Optimized gene transduction in human lung organoids: A high-efficiency method for advanced research applications.
Jasmin Khateeb, Jady Liang, Yuchong Li, Thenuka Thanabalasingam, Julie Khang, Mirjana Jerkic, Giovanna Pellecchia, Bhooma Thiruv, Ya-Wen Chen, Ori Rotstein, Arthur S Slutsky, Haibo Zhang
Author Information
Jasmin Khateeb: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada. ORCID
Jady Liang: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Yuchong Li: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Thenuka Thanabalasingam: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Julie Khang: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Mirjana Jerkic: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Giovanna Pellecchia: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada. ORCID
Bhooma Thiruv: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada. ORCID
Ya-Wen Chen: Black Family Stem Cell Institute, Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York city, New York, USA. ORCID
Ori Rotstein: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Arthur S Slutsky: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada.
Haibo Zhang: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, Toronto, ON, Canada. haibo.zhang@unityhealth.to. ORCID
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.
