Human pluripotent stem cell-derived β cells: Truly immature islet β cells for type 1 diabetes therapy?

Table 1 A summary of several differentiation protocols for generating stem cell-derived β cells ex vivo

Differentiation protocol	Marker expression	Characterization	Ref.
The stem cell-derived β cell protocol is a 6-stage differentiation protocol using specific inducing factors for each stage (11 factors) to produce SC-islets from hPSCs, in a 3D suspension-based cell culture system (4-5 wk)	Pancreas progenitor marker: PDX1 (about 90% at stage 3). Endocrine markers: C-peptide, CHGA and NKX6-1. SC-β cell markers: INS, NKX6-1, ISL1, and others	Immunofluorescence. Ultrastructure. Insulin packaging into secretory granules. qRT-PCR. Static and dynamic GSIS. Functional test. Glucose-responsive Ca2+ flux, KCl depolarisation, ameliorate hyperglycaemia in diabetic mice. scRNA-seq. Purified SC-β cells with anti-CD49a	Pagliuca et al[73], 2014; Veres et al[18], 2019
Optimised 7 stage in vitro differentiation protocol of serial culture steps using factors such as vitamin C, ALK5 inhibitor, TGF-β receptor inhibitors, thyroid hormone (T3), R428 (AXL inhibitor), N-acetyl cysteine, Notch and BMP signalling inhibitors	Pancreas progenitor marker: PDX1 (about 90% at stage 3), NKX6-1. Endocrine markers: C-peptide, CHGA, NEUROD1, NKX2-2 and NKX6-1. β cell markers: INS, NKX6-1, ISL1, MAFB. Mature β cell markers: MAFA, ABCC8, IAPP, HOPX, NEFM, SIX2, G6PC2	Immunofluorescence. Transmission electron microscopy. qRT-PCR. Static and dynamic GSIS. Functional test in diabetic mice. Flow cytometry. Immunohistochemistry. Diabetes reversal within 40 d in mice. Metabolic analysis. Perifusion assay. Calcium imaging.	Rezania et al[78], 2014
Scalable 3D suspension culture system based on previous methods[101,102], with the addition of retinoic acid, cyclopamine (SHH inhibitor), Noggin (BMP inhibitor), then treatment with epidermal growth factor, KGF and Noggin (EKN). Followed by exposure to a cocktail of factors e.g., TBP, ALK inhibitor, Noggin, TANK	Pancreas progenitor marker: PDX1 and NKX6-1 (90%). Endocrine markers: C-peptide, NEUROG3, NKX2-2 and NKX6-1. β cell markers: INS, NKX6-1, NKX2-2, PDX1. Mature β cell markers: MAFA, MAFB, PDX1, NKX6.1, NXK2.2, ISL1, PAX6, NEUROD1, and CHGA	Immunofluorescence. Transmission electron microscopy. qRT-PCR. Static and dynamic GSIS. Insulin biosynthesis and glucose metabolism, blood glucose reduction but not reversal of diabetes in mice. Flow cytometry. Western blot analysis. Statistical analysis	Russ et al[74], 2015
Six-stage differentiation strategy modulating TGF-β signaling by modulating Alk5i exposure, combined with controlling cell cluster size and use of enriched serum-free media culture	Pancreas progenitor marker: PDX1. Endocrine markers: CHGA (96%), C-peptide (73%), NKX6-1. SC-β cell markers: INS, CHGA, NKX2-2, PDX1, NKX6-1, MAFB, GCK, and GLUT1	Immunofluorescence. qRT-PCR. Static and dynamic GSIS. Flow cytometry. Light microscopy. Glucose responsive, first- and second-phase insulin release, improved glucose tolerance in mice. Western blot. Perifusion assay. Glucose tolerance test	Velazco-Cruz et al[19], 2019
Optimised differentiation protocol combining previous protocols. Changes made: Differentiation of hPSCs in adherent conditions until pancreatic progenitor stage. Then optimised with nicotinamide, epidermal growth factor, activin A and a ROCK inhibitor; a microwell aggregation step; and a final maturation step in suspension culture	Pancreas progenitor marker: PDX1. Endocrine markers: C-peptide. SC-β cell markers: INS. Mature β cell markers: INS, G6PC2, SIX2, GLIS3, RBP4, SIX3. HOPX, UCN3, IAPP, CPE and FXYD2 upregulated post engraftment. CHGB and MAFA upregulated 6 mo post-engraftment. Β cell differentiation: SIX2, HOPX, ZBTB20. Insulin secretion genes: PCSK1, CPE, CHGB, ABCC8, FXYD2, GABRA2	Immunohistochemistry. Flow cytometry. Perifusion assay. Respirometry. Transmission electron microscopy. Electrophysiology. Exocytosis imaging. [Ca2+]i imaging. [cAMP]m imaging. Metabolite tracing analysis. Ratiometric analysis. Transplantation study. scRNA-seq transcriptomic profiling. Glucose responsive biphasic insulin secretion. Glucose tolerance test	Balboa et al[20], 2022
Differentiation protocol using hCiPSC-islets by optimising pancreatic progenitor to β cells fate commitment by modulating signaling pathways and reconstructing islet spatial structure through 3D cell aggregates of posterior foregut-committed cells and combination of ISX9 and Wnt-C59 at stage 5	Pancreas progenitor marker: PDX1. Endocrine markers: C-peptide, CHGA and NKX6-1. β cell markers: PDX1, NKX6.1 and NKX2.2. Mature β cell markers: MAFA, UCN3	Immunofluorescence. qRT-PCR. Transmission electron microscopy. Static and dynamic GSIS. Glucose-stimulated calcium flux assay. Flow cytometry. scRNAseq. Glucose responsive biphasic insulin secretion, decrease HbA1c, restore endogenous C-peptide secretion. Glucose tolerance tests. Preclinical diabetic non-human primate transplantation study. Fasting blood glucose levels. Glycated HbA1c. scRNA-seq. Teratoma assay. Karyotype analysis. Calcium imaging. Cryo-electron microscopy. ELISA	Du et al[21], 2022

