased blood amino acid concentration | |
Decreases hepatic output of glucose by | Increased free fatty acid concentration | |
inhibiting.gluconeogenesis | ||
Intestinal hormones (e.g., gastrin, cholecystokinin, | ||
secretin, glucagons-like peptide 1, and glucose-. | ||
dependent insulinotropic polypeptide) | ||
Increases fatty acids and triglyceride synthesis by liver | Glucagon | |
Increases entry of fatty acids from blood into adipose. | Growth hormone | |
tissues | ||
Inhibits lipolysis, decreasing release of fatty acids from | Cortisol | |
adipose tissue | ||
Insulin resistance | ||
Increases active transport of amino acids into target cells | Obesity | |
(e.g., muscle cells) | ||
Increases protein synthesis | ||
Inhibits protein catabolism | Decreased blood glucose concentration | |
Fasting | ||
Sympathetic stimulation | ||
Somatostatin | ||
Leptin | ||
Glucagon | ||
Increases glycogenolysis | Decreased blood glucose concentration | |
Increases gluconeogenesis | Increased blood amino acid concentration | |
Increased catecholamines | ||
Increases lipolysis, making increased amounts of fatty acids available to the body | Sympathetic stimulation | |
Exercise | ||
Increases amino acid uptake by liver cells | Increased blood glucose concentration | |
Increases conversion of amino acid to glucose by | Increased blood free fatty acid concentration | |
gluconeogenesis in the liver | ||
Somatostatin | ||
Insulin |
Diabetes mellitus is a group of metabolic diseases with hyperglycaemia as a hallmark. It is due to defects in insulin secretion or action, or both. Chronic hyperglycaemia is associated with many debilitating complications involving multiple organs such as the eyes, kidneys, heart, nerves, and blood vessels [
The number of people with diabetes mellitus is ever increasing. Some contributing factors to this increase include population growth, aging, urbanisation, increasing prevalence of obesity and physical inactivity [
The treatment of diabetes mellitus mainly revolves around oral hypoglycaemic drugs and insulin replacement therapy. Pancreatic islets transplantation is an alternative, but its use is restricted by a shortage of donated organs, immune rejection, and the need of life-long immunosuppression [
To date, many different types of human or animal stem/progenitor cells have been used for the generation of IPCs
Embryonic stem cells (ESCs) are defined as self-renewing pluripotent cells derived from the inner cell mass of blastocysts. ESCs are unique in that they can be indefinitely cultured in an undifferentiated state and be differentiated into cells of the three embryonic germ layers, namely, the ectoderm, mesoderm, and endoderm [
Adult stem cells differ from embryonic stem cells in that they are restricted to differentiate into a variety of cell types with a defined lineage. Therefore, differentiation of stem cells into cells of a different lineage is considered a form of transdifferentiation. Several studies have looked into such transdifferentiation of adult stem cells into IPCs using adult stem cells of both pancreatic and non-pancreatic origins.
For many years, scientists were misled to think that beta cells did not replicate based on the commonly accepted concept that terminally differentiated cells do not replicate. It is now clear that new beta cells are formed in the adult pancreas [
Besides stem or progenitor cells of pancreatic origin, other adult stem cells of both animal and human origins have been shown to transdifferentiate into IPCs. The possible pathways for the generation of differentiated cells from a different tissue include (1) true transdifferentiation of a differentiated cell into another differentiated cell, (2) de-differentiation of a differentiated cell into a common progenitor cell type, followed by differentiation into another cell type which is different from that of the original cell, (3) de novo differentiation of pluripotent cells which have persisted in adult tissues, and (4) fusion of a pluripotent cell with another already differentiated cell (Figure
Possible pathways of generation of differentiated cells from another cell type. (a) True differentiation of one cell type into another. (b) De-differentiation of one cell type into a common progenitor cell followed by differentiation into another cell type. (c) De novo differentiation of a pluripotent stem cell in adult tissue into another cell type. (d) Fusion of one cell type with a pluripotent stem cell giving rise to another cell type.
The vast variety of stem cells used makes comparison of methods difficult. Different types of stem cells require different culture and induction media for differentiation of IPCs to take place. However, some common themes seem to appear in various induction methods.
