Small-molecule compounds promote corneal epithelial cell differentiation from human embryonic stem cells
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摘要:
背景 角膜缘干细胞缺乏是角膜盲的主要病因，种子细胞来源有限是细胞移植疗法的瓶颈。 目的 诱导人胚胎干细胞(human embryonic stem cells，hESCs)分化为角膜上皮祖细胞和成熟的角膜上皮细胞(corneal epithelial cells，CECs)。 方法 采用拟胚体联合小分子化合物(SB505124和IWP-2)及碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)对人胚胎干细胞H9系(hESCs H9)进行诱导，分别于诱导10 d、15 d和20 d时观察细胞形态，用免疫荧光法和实时定量PCR法对特征性标志物OCT4、SOX2、ΔNp63、CK14、CK3和CK12进行基因及蛋白水平的检测。 结果 小分子化合物诱导后胚胎干细胞形态改变，类似角膜上皮细胞形态。诱导后干性标志物OCT4和SOX2表达下调(P均＜0.01)，角膜上皮祖细胞及角膜上皮细胞标志物ΔNp63、CK14、CK3和CK12表达均上调(P均＜0.05)。 结论 通过拟胚体联合小分子化合物的方法，可以将ESCs诱导为角膜上皮样细胞。 Abstract:Background Limbal stem cell deficiency is the main cause of corneal blindness, and the limited source of seed cells is the bottleneck of cell transplantation therapy. Objective To induce human embryonic stem cells (hESCs) to differentiate into corneal epithelial progenitor cells and mature corneal epithelial cells. Methods The induction of human embryonic stem cell line H9 (hESCs H9) involved embryonic body (EB) formation and the addition of small molecular compounds (SB505124 and IWP-2) and basic fibroblast growth factor (bFGF). After observation of cell morphology, the cells were collected at day 10, 15 and 20 of induction, respectively, and the mRNA and protein expression of characteristic markers OCT4, SOX2, ΔNp63; CK14, CK3, CK12 were detected by immunofluorescence assay and quantitative real-time PCR. Results The morphology of induced cells was different from that of hESCs and similar to that of corneal epithelial cells (CECs). Compared with hESCs, the expressions of stem cell markers OCT4 and SOX2 were down-regulated (P＜0.01, respectively), and the expressions of corneal epithelial progenitor cells and corneal epithelial cell markers ΔNp63, CK14, CK3, CK12 were up-regulated after induction (all P＜0.05). Conclusion This small-molecule compound promotes corneal epithelial cell differentiation from human embryonic stem cells. -
Key words:
- human embryonic stem cells /
- small molecular compounds /
- corneal epithelial cells /
- embryonic body /
- CK3
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图 1 诱导人胚胎干细胞分化为角膜上皮样细胞方法示意图
Figure 1. Schematic diagram of method used to induce human embryonic stem cells to differentiate into corneal epithelial cell
图 2 不同阶段光镜下细胞形态(标尺=100 μm)
A：ESCs；B：拟胚体；C：诱导第7天的细胞；D：诱导第10天的细胞；E：诱导第15天的细胞；F：诱导第20天的细胞
Figure 2. Cell morphology images at different stages under light microscope (bar=100μm)
A: ESCs; B: embryonic bodies; C: induced cells on day 7; D: induced cells on day 10; E: induced cells on day 15; F: induced cells on day 20
图 3 ESCs和诱导细胞相关标志物mRNA的表达(aP＜0.05，bP＜0.01，vs ESCs)
Figure 3. mRNA expressions of related markers in ESCs and induced cells (aP＜0.05, bP＜0.01, vs ESCs)
图 4 共聚焦显微镜下ESCs和诱导细胞相关标志物的表达(标尺=75 μm) A：OCT4；B：ΔNp63；C：CK3
Figure 4. Expressions of related markers in ESCs and induced cells under confocal microscope (bar=75 μm) A: OCT4; B: ΔNp63; C: CK3
表 1 引物序列
Table 1. Primer sequence
Primers name Sequence(5’-3’) Size/bp GAPDH F: GGAAGCTTGTCATCAATGGAAATC; R: TGATGACCCTTTTGGCTCCC 168 OCT4 F: TCTATTTGGGAAGGTATTCAGCC; R: CCTCTCACTCGGTTCTCGATACTG 210 SOX2 F: GCGGCAACCAGAAAAACAG; R: CAAAAGTTTCCACTCGGCG 155 TP63 F: ATCCCATCACAGGAAGACAGAGT; R: TATCTTCATCCGCCTTCCTGTC 240 CK14 F: CGGCCTGCTGAGATCAAAGAC; R: CTCTGTCTCATACTTGGTGCGG 159 CK3 F: GCCAAAGTGGATGCCTTGATAG; R: CAGGTCCAGGGAGCGATTAT 135 CK12 F: CTCTCCTCGCAGAGTGTGATA; R: AACTAGAACCAAACATGGAAGCA 119 GAPDH: glyceraldehyde-phosphate dehydrogenase; F: forward; R: reverse. 
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