诱导小鼠颊黏膜成纤维细胞形成诱导性多潜能干细胞

doi:10.3877/cma.j.issn.1674-1366.2017.06.006

目的

研究小鼠颊黏膜成纤维细胞（BF）重编程形成诱导性多潜能干细胞（iPSC）的诱导方法。

方法

应用携带有Oct4、Sox2和Klf4基因的逆转录病毒诱导小鼠BF重编程形成iPSC。观察iPSC克隆形态；进行碱性磷酸酶（ALP）染色、核型分析；实时荧光定量聚合酶链反应（PCR）及细胞免疫荧光术检测内/外源性多能基因和干细胞标志物的表达；体外、体内分化实验检测iPSC多向分化潜能。

结果

iPSC呈典型胚胎干细胞（ESC）样克隆生长，边界清晰；细胞ALP染色阳性，核型正常。内源性Dppa3、Nanog、Rex1、Sox2、Klf4、Oct4、c-Myc基因表达量与ESC接近，未检测到外源性Sox2、Klf4、Oct4、c-Myc基因表达。干细胞标志物Oct4、Sox2和Nanog表达呈阳性。在体外，iPSC可被诱导向成骨、成脂、成软骨方向分化；体内可分化成多种组织，形成畸胎瘤。

结论

成功诱导BF重编程形成iPSC，为牙再生医学相关研究提供种子细胞来源。

关键词: 诱导多功能干细胞, 成纤维细胞, 细胞重编程, 再生

Objective

To generate induced pluripotent stem cells (iPSCs) from mouse buccal mucosa fibroblasts (BFs) by genetic reprogramming.

Methods

iPSCs were established from mouse BFs via retroviral gene transfer with three reprogramming factors (Oct4, Sox2, and Klf4) . The properties of iPSCs were characterized by alkaline phosphatase staining assay, karyotype analysis, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) , immunofluorescence and bisulfite genomic sequencing. In vitro and in vivo studies were also performed to test their differentiation and pluripotent capability.

Results

The resulting iPSCs were found to resemble mouse embryonic stem cells (ESCs) with similar clone growth and high positive stain of alkaline phosphatase. They had normal karyotype, and expressed high levels of ESCs-like genes (Dppa3, Nanog, Rex1, Sox2, Klf4, Oct4 and c-Myc) and protein markers (Oct4, Sox2, and Nanog) . Bisulfite sequencing demonstrated that the methylation of the endogenous Oct4 and Nanog promoters in iPSCs and the control ESCs clones was low or absent, while a high methylation level was found in mouse embryonic fibroblasts (MEFs) . By in vitro induction, iPSCs could produce mineralized nodules, form adipocyte-like cells with lipid droplets, and change to chondrocytes. Moreover, they can differentiate into teratomas composed of tissues of the three germ layers in vivo.

Conclusions

iPSCs were successfully established from the mouse BFs with ESCs-like properties and their pluripotency was verified. These iPSCs may serve as seed cells for tooth regeneration.

Key words: Induced pluripotent stem cells, Fibroblasts, Cellular reprogramming, Regeneration

表1 实时荧光定量PCR引物序列

基因名称	引物序列
Gapdh	F:5′-AACTTTGGCATTGTGGAAGGGCTCA-3′
?	R:5′-TTGGCAGCACCAGTGGATGCAGGGA-3′
Dppa3	F:5′-TGTGGAGAACAAGAGTGA-3′
?	R:5′-CTCAATCCGAACAAGTCTT-3′
Nanog	F:5′-CTCAAGTCCTGAGGCTGACA-3′
?	R:5′-TGAAACCTGTCCTTGAGTGC-3′
Rex1	F:5′-CAGCCAGACCACCATCTGTC-3′
?	R:5′-GTCTCCGATTTGCATATCTCCTG-3′
endo-Sox2	F:5′-AGGGCTGGGAGAAAGAAGAG-3′
?	R:5′-CCGCGATTGTTGTGATTAGT-3′
endo-Klf4	F:5′-AACATGCCCGGACTTACAAA-3′
?	R:5′-TTCAAGGGAATCCTGGTCTTC-3′
endo-Oct4	F:5′-TAGGTGAGCCGTCTTTCCAC-3′
?	R:5′-GCTTAGCCAGGTTCGAGGAT-3′
endo-c-Myc	F:5′-TAACTCGAGGAGGAGCTGGA-3′
?	R:5′-GCCAAGGTTGTGAGGTTAGG-3′
exo-Sox2	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-GGGCTGTTCTTCTGGTTG-3′
exo-Klf4	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-GCTGGACGCAGTGTCTTCTC-3′
exo-Oct4	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-CCAGGTTCGAGAATCCAC-3′
exo-c-Myc	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-CCTCGTCGCAGATGAAATAG-3′