Firstly, induction of stem or progenitor cells into IPCs usually requires a multistage protocol. Most protocols require at least a two-stage protocol [
Secondly, the induction period varies greatly with the type of cells used. It may last from several days to several months. For example, in a study carried out by Chen et al., the protocol involved a two-stage protocol during which rat bone marrow mesenchymal stem cells were preinduced for 24 hours and reinduced for an additional 10 hours. The entire induction process took less than 48 hours [
Summary of induction protocols used in insulin-producing cell generation.
Summary of protocol | Stem cell used in induction | Duration of induction | Remarks | Author |
---|---|---|---|---|
Stage 1: culture of undifferentiated human embryonic stem cells (hES)—DMEM, 20% knockout serum replacement, glutamine, nonessential amino acid, | Stage 1: Cells were dissociated after 30 minutes | Differentiated cells showed enhanced expression of pancreatic genes. Immunofluorescence and | ||
Stage 2: generation of embryoid bodies 80% knockout DMEM, 20% FBS, glutamine, and non-essential amino acids | Stage 2: 7 days | |||
Stage 3: culture of embryoid bodies in DMEM/F12 medium with insulin-transferrin-selenium-fibronectin | Stage 3: 7 days | Segev et al., 2004 [ | ||
Stage 4: Expansion of pancreatic progenitor cells in DMEM/F12 medium with N2 & B27 supplement, bFGF | Human embryonic stem cells | Stage 4: 7 days | ||
Stage 5: withdrawal of bFGF, addition of nicotinamide, and reduction of glucose concentration | Stage 5: 4 days | |||
Stage 6: formation of clusters in suspension | Stage 6: cluster formation | |||
Total: 25 days or longer | ||||
Stage 1: Preinduction in L-DMEM medium with | Rat marrow mesenchymal stem cells | Stage 1: 24 hours | Islet-like clusters were observed showing positive insulin mRNA and protein expressions. Differentiated cells responded to glucose challenge |
Chen et al., 2004 [ |
Stage 2: Reinduction in serum-free HDMEM medium with nicotinamide, | Stage 2: 10 hours | |||
Total: 34 hours | ||||
Stage 1: RPMI medium, 10% FCS | Murine bone marrow-derived cells | Stage 1: 2 to 4 months | Differentiated cells expressed multiple genes related to pancreatic beta cell development and function. Insulin and C-peptide production was confirmed by immunocytochemistry and electron microscopy. | |
Stage 2: RPMI medium, glucose, 5% FCS, and nicotinamide | Stage 2: 7 days |
Tang et al., 2004 [ | ||
Stage 3: RPMI medium, 5% FCS, glucose, nicotinamide, and exandin-4 | Stage 3: 5–7 days | Transplantation of differentiated cells showed reversal of hyperglycaemia in streptozotocin-induced diabetic mice. | ||
Total: variable | ||||
H-DMEM serum-free medium, insulin, transferring, selenium, activin A, betacellulin, exendin-4, and hepatocyte growth factor | Human liver-derived fetal cells (FH-B-TPN) | Manipulation of culture conditions in various experimental settings | Cells cultured with activin A and betacellulin serum-free medium showed upregulation of NeuroD, Nkx22, glucokinase, prohormone convertase 1/3 and downregulation of Pax6, pancreatic polypeptide, glucagon, and liver markers. Insulin content of cultured cells increased 33-fold that of normal beta cells. | Zalzman et al., 2005 [ |
Stage 1: neurosphere cell line cultured in expanded in medium with X-VIVO15, N2 supplement, heparin, leukaemia inhibitory factor, EGF, and bFGF | Human neurospheres cell lines | Stage 1: cells were frozen after expansion | Formation of glucose-responsive, insulin-producing cells in clusters. | |
Stage 2: DMEM/F12 medium with, bovine serum albumin, N2 supplement, heparin, leukaemia inhibitory factor, EGF, and bFGF | Stage 2: 14 days | |||
Stage 3: L-DMEM/F12 medium, apo-transferrin, glucose, bovine insulin, sodium selenite, and retinoic acid | Stage 3: 14 days | Transplantation of differentiated cells into immunocompromised mice showed release of insulin C-peptide upon glucose challenge transplanted cells did not differentiate further and did not form tumours. | Hori et al., 2005 [ | |
Stage 4: N2 medium, nicotinamide, insulin-like growth factor-1, and glucose | Stage 4: 6 days | |||
Total: 34 days (excluding cell expansion in stage 1) | ||||
