CN112143706A - 一种将尿液细胞重编程为诱导性多能干细胞的方法 - Google Patents
一种将尿液细胞重编程为诱导性多能干细胞的方法 Download PDFInfo
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Abstract
本发明提供了一种将尿液细胞重编程为诱导性多能干细胞的方法，所述方法包括：将表达四个转录因子和miRNA的载体导入尿液细胞中，包裹到多肽水凝胶中之后再置于细胞培养基中培养；所述转录因子包括OCT4、SOX2、SV40LT和KLF4；所述miRNA包括miRNA302和miRNA367。本发明利用三维多肽水凝胶替代传统的Matrigel，将表达转录因子和miRNA的尿液细胞重编程为诱导性多能干细胞，避免了使用动物源性的Matrigel，尿液细胞均一培养分布于多肽水凝胶中，不贴壁培养，有利于实现自动化吸取细胞克隆，质量可控、成分安全，扩大了诱导性多能干细胞的临床应用范围。
Description
技术领域
本发明属于体细胞重编程技术领域，涉及一种将尿液细胞重编程为诱导性多能干细胞的方法。
背景技术
人的多种体细胞包括尿液细胞可以通过导入转录因子OCT4、SOX2、SV40LT、KLF4和micro-RNA的方式被重编程为诱导性多能干细胞(iPSCs)(Xue,Y.；Cai,X.；Wang,L.；Liao,B.；Zhang,H.；Shan,Y.；Chen,Q.；Zhou,T.；Li,X.；Hou,J.；Chen,S.；Luo,R.；Qin,D.；Pei,D.；Pan,G.,Generating a non-integrating human induced pluripotent stem cellbank from urine-derived cells.PLoS One 2013,8(8),e70573.)。
但是，目前的重编程技术是将尿液细胞置于二维Matrigel包被的培养板中进行重编程的。Matrigel是从EHS Engelbreth-Holm-Swarm小鼠肉瘤中得到的可溶性基底膜抽提物，含有不可靠的动物来源成分，且存在难以控制的批次效应，临床应用于重编程诱导性多能干细胞存在诸多问题。
因此，有必要找到一种成分确定、非动物源性来源的尿液细胞重编程环境，更好地制备诱导性多能干细胞并应用于临床。
发明内容
针对现有技术的不足和实际需求，本发明提供了一种将尿液细胞重编程为诱导性多能干细胞的方法，利用三维多肽水凝胶替代传统的Matrigel，将尿液细胞重编程为诱导性多能干细胞，避免了使用动物源性的Matrigel，扩大了诱导性多能干细胞的临床应用范围。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种将尿液细胞重编程为诱导性多能干细胞的方法，所述方法包括：将表达转录因子和miRNA的尿液细胞包裹于多肽水凝胶中，置于细胞培养基中培养；
所述转录因子包括OCT4、SOX2、SV40LT和KLF4；
所述miRNA包括miR302和miR367。
本发明中，将表达转录因子OCT4、SOX2、SV40LT、KLF4和miR302、miR367的尿液细胞培养于非动物性来源的含有16个氨基酸的多肽水凝胶中进行重编程，将尿液细胞高效诱导为多能性干细胞，替代了动物源性的Matrigel，得到的多能性干细胞质量可控、成分安全，有利于应用于临床。
本发明的三维多肽水凝胶不含有细胞外基质成分，能够抑制尿液细胞膜上integrinβ1(ITGB1)的表达，从而降低了focal adhesion kinase(FAK)的磷酸化，显著提高了可诱导多能干细胞的效率，且尿液细胞均匀分散于多肽水凝胶中、均一性好，不贴壁培养，有利于实现自动化吸取细胞克隆。
优选地，所述尿液细胞通过电转质粒的方式表达OCT4、SOX2、SV40LT、KLF4、miR302和miR367。
优选地，所述多肽水凝胶包括精氨酸、丙氨酸或天冬氨酸中的任意一种或至少两种的组合，优选为精氨酸、丙氨酸和天冬氨酸的组合。
优选地，所述多肽水凝胶包括SEQ ID NO:1所示的氨基酸序列；
SEQ ID NO:1：RARADADARARADADA。
