Extrinsic Factors Involved in the Differentiation of Stem Cells into Insulin-Producing Cells: An Overview

Table 2

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, β-mercaptoethanol, and bFGF. Cells were dissociated after 30 minutes		Stage 1: Cells were dissociated after 30 minutes	Differentiated cells showed enhanced expression of pancreatic genes. Immunofluorescence and in situ hybridization showed a high percentage of insulin-producing cells in clusters, with most cells co-expressing somatostatin or glucagons, resembling immature pancreatic cells.
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 [3]
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 β-mercaptoethanol and nicotinamide	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 in vitro and could downregulate glucose in streptozotocin-induced diabetic rats.	Chen et al., 2004 [44]
Stage 2: Reinduction in serum-free HDMEM medium with nicotinamide, β-mercaptoethanol		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. In vitro insulin release was glucose stimulated.
Stage 2: RPMI medium, glucose, 5% FCS, and nicotinamide		Stage 2: 7 days		Tang et al., 2004 [45]
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. In vitro insulin release responded to physiological glucose levels.; Transplanted cultured cells in diabetic mice resulted in restoration of stable euglycaemia with continued in vivo insulin expression and no cell replication	Zalzman et al., 2005 [40].
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.; Expression of various genes involved in pancreatic development
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 [43]
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 [38]
		Total: 3 days
Stage 1: chemically defined medium (CDM): 50% ICDM + 50% F12 NUT-MIX, insulin-transferrin-selenium-A, monothioglycerol, albumin fraction V, and β-mercaptoethanol	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 in vitro.
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 [27]
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, β-mercaptoethanol	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 in vitro
Stage 2: DMEM medium, bFGF, EGF, B27, and non-essential amino acids		Stage 2: 8 days		Sun et al., 2007 [35]
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 [46]
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 in vitro.; After transplantation of islet-like clusters in diabetic rats, islet-like cells expressed islet hormones and lowered glucose levels of diabetic rats during day 6 to day 20
Stage 2: addition of nicotinamide to the above medium		Stage 2: 7 days		Wu et al., 2007 [47]
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 in vitro glucose secretion in a dose-response manner when challenged with increasing glucose concentrations
Stage 2: H-DMEM medium, 10% FBS, pancreatic extract		Stage 2: 7 days		Gabr et al., 2008 [36]
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 [37]
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 in vitro and in vivo
Stage 2: generation of nestin positive cells in DMEM/F12 medium, 2% FBS, nicotinamide, and B27		Stage 2: 7 days		Chao et al., 2008 [48]
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.