表1 实时荧光定量PCR引物序列

基因名称	引物序列
Gapdh	F:5′-AACTTTGGCATTGTGGAAGGGCTCA-3′
?	R:5′-TTGGCAGCACCAGTGGATGCAGGGA-3′
Dppa3	F:5′-TGTGGAGAACAAGAGTGA-3′
?	R:5′-CTCAATCCGAACAAGTCTT-3′
Nanog	F:5′-CTCAAGTCCTGAGGCTGACA-3′
?	R:5′-TGAAACCTGTCCTTGAGTGC-3′
Rex1	F:5′-CAGCCAGACCACCATCTGTC-3′
?	R:5′-GTCTCCGATTTGCATATCTCCTG-3′
endo-Sox2	F:5′-AGGGCTGGGAGAAAGAAGAG-3′
?	R:5′-CCGCGATTGTTGTGATTAGT-3′
endo-Klf4	F:5′-AACATGCCCGGACTTACAAA-3′
?	R:5′-TTCAAGGGAATCCTGGTCTTC-3′
endo-Oct4	F:5′-TAGGTGAGCCGTCTTTCCAC-3′
?	R:5′-GCTTAGCCAGGTTCGAGGAT-3′
endo-c-Myc	F:5′-TAACTCGAGGAGGAGCTGGA-3′
?	R:5′-GCCAAGGTTGTGAGGTTAGG-3′
exo-Sox2	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-GGGCTGTTCTTCTGGTTG-3′
exo-Klf4	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-GCTGGACGCAGTGTCTTCTC-3′
exo-Oct4	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-CCAGGTTCGAGAATCCAC-3′
exo-c-Myc	F:5′-GGGTGGACCATCCTCTAGAC-3′
?	R:5′-CCTCGTCGCAGATGAAATAG-3′

表2 Nanog、Oct4启动子PCR引物序列

基因名称	引物序列
mNanog-L4	5′-TACTTCCTCAAAACTAAACAAACTAT-3′
mNanog-U1	5′-GAGGATGTTTTTTAAGTTTTTTTT-3′
mNanog-U2	5′-AATGTTTATGGTGGATTTTGTAGGT-3′
mNanog-L3	5′-CCCACACTCATATCAATATAATAAC-3′
mOct-U1	5′-GAGGATTGGAGGTGTAATGGTTGTT-3′
mOct-L4	5′-CTACTAACCCATCACCCCCACCTA-3′
mOct-U2	5′-TGGGTTGAAATATTGGGTTTATTT-3′
mOct-L3	5′-CTAAAACCAAATATCCAACCATA-3′

表2 Nanog、Oct4启动子PCR引物序列

基因名称	引物序列
mNanog-L4	5′-TACTTCCTCAAAACTAAACAAACTAT-3′
mNanog-U1	5′-GAGGATGTTTTTTAAGTTTTTTTT-3′
mNanog-U2	5′-AATGTTTATGGTGGATTTTGTAGGT-3′
mNanog-L3	5′-CCCACACTCATATCAATATAATAAC-3′
mOct-U1	5′-GAGGATTGGAGGTGTAATGGTTGTT-3′
mOct-L4	5′-CTACTAACCCATCACCCCCACCTA-3′
mOct-U2	5′-TGGGTTGAAATATTGGGTTTATTT-3′
mOct-L3	5′-CTAAAACCAAATATCCAACCATA-3′

图1 小鼠颊黏膜成纤维细胞来源诱导性多潜能干细胞的诱导形成

图2 小鼠诱导性多潜能干细胞鉴定

图3 小鼠诱导性多潜能干细胞体外多向诱导分化

图4 诱导性多潜能干细胞体内分化形成畸胎瘤

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Generation of induced pluripotent stem cells from mouse buccal mucosa fibroblasts

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Generation of induced pluripotent stem cells from mouse buccal mucosa fibroblasts