Reconstruction of Dynamic Mammary Mini Gland in vitro for Normal Physiology and Oncogenesis

Organoid culture has been extensively exploited for normal tissue reconstruction and disease modeling, however, it is still challenging to establish physiologically relevant architecture, size and functions in homeostasis. Here, we describe the development of a long-term adult stem cell derived mammary mini-gland culture system that supports robust 3D outgrowths recapitulating the morphology, cellular context, transcriptional heterogeneity of the normal mammary gland. The self-organization ability of stem cells and the stability of the outgrowths were determined by a coordinated combination of extracellular matrix, environmental signalings and dynamic physiological cycles. These mini glands are hormone responsive and reproduce the entire postnatal mammary development including puberty, estrous cycle, lactation and involution. Most intriguingly, these mini glands maintained the mammary stem cells and represented the fate transition from embryonic bipotency to postnatal unipotency in lineage tracing assays. In addition, induced oncogene expression in the mini glands drove the clinically relevant cancer initiation that can be monitored chronologically. Together, this study provides an experimental system fostering a dynamic organ miniature that can be studied chronologically and spatially for physiologically relevant biological processes with complexity. Single cell RNA profiles of mammary gland basal cells and luminal cells from primary mammary gland and mammary mini gland类器官培养（organoid culture）已被广泛应用于正常组织重建与疾病建模，但在稳态（homeostasis）条件下构建具备生理相关性的结构、尺寸与功能仍颇具挑战。本文报道了一种长期体外培养成人干细胞来源的乳腺迷你腺体（mammary mini-gland）培养体系，该体系可支持稳定的三维（3D）扩增类器官，重现正常乳腺的形态结构、细胞微环境与转录异质性（transcriptional heterogeneity）。干细胞的自组织能力与扩增类器官的稳定性，由细胞外基质（extracellular matrix）、环境信号通路与动态生理周期的协同调控所决定。该迷你腺体可响应激素信号，并完整重现出生后乳腺发育的全过程，包括青春期、动情周期（estrous cycle）、泌乳与乳腺退化（involution）。最引人关注的是，该迷你腺体可维持乳腺干细胞群，并在谱系示踪（lineage tracing）实验中重现了从胚胎期双潜能性到出生后单潜能性的细胞命运转变过程。此外，在迷你腺体中诱导癌基因（oncogene）表达后，可驱动具有临床相关性的肿瘤发生过程，且该过程可被时序性监测。综上，本研究构建了一套可动态培养的器官微型化实验体系，能够在时间与空间维度上研究具备生理相关性的复杂生物学过程。原代乳腺组织与乳腺迷你腺体来源的乳腺基底细胞（basal cells）与腔上皮细胞（luminal cells）的单细胞RNA表达谱（single cell RNA profiles）