PDX1 expressing endoderm

a technology of endoderm and pdx1 is applied in the field of cell biology and medicine, which can solve the problems of limited pdx1-positive production, unique challenges, and inability to generate an insulin-producing -cell from hescs, and achieve the effects of increasing the production of pdx1-positive, and increasing the differentiation of pdx1-negative definitive endoderm to pdx1

Inactive Publication Date: 2005-12-01
CYTHERA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0011] Further embodiments described herein relate to methods of producing PDX1-positive foregut endoderm cells by providing a cell culture or cell population comprising definitive endoderm cells which do not substantially express PDX1 (PDX1-negative definitive endoderm cells) with a foregut differentiation factor, such as a retinoid. The retinoid, for example RA, can be supplied in a concentration ranging from about 0.01 μM to about 50 μM. In some embodiments, the differentiation of PDX1-negative definitive endoderm to PDX1 positive foregut endoderm is increased by providing the cell culture or cell population with FGF-10 and/or B27. FGF-10 can be supplied in a concentration ranging from about 5 ng/ml to about 1000 ng/ml. In some embodiments, B27 is supplied to the cell culture or cell population at a concentration ranging from about 0.1% to about 20%. FGF-10 and/or B27 can be added to the cell culture or cell population at about the same time as the retinoid or each of the factors may be added separately with up to several hours between each addition. In certain embodiments, the retinoid is added to an approximately 4-day-old PDX1-negative definitive endoderm culture. In some embodiments, the retinoid is added to an approximately 5-day-old PDX1-negative definitive endoderm culture.
[0012] Still other embodiments relate to methods of using a foregut differentiation factor to further increase the production of PDX1-positive f

Problems solved by technology

However, presently it is not known how to generate an insulin-producing β-cell from hESCs.
As such, current cell therapy treatments for diabetes mellitus, which utilize islet cells from donor pancreases, are limited by the scarci

Method used

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Examples

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example 1

Human ES Cells

[0262] For our studies of endoderm development we employed human embryonic stem cells, which are pluripotent and can divide seemingly indefinitely in culture while maintaining a normal karyotype. ES cells were derived from the 5-day-old embryo inner cell mass using either immunological or mechanical methods for isolation. In particular, the human embryonic stem cell line hESCyt-25 was derived from a supernumerary frozen embryo from an in vitro fertilization cycle following informed consent by the patient. Upon thawing the hatched blastocyst was plated on mouse embryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non essential amino acids, beta-mercaptoethanol, ITS supplement). The embryo adhered to the culture dish and after approximately two weeks, regions of undifferentiated hESCs were transferred to new dishes with MEFs. Transfer was accomplished with mechanical cutting and a brief digestion with dispase, followed by mechanical removal of the cell clusters, wa...

example 2

hESCyt-25 Characterization

[0264] The human embryonic stem cell line, hESCyt-25 has maintained a normal morphology, karyotype, growth and self-renewal properties over 18 months in culture. This cell line displays strong immunoreactivity for the OCT4, SSEA-4 and TRA-1-60 antigens, all of which, are characteristic of undifferentiated hESCs and displays alkaline phosphatase activity as well as a morphology identical to other established hESC lines. Furthermore, the human stem cell line, hESCyt-25, also readily forms embryoid bodies (EBs) when cultured in suspension. As a demonstration of its pluripotent nature, hESCyt-25 differentiates into various cell types that represent the three principal germ layers. Ectoderm production was demonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC) for nestin and more mature neuronal markers. Immunocytochemical staining for β-III tubulin was observed in clusters of elongated cells, characteristic of early neurons. Previously, we treated ...

example 3

Production of SOX17 Antibody

[0265] A primary obstacle to the identification of definitive endoderm in hESC cultures is the lack of appropriate tools. We therefore undertook the production of an antibody raised against human SOX17 protein.

[0266] The marker SOX17 is expressed throughout the definitive endoderm as it forms during gastrulation and its expression is maintained in the gut tube (although levels of expression vary along the A-P axis) until around the onset of organogenesis. SOX17 is also expressed in a subset of extra-embryonic endoderm cells. No expression of this protein has been observed in mesoderm or ectoderm. It has now been discovered that SOX17 is an appropriate marker for the definitive endoderm lineage when used in conjunction with markers to exclude extra-embryonic lineages.

[0267] As described in detail herein, the SOX17 antibody was utilized to specifically examine effects of various treatments and differentiation procedures aimed at the production of SOX17 p...

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Abstract

Disclosed herein are cell cultures comprising PDX1-positive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified PDX1-positive endoderm cells as well as methods for enriching, isolating and purifying PDX1-positive endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of endoderm cells, such as PDX1-positive foregut endoderm cells and PDX1-negative definitive endoderm cells, are also disclosed.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 11 / 021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, which claims priority under 35 U.S.C. § 119(e) to the following three provisional patent applications: U.S. Provisional Patent Application No. 60 / 566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent Application No. 60 / 587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14, 2004; and U.S. Provisional Patent Application No. 60 / 586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004. The disclosure of each of the foregoing applications is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention relates to the fields of medicine and cell biology. In particular, the present invention relates to compositions comprising mammalian PDX1-posit...

Claims

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Application Information

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IPC IPC(8): C07K14/71C12N5/00C12N5/071C12N5/073C12N5/0735
CPCA61K35/12C12N5/0608C12N5/0603C12N5/0606C12N5/0676C12N5/0679C12N15/1086C12N2500/99C12N2501/115C12N2501/119C12N2501/155C12N2501/16C12N2501/385C12N2501/415C12N2502/02C12N2503/00C12N2506/02C12N2510/00C12Q1/6881C12N2502/13C12N5/0018C12N2500/90C12Q2600/158
Inventor D'AMOUR, KEVINAGULNICK, ALANELIAZER, SUSANBAETGE, EMMANUEL
Owner CYTHERA
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