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. 2006 May 2;103(18):6907-12.
doi: 10.1073/pnas.0602280103. Epub 2006 Apr 21.

Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions

Shuyuan Yao  1 , Shuibing Chen, Julie Clark, Ergeng Hao, Gillian M Beattie, Alberto Hayek, Sheng Ding
Affiliations

Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions

Shuyuan Yao et al. Proc Natl Acad Sci U S A. .

Abstract

Chemically defined medium (CDM) conditions for controlling human embryonic stem cell (hESC) fate will not only facilitate the practical application of hESCs in research and therapy but also provide an excellent system for studying the molecular mechanisms underlying self-renewal and differentiation, without the multiple unknown and variable factors associated with feeder cells and serum. Here we report a simple CDM that supports efficient self-renewal of hESCs grown on a Matrigel-coated surface over multiple passages. Expanded hESCs under such conditions maintain expression of multiple hESC-specific markers, retain the characteristic hESC morphology, possess a normal karyotype in vitro, as well as develop teratomas in vivo. Additionally, several growth factors were found to selectively induce monolayer differentiation of hESC cultures toward neural, definitive endoderm/pancreatic and early cardiac muscle cells, respectively, in our CDM conditions. Therefore, this CDM condition provides a basic platform for further characterization of hESC self-renewal and directed differentiation, as well as the development of novel therapies.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
N2 and N2/B27-CDM plus 20 ng/ml bFGF support long-term self-renewal of hESCs. (A) Serially passaged H1 and HSF6 hESCs grown on a Matrigel-coated surface in 20 ng/ml bFGF-supplemented N2-CDM (Upper) or N2/B27-CDM (Lower) form large compact colonies. (B) H1 and HSF6 hESCs cultured in the above conditions show positive staining of Tra-1-81, Oct4, Tra-1-60, and SSEA-4. (C) RT-PCR analysis shows that the serially passaged H1 hESCs cultured in the above CDM conditions express Nanog, Oct4, and Sox2.
Fig. 2.
Fig. 2.
The N2/B27-CDM expanded hESCs maintain normal karyotype and full differentiation potential. (A) Karyotype analysis of passage 8 and 22 H1-hESCs cultured under the feeder-free condition in the N2/B27-CDM plus 20 ng/ml bFGF using G-banding. A normal karyotype was observed for all analyzed nuclei (two representative examples are shown). (B) Complex differentiation developed in the hESC-derived teratomas 4–5 weeks after inoculation in nude mice. Typical differentiated tissues are shown. a, Glandular acinus-forming epithelium; b, osteoid-forming cells; c, neuroepithelial (ependymal) rosette; d, pigmented retinal epithelium; e, neuroepithelial rosette and glandular-type epithelium; f, columnar epithelium.
Fig. 3.
Fig. 3.
Noggin induced neural differentiation of hESCs cultured in monolayer in the N2/B27-CDM. (A) Four-day-old undifferentiated hESC colonies were treated with 100 ng/ml Noggin for 8 days in the N2/B27-CDM. Noggin blocked the differentiation in the center of the hESC colonies that are typically seen after withdrawal of bFGF but induced formation of multiple neural rosette-like structures over 8 days. (B) Cells within these early rosette-like structures immunostain positive for Pax6 but not Sox1. (C) After the early neural rosettes are split into single cells in the N2/B27-CDM plus bFGF, they further differentiate into Pax6+/Sox1+ double- positive neural cells. (D) RT-PCR analysis confirmed that the late neural cells express both Pax-6 and Sox1.
Fig. 4.
Fig. 4.
N2/B27-CDM plus activin A induced definitive endoderm/pancreatic differentiation of hESCs. (A) Four-day-old undifferentiated hESC colonies were treated with 100 ng/ml activin A for 9 days in the N2/B27-CDM. Activin A blocked the differentiation in the center of the hESC colonies that are typically seen after withdrawal of bFGF but induced definitive endoderm differentiation. (B) Immunostaining of hESCs after 5 and 9 days of activin A treatment. At day 5, >80% of the cells express Brachyury, whereas <50% of the cells express Sox17. At day 9, little expression of Brachyury was detected, but Sox17, FOXA2, and PDX-1 were expressed in >80% of the cells. (C) RT-PCR analysis confirmed the expression of Brachyury, Sox17, FOXA2, and PDX-1 at days 5 and 9.
Fig. 5.
Fig. 5.
N2/B27-CDM plus activin A and BMP induced marker expression associated with cardiac muscle lineage. (A) Four-day-old undifferentiated hESC colonies were treated with 50 ng/ml activin A and 50 ng/ml BMP-4 for 4 days in the N2/B27-CDM and further cultured for an additional 8–10 days in the basal N2/B27-CDM. (B) Immunostaining shows expression of specific cardiomyocyte makers: αMHC, cardiac Troponin I, MEF-2, and GATA-4. (C) RT-PCR shows the expression of αMHC, cardiac Troponin I, Nkx-2.5, and ANF.
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