优选地，所述尿液细胞在所述多肽水凝胶的浓度为1×105～1×106/mL，即1×105～1×106个尿液细胞包裹于1mL多肽水凝胶中。
优选地，所述培养基包括人多能干细胞培养基，优选为mTesr培养基。
优选地，所述mTesr培养基含有抑制剂。
优选地，所述抑制剂包括A8301、PD032590、CHIR99021或thiazovivin中的任意一种或至少两种的组合，优选为A8301、PD032590、CHIR99021和thiazovivin的组合。
优选地，所述培养基包括含有0.1～1μM A8301、0.1～1μM PD032590、1～5μMCHIR99021和0.1～1μM thiazovivin的mTesr培养基，和不含抑制剂的mTesr培养基。
优选地，所述培养的温度为35～38℃，例如可以是35℃、36℃、37℃或38℃，优选为37℃。
优选地，所述培养的CO2浓度为3％～6％，例如可以是3％、4％、5％或6％，优选为5％。
优选地，所述培养的时间为15～25天，例如可以是15天、16天、17天、18天、19天、20天、21天、22天、23天、24天或25天，优选为20天。
优选地，所述尿液细胞包裹于多肽水凝胶中后，置于含有0.1～1μM A8301、0.1～1μM PD032590、1～5μM CHIR99021和0.1～1μM thiazovivin的mTesr培养基培养8～12天，随后更换培养基为mTesr培养基继续培养8～12天。
作为优选技术方案，所述将尿液细胞重编程为诱导性多能干细胞的方法，包括以下步骤：
(1)将OCT4、SOX2、SV40LT、KLF4重组质粒和miR302、miR367重组质粒电转导入尿液细胞；
(2)将电转后的尿液细胞重悬到氨基酸序列如SEQ ID NO:1所示的多肽水凝胶溶液中，加入尿液培养基瞬间成胶，置于35～38℃、3％～6％CO2中培养形成三维半固体水凝胶；
(3)更换培养基为含有0.1～1μM A8301、0.1～1μM PD032590、1～5μM CHIR99021和0.1～1μM thiazovivin的mTesr培养基培养8～12天，随后更换培养基为mTesr培养基继续培养8～12天，得到所述诱导性多能干细胞。
第二方面，本发明提供了一种诱导性多能干细胞，所述诱导性多能干细胞由第一方面所述的方法制备得到。
与现有技术相比，本发明具有如下有益效果：
(1)本发明采用成分确定的非动物来源的多肽水凝胶代替Matrigel用于尿液细胞的重编程，三维多肽水凝胶不含有细胞外基质成分，抑制了尿液细胞膜上integrinβ1(ITGB1)的表达，降低了focal adhesion kinase(FAK)的磷酸化，将尿液细胞高效诱导为多能性干细胞；
(2)本发明采用三维多肽水凝胶培养尿液细胞，尿液细胞均匀分散于多肽水凝胶中、均一性好，不贴壁培养，有利于实现自动化吸取细胞克隆；
(3)本发明的三维多肽水凝胶成分简单、可靠，培养得到的多能性干细胞质量可控、成分安全，具有广泛的临床应用前景。
附图说明
图1为尿液细胞在二维Matrigel和在三维多肽水凝胶中的重编程过程；
图2为碱性磷酸酶染色鉴定的二维Matrigel和三维多肽水凝胶中的克隆；
图3为免疫荧光染色鉴定内源多能干性基因(Oct4、Sox2、SSEA4、TRA-1-60)在二维Matrigel和三维多肽水凝胶中克隆中的表达；
图4A为Oct4、DPPA4在所有克隆中和单克隆中的表达情况，图4B为2D Matriel和3D-PM中克隆的相关性统计，图4C为胚胎干细胞多能性相关基因在2D Matriel和3D-PM中克隆的表达谱，图4D为在2D Matriel和3D-PM中克隆的上调及下调基因分析，图4E为对2DMatriel和3D-PM中克隆的聚类分析；
图5A为对2D Matriel和3D-PM中克隆的单细胞进行无标记分类，图5B为在2DMatriel和3D-PM中克隆单细胞标记的iPSCs的数量分析，图5C为多能干性基因Oct4在2DMatriel和3D-PM中克隆单细胞的表达谱；
