Cell-membrane permeant proteins to directly generate human and mouse iPS cells

细胞膜渗透蛋白可直接生成人和小鼠 iPS 细胞

• 批准号: 8746658
• 负责人: NEAL DAVID EPSTEIN
• 金额: $ 53.85万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746658
• 关键词: Adopted; Buffers; Cell Nucleus; Cell membrane; Cells; Development; Embryo; Epigenetic Process; Escherichia coli; Excision; Exogenous Factors; Failure; Fibroblasts; Future; Genes; Genomics; Human; Inclusion Bodies; Infection; Insecta; Insertional Mutagenesis; Lead; Malignant Neoplasms; Mediating; Methods; Mus; Octamer Transcription Factor-3; Platelet Factor 4; Pluripotent Stem Cells; Process; Proteins; Publications; Race; Recombinants; Research; Solid; Solubility; Solutions; Somatic Cell; Staining method; Stains; Stem cells; System; Techniques; Therapeutic; Time; Urea; Viral; Virus; Western Blotting; Work; base; c-myc Genes; design; embryonic stem cell; improved; metaplastic cell transformation; novel; protein purification; research study; retroviral-mediated; transcription factor

The field of cell reprogramming changed dramatically with the breakthrough publication by Yamanaka in 2006 in which he showed that mouse Embryonic Fibroblasts (MEFs) could be dedifferentiated into pluripotent stem cells through the forced retroviral mediated expression of four transcription factors (Oct 4, Sox 2, Klf 4, and c-Myc). With the promise of pluripotent cells while avoiding politically and ethically challenging Embryonic Stem (ES) Cells, the race was on to demonstrate the technique on human cells. Within one year, both Yamanakas and James Thompsons groups had demonstrated similar techniques with human fibroblasts. Whereas, Yamanaka used the same 4 factors, Thompson used Oct 4, Sox 2, Nanog and Lin 28 to reprogram the human somatic cells. The process of dedifferentiation of somatic cells was observed to involve an epigenetic based suppressed expression of the four exogenous transcription factors together with a lower level constitutive expression of the concomitant endogenous genes. Surprisingly, the failure of epigenetic suppression of the exogenous factors was already known to be associated with the failure of cellular reprogramming. However, despite the epigenetic suppression, the original mice generated by Yamanaka had a propensity to develop cancers associated with reactivation of the c-Myc gene. Furthermore, since genomic integration of viral LTR sequence can cause cellular transformation and Oct 4, and Nanog are sometimes elevated in cancers, it seemed unlikely that viral mediated iPS cells would ever be used for targeted stem cell mediated therapeutics. More recent work has shown that despite the continued suppression of the viral integrated exogenous transcription factors in iPS cells, the iPS cells remain less pluripotent than if the viral integrants are removed. The removal of these integrants has not proved possible to date, in any practical manner. For this reason, it would seem that the direct addition of the transcription factor proteins themselves to the cells (for a limited period of time) would be a perfect solution. The fly in the ointment here is the great difficulty in expressing these proteins in bioactive forms, especially Oct4, which is acknowledged as the one most critical factor. When expressed in E. coli., these transcription factors are sequestered in inclusion bodies as insoluble inactive proteins. Purification of these proteins requires denaturation in 8M urea which totally inactivates the protein and to date, renaturation has involved fast 20-fold dilution in urea free buffer. We have replicated this process and showed how this technique yields degraded and mostly inactive Oct4 and Sox2 protein. To remedy this, we have developed an automated process to refold these proteins expressed in E. coli., in a carefully controlled manner on solid support. We have used these proteins, renatured by this novel process to generate murine and human iPS colonies, which stain correctly but are not robust enough to serially passage. We attribute this to inadequate refolding fidelity of the Oct4 protein. We are continuing to refine the process while at the same time we are submitting the work to date for publication. In parallel experiments, we have succeeded in producing Oct4 and Sox2, in a Baculoviral based system, which, to our knowledge, has not been accomplished by any other group in the world to date. This protein, grown in insect SF-9 cells, is found exclusively in the nucleus of the cells expressing the protein, indicative of its solubility and bioactivity. Recently, we have purified this bioactive protein and identified it by Western blot. This year Nanog has been purified and refolded. We intend to use these 3 proteins to reprogram human CD-34 cells into iPS cells. We will also try and use the recombinant Baculo-viruses themselves to generate iPS cells.

通过Yamanaka的突破性出版物在2006年的突破性出版物中发生了巨大变化，他表明可以将小鼠胚胎成纤维细胞（MEFS）通过四个转录因子的强制逆转录病毒介导的表达（10月4日，Sox 2，Klf 4，klf 4和c-Myc）推迟到多能干细胞中。 有了多能细胞的承诺，同时避免了政治和伦理上具有挑战性的胚胎（ES）细胞，该种族正在展示人类细胞的技术。 在一年之内，Yamanakas和James Thompsons群体都表现出具有人类成纤维细胞的类似技术。 Yamanaka使用了相同的4个因素，而汤普森则使用了10月4日，Sox 2，Nanog和Lin 28来重新编程人类体细胞。 观察到体细胞的去分化的过程涉及基于表观遗传的四个外源转录因子的抑制表达，以及伴随性内源基因的较低水平的本构表达。 令人惊讶的是，已经知道对外源性因子的表观遗传抑制的失败与细胞重编程的失败有关。 然而，尽管表观遗传抑制，Yamanaka产生的原始小鼠仍倾向于发展与C-MYC基因重新活化相关的癌症。 此外，由于病毒LTR序列的基因组整合会导致细胞转化和10月4日，并且有时在癌症中升高了纳米，因此似乎不太可能将病毒介导的IPS细胞用于靶向干细胞介导的疗法。 最近的工作表明，尽管IPS细胞中病毒整合的外源转录因子的持续抑制，但与去除病毒整合体的情况相比，IPS细胞的多能量较少。迄今为止，以任何实际的方式去除这些整合物。 因此，似乎直接添加转录因子蛋白本身（在有限的时间段内）将是一个完美的解决方案。 油膏中的苍蝇是以生物活性形式表达这些蛋白质的极大困难，尤其是OCT4，这被认为是最关键的因素。 当在大肠杆菌中表达时，这些转录因子在包容体中被隔离为不溶性无活性蛋白。 这些蛋白质的纯化需要在8M尿素中变性，这使蛋白质完全失活，并且迄今为止，恢复饱和涉及尿素无尿素缓冲液中的20倍稀释。 我们已经复制了此过程，并展示了该技术如何产生降解，并且大多是非活性的OCT4和SOX2蛋白。 为了解决这一问题，我们开发了一个自动化过程，以仔细控制的方式以固体支持方式重新折叠这些蛋白质。 我们已经使用了这些蛋白质，随着这种新过程的重生来产生鼠和人类IPS菌落，它们正确染色，但不足以串行通道。 我们将其归因于OCT4蛋白的不足性递减忠诚度。我们将继续完善该过程，同时我们提交了迄今为止的作品进行出版。 在平行的实验中，我们在基于baculoviral的系统中成功地生产了Oct4和Sox2，据我们所知，迄今为止，世界上任何其他群体都没有完成。 这种蛋白质在昆虫SF-9细胞中生长，仅在表达蛋白质的细胞核中发现，表明其溶解度和生物活性。 最近，我们纯化了这种生物活性蛋白，并通过Western印迹将其鉴定出来。今年的纳米被净化和重新折叠。 我们打算使用这3种蛋白质将人CD-34细胞重新编程为IPS细胞。 我们还将尝试使用重组杆状病毒本身来产生IPS细胞。