TAT蛋白转运结构域介导的重组蛋白诱导成体细胞重编程
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摘要
诱导干细胞(induced pluripotent stem cell,iPS或iPSCs)的建立在再生医学领域有着极大的应用潜力。但是,在该技术应用于临床之前,安全与效率是其必须应对的重要问题。
众多研究致力于推进以上问题的解决：介导重编程因子转染供体细胞的方法由最初的整合型病毒(逆转录病毒、慢病毒)到非整合型病毒(腺病毒),继而发展到无病毒(如PB转座子系统、episome),直至穿膜肽介导的目的因子融合蛋白直接转运至细胞内。09年4月,Zhou等人利用11精氨酸穿膜肽介导四因子融合蛋白建立了小鼠蛋白iPS细胞系(protin-iPS, p-iPS),同年5月Kim等人利用同方法建立了人p-iPS。由于蛋白诱导iPS彻底排除了外源遗传物质,被认为是目前为止最安全的iPS诱导方法。但是,以上的非整合型的诱导方法效率低、耗时长、不稳定,增加了细胞变异的可能,限制了iPS在临床和研究方面的应用。
TAT-PTD是迄今为止应用最为广泛的转膜肽,大量研究表明该系统能够介导多种融合蛋白高效转染不同种类的细胞且重折叠后的外源蛋白多具有生物学活性。我们尝试检测TAT-PTD转导系统是否可以促进蛋白iPS细胞系的建立。在本工作中,我们运用TAT蛋白转导结构域构建了TAT-PTD介导的蛋白转运系统。通过原核表达His-TAT-Sox2/Oct4/Klf4/c-Myc/Nanog等多能因子融合蛋白；从TAT融合蛋白转染条件、工作浓度、孵育时间等方面优化转染效果,并检测融合蛋白在细胞内的稳定性；在多细胞系上测试TAT-目的蛋白的转染效果以测定所得优化条件的稳定性和通用性。在所得条件下,于成体细胞重编程系统中比较TAT和11精氨酸(11R)蛋白转导结构域的异同,并探索基于蛋白转导结构域的重编程因子(reprogramming factor, RF)重组蛋白诱导iPS的条件。我们发现：TAT和11R融合重编程因子具有转录活性,在荧光素报告系统中能够激活其对应的下游靶基因的报告基因。TAT-RFs在转录活性上要优于相应的11R-RFs,但弱于相应的病毒因子。在我们构建的“3病毒+1蛋白”重编程系统中,TAT-RF能够一定程度上替代相应病毒因子的作用,同时利用该实验系统最终确定各TAT-转录因子在重编程过程中的工作条件,确定了TAT-转录因子融合蛋白(4因子)诱导iPS的诱导策略和具体条件：四个TAT-RFs因子(各50nM工作浓度,每次孵育2h)每48h孵育HFFs细胞至第17天。遗憾的是,在以上实验条件下,我们并没有获得iPS状克隆(OCT4, NANOG和AP阳性)。这个结果暗示TAT-RFs的转录活性或许相较对应病毒因子较弱。我们又进行了进一步的探索：增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性。在此方法下,两周即可出现与iPS形态相似的克隆。经检测,这些克隆具iPS形态学特征,呈AP染色阳性,在转录水平和蛋白水平上表达多能相关基因。这说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。
我们的工作的兴趣点在于：利用不同系统中(如从荧光素报告系统,“3病毒+1蛋白”重编程系统)摸索PTD-融合蛋白转染条件、工作浓度、孵育时间等方面的合适条件,探索优化蛋白iPS诱导策略。针对蛋白转录因子转录活性可能低于相应病毒转录因子这一制约蛋白-iPS应用的瓶颈,我们增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性,从而在细胞水平上(AP染色,多能因子在转录水平和蛋白水平的表达)证明说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。
我们的工作对TAT融合重编程因子在重编程过程中的系统检测,可以为建立具临床应用价值和不含外源遗传物质的人iPSC的获得提供依据。
Successful establishment of Induced pluripotent stem cells (iPSCs) is thought to herald the revolutionaries of regenerative medicine, pharmaceutic screening, disease models and basic researches. Before this promising technique could be fulfilled in the therapeutic use, the efficiency of iPSC generation and safety of iPSCs, as the clinical prerequisites, are required to be satisfied.
