利用擴展多能干細胞培養系統產生豬誘導多能干細胞

2019年6月，美國德州大學西南醫學中心分子生物系，云南省動物基因編輯與克隆重點實驗室，生物科學學院農業生物技術國家重點實驗室

（Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province, Kunming 650201, China State Key Laboratory for Agrobiotechnology, College of Biological Sciences,） Hongjiang Wei and Jianyong Han研究團隊，在《Stem Cell Research & Therapy》發表論文，標題為：“Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system”

“利用擴展多能干細胞培養系統產生豬誘導多能干細胞”

Abstract

Background: Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models.   In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials.   Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus.   Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.

Methods: iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium.   The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities.   Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.

Results: In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs.   Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture.   The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint).   PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro.   The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively.   Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance.   And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways.   Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses.   Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.

Conclusions: Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo.   However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

摘要

背景:豬已經成為生物醫學研究中****的大型動物模型之一，在許多情況下，豬被認為是嚙齒動物模型的優越選擇。此外，使用豬多能干細胞衍生物的移植研究可以作為人體試驗之前安全性和有效性的測試平臺。最近有研究表明，小鼠和人PS細胞在LCDM(重組人LIF、CHIR 99021、(S)-(+)-馬酸二甲基丁烯、鹽酸米諾環素)培養基中培養，表現出擴展的發育潛力(被稱為擴展多能干細胞，或EPS細胞)，可以在嵌合小鼠受孕過程中產生胚胎和胚胎外組織。LCDM培養基能否產生穩定的豬誘導多能干細胞及其嵌合能力尚不清楚。

方法:用編碼Oct4、Sox2、Klf4和cMyc重編程因子的逆轉錄病毒載體感染豬周細胞(PC)和胚胎成纖維細胞(PEFs)產生iPS細胞，然后在改良的LCDM培養基中培養。通過檢測多能性相關轉錄因子和表面標記物的表達、轉錄組分析以及體外和體內分化能力來表征PC-iPS和PEF-iPS細胞的多能性。用熒光顯微鏡、流式細胞術和PCR分析評估PC-iPS細胞對小鼠和豬受孕的嵌合貢獻。

結果:在本研究中，使用改良版的LCDM培養基，我們成功地從pc和pef中生成了iPS細胞。PC-iPS和PEF-iPS細胞在長期培養后均保持穩定的“圓頂”形態和基因組穩定性。免疫細胞化學分析顯示，PC-iPS和PEF-iPS細胞均表達OCT4、SOX2和SALL4，但PC-iPS細胞僅表達NANOG和TRA-1-81(微弱)。PC-iPS和PEF-iPS細胞在體外均可分化為三種原胚層的細胞衍生物。轉錄組分析顯示，PEF-iPS細胞和PC-iPS細胞與豬ICM、Heatmap和volcano圖聚集，結果顯示PC-iPS細胞與PEF-iPS細胞之間差異表達基因(deg)為1475個(調整p值< 0.1)，PC-iPS細胞中差異表達基因的數量分別為755個和720個。上調的基因被富含氧化石墨烯，包括干細胞分化、增殖、發育和維持的調控。KEGG通路中表達上調基因的富集揭示了Wnt、Jak-STAT、TGF-β、P53、MAPK等干細胞信號通路。利用豬mtdna特異性引物和GFP引物進行熒光顯微鏡和基因組PCR分析表明，PC-iPS細胞衍生物可以在小鼠和豬著床前囊胚和著床后胚胎中檢測到。流式細胞術定量分析顯示，豬PC-iPS細胞在小鼠受孕中的嵌合貢獻高達0.04%。

結論:我們的研究結果表明，在LCDM培養基中可以產生穩定的iPS細胞，在體內可以產生胚胎細胞和胚胎外細胞。然而，豬LCDM-iPS細胞嵌合的效率和水平較低。

該論文中，誘導多能干細胞實驗（豬周細胞(PC)和胚胎成纖維細胞(PEFs)產生iPS細胞）的體外培養是使用Ausbian特級胎牛血清完成的。欲了解或購買Ausbian特級胎牛血清可以聯系北京締一生物400-166-8600.