2026 Theses Doctoral

Engineering a human intestinal model for anaerobe co-culture and epithelial reprogramming

Zhou, Jessica

This dissertation addresses a limitation in many intestinal in vitro models, namely the difficulty of studying human epithelial biology together with obligate anaerobic gut commensals under controlled, reproducible conditions. Conventional organoids capture key features of intestinal epithelium but restrict luminal access, lack defined gradients, and show substantial donor and maturation variability, while most co-culture systems struggle to maintain stable anaerobic conditions without compromising epithelial viability. This work therefore pursued two complementary engineering goals: to build a human intestinal platform that supports anaerobe co-culture at an anoxic luminal interface with an oxygenated basolateral compartment, enabling isolated culture of Candidatus Cibiobacter qucibialis with human colon epithelium, and to develop a transcription factor driven strategy that can generate more standardized intestinal epithelia from pluripotent stem cells.

The device was fabricated by soft-lithography to generate reusable PDMS stamps and then used to micromold a crypt scale collagen scaffold within a modified cell culture insert, creating separated luminal and basal compartments. Briefly, colon pattern photomasks were designed in CAD and used to fabricate SU-8 features on silicon wafers, followed by PDMS casting to produce stamps that transfer an array of circular crypt-like openings into neutralized collagen. The patterned collagen was crosslinked to stabilize the topography, soaked to remove residual crosslinker, and sterilized before seeding epithelial cells.

To enable anaerobe co culture, a polycarbonate diffusion barrier was incorporated to reduce oxygen ingress into the luminal compartment and the apical chamber was sealed with an oxygen impermeable butyl rubber lid while the basal chamber remained oxygenated. Functional validation of the anoxic-oxic configuration was performed by inoculating obligate anaerobes onto the apical side of confluent epithelialized devices, confirming bacterial identity and purity by 16S rRNA gene sequencing and observing robust anaerobe growth over the culture period, consistent with maintenance of a sufficiently low oxygen luminal environment while preserving epithelial integrity.

For host microbe experiments, de-identified human sigmoid colon biopsies were processed to isolate epithelial crypts, which were expanded on collagen scaffolds and seeded onto compartmentalized devices. Ca. qucibialis was grown anaerobically and applied to the apical compartment in defined media conditions. Host and bacterial RNA were harvested after co-culture for RNA sequencing. Host transcriptional responses across donors were analyzed using differential expression and gene set enrichment analysis, followed by cross-donor integration through meta-analysis and Fisher-combined significance. Shared pathways were organized into modules by clustering on Jaccard similarity of consensus leading-edge gene sets. Bacterial transcriptional changes were interpreted using a tissue-media attribution design that separates tissue exposure effects from media formulation effects, followed by functional categorization of differentially expressed bacterial genes.

The results establish an ex vivo human colon co-culture approach that supports anaerobe exposure and enables coordinated host and bacterial transcriptome profiling. Epithelial responses resolved into shared modules that included cilium associated programs, cytoskeletal remodeling, and Ca2+ linked signaling and regulated secretion adjacent annotations, alongside downregulated glycolysis and cellular housekeeping pathways, while donor to donor variation organized around a small set of recurring response axes rather than unrelated profiles. Bacterial differential expression was driven primarily by tissue exposure and highlighted host facing nutrient and interface programs, including iron related signatures and induction of corrinoid biosynthesis genes.

Finally, a doxycycline inducible transcription factor strategy in hESC-derived definitive endoderm supported the generation and maintenance of intestinal organoids with minimal morphogen exposure, providing a foundation for more reproducible epithelial starting states. Briefly, H9 hESCs were engineered with a doxycycline-inducible CRISPRa system by PiggyBac integration of TRE-dCas9-VPR with an mCitrine reporter, followed by blasticidin selection and monoclonal line isolation. Clones were then transduced with modified CROP-seq lentiviral vectors to deliver sgRNAs with neomycin selection. In parallel, DOX-inducible ORF constructs for HNF4A and FOXA3 (pCW57) were delivered by lentivirus and selected to generate monoclonal or polyclonal populations.

Reprogrammed organoids were validated by combining early qPCR fate signals with longer term phenotyping after 3D embedding. CDX2 and OTX2 were used to assess posterior intestinal bias versus anterior drift across patterning conditions, and organoids generated under DOX only TF induction were expanded for at least one month and evaluated by immunofluorescence for epithelial organization and lineage marker expression. Across conditions, staining supported formation of epithelial organoids with intestinal associated markers and persistence of DOX dependent Cas9 signal within the epithelial lining, indicating that the inducible program remained active during organoid maintenance and that stable intestinal like structures can be sustained without continuous exogenous gut patterning cues beyond definitive endoderm.

Collectively, this work outlines a practical experimental platform and analysis framework for mechanistic, human relevant studies of anaerobic commensals and for future work that links transcriptomic motifs to functional measures of barrier biology, nutrient exchange, and epithelial state.