Yonsei Med J.  2005 Oct;46(5):693-699. 10.3349/ymj.2005.46.5.693.

Hematopoietic Differentiation of Embryoid Bodies Derived from the Human Embryonic Stem Cell Line SNUhES3 in Co-culture with Human Bone Marrow Stromal Cells

• Kim SJ
• Kim BS
• Ryu SW
• Yoo JH
• Oh JH
• Song CH
• Kim SH
• Choi DS
• Seo JH
• Choi CW
• Shin SW
• Kim YH
• Kim JS

• KMID: 2158151
• DOI: http://doi.org/10.3349/ymj.2005.46.5.693

Abstract

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.

Keyword

Embryonic stem cells; embryoid body; differentiation

MeSH Terms

Stromal Cells/physiology
Stem Cells/*cytology
Humans
Hematopoietic Stem Cells/*cytology
Embryo/*cytology
Coculture Techniques
Cells, Cultured
*Cell Differentiation
Bone Marrow Cells/*cytology
Antigens, CD45/analysis
Antigens, CD38/analysis
Antigens, CD34/analysis

Figure

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  Fig. 1 Schematic for the comparison of the EB, EB/high FBS, and EB/BM co-culture groups. Undifferentiated human ES cells at confluence were scraped off and transferred to low-attachment plates in differentiation medium. In the EB group, human ES cells were cultured for EB formation in differentiation medium for 10 days. The EB/high FBS group was cultured for 10 days in the same culture media supplemented with 20% FBS. The EB/BM co-culture group was a co-culture of EBs with human BM stromal cells.

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  Fig. 2 The percentage of CD34+/CD45- cells (top) and CD34+/CD38- cells (bottom) was significantly higher in the EB/BM co-culture group than in the EB and EB/FBS groups (p < 0.05).

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  Fig. 3 Flow cytometry of CD34+/CD38- cells shows the number of CD34+/CD 38- cells increased on day 5 and 10 in the EB/BM co- culture group.

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  Fig. 4 The number of CFCs per 105 cells was increased in the EB/BM co-culture group on days 7 and 10 (11.0 ± 5.14, 20.6 ± 7.40, respectively), while there was no change in the EB and EB/high FBS groups.

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