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