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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 islets1,2. 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.......

Representative Results

The results show that optimized protocol P2-D (Figures 1A) enhanced pancreatic progenitor differentiation efficiency by upregulating PDX1 and NKX6.1 co-expression (Figure 2A,B, and Figure 3A). In particular, the results showed that dissociation of endodermal cells and their replating on fresh membrane matrix along with a longer duration of Stage 3 enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors (op.......

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 .......

Acknowledgements

This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).

....

Materials

NameCompanyCatalog NumberComments
15 mL, conical, centrifuge tubesThermo Scientific339651
20X TBS Tween 20Thermo Scientific28360
24-well culture plates, flat bottom with lidCostar3524
50 mL, conical, centrifuge tubesThermo Scientific339652
6- well culture plates, multidishThermo Scientific140685
AccutaseStem Cell Technologies0-7920
Activin AR&D338-ACReconstituted in 4 mM HCl
Anti NKX6.1 antibody, mouse monoclonalDSHBF55A12-CDiluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
Anti-PDX1 antibody, guinea pig polyclonalAbcamab47308Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining
B27 minus Vit AThermoFisher12587010
Bovine serum albumin, heat shock fraction, fatty acid freeSigmaA7030
CHIR 99021Tocris4423Reconstituted in DMSO
DMEM, high glucoseThermoFisher41965047
Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568InvitrogenA10037
Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488A-21206
DPBS 1XThermoFisher14190144
EGFThermoFisherPHG0313Reconstituted in 0.1% BSA in PBS
FGF10R&D345-FGReconstituted in PBS
GlucoseSigma AldrichG8644
Hoechst 33258Sigma23491-45-4
Inverted microscopeOlympusIX73
KnockOut DMEM/F-12 (1X)Gibco12660-012
KnockOut SR serum replacementGibco10828-028
L-Ascorbic acid (vitamin C)SigmaA92902Reconstituted in distilled water
Matrigel Growth Factor Reduced (GFR) Basement Membrane MatrixCorning354230Aliquot the thawed stock and freeze at -20C.
MCDB131ThermoFisher10372019
Mouse anti-SOX17ORIGENECF500096Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
mTeSR PlusStem Cell Technologies85850Mix 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 PESThermoFisher566-0020
NicotinamideSigma72340Reconstituted in distilled water
NOGGINR&D6057-NGReconstituted in 0.1% BSA in PBS
Paraformaldehyde solution 4% in PBSChemCruzsc-281692
Penicillin-Streptomycin (10,000 U/mL)ThermoFisher15140122
Portable vacuum aspirator
Rabbit anti-FOXA2Cell signaling technology3143Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining
Retinoic AcidSigma AldrichR2625Reconstituted in DMSO
Rock inhibitor (Y-27632)ReproCell04-0012-02Reconstituted in DMSO
Round Bottom Polystyrene FACS Tubes with Caps, STERILEStellar ScientificFSC-9010
SANT-1Sigma AldrichS4572Reconstituted in DMSO
Sodium bicarbonateSigmaS5761-500G
StemFlexThermoFisherA3349401Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use
TALI Cellular Analysis SlideInvitrogenT10794
Tali image-based cytometer automated cell counterInvitrogenT10796
Triton X-100Sigma9002-93-1
TrypLE 100 mLThermoFisher12563011
Tween 20SigmaP2287
UltraPure 0.5 M EDTA, pH 8.0Invitrogen15575-038

References

  1. Rickels, M. R., Robertson, R. P. Pancreatic islet transplantation in humans: Recent progress and future directions. Endocrine Reviews. 40 (2), 631-668 (2019).
  2. Latres, E., Finan, D. A., Greenstein, J. L., Kowalski, A., Kieffer, T. J.

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