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Compositions and methods of obtaining and using endoderm and hepatocyte cells

A technology of endoderm cells and hepatocytes, applied in biochemical equipment and methods, hepatocytes, embryonic cells, etc., can solve problems such as low efficiency

Inactive Publication Date: 2015-02-11
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pluripotency of stem cells also presents unique challenges for studying and manipulating these cells and their derivatives
Due to the large number of cell types that can arise in differentiated stem cell cultures, most cell types are generated with very low efficiency

Method used

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  • Compositions and methods of obtaining and using endoderm and hepatocyte cells
  • Compositions and methods of obtaining and using endoderm and hepatocyte cells
  • Compositions and methods of obtaining and using endoderm and hepatocyte cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0393] Example 1: Methods and materials for endoderm differentiation

[0394] endoderm markers

[0395] A variety of cell type-specific markers were used to monitor differentiation of stem cells into endoderm cells in flow cytometry, fluorescence imaging, and immunoassay experiments as described below. To detect endoderm transformation, hESC-derived cell samples were stained for the expression of SOX17, FoxA2 and CXCR4 proteins. SOX17, FoxA2 and CXCR4 are proteins expressed by endoderm cells but not stem cells. To detect stem cells, cell samples were stained for expression of OCT4, a protein expressed by stem cells but not endoderm cells.

[0396] Endoderm Differentiation Protocol Using hESCs and Matrigel

[0397] in TesR TM 2 Medium (STEMCELL TM 40,000 cells / cm on qualified Matrigel (BD, #354277) in Technologies #05860) 2 Density maintenance of undifferentiated human embryonic stem cells (hES). Cultures were manually passaged every two weeks. To prepare for end...

Embodiment 2

[0421] Example 2: Endoderm differentiation using hESCs

[0422] Comparison of the effects of various commercially available PI3K inhibitors on endoderm differentiation. hESC cell samples were prepared as described above and in basal medium or supplemented with Activin A; Activin A and 50 μg / ml human Wnt3a (R&D, #5036-WN-010); or Activin A, Wnt3A and Table 1 below. Differentiate in basal medium with one of the listed PI3K inhibitors.

[0423] Table 1

[0424] PI3K inhibitors

Concentrations used in Example 2

Compound A

500nM

Wortmannin

250nM

PIK90

500nM

LY294002

5μM

[0425] Except for Compound A, the compounds shown in Table 1 are not isoform-selective PI3K inhibitors.

[0426] The structure of Compound A is provided below:

[0427]

[0428] After 3 days of treatment, cells were harvested and stained for flow cytometry analysis with anti-SOX17 antibody in preparation as described above. exist figure 1 Th...

Embodiment 3

[0429] Example 3: Wnt3a is not essential for differentiation into endoderm

[0430] Experiments were performed to determine whether the growth factor Wnt3a is required for endoderm differentiation. hESC cell samples were prepared as described above and differentiated in basal medium; basal medium supplemented with Activin A, 50 μg / ml Wnt3a and Compound A, or Activin A and 750 nM Compound A alone. After 3 days of treatment, cells were harvested as described above and stained with anti-SOX17 antibody in preparation for flow cytometry analysis. exist figure 2 The results of flow cytometry analysis are shown in . These results indicate that hESC-derived cells cultured with Compound A and Activin A in the absence of Wnt3a convert to endoderm at slightly lower, but comparable, efficiency to cells cultured with Compound A, Activin A and Wnt3a. Such as image 3 As shown in , this effect is independent of the base medium used.

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Abstract

The invention provides for efficient methods for generating populations of endoderm cells and / or differentiated cells derived from endoderm cells (e.g., hepatic cells, pancreatic precursor cells, pancreatic cells, intestinal progenitor cells, intestinal cells, lung progenitor cells, lung cells, etc.). Also provided are compositions of endoderm cells and differentiated cells derived from endoderm cells (e.g., hepatic cells, pancreatic precursor cells, pancreatic cells, intestinal progenitor cells, intestinal cells, lung progenitor cells, lung cells, etc.) and methods of using such cells.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 61 / 650,762, filed May 23, 2012, the contents of which are hereby incorporated by reference in their entirety. field of invention [0003] The present invention is in the field of stem cells, endoderm cells, pancreatic progenitor cells, hepatocytes and other cells derived from endoderm cells. In general, the present invention relates to compositions and methods of making and using endoderm cells, pancreatic progenitor cells, hepatocytes, and / or other cells derived from endoderm cells. Background of the invention [0004] Two properties that make stem cells uniquely suited for cell therapy applications are pluripotency and the ability to maintain these cells in culture for extended periods of time. Pluripotency is defined by the ability of stem cells to differentiate into derivatives of all three primitive germ layers (endoderm, mesoderm, ectoderm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/071
CPCC12N5/0678C12N2501/727C12N5/0672C12N2506/45C12N2506/02C12N5/0603C12N2501/415C12N2501/16C12N5/067C12N2501/999A61P1/00A61P1/04A61P1/16A61P1/18A61P29/00A61P35/00A61P43/00C12N5/06C12N5/0606C12N5/0613C12N5/0676A61K35/39A61K35/407
Inventor E·杜代蒙特H·乌帕尔
Owner F HOFFMANN LA ROCHE & CO AG
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