The exploration for technologies of reprograming pluripotency without ectopic integration, aiming at circumventing of transgene reactivation or insertional mutagenesis, has advanced significantly. So far integration-free iPSCs have been derived via excisable (transposon and floxed lentiviral), nonintegrating vectors (plasmid, episomal, and adenovirus vectors), and DNA-free (recombination proteins incorporating cell-penetrating peptide, Sendai virus and Synthetic Modified mRNA) approaches. Among these, generation of iPSCs through recombinant proteins is the exclusive method to date without the use of genetic materials. Despite such advantages, protein based iPS-generations have so far suffered with an extremely low efficiency and a long conversion process, which limit its further application. Nevertheless, protein based iPSCs generation deserves further research.
In this study, we explore a simple, genetic materials-free strategy for somatic cell reprogramming based on fusion proteins with protein transduction domain TAT. We purify TAT-fusion proteins of reprogramming factors (Oct4/Sox2/c-Myc/Klf4) with bio-functions from E. coli and explorate the optimized conditions for TAT-RF transductions, including working concentrations, incubation time. Also, staibilities of TAT-RFs in vivo are exaimed. We demonstrate that TAT-RFs could transduce multiple human and mouse cell lines and activate their corresponding reporter genes. In addition, we show that these fusion proteins can substitute their corresponding retrovirus to promote generation of iPSCs in the reprogramming process using '3virus+1protein' assays. Based on above results, we produce a protocol for4TAT-RF induced iPS generations:human forskin fibroblast cells are treated with4TAT-RFs (50nM each, incubation time:2h)2days one round for17days in total. Unfortrunately, no iPS-like colony is emerged under this protocol. We hypotheses Transduction of TAT-fusion proteins generates iPS-like colonies from human fibroblast cells and mouse fetal neural progenitor cells with relatively high efficiency and quick dynamics based on the result of alkaline phosphatase staining. Additionally, our work suggests that the relative low level of protein transduction is likely the key barrier to the success of protein-based iPSCs.
In conclusion, our study provides new information of using TAT-based protein transduction approach for the generation of clinic relevant and genetic material-free human iPSCs.
众多研究致力于推进以上问题的解决：介导重编程因子转染供体细胞的方法由最初的整合型病毒(逆转录病毒、慢病毒)到非整合型病毒(腺病毒),继而发展到无病毒(如PB转座子系统、episome),直至穿膜肽介导的目的因子融合蛋白直接转运至细胞内。09年4月,Zhou等人利用11精氨酸穿膜肽介导四因子融合蛋白建立了小鼠蛋白iPS细胞系(protin-iPS, p-iPS),同年5月Kim等人利用同方法建立了人p-iPS。由于蛋白诱导iPS彻底排除了外源遗传物质,被认为是目前为止最安全的iPS诱导方法。但是,以上的非整合型的诱导方法效率低、耗时长、不稳定,增加了细胞变异的可能,限制了iPS在临床和研究方面的应用。
TAT-PTD是迄今为止应用最为广泛的转膜肽,大量研究表明该系统能够介导多种融合蛋白高效转染不同种类的细胞且重折叠后的外源蛋白多具有生物学活性。我们尝试检测TAT-PTD转导系统是否可以促进蛋白iPS细胞系的建立。在本工作中,我们运用TAT蛋白转导结构域构建了TAT-PTD介导的蛋白转运系统。通过原核表达His-TAT-Sox2/Oct4/Klf4/c-Myc/Nanog等多能因子融合蛋白；从TAT融合蛋白转染条件、工作浓度、孵育时间等方面优化转染效果,并检测融合蛋白在细胞内的稳定性；在多细胞系上测试TAT-目的蛋白的转染效果以测定所得优化条件的稳定性和通用性。在所得条件下,于成体细胞重编程系统中比较TAT和11精氨酸(11R)蛋白转导结构域的异同,并探索基于蛋白转导结构域的重编程因子(reprogramming factor, RF)重组蛋白诱导iPS的条件。我们发现：TAT和11R融合重编程因子具有转录活性,在荧光素报告系统中能够激活其对应的下游靶基因的报告基因。TAT-RFs在转录活性上要优于相应的11R-RFs,但弱于相应的病毒因子。在我们构建的“3病毒+1蛋白”重编程系统中,TAT-RF能够一定程度上替代相应病毒因子的作用,同时利用该实验系统最终确定各TAT-转录因子在重编程过程中的工作条件,确定了TAT-转录因子融合蛋白(4因子)诱导iPS的诱导策略和具体条件：四个TAT-RFs因子(各50nM工作浓度,每次孵育2h)每48h孵育HFFs细胞至第17天。遗憾的是,在以上实验条件下,我们并没有获得iPS状克隆(OCT4, NANOG和AP阳性)。这个结果暗示TAT-RFs的转录活性或许相较对应病毒因子较弱。我们又进行了进一步的探索：增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性。在此方法下,两周即可出现与iPS形态相似的克隆。经检测,这些克隆具iPS形态学特征,呈AP染色阳性,在转录水平和蛋白水平上表达多能相关基因。这说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。
