Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.
Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.
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DOI：
10.1186/scrt246
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发表时间：
2013-07-26
影响因子：
7.5
通讯作者：
Byrne JA
中科院分区：
文献类型：
--
作者：
Awe JP;Lee PC;Ramathal C;Vega-Crespo A;Durruthy-Durruthy J;Cooper A;Karumbayaram S;Lowry WE;Clark AT;Zack JA;Sebastiano V;Kohn DB;Pyle AD;Martin MG;Lipshutz GS;Phelps PE;Pera RA;Byrne JA
将患者的体细胞重新编程为诱导多能干细胞（iPSC）为未来的自体细胞治疗带来了重大希望。然而，重编程后潜在致癌转基因元件的持续存在代表了临床应用前应解决的安全性问题。多顺反子干细胞盒（STEMCCA）是一种可切除的慢病毒重编程载体，在我们手中提供了解决这一安全问题的最一致的重编程方法。然而，大多数病毒整合发生在基因中，并且STEMCCA重编程载体的整合、表观遗传重编程和切除如何影响这些基因以及这些细胞是否仍然具有临床潜力尚不清楚。在这项研究中，我们使用微阵列和敏感的实时PCR来研究在从成人皮肤成纤维细胞产生人iPSC期间基于内含子的重编程和切除STEMCCA盒后的基因表达变化。使用非限制性线性扩增PCR进行整合位点分析。通过免疫细胞化学、核型分析和畸胎瘤形成对无转基因iPSC进行了充分表征，并实施了当前的方案用于指导分化。我们还利用当前的良好生产实践指南和生产设施将我们的iPSC转化为推定的临床级条件。我们发现，与切除后的干细胞相比，STEMCCA衍生的iPSC系包含单个整合，发现位于活跃转录基因PRPF39的内含子位置，显示出显著增加的表达。通过Cre重组酶切除STEMCCA返回PRPF 39的基础表达水平。这些细胞也显示出具有适当的剪接模式和PRPF 39基因序列。我们还充分表征了切除后的iPSC，将其分化为多种临床相关的细胞类型（包括少突胶质细胞，肝细胞和心肌细胞），并使用美国食品和药物管理局先前批准的将人类胚胎干细胞从研究级转化为临床级状态的相同方法将其转化为推定的临床级条件。这些研究第一次为产生完全表征的无转基因人类iPSC提供了原理证明，并且鉴于当前良好生产规范细胞生产设施的有限可用性，突出了将研究级细胞系转化为用于个性化细胞治疗的临床级生物制剂的有吸引力的潜在机制。
The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
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影响因子：
14.8
作者：
通讯作者：
--
影响因子：
23.9
作者：
Loh YH;Hartung O;Li H;Guo C;Sahalie JM;Manos PD;Urbach A;Heffner GC;Grskovic M;Vigneault F;Lensch MW;Park IH;Agarwal S;Church GM;Collins JJ;Irion S;Daley GQ
通讯作者：
Daley GQ
影响因子：
23.9
作者：
Kim D;Kim CH;Moon JI;Chung YG;Chang MY;Han BS;Ko S;Yang E;Cha KY;Lanza R;Kim KS
通讯作者：
Kim KS
影响因子：
12.3
作者：
Langmead B;Trapnell C;Pop M;Salzberg SL
通讯作者：
Salzberg SL
影响因子：
48
作者：
Jia, Fangjun;Wilson, Kitchener D.;Sun, Ning;Gupta, Deepak M.;Huang, Mei;Li, Zongjin;Panetta, Nicholas J.;Chen, Zhi Ying;Robbins, Robert C.;Kay, Mark A.;Longaker, Michael T.;Wu, Joseph C.
通讯作者：
Wu, Joseph C.