图6A为2D Matriel和3D-PM中的克隆中Integrin家族的表达，图6B为qRT-PCR验证2D Matriel和3D-PM中的克隆中ITGB1的表达，图6C为Western Blot验证2D Matriel和3D-PM中的克隆中ITGB1、FAK和磷酸化FAK的蛋白水平表达，图6D为qRT-PCR验证2D Matriel和3D-PM中的尿液细胞ITGB1的表达，图6E为Western Blot验证2D Matriel和3D-PM中的尿液细胞ITGB1的蛋白水平表达。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1构建用于尿液细胞重编程的重组质粒
本实施例以pCEP4为载体，构建含有四种转录因子(OCT4、SOX2、SV40LT、KLF4)的质粒，和含有miR302、miR367前体的质粒。
步骤为：首先利用PCR扩增基因OCT4、SOX2、SV40LT、KLF4的CDS区以及miR302、miR367的前体序列，将扩增产物采用限制性内切酶进行酶切后，连接入经同样的限制性内切酶酶切的线性化pCEP4载体上。
实施例2重组质粒的电转
(1)收集人的250mL尿液，250g离心10min，用PBS润洗三次提取出尿液细胞，置于REGM+MEF培养基中扩增到第2～3代，进行细胞计数；
(2)将5×105～1×106个尿液细胞用0.25％胰蛋白酶消化后，采用AmaxaTM BasicNucleofectorTM试剂盒将表1所示的质粒体系电转入尿液细胞中，电转程序为Lonza公司AAD1001S型号电转仪T-020程序。
表1
实施例3尿液细胞重编程为诱导性多能干细胞
采用0.25％胰蛋白酶消化收集电转后的尿液细胞，将尿液细胞以1×106/mL的密度重悬到多肽水凝胶(SEQ ID NO:1)溶液中，并以300μL的体积加入到48孔板的一个孔中，与尿液培养基混合后置于37℃、5％CO2的培养箱中培养形成三维半固体水凝胶；
诱导第二天换入mTesR1培养液(含有0.5μM A8301，0.5μM PD032590，3μMCHIR99021，0.5μM thiazovivin)到尿液细胞和三维多肽水凝胶的复合体中，诱导10天后撤掉抑制剂，采用mTesR1培养液继续培养，并隔天换液；
在诱导后第20天采用玻璃针将克隆挑取出来，进行iPSCs的分离和鉴定。
实施例4多能性干细胞的鉴定
尿液细胞在二维Matrigel(2D Matrigel)和在三维多肽水凝胶(3D PM)中的重编程过程如图1所示，说明尿液细胞在三维多肽水凝胶中可以重编程为克隆状的诱导性多能干细胞。
经碱性磷酸酶染色鉴定，如图2所示，三维多肽水凝胶里产生的克隆同样含有诱导性多能干细胞(蓝色)。
将二维Matrigel和三维多肽水凝胶中的克隆挑取出来，进行多能干性基因免疫荧光鉴定，如图3所示，发现在三维多肽水凝胶中的克隆含有与二维Matrigel上相同的诱导性多能干细胞。
将二维Matrigel和三维多肽水凝胶中的克隆挑取出来提取RNA，进行RNA-seq，如图4A、图4B、图4C、图4D和图4E所示，发现在三维多肽水凝胶中产生诱导性多能干细胞的效率更高。
并对RNA进行单细胞测序，如表2、图5A、图5B和图5C所示，同样发现在三维多肽水凝胶中产生诱导性多能干细胞的效率更高，与RNA-seq结果吻合。
表2
| 10<sup>4</sup>二维细胞百分比 | 10<sup>4</sup>三维细胞百分比 | 3维细胞/2维细胞 | |
| Cluster 0 | 0.442889684 | 0.231625389 | 0.522986643 |
| Cluster 1 | 0.161622736 | 0.184798957 | 1.143397034 |
| Cluster 2 | 0.13407094 | 0.111400782 | 0.830909234 |
| Cluster 3 | 0.111834255 | 0.123132458 | 1.101026318 |
| Cluster 4 | 0.0378566 | 0.161135065 | 4.256458979 |
| Cluster 5 | 0.024840004 | 0.076506568 | 3.079974061 |
| Cluster 6 | 0.014969086 | 0.072796551 | 4.863125978 |
| Cluster 7 | 0.049463065 | 0.019853605 | 0.401382425 |
