Differentiation to insulin-positive cells from human amnion epithelial cells using a pancreatic development mimicry protocol

Abstract

Diabetes mellitus (DM) is characterized by the loss or dysfunction pancreatic β-cells. Human amniotic epithelial cells (hAEC), which retain pluripotency markers and are readily obtainable from term placentas, represent a promising alternative source of stem cells. We investigated whether hAECs can be guided through pancreatic ontogeny to generate insulin-producing β-like cells. hAEC from uncomplicated term deliveries were expanded to passage 1 and exposed to a four-stage differentiation sequence that sequentially modulated Activin/WNT, KGF/TGF-β, retinoic-acid/hedgehog, and EGF/Noggin signaling. Stage progression was monitored by end-point RT-PCR and quantitative immunofluorescence for hallmark transcription factors. After definitive endoderm induction, 64% of cells were Brachyury positive and 71% were WNT3A positive; primitive-gut specification yielded 57% HNF1B⁺ cells. Posterior foregut commitment produced 75% PDX1⁺ and 60% Sox9⁺ nuclei and the final endocrine stage generated 74% NKX2.2⁺ cells, with 71% showing cytoplasmatic insulin and 51% C-peptide staining; insulin/C-peptide co-localization was confirmed by double labelling. Thus a chemically defined, four-step protocol can convert term-derived hAEC into insulin-producing β-like cells, supporting their use as an accessible platform for diabetes modelling and future cell-replacement therapies.

Keywords: Differentiation; Human amniotic epithelial cells; Pancreatic beta cells.

Fig. 1

Schematic of four-stage differentiation protocol from human amniotic epithelial cells (hAEC) into definitive endoderm, primitive gut tube, posterior foregut, and mixed population comprising pancreatic endoderm and endocrine precursor cells

Fig. 2

Reverse transcription polymerase chain reaction (RT-PCR) analysis of pluripotency marker gene expression in hAEC, including OCT-4, NANOG, and SOX-2. Positive control; human embryonic stem cell line H9 (CTR+). Negative control (CTR−): reactions without cDNA

Fig. 3

Microphotographs of hAEC positive for pluripotency markers OCT-4, NANOG, and SOX-2 (red), E-cadherin (green) and DAPI (blue) staining the nuclei. Image taken at ×20. Scale bar = 100 μm

Fig. 4

RT-PCR analysis of gene expression in hAEC at the end of stage 1 of differentiation. Expression of constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in undifferentiated hAEC and hAEC at the end of the protocol (differ). The gels show the RT-PCR products for BRACHYURY (122 bp), SOX17 (151 bp), and GADPH (229 bp). Negative control (CTR): reactions without cDNA

Fig. 5

RT-PCR analysis of gene expression in hAEC at the end of stage 4 of differentiation protocol into insulin-positive cells. Expression of somatostatin (SST), glucagon (GCG), insulin (INS) and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in undifferentiated and differentiated (differ) hAEC. The gels shown RT-PCR products for SST (108 bp), CGC (91 bp), INS (58 bp), and GAPDH (229 bp). Negative control (CTR): reactions without cDNA

Fig. 6

Immunofluorescence of hAEC at stage 1 of differentiation. Representative images show cells positive for definitive endoderm markers; FGF-4 (red), BRACHYURY (BRA, green), SOX-17 (red), WNT3A (green), FOXA2 (red), and DAPI (blue) staining the nuclei. Image taken at ×20. Scale bar = 100 μm

Fig. 7

Immunofluorescence of hAEC at stage 2 of differentiation. Representative images show cells positive for primitive gut tube markers; HNF1B (red), HNF4A (red), and DAPI (blue) staining the nuclei. Image taken at ×20. Scale bar = 100 μm

Fig. 8

Immunofluorescence of hAEC at stage 3 of differentiation. Representative images show cells positive for posterior foregut markers; PDX-1 (red), HNF-6 (red), SOX-9 (red), and DAPI (blue) staining the nuclei. Image taken at ×20. Scale bar = 100 μm

Fig. 9

Immunofluorescence of hAEC at stage 4 of differentiation. Representative images show cells positive for pancreatic progenitor and endocrine precursor markers; NKX2 (red), glucagon, (GCG, green), C-peptide (C-PEP, red), insulin (INS, green), and DAPI (blue) staining the nuclei. Image taken at ×20. Scale bar = 100 μm

Fig. 10

Quantification of marker-positive cells at each stage of the differentiation protocol from human amniotic epithelial cells (hAEC)