我们的工作的兴趣点在于：利用不同系统中(如从荧光素报告系统,“3病毒+1蛋白”重编程系统)摸索PTD-融合蛋白转染条件、工作浓度、孵育时间等方面的合适条件,探索优化蛋白iPS诱导策略。针对蛋白转录因子转录活性可能低于相应病毒转录因子这一制约蛋白-iPS应用的瓶颈,我们增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性,从而在细胞水平上(AP染色,多能因子在转录水平和蛋白水平的表达)证明说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。
我们的工作对TAT融合重编程因子在重编程过程中的系统检测,可以为建立具临床应用价值和不含外源遗传物质的人iPSC的获得提供依据。
Successful establishment of Induced pluripotent stem cells (iPSCs) is thought to herald the revolutionaries of regenerative medicine, pharmaceutic screening, disease models and basic researches. Before this promising technique could be fulfilled in the therapeutic use, the efficiency of iPSC generation and safety of iPSCs, as the clinical prerequisites, are required to be satisfied.
The exploration for technologies of reprograming pluripotency without ectopic integration, aiming at circumventing of transgene reactivation or insertional mutagenesis, has advanced significantly. So far integration-free iPSCs have been derived via excisable (transposon and floxed lentiviral), nonintegrating vectors (plasmid, episomal, and adenovirus vectors), and DNA-free (recombination proteins incorporating cell-penetrating peptide, Sendai virus and Synthetic Modified mRNA) approaches. Among these, generation of iPSCs through recombinant proteins is the exclusive method to date without the use of genetic materials. Despite such advantages, protein based iPS-generations have so far suffered with an extremely low efficiency and a long conversion process, which limit its further application. Nevertheless, protein based iPSCs generation deserves further research.
In this study, we explore a simple, genetic materials-free strategy for somatic cell reprogramming based on fusion proteins with protein transduction domain TAT. We purify TAT-fusion proteins of reprogramming factors (Oct4/Sox2/c-Myc/Klf4) with bio-functions from E. coli and explorate the optimized conditions for TAT-RF transductions, including working concentrations, incubation time. Also, staibilities of TAT-RFs in vivo are exaimed. We demonstrate that TAT-RFs could transduce multiple human and mouse cell lines and activate their corresponding reporter genes. In addition, we show that these fusion proteins can substitute their corresponding retrovirus to promote generation of iPSCs in the reprogramming process using '3virus+1protein' assays. Based on above results, we produce a protocol for4TAT-RF induced iPS generations:human forskin fibroblast cells are treated with4TAT-RFs (50nM each, incubation time:2h)2days one round for17days in total. Unfortrunately, no iPS-like colony is emerged under this protocol. We hypotheses Transduction of TAT-fusion proteins generates iPS-like colonies from human fibroblast cells and mouse fetal neural progenitor cells with relatively high efficiency and quick dynamics based on the result of alkaline phosphatase staining. Additionally, our work suggests that the relative low level of protein transduction is likely the key barrier to the success of protein-based iPSCs.
In conclusion, our study provides new information of using TAT-based protein transduction approach for the generation of clinic relevant and genetic material-free human iPSCs.
引文
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