Gene expression profiles of human patient derived ectocervical stem cells and 3D organoids with and without Chlamydia trachomatis infection

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modelled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renew capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrate the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression. Patient-derived human ectocervical epithelial stem cells were cultured in 2D and as 3D organoids as described in Chumduri et al. 2021, Nature Cell Biology. The 2D stem cells were genetically manipulated to introduce HPV16 E6E7 oncogenes into their genome to mimic HPV persistence in vivo. The HPV negative and positive 2D stem cells and 3D organoids were either uninfected or infected with Chlamydia trachomatis for 2 days or 5days, respectively as described in this manuscript. Total RNA was extracted from samples and hybridized on Agilent-8x60k Human custom arrays as single-color hybridizations.