Abstract Stem cells self-renew and differentiate into various cell lineages to sustain tissue homeostasis and functions. Most tissue microenvironments comprise multiple components, including epithelial, stromal, immune, and endothelial cells, that interact with each other to coordinate response to intrinsic and extrinsic stimuli. There remains an unmet need for in vitro models that can capture tissue heterogeneity and enable high-throughput studies. The PI developed Micro-OrganoSpheres (MOS) that can be established rapidly and efficiently from small amounts of primary tissues. Allowing self-renewal and differentiation of adult stem cells to recapitulate key tissue characteristics, MOS also sustain the original tissue microenvironment, enabling both ultra-high-throughput assays aided by deep-learning algorithms and direct microbial-host interactions. For the next R35 period, MOS will be leveraged as a fundamental research tool for understanding interactive processes in heterogeneous tissues in a systematic and scalable manner. Three projects are proposed: (1) We will set up a high-throughput MOS screen to systematically study how different cell types in a heterogenous tissue respond to different microbes. (2) We will develop a MOS combinatorial indexing technique to probe how cell-cell interactions in local microenvironments lead to spatially heterogenous tissue responses. (3) We will use MOS engraftment and intravital imaging to understand cell fate decisions and progression of stem cell niche from development to adulthood. The success of these projects will elucidate the mechanisms by which 1) different cells coordinate locally to respond to stimuli and 2) the stem cell niche progresses during growth. Furthermore, this research will provide the broader scientific community with basic research techniques that can be generalized to understand a wide variety of tissue and stem cell functions.