PDX1: Pancreatic and duodenal homeobox 1; SC-islets: Stem cell-derived islets; hPSC: Human pluripotent stem cells; C-peptide: Connecting peptide; CHGA: Chromogranin A; NKX6-1: NK6 homeobox 1; qRT-PCR: Quantitative real time-polymerase chain reaction; GSIS: Glucose-stimulated insulin secretion; SC-β cell: Stem cell-derived β cell; INS: Insulin gene; ISL1: ISL LIM homeobox 1; Ca²⁺: Calcium; KCl: Potassium chloride; scRNA-seq: Single-cell RNA sequencing; anti-CD49a: CD49a (Integrin alpha 1) antibody; TGF-β receptor inhibitor/ALK5i: Transforming growth factor β-receptor I/activin receptor-like kinase 5 inhibitor; R428 (AXL inhibitor): A selective small-molecule inhibitor of AXL (bemcentinib, BGB324); BMP: Bone morphogenetic protein; NEUROD1: Neurogenic differentiation 1; NKX2-2: NK2 homeobox 2; MAFA: MAF BZIP transcription factor A; MAFB: MAF BZIP transcription factor B; ABCC8: ATP Binding Cassette Subfamily C Member 8; IAPP: Islet amyloid polypeptide; HOPX: HOP homeobox; NEFM: Neurofilament medium chain; SIX2: SIX homeobox 2; G6PC2: Glucose-6-phosphatase catalytic subunit 2; KGF: (FGF7) Keratinocyte growth factor; TBP: TATA-binding protein; TANK: TRAF family member associated NFKB activator; PAX6: Paired box 6; GCK: Glucokinase; GLUT1: Glucose transporter 1; ROCK inhibitor: Rho-kinase inhibitor; GLIS3: GLI-similar 3/zinc finger 3; RBP4: Retinol binding protein 4; SIX3: SIX homeobox 3; UCN3: Urocortin 3; CPE: Carboxypeptidase E; FXYD2: FXYD domain containing ion transport regulator 2; cAMP: Cyclic adenosine monophosphate; ZBTB20: Zinc finger and BTB domain containing 20; PCSK1: Proprotein convertase subtilisin/kexin type 1; GABRA2: Gamma-aminobutyric acid type A receptor subunit alpha-2; hCiPSC: Human chemically induced pluripotent stem cells; ISX9: Isoxazole-9; Wnt-C59: Nanomolar inhibitor of mammalian PORCN acyltransferase activity and blocks activation of all evaluated human Wnts; HbA1c: Hemoglobin A1c; ELISA: Enzyme-linked immunosorbent assay.