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Embryonic stem cells

Inactive Publication Date: 2005-07-28
REUBINOFF BENJAMIN +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0044] In another aspect of the invention there is provided a method of induction of differentiation of stem cells. This method involves cultivation under conditions which limit stem cell renewal but do not result in stem cell death or unidirectional differentiation into extraembryonic lineages such as extraembryonic endoderm. The method also facilitates the derivation of committed lineage progenitor cells which are no longer pluripotent but may give rise to mature somatic cells. Preferably the method provides for induction of somatic cells from embryonic stem cells.

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 coworkers had the morphology expected of pluripotent stem cells, but these early studies did not employ feeder cell support, and it was impossible to achieve long term maintenance of the cultures.

Method used

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Examples

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Effect test

example 1

Derivation of Cell Lines HES-1 and HES-2

[0201] 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. 3A). 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. 3B, 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

[0202] 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. 4A). Immunophenotyping 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. 4B, C). 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. 4D). Like human EC cells, human ES cells did not express SSEA-1, a marker for mouse ES cells. Both cell lines were karyotypically norma...

example 3

Differentiation of Human ES Cells in Vitro

[0204] 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. 5, 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 char...

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Abstract

The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation, production of differentiated cells and in particular the production of human embryonic stem cells capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells capable of giving rise to mature somatic cells and uses thereof. The present invention also provides a purified preparation of undifferentiated human embryonic stem cells capable of proliferation in vitro. Furthermore, the present invention provides a somatic cell differentiated in vitro from an undifferentiated embryonic stem cell. There is also provided a committed progenitor cell capable of giving rise to mature somatic cells.

Description

[0001] The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation, production of differentiated cells and in particular the production of human ES capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells capable of giving rise to mature somatic cells and uses thereof. [0002] 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 transplantation or drug screening and drug discovery in vitro. [0003] In general, stem cells are undifferentiated cells which can give rise to a succession of mature functional cells. For example, a haematopoietic stem cell may give rise to any of the diff...

Claims

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Application Information

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IPC IPC(8): A61K35/12C12N15/09C12N5/02C12N5/0735C12N5/077C12N5/10C12R1/91
CPCA61K35/12C12N5/0606C12N2506/02C12N2502/13C12N5/0652
Inventor REUBINOFF, BENJAMINPERA, MARTINFONG, CHUI-YEETROUNSON, ALANBONGSO, ARIFFEEN
Owner REUBINOFF BENJAMIN
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