Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Summary

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Abstract

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling, however, with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell's physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

Introduction

Diabetes is a complex metabolic disorder affecting millions of people globally. Supplementation of insulin is considered the only treatment option for diabetes. More advanced cases are treated with beta cell replacement therapy, achieved through transplantation of either whole cadaveric pancreas or islets^1,2. Several issues surround transplantation therapy, such as limitation with the availability and quality of the tissue, invasiveness of transplantation procedures in addition to the continuous need for immunosuppressants. This necessitates the need for discovering novel and alternative options for beta cell ....

Protocol

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and Qatar Biomedical Research Institute (QBRI) (no. 2016-003). This work is optimized for hESCs such as H1, H9, and HUES8. Blood samples were obtained from healthy individuals from Hamad Medical Cor.......

Discussion

This work describes an enhanced protocol for generating pancreatic progenitors from hPSCs with a high co-expression of PDX1 and NKX6.1. Dissociation and replating of the hPSC-derived endoderm at half density on fresh matrix resulted in higher PDX1 and NKX6.1 in hPSC-derived pancreatic progenitors.

Although the growth factor cocktail for each stage is highly similar to P1-ND²⁷, it has been shown that a more extended Stage 3 treatment including FGF and retinoid signaling .......

Materials

Name	Company	Catalog Number	Comments
15 mL, conical, centrifuge tubes	Thermo Scientific	339651
20X TBS Tween 20	Thermo Scientific	28360
24-well culture plates, flat bottom with lid	Costar	3524
50 mL, conical, centrifuge tubes	Thermo Scientific	339652
6- well culture plates, multidish	Thermo Scientific	140685
Accutase	Stem Cell Technologies	0-7920
Activin A	R&D	338-AC	Reconstituted in 4 mM HCl
Anti NKX6.1 antibody, mouse monoclonal	DSHB	F55A12-C	Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
Anti-PDX1 antibody, guinea pig polyclonal	Abcam	ab47308	Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining
B27 minus Vit A	ThermoFisher	12587010
Bovine serum albumin, heat shock fraction, fatty acid free	Sigma	A7030
CHIR 99021	Tocris	4423	Reconstituted in DMSO
DMEM, high glucose	ThermoFisher	41965047
Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	Invitrogen	A10037
Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488		A-21206
DPBS 1X	ThermoFisher	14190144
EGF	ThermoFisher	PHG0313	Reconstituted in 0.1% BSA in PBS
FGF10	R&D	345-FG	Reconstituted in PBS
Glucose	Sigma Aldrich	G8644
Hoechst 33258	Sigma	23491-45-4
Inverted microscope	Olympus	IX73
KnockOut DMEM/F-12 (1X)	Gibco	12660-012
KnockOut SR serum replacement	Gibco	10828-028
L-Ascorbic acid (vitamin C)	Sigma	A92902	Reconstituted in distilled water
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix	Corning	354230	Aliquot the thawed stock and freeze at -20C.
MCDB131	ThermoFisher	10372019
Mouse anti-SOX17	ORIGENE	CF500096	Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
mTeSR Plus	Stem Cell Technologies	85850	Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use
Nalgene filter units, 0.2 µm PES	ThermoFisher	566-0020
Nicotinamide	Sigma	72340	Reconstituted in distilled water
NOGGIN	R&D	6057-NG	Reconstituted in 0.1% BSA in PBS
Paraformaldehyde solution 4% in PBS	ChemCruz	sc-281692
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher	15140122
Portable vacuum aspirator
Rabbit anti-FOXA2	Cell signaling technology	3143	Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining
Retinoic Acid	Sigma Aldrich	R2625	Reconstituted in DMSO
Rock inhibitor (Y-27632)	ReproCell	04-0012-02	Reconstituted in DMSO
Round Bottom Polystyrene FACS Tubes with Caps, STERILE	Stellar Scientific	FSC-9010
SANT-1	Sigma Aldrich	S4572	Reconstituted in DMSO
Sodium bicarbonate	Sigma	S5761-500G
StemFlex	ThermoFisher	A3349401	Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use
TALI Cellular Analysis Slide	Invitrogen	T10794
Tali image-based cytometer automated cell counter	Invitrogen	T10796
Triton X-100	Sigma	9002-93-1
TrypLE 100 mL	ThermoFisher	12563011
Tween 20	Sigma	P2287
UltraPure 0.5 M EDTA, pH 8.0	Invitrogen	15575-038