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. 2022 Jul 28;13:927324. doi: 10.3389/fendo.2022.927324

Table 1.

Overview of protocols for the generation of stem cell-derived β-cells.

Sources Cultures Improvements Outcomes and significance Reference
hESCs Planar culture and air-liquid interface

  • -Supply of Vitamin C to downregulate NGN3 during S2-4

  • -Transferring to an air-liquid interface plus the addition of ALK5iII and T3 during S5

  • -The combination of ALK5iII, T3, LDN and GSiXX during S6

  • -Addition of N-Cys during S7

  • -Elevated expression of key markers of mature pancreatic β-cells such as MAFA

  • -Ameliorating hyperglycemia in diabetic mice within 40 days

  • -Establishment of a standardized and classical 7-stage protocol for the generation of β-like cells

 (14)
hESCs and iPSCs Suspension culture

  • -Extension of time during PP2 with KGF, SANT1 and retinoic acid

  • -A distinct combination of 11 factors and molecules including PdbU to differentiate PPs

  • -Similar calcium flux in response to glucose

  • -Secreting quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro

  • -Higher proportion of mice that survived when transplanted with SC-β cells within 8 weeks

 (15)
hESCs and iPSCs Suspension culture

  • -Regulation of Alk5i to inhibit or permit TGF-β signaling pathway in S6

  • -Use of ESFM in S6 to create a serum-free protocol

  • -Increased insulin secretion with glucose rising

  • -Mostly expressed CHGA and C-peptide

  • -Improved β cell markers expression

  • -Improved glucose tolerance in vivo

  • -Rapidly controlling glucose in STZ mice within 10 days

  • -Functional improvements compared with previous SC-β cells

 (16)
hESCs Suspension culture

  • -Use of the INSGFP/W cell line to optimize previous protocol

  • -Addition of a re-aggregation step for the cluster of immature β cells

  • -Co-localization with TOMM20 and measuring rhodamine-123 to reveal the mechanisms of mitochondrial function to the maturity of β cells

  • -Mostly expressed key markers of β cells and C-peptide

  • -Improved maturation of target cells

  • -Calcium flux in response to glucose

  • -Sulfonylurea reaction

 (17)
hESCs 3D culture

  • -Re-aggregation technique to deplete non-endocrine cells

  • -Identifying CD49a (also known as ITGA1) as a surface marker of the β-cell population

  • -Use of magnetic microbeads with anti-CD49a for efficient sorting of SC-β-cells

  • -Stabilized glucose responsiveness

  • -Generation of high purity SC-β-cell clusters

  • -Revelation a CD49a as a surface marker for enriching β cells in vitro

  • -As a reference for future studies on the differentiation of β-cells

 (18)
iPSCs Gellan-gum-based 3D culture

  • -Inclusion of hADSCs and HUVECs in the formation of multicellular spheroids (MCSs) from PPs

  • -Addition of WNT4 in the functional maturation process of HILOs

  • -Improved oxidative metabolism and in vitro GSIS

  • -Ameliorating hyperglycemia in mice and maintaining normoglycemia for more than 6 weeks

  • -Identifying the non-canonical WNT signaling as a necessity to the metabolic maturation of HILOs

 (19)
hESCs and iPSCs Suspension and 2D culture

  • -Dispersing spheroids into single cells and interacting with basement membrane proteins such as laminin 511 for 3 days on monolayer culture

  • -Enforced β-like cell polarity

  • -Reduced basal insulin secretion and increased stimulated insulin secretion

  • -Uncovering the role of basement membrane proteins during differentiation and maturation via cell polarization

 (20)
hESCs and iPSCs Planar culture

  • -Treatment with 1 μM latrunculin A for the first 24h at S5

  • -Elimination of the need for suspension culture

  • -Similar expression of pancreatic β-cell markers including MAFB and a smaller proportion of somatostatin and glucagon positive cells