Serum free DMEM/F12 medium, glucose, nicotinamide, activin-A, exendin-4, hepatocyte growth factor, pentagastrin, B27 supplement, and N2 supplement | Human adipose-derived mesenchymal stem cells | Gene expression profile was analyzed every 24 hours for 3 days. | Down-regulation of ABCG-2 and up-regulation of pancreatic developmental transcription factors (Isl-1, Ipf-1 and Ngn3) were observed, together with induction of islet hormones insulin, glucagon, and somatostatin. | Timper et al., 2006 [ |
Total: 3 days | ||||
Stage 1: chemically defined medium (CDM): 50% ICDM + 50% F12 NUT-MIX, insulin-transferrin-selenium-A, monothioglycerol, albumin fraction V, and | Human embryonic stem cells | Stage 1: 2 days | Activin A induced definitive endoderm differentiation from human embryonic stem cells with detection of the expression of definitive endoderm markers Sox17 and Brachyury. Retinoic acid promoted pancreatic differentiation, indicated by the expression of early pancreatic transcription factors Pdx1 and Hlxb9; bFGF and nicotinamide helped the differentiated cells to express islet specific markers such as C-peptide, insulin, glucagon, and glut2. Differentiated cells were able to secrete insulin in response to glucose stimulation | |
Stage 2: CDM, activin A | Stage 2: 4 days | |||
Stage 3: induced cells transferred into CDM with retinoic acid | Stage 3: 4 days | Transplanted cells in streptozotocin-induced nude mice survived and maintained expression of beta cell marker genes (C-peptide, Pdx-1, glucokinase, Nkx6.1, IAPP, Pax6, and Tcf1). 30% of mice showed restoration of stable euglycaemia for more than 6 weeks | Jiang et al., 2007 [ | |
Stage 4: maturation medium (DMEM/F12, insulin-transferrin-selenium-A, albumin fraction V, bFGF | Stage 4: 3 days | |||
Stage 5: addition of nicotinamide, removal of bFGF | ||||
Stage 5: 5 days | ||||
Total: 18 days | ||||
Stage 1: serum free H-DMEM medium, | Human bone marrow-derived mesenchymal stem cells from diabetic patients | Stage 1: 2 days | Transdifferentiated cells tested positive for dithizone and immunohistochemistry for insulin, PDX-1, Neurogenin3, Pax4, insulin, glucagon by RT-PCR; they also responded to glucose stimulation | |
Stage 2: DMEM medium, bFGF, EGF, B27, and non-essential amino acids | Stage 2: 8 days | Sun et al., 2007 [ | ||
Stage 3: DMEM medium, betacellulin, activin A, nicotinamide, B27 | Stage 3: 8 days | |||
Total: 18 days | ||||
Stage 1: DMEM/F12 medium, 15% FCS, progesterone, putrescine, laminin, insulin, sodium selenite, nicotinamide, transferring, and fibronectin | Human umbilical cord blood-derived stem cells with embryonic stem cell phenotype | Stage 1: 24 hours | Insulin-producing islet-like structures that co-expressed insulin and C-peptide were observed |
Sun et al., 2007 [ |
Stage 2: H-DMEM medium, 15% FCS, progesterone, putrescine, laminin, insulin, sodium selnite, nicotinamide, transferring, and fibronectin | Stage 2: pancreatic islet-like structure started to appear after 5–7 days of induction | |||
Total: up to 7 days | ||||
Stage 1: H-DMEM medium, 5% FBS | Bone-marrow mesenchymal stem cells from Sprague-Dawly rats | Stage 1: 14 days | Islets like clusters were observed at the end of induction. Electron microscopy showed increased cytoplasmic secretory granules in differentiated cells. Differentiated cells insulin secretion increased by 1.5-fold after glucose challenge | |
Stage 2: addition of nicotinamide to the above medium | Stage 2: 7 days | Wu et al., 2007 [ | ||
Stage 3: addition of exendin-4 | Stage 3: 7 days | |||
Total: 28 days | ||||
Stage 1:serum free DMEM medium, DMSO | Adult bone marrow stem cells from the long bones of rats | Stage 1: 3 days | Observation of islet-like clusters stained positive for dithizone. Differentiated cells showed expression of insulin and endocrine-specific genes. Differentiated cells showed | |
Stage 2: H-DMEM medium, 10% FBS, pancreatic extract | Stage 2: 7 days | Gabr et al., 2008 [ | ||
Stage 3: L-DMEM medium, 5% FBS, nicotinamide, and exendin-4 | Stage 3: 7 days | |||
Total: 17 days | ||||
