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

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.

Differentiation Culture of Hematopoietic Stem Cells from Embryonic Stem Cells / 대한혈액학회지

BACKGROUND: This study was designed to verify the effective culture condition for the differentiation of human hematopoietic stem cells from SNU-3 embryonic stem cell line. METHODS: Control group was that of which embryonic stem cells were directly cultured to LTC-IC assay. Study group was that of which an embryonic body manufactured from embryonic stem cells was cultured to LTC-IC assay. CD34+ cells were separated by MACS method at 1, 3, 5, 7, 10, 14, 21 days of LTC-IC assay in both groups. Thereafter, CD34+ cell were cultured to semisolid methyl-cellulose media to count CFUs. CD34+CD45- cell percentage was measured with FACS method at 5 days of LTC-IC assay. This study was repeated for 14 times. RESULTS: In control group, CD34+ cells were hardly separated in any period of LTC-IC assay. In study group, the median count of CD34+ cells was 0.7 (0.4-1.2)x10(4), 1.8 (1.4-2.6)x10(4), 0.6 (0.5-0.7)x10(4) and the median count of CFUs was 0.5 (0.2-0.8)x10(2), 1.0 (0.6-1.3)x10(2), 0.2(0.1-0.4)x10(2) at 3, 5, 7 days of LTC-IC assay, respectively. Median CFUs count per CD34+ cell was 0.0071, 0.0056, 0.0033 at 3, 5, 7 days of LTC-IC assay, respectively. In study group, the count of CD34+ cells and CFUs was significantly higher at 5 days of LTC-IC assay than at any other days (P<0.01). CFUs count per CD34+ cell was significantly higher at 3 days of LTC-IC assay than at any other days (P<0.01). CD34+CD45- cells detected by FACS method of study group(1.16%(0.92-1.97) was significantly higher than that of control group (0.09% (0.00-0.23)) (P<0.01). CONCLUSION: The differentiation of hematopoietic stem cells from SNU-3 embryonic stem cell line is effective in the condition of which an embryonic body manufactured from embryonic stem cells is cultured to LTC-IC assay. The period of which embryonic stem cells differentiate to hematopoietic stem cells is between 3 to 7 days of LTC-IC assay.