Wang Jian, Lin Ge, Zhao Hui-ping, Lu Guang-xiu
Institute of Reproductive and Stem Cell Engineering of Central South University, Human Stem Cell Engineering Research Center of China, Changsha 410078, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Apr;29(4):602-5.
To characterize the time course of spontaneous differentiation of in vitro cultured human embryonic stem cells (hESCs) into hematopoietic cells to provide experimental evidence for induction of hematopoietic commitment of hESCs.
In human embryoid bodies (hEBs) derived from spontaneous differentiation of chESC3, a hESC cell line we established previously, the expressions of such genes as KDR, Bmi1, Scl and gata2 were detected by RT-PCR every other day during the 12-day differentiation to monitor the process of the hematopoiesis. The hematopoietic stem cell marker CD34 was examined using flow cytometry to evaluate the efficiency of hematopoietic differentiation of the cells on days 6, 8, 10 and 12. The spontaneously differentiated hESCs were seeded in the hematopoietic colony culture system to study the hematopoietic colony forming ability. Immunocytochemical staining for CD45 was performed on the hEBs to examine the emergence of mature hematopoietic cells.
The expressions of the hematopoietic stem cell-related genes KDR and Bmi-1 were detected in the hESCs, and on days 4 to 6, the two genes were upregulated with prolonged cuture of the hEBs. Scl and gata2 gene expressions were detected since 6-8 days of culture and maintained high expressions till day 12. Flow cytometry revealed a gradual increase in CD34-positive cells in the culture, with positivity rates on days 6, 8, 10, and 12 of (1.4-/+0.4)%, (3.4-/+1.3)%, (5.5-/+2.2)%, and (5.1-/+1.7)%, respectively. The numbers of CD43-positive cell colonies on days 6, 8, 10, and 12 were 0, 7-/+2, 37-/+11, and 89-/+29 in each 10(5) cells, respectively. Immunocytochemical staining identified CD45-positive cells on days 10, 12, 15, and 18 in the cell colonies, with the positive cell numbers of 0, 40.5-/+15.09, 178.6-/+55.89, and 253.0-/+52.04, respectively.
The hESCs undergo spontaneous hematopoietic differentiation in 3 stages, including the differentiation into germ layer-specific cells (days 6-8), expansion period of the hematopoitic progenitors (days 8-12), and maturation of the hematopoietic cells (after day 15).
明确体外培养的人胚胎干细胞（hESCs）向造血细胞自发分化的时间进程，为诱导hESCs造血定向分化提供实验依据。
在由我们先前建立的hESC细胞系chESC3自发分化形成的人胚状体（hEBs）中，于12天的分化过程中每隔一天通过逆转录聚合酶链反应（RT-PCR）检测KDR、Bmi1、Scl和gata2等基因的表达，以监测造血过程。在第6、8、10和12天使用流式细胞术检测造血干细胞标志物CD34，以评估细胞的造血分化效率。将自发分化的hESCs接种于造血集落培养体系中，研究其造血集落形成能力。对hEBs进行CD45免疫细胞化学染色，以检测成熟造血细胞的出现情况。
在hESCs中检测到造血干细胞相关基因KDR和Bmi-1的表达，在第4至6天，随着hEBs培养时间延长，这两个基因上调。培养6 - 8天后检测到Scl和gata2基因表达，并维持高表达直至第12天。流式细胞术显示培养物中CD34阳性细胞逐渐增加，第6、8、10和12天的阳性率分别为（1.4±0.4）%、（3.4±1.3）%、（5.5±2.2）%和（5.1±1.7）%。每10⁵个细胞在第6、8、10和12天的CD43阳性细胞集落数分别为0、7±2、37±11和89±29。免疫细胞化学染色在细胞集落的第10、12、15和18天鉴定出CD45阳性细胞，阳性细胞数分别为0、40.5±15.09、178.6±55.89和253.0±52.04。
hESCs经历三个阶段的自发造血分化，包括向胚层特异性细胞的分化（第6 - 8天）、造血祖细胞的扩增期（第8 - 12天）以及造血细胞的成熟（第15天后）。