Stage 1: H-DMEM medium, 10% FBS, retinoic acid (24 hours), H-DMEM medium, 10% FBS (2 days) | Human umbilical cord blood-derived mesenchymal stem cells | Stage 1: 3 days | Islet-like cell clusters appeared 9 days after pancreatic differentiation. Insulin-secreting cells accounted for approximately 25% of the induced cells. Induced cells expressed islet-related genes and hormones but were not responsive to glucose challenge. Induced cells that were cultured without extracellular matrix gel failed to form clusters, and functional islet proteins were absent | |
Stage 2: L-DMEM medium, 10% FBS, nicotinamide, EGF seeded in wells with extracellular matrix gel | Stage 2: 6 days | Gao et al., 2008 [ | ||
Stage 3: L-DMEM medium, 10% FBS, exendin | Stage 3: 6 days | |||
Total: 15 days | ||||
Stage 1: expansion of human umbilical cord mesenchymal cells in neuronal conditioned medium | Mesenchymal stem cells in Wharton’s jelly of human umbilical cord | Stage 1: 7 days | Transdifferentiated cells formed islet-like clusters. RT-PCR showed expression of Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2. Islet-like clusters capable of producing insulin both | |
Stage 2: generation of nestin positive cells in DMEM/F12 medium, 2% FBS, nicotinamide, and B27 | Stage 2: 7 days | Chao et al., 2008 [ | ||
Stage 3: differentiation of premature clusters in DMEM/F12 medium, 2% FBS, nicotinamide, B27, and stem cell conditioned medium | Stage 3: 14 days | |||
Stage 4: maturation of insulin-secreting cells | ||||
Total: 28 days (excluding stage 4) |
FBS: foetal bovine serum, FCS: foetal calf serum, H-DMEM: high-glucose DMEM, L-DMEM: low-glucose DMEM.
Lastly, addition and withdrawal of a combination of extrinsic factors in a stage-wise manner are common to most protocols. Many extrinsic factors have been shown to promote beta-cell proliferation and differentiation and increase insulin content of IPCs. A number of these factors have been commonly observed in induction protocols. Careful use of serum and glucose in the induction media has also been indicated for successful generation of IPCs. A summary of these extrinsic factors and their effects is given in Table
Extrinsic factors involved in insulin-producing cell generation.
Extrinsic factor | Effect | Author |
---|---|---|
bFGF | Beta-cell differentiation | Dalvi et al., 2009 [ |
Potent chemoattractant. May be useful in cluster formation | Hardikar et al., 2003 [ | |
EGF | High concentration may be inhibitory to beta-cell differentiation | Cras-Méneur et al. 2001 [ |
bFGF and EGF | Differentiation of embryonic stem cells into IPCs | Lumelsky et al., 2001 [ |
Differentiation of human bone marrow-derived mesenchymal stem cells into IPCs | Sun et al., 2007 [ | |
Betacellulin | Formation of islet-like clusters |
Demeterco et al., 2000 [ |
Induction of beta cell differentiation | ||
Activin A | Increase in insulin content | Demeterco et al., 2000 [ |
Betacellulin and activin A | Differentiation of pancreatic acinar AR42J cells into IPCs | Mashima et al., 1996 [ |
Combined effect may be weaker than that of either factor alone | Demeterco et al., 2000 [ | |
Differentiation of human liver-derived insulin-producing cells toward the beta-cell phenotype | Zalzman et al., 2005 [ | |
Differentiation of human bone marrow-derived mesenchymal stem cells into IPCs | Sun et al., 2007 [ | |
Nicotinamide | Differentiation of stem cells of various origins into IPCs | Chen et al., 2004 [ |
Increase in insulin content, DNA content, expression of insulin, glucagon, and somatostatin genes | Otonkoski et al., 1993 [ | |
Exendin-4 | Differentiation of murine bone marrow stem cells into IPCs | Tang et al., 2004 [ |
Formation of insulin-expressing cells generated from adipose tissue-derived mesenchymal stem cells | Timper et al., 2006 [ | |
Differentiation of rat bone marrow-derived mesenchymal stem cells into IPCs | Wu et al., 2007 [ | |
Differentiation of rat bone marrow-derived stem cells into IPCs | Gabr et al., 2008 [ | |
Increase in insulin release by IPCs generated from mouse embryonic stem cells | Li et al., 2010 [ | |
Hepatocyte growth factor | Differentiation of pancreatic acinar cells into IPCs | Mashima et al., 1996 [ |
Increase in the number of IPCs in cultured human islets | Otonkoski et al., 1996 [ | |
Gastrin | Stimulation of islet differentiation and islet growth | Wang et al., 1993 [ |