| Cluster 8 | 0.022453628 | 0.018750627 | 0.835082286 |
将二维Matrigel和三维多肽水凝胶中的克隆进行基因表达分析和蛋白标志物分析，结果如图6A、图6B、图6C、图6D和图6E所示，说明尿液细胞培养于三维水凝胶中，ITGB1的表达得到抑制，同时降低了磷酸化FAK的表达水平。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
序列表
<110> 中国科学院广州生物医药与健康研究院
<120> 一种将尿液细胞重编程为诱导性多能干细胞的方法
<130> 20200927
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> 人工序列()
<400> 1
Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala
1 5 10 15
Claims (10)
1.一种将尿液细胞重编程为诱导性多能干细胞的方法，其特征在于，所述方法包括：将表达转录因子和miRNA的尿液细胞包裹于多肽水凝胶中，置于细胞培养基中培养；
所述转录因子包括OCT4、SOX2、SV40LT和KLF4；
所述miRNA包括miR302和miR367。
2.根据权利要求1所述的方法，其特征在于，所述尿液细胞通过电转质粒的方式表达OCT4、SOX2、SV40LT、KLF4、miR302和miR367。
3.根据权利要求1或2所述的方法，其特征在于，所述多肽水凝胶包括精氨酸、丙氨酸或天冬氨酸中的任意一种或至少两种的组合，优选为精氨酸、丙氨酸和天冬氨酸的组合；
优选地，所述多肽水凝胶包括SEQ ID NO:1所示的氨基酸序列。
4.根据权利要求1-3任一项所述的方法，其特征在于，所述尿液细胞在所述多肽水凝胶的浓度为1×105～1×106/mL。
5.根据权利要求1-4任一项所述的方法，其特征在于，所述培养基包括人多能干细胞培养基，优选为mTesr培养基；
优选地，所述mTesr培养基含有抑制剂；
优选地，所述抑制剂包括A8301、PD032590、CHIR99021或thiazovivin中的任意一种或至少两种的组合，优选为A8301、PD032590、CHIR99021和thiazovivin的组合；
优选地，所述培养基包括含有0.1～1μM A8301、0.1～1μM PD032590、1～5μMCHIR99021和0.1～1μM thiazovivin的mTesr培养基，和不含抑制剂的mTesr培养基。
6.根据权利要求1-5任一项所述的方法，其特征在于，所述培养的温度为35～38℃。
7.根据权利要求1-6任一项所述的方法，其特征在于，所述培养的CO2浓度为3％～6％。
8.根据权利要求1-7任一项所述的方法，其特征在于，所述培养的时间为15～25天；
优选地，所述尿液细胞包裹于多肽水凝胶中后，置于含有0.1～1μM A8301、0.1～1μMPD032590、1～5μM CHIR99021和0.1～1μM thiazovivin的mTesr培养基培养8～12天，随后更换培养基为mTesr培养基继续培养8～12天。
9.根据权利要求1-8任一项所述的方法，其特征在于，所述方法包括以下步骤：
(1)将OCT4、SOX2、SV40LT、KLF4重组质粒和miR302、miR367重组质粒电转导入尿液细胞；
(2)将电转后的尿液细胞重悬到氨基酸序列如SEQ ID NO:1所示的多肽水凝胶溶液中，加入尿液培养基瞬间成胶，置于35～38℃、3％～6％CO2中培养形成三维半固体水凝胶；
(3)更换培养基为含有0.1～1μM A8301、0.1～1μM PD032590、1～5μM CHIR99021和0.1～1μM thiazovivin的mTesr培养基培养8～12天，随后更换培养基为mTesr培养基继续培养8～12天，得到所述诱导性多能干细胞。
10.一种诱导性多能干细胞，其特征在于，所述诱导性多能干细胞由权利要求1-9任一项所述的方法制备得到。
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