In vitro pancreatic differentiation of pluripotent mammalian cells

A pluripotent cell and cell technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc.

Active Publication Date: 2015-07-01
CAMBRIDGE ENTERPRISE LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite this extensive knowledge, the molecular mechanisms that orchestrate the extracellular signaling pathways of transcriptional networks in pancreatic or hepatic progenitor cells, especially in humans, remain to be elucidated, and hPSCs may be uniquely positioned to accomplish this. a main task

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  • In vitro pancreatic differentiation of pluripotent mammalian cells
  • In vitro pancreatic differentiation of pluripotent mammalian cells
  • In vitro pancreatic differentiation of pluripotent mammalian cells

Examples

Experimental program
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Embodiment Construction

[0201] experiment

[0202] hESC and hIPSC culture conditions.

[0203] hESCs (H9 purchased from WiCell) and hIPSCs (BBHX8, A1ATD-1, JRO1D) (Rashid S et al. (2010) J. Clin. Invest. 120: 3127-3136) were grown in defined media (Brons et al. ( 2007) Nature 448:191-195). Cells were passaged weekly with collagenase IV and maintained in chemically defined medium (CDM) supplemented with activin A (10 ng / ml) and FGF2 (12 ng / ml) as previously described (Brons et al. )middle. differentiated as figure 1 in the manner described. Medium was changed daily throughout the differentiation process. After the DE stage (Stage 1), the cells were treated with SB-431542 (10 μM; Tocris), FGF10 (50 ng / ml; AutogenBioclear), all-trans retinoic acid (RA, 2 μM; Sigma) and Tau Protein (50ng / ml; R&D Systems) was cultured in advanced DMEM (Invitrogen) for 3 days. In stage 3, the cells were incubated with high-grade DMEM+human FGF10 (50ng / ml; AutogenBioclear), all-trans retinoic acid (RA, 2μM; Sigma...

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Abstract

This invention relates to the in vitro differentiation of pluripotent cells into pancreatic progenitors by i) culturing pluripotent cells in a definitive endoderm (DE) medium comprising a TGFp ligand, fibroblast growth factor ( FGF), bone morphogenetic protein (BMP), a PI3K inhibitor and optionally a GSK3 β inhibitor to produce a population of definitive endoderm cells, ii) culturing the definitive endoderm cells in a first pancreatic medium comprising an activin antagonist; FGF; retinoic acid; and a BMP inhibitor to produce a population of dorsal foregut cells; iii) culturing the dorsal foregut cells in a second pancreatic medium comprising FGF, retinoic acid, a BMP inhibitor, and a hedgehog signalling inhibitor, and; iv) culturing the endoderm cells in a third pancreatic medium comprising FGF. The progenitor cells thus produced may be further differentiated into pancreatic endocrine cells. These methods may be useful, for example, in producing pancreatic cells for therapy or disease modelling.

Description

technical field [0001] The present invention relates to inducing pancreatic differentiation in pluripotent mammalian cells in vitro. Background technique [0002] The generation of pancreatic β cells represents a major goal of regenerative medicine. Indeed, a large supply of these cells would enable the development of cell-based diabetes therapies, currently limited by a lack of donor organs and the difficulty of expanding insulin-secreting cells in vitro. Human pluripotent stem cells (hPSCs) of embryonic origin (human embryonic stem cells or hESCs) [1] or generated from reprogrammed somatic cells (human induced pluripotent stem cells or hIPSCs) [2] offer the prospect of bypassing these limitations. In fact, these cells are capable of unlimited proliferation in vitro while maintaining the ability to differentiate into various cell types, including pancreatic progenitors [3-6]. However, robust methods that allow the generation of homogenous populations of these cells under ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N2506/02C12N2501/16C12N2501/115C12N5/0676C12N2501/155A61P1/18A61P3/10A61K35/39C12N5/0678C12N2500/30C12N2500/38C12N2501/117C12N2501/40C12N2501/41C12Q1/6881C12Q2600/158C12Q2600/16G01N33/5038G01N33/507G01N33/74
Inventor 鲁多维奇·瓦利耶卓欣樺
Owner CAMBRIDGE ENTERPRISE LTD
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