Qiu Caihong, Hanson Eric, Olivier Emmanuel, Inada Mari, Kaufman Dan S, Gupta Sanjeev, Bouhassira Eric E
Einstein Center for Human Embryonic Stem Cell Research, Department of Medicine, Division of Hematology and Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA.
Exp Hematol. 2005 Dec;33(12):1450-8. doi: 10.1016/j.exphem.2005.09.003.
To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells.
FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction.
Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined.
FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.
寻找一种能够支持人类胚胎干细胞（hESCs）分化为造血细胞的人类细胞系。详细确定hESC来源的红系细胞中β样珠蛋白基因的表达谱。
将人胎肝来源的细胞系FH-B-hTERT和小鼠骨髓基质细胞系S17用作基质，诱导hESC分化为造血细胞。在时间进程实验中，使用集落测定法和流式细胞术监测造血祖细胞数量和表面抗原表达。通过实时定量逆转录聚合酶链反应确定单个红系集落中的珠蛋白表达模式。
hESCs与FH-B-hTERT或S17细胞共培养的比较显示，与S17相比，FH-B-hTERT接种的每250,000个细胞中CD34(+)细胞的比例和克隆祖细胞的数量更高。对单个红系爆式集落形成单位和红系集落形成单位集落中β样珠蛋白表达的分析表明，在FH-B-hTERT或S17上共培养8至21天的hESC来源的红系细胞产生的ε-和γ-珠蛋白mRNA量相似。随着共培养时间的增加，在S17和FH-B-hTERT基质上γ/ε的平均比值均增加了10倍以上。重要的是，在所检测的所有时间点几乎都检测不到β-珠蛋白的表达。
FH-B-hTERT比S-17能更有效地诱导hESCs分化为造血细胞。hESCs的体外分化重现了ε-珠蛋白向γ-珠蛋白的转换，但没有重现出生前后发生的γ-珠蛋白向β-珠蛋白的转换。该实验系统将有助于研究人类早期造血过程中珠蛋白基因表达的调控。
