Khateeb Jasmin, Liang Jady, Li Yuchong, Thanabalasingam Thenuka, Khang Julie, Jerkic Mirjana, Pellecchia Giovanna, Thiruv Bhooma, Chen Ya-Wen, Rotstein Ori, Slutsky Arthur S, Zhang Haibo
Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Pulmonary Institute, Rambam Health Care Campus, Haifa, Israel; Ruth & Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel.
Commun Biol. 2025 Feb 3;8(1):164. doi: 10.1038/s42003-025-07461-w.
Human induced pluripotent stem cell (iPSC)-derived lung organoids, engineered to carry targeted genes, offer a robust platform for investigating mechanistic insights in lung research. Although lentiviral vectors (LVVs) are highly effective for stable expression due to their integrative properties, achieving efficient transduction in human iPSC-derived lung organoids poses a significant technical challenge, likely due to the complex structure of these organoids. In this study, we optimized a method to enhance LVV transduction efficiency by physically disrupting the organoids to increase surface area, followed by spinoculation to apply shear force during cell dissociation. This approach, combined with the use of an optimized culture medium, significantly improved transduction efficiency. The success of this method was validated at both the gene and protein levels using single-cell RNA sequencing (scRNA-seq) and various cellular and molecular assays. Our optimized transduction protocol may provide a valuable tool for investigating specific cellular and molecular mechanisms in development and disease models using human iPSCs-derived lung organoids.
经过基因工程改造携带靶向基因的人诱导多能干细胞（iPSC）来源的肺类器官，为深入了解肺部研究的机制提供了一个强大的平台。尽管慢病毒载体（LVV）因其整合特性对稳定表达非常有效，但要在人iPSC来源的肺类器官中实现高效转导面临重大技术挑战，这可能是由于这些类器官的结构复杂所致。在本研究中，我们优化了一种方法，通过物理破坏类器官以增加表面积来提高LVV转导效率，随后在细胞解离过程中采用离心接种以施加剪切力。这种方法与使用优化的培养基相结合，显著提高了转导效率。使用单细胞RNA测序（scRNA-seq）以及各种细胞和分子检测方法，在基因和蛋白质水平验证了该方法的成功。我们优化的转导方案可能为利用人iPSC来源的肺类器官研究发育和疾病模型中的特定细胞和分子机制提供一个有价值的工具。
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