Embryonic stem cells and neural progenitor cells derived therefrom

Inactive Publication Date: 2005-11-24
REUBINOFF BENJAMIN +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0060] In an additional aspect of the invention method may be used for directing stem cells to differentiate toward a somatic lineage. Furthermore, the method a

Problems solved by technology

EC cells however had limitations.
They often contained chromosomal abnormalities, and their ability to differentiate into multiple tissue types was often limited.
The cells isolated by Bongso and c

Method used

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  • Embryonic stem cells and neural progenitor cells derived therefrom
  • Embryonic stem cells and neural progenitor cells derived therefrom
  • Embryonic stem cells and neural progenitor cells derived therefrom

Examples

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example 1

Derivation of Cell Lines HES-1 and HES-2

[0400] The outer trophectoderm layer was removed from four blastocysts by immunosurgery to isolate inner cell masses (ICM), which were then plated onto a feeder layer of mouse embryo fibroblasts (FIG. 1A). Within several days, groups of small, tightly packed cells had begun to proliferate from two of the four ICM. The small cells were mechanically dissociated from outgrowths of differentiated cells, and following replating they gave rise to flat colonies of cells with the morphological appearance of human EC or primate ES cells (FIG. 1B, C stem cell colonies). These colonies were further propagated by mechanically disaggregation to clumps which were replated onto fresh feeder cell layers. Growth from small clumps of cells (<10 cells) was not possible under the conditions of these cultures. Spontaneous differentiation, often yielding cells with the morphological appearance of early endoderm, was frequently observed during routine passage of th...

example 2

Marker Expression and Karyotype of the Human ES Cells

[0401] Marker and karyotype analysis were performed on HES-1 at passage levels 5-7, 14-18, 24-26 and 44-46, and on HES-2 at passage levels 6-8. ES cells contained alkaline phosphatase activity (FIG. 2A). Imiunophenotyping of the ES cells was carried out using a series of antibodies which detect cell surface carbohydrates and associated proteins found on human EC cells. The ES cells reacted positively in indirect immunofluorescence assays with antibodies against the SSEA-4 and TRA 1-60 carbohydrate epitopes, and the staining patterns were similar to those observed in human EC cells (FIG. 2B, 2C). ES cells also reacted with monoclonal antibody GCTM-2, which detects an epitope on the protein core of a keratan sulphate / chondroitin sulphate pericellular matrix proteoglycan found in human EC cells (FIG. 2D). Like human EC cells, human ES cells did not express SSEA-1, a marker for mouse ES cells. Both cell lines were karyotypically norm...

example 3

Differentiation of Human ES Cells in vitro

[0403] Both cell lines underwent spontaneous differentiation under standard culture conditions, but the process of spontaneous differentiation could be accelerated by suboptimal culture conditions. Cultivation to high density for extended periods (4-7 weeks) without replacement of a feeder layer promoted differentiation of human ES cells. In high density cultures, expression of the stem cell marker Oct-4 was either undetectable or strongly downregulated relative to the levels of the housekeeping gene beta actin (FIG. 3A-3B, lanes 5-7). Alphafetoprotein and human chorionic gonadotrophin were readily detected by immunoassay in the supernatants of cultures grown to high density. Alphafetoprotein is a characteristic product of endoderm cells and may reflect either extraembryonic or embryonic endodermal differentiation; the levels observed (1210-5806 ng / ml) are indicative of extensive endoderm present. Human chorionic gonadotrophin secretion is ...

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Abstract

The present invention provides undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, and committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof. The invention also provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model and the cells generated along the pathway of neural differentiation may be used for the study of the cellular and molecular biology of human neural development, for the discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, for drug discovery and for the development of screening assays for teratogenic, toxic and neuroprotective effects.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of U.S. patent application Ser. No. 09 / 808,382, filed Mar. 14, 2001. [0002] The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and / or glial cells and uses thereof.INTRODUCTION [0003] The production of human embryonic stem cells which can be either maintained in an undifferentiated state or directed to undergo differentiation into extraembryonic or somatic lineages in vitro allows for the study of the cellular and molecular biology of early human development, functional genomics, generation of differentiated cells from the stem cells for use in ...

Claims

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Application Information

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IPC IPC(8): A61K35/12C12N15/09A61K48/00A61P9/00A61P17/02A61P25/00A61P25/28A61P37/00A61P43/00C12N5/0735C12N5/079C12N5/0793C12N5/0797
CPCA61K35/12C12N2502/13C12N5/0619C12N5/0622C12N5/0623C12N2500/32C12N2501/11C12N2501/115C12N2501/135C12N2501/155C12N2501/385C12N2501/39C12N2501/91C12N2506/02C12N5/0606A61P17/02A61P25/00A61P25/28A61P37/00A61P43/00A61P9/00
Inventor REUBINOFF, BENJAMINPERA, MARTINBEN-HUR, TAMIR
Owner REUBINOFF BENJAMIN
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