Glucose | Low concentration (5 mM) increased insulin content.High concentrations (20–30 mM) increased beta cell replication | Bonner-Weir et al., 1989 [ |
EGF is a growth factor belonging to the EGF family of proteins. It plays an important role in cellular proliferation, differentiation, and survival [
Fibroblast growth factor (FGF) was first discovered as an activity in extracts of pituitary and brain which had a stimulatory effect on the growth of mouse fibroblast cells. It was later shown that the activity was due to two proteins, namely, acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) [
Low concentrations of EGF and bFGF are used in the culture of stem cells, especially the MSCs. Interestingly, high concentrations of EGF and bFGF, either used alone, or in combination, have been shown to be useful in IPC differentiation [
Activin proteins are members of the transforming growth factor-beta (TGF-
Betacellulin is a member of the EGF family isolated as a 32 kDa glycoprotein from the conditioned medium of a mouse pancreatic insulinoma cell line [
The use of betacellulin and activin A, either alone or in combination, has resulted in differentiation of stem cells into IPCs [
Nicotinamide is also called niacinamide or nicotinic acid amide. It is the amide of nicotinic acid or vitamin B3. Nicotinamide represents another commonly used extrinsic induction factor. As early as the 1990s Otonkoski et al. had used nicotinamide as an inducer of endocrine differentiation in cultured human foetal pancreatic cells [
Exendin-4 is a 39 amino acid protein isolated from the venom of the lizard
HGF is a heparin-binding protein secreted by mesenchymal cells. It is an inducer of cell proliferation, cell motility, and morphogenesis in many cell types. HGF has been reported to stimulate T cell adhesion to endothelium and migration as well as to enhance neuron survival. It has also been found to play a role in the regulation of erythroid differentiation and has an inhibitory effect on cell growth [
Gastrin is a hormone produced by specialised cells called G cells in the antral part of gastric mucosa. Small amounts of gastrin are produced by the duodenum and the pancreas. It is also found in the pancreatic islets in foetal life. Its principle physiologic role is to stimulate gastric acid and pepsin secretion and to stimulate growth of the mucosa of the stomach and small and large intestine [
Interestingly, the concentration of glucose in the induction medium seemed to play a role in IPC differentiation. Bonner-Weir et al. demonstrated that low concentrations of glucose (5 mM) increased insulin content in islet-like clusters while higher concentrations (20–30 mM) increased beta cell replication
Although several studies have demonstrated stem cells of various origins can be differentiated into IPCs, the small amounts of insulin secreted by these IPCs
Some of the challenges faced by researchers include (1) the choice of cell and an induction method which can consistently produce functional, beta islet-like IPCs and (2) generation of IPCs that can consistently produce clinically significant amounts of insulin. Although several studies have demonstrated that IPCs generated
Stem cells of various origins may be an important source of IPCs. The abundance of literature suggests that the generation of stem cells into IPCs is feasible and promising. However, the wide variations in induction techniques and sources of stem cell used may be a challenge to researchers as there is no standard method for IPC generation. A careful choice of stem cell and induction method is necessary for successful IPC differentiation. Further exploration is necessary for the generation of sufficient IPCs that can produce sufficient insulin for clinical use and the mechanism of such differentiation and how these differentiated cells correct hyperglycaemia need to be established before they can be used in human subjects for the treatment of diabetes mellitus.
The author declares that there is no conflict of interests.
The author would like to acknowledge the International Medical University, Malaysia and the Malaysia Toray Science Foundation for funding research that led to the understanding and writing of this paper (Grant numbers IMU 190/2009 and IMU R049/2009, resp.).