  • -Similar insulin content and increased biphasic GSIS

  • -Similar rates of reconstructing normoglycemia in STZ-induced mice and maintaining the state for at least 9 months

  • -Compatible to various cell lines

  • -Revelation of the connection between actin cytoskeleton and the differentiation of pancreatic cells

 (21)
hESCs V-bottom plate and air-liquid interface

  • -Insertion of GFP and NLS into NKX6.1 gene

  • -Use of V-bottom plate to promote the 3D structure aggregation of PPS

  • -A combination of ten chemicals (PP-10C) to maintain NKX6.1

  • expression and 3D structure

  • -Three combinations of chemicals and factors for the stepwise induction of β-cells

  • -Late-stage readout strategy in the screening process

  • -Higher efficiency for the generation β-cells from multiple cell lines and enrichment for the desired cell types

  • -Similar C-peptide level and insulin secretion in response to high-concentration glucose

  • -Restoring normoglycemia in STZ-induced mice within ~2 weeks

 (22)
hESCs and iPSCs Planar culture

  • -Treatment with latrunculin A at the start of S5, with dosage and timing depending on different cell lines

  • -Elimination of the need for suspension culture

  • -Establishment of a six-stage planar differentiation protocol with expanded accessibility and simplified and economic generation procedures

  • -Robust production of functional β-like cells with double or triple the number of cells per volume compared to suspension protocols, displaying static and dynamic GSIS

  • -Compatible to various cell lines

 (23)
hESCs and iPSCs Planar culture or suspension culture

  • -Replacing Activin A with dorsomorphin (DM)

  • -RPMI basal medium with HAS plus Vc

  • -Increased population of DE with up to 87% efficiency of SOX17 and FOXA2 double positive

  • -Enhanced cell survival and decreased costs

  • -Establishment of a xeno-free “GiBi” protocol for the generation of PPs which possess the capability of differentiating into pancreatic lineages

  • -More insulin secretion in response to high-level glucose

  • -Identifying the inhibition of BMP signaling to improve the efficiency of DE induction

 (24)
hESCs and iPSCs Monolayer, microwells, and suspension culture

  • -Addition of nicotinamide, epidermal growth factor, Activin A and ROCKi during S4

  • -Aggregation during S5 in microwells

  • -Maturation in suspension culture replacing ALK5i with ZM447439, T3, and NAC

  • -Completion of a comprehensive analysis of the functional maturation process in vitro and in vivo, including lower proportion of undesired cell types, downregulation of markers related to proliferation, reorganization of cytoarchitecture, increased insulin content, and analogous biphasic GSIS.

 (25)
hESCs Basal and suspension culture

  • -Coculturing mesenchymal and endothelial cells (M-E cells) with PPs for a week

  • -Addition of WNT5A to EPs for 3 days

  • -Increased number of insulin positive and C-peptide positive cells

  • -Sensitive insulin and C-peptide secretion in response to high-concentration glucose and KCl in vitro

  • -Discovery of WNT5A as an inducer to promote differentiation and maturation of pancreatic β-cells by activation of non-canonical WNT5A/JNK signaling and inhibition of BMP signaling

 (26)
hESC Matrigel 3D culture and microwell chips

  • -Coculturing PPs with hFP-MC at a ratio of 1:1 in microwell chips for 2 days and transferring to static suspension culture for 4 days to produce DC-PA

  • -Upregulation of NGN3 and INSULIN genes

  • -Consistent sized, spheroid-like structure and stability of DC-PA

  • -Proposing a scalable strategy to produce islet organoids

 (27)
hESCs and iPSCs 3D culture

  • -7-days treatment of the bromodomain and extraterminal domain (BET) inhibitor I-BET151

  • -Use of EF6I (the combination of EF6 medium and I-BET151) medium

  • -Promoting the expansion of PPs

  • -Revelation of the molecular mechanisms of I-BET151

  • -Lowering significantly glucose within 2 weeks and maintain normal levels after 3 weeks in STZ-induced mice

 (28)