Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1

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Abstract

Background: Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). Methods: In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Results: Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (∼90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. Conclusions: We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1- population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.

Original languageEnglish
Article number0759
JournalStem Cell Research and Therapy
Volume9
Issue number1
DOIs
Publication statusPublished - 23 Jan 2018

Fingerprint

Pluripotent Stem Cells
Stem cells
Plating
Monolayers
Transcription Factors
Fibroblast Growth Factor 10
Retinoids
Specifications
Population Characteristics
Population
Cell Proliferation
Liver

Keywords

  • Beta cells
  • Diabetes
  • Differentiation
  • hPSCs
  • Pancreatic epithelium
  • Transcription factors

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Molecular Medicine
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Cell Biology

Cite this

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title = "Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1",
abstract = "Background: Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). Methods: In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Results: Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (∼90{\%}) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. Conclusions: We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1- population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.",
keywords = "Beta cells, Diabetes, Differentiation, hPSCs, Pancreatic epithelium, Transcription factors",
author = "Bushra Memon and Manale Doldur and Sara Al-Khawaga and Essam Mohamed",
year = "2018",
month = "1",
day = "23",
doi = "10.1186/s13287-017-0759-z",
language = "English",
volume = "9",
journal = "Stem Cell Research and Therapy",
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TY - JOUR

T1 - Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1

AU - Memon, Bushra

AU - Doldur, Manale

AU - Al-Khawaga, Sara

AU - Mohamed, Essam

PY - 2018/1/23

Y1 - 2018/1/23

N2 - Background: Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). Methods: In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Results: Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (∼90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. Conclusions: We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1- population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.

AB - Background: Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). Methods: In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Results: Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (∼90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. Conclusions: We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1- population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.

KW - Beta cells

KW - Diabetes

KW - Differentiation

KW - hPSCs

KW - Pancreatic epithelium

KW - Transcription factors

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U2 - 10.1186/s13287-017-0759-z

DO - 10.1186/s13287-017-0759-z

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