Yu-Hwai Tsai

Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells

Summary We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.

Identification, isolation, and characterization of human LGR5-positive colon adenoma cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. Summary: Immunohistochemical and transcriptomic analyses of organoids generated from precancerous adenoma, colon adenocarcinoma and normal human tissue shows that the intestinal stem cell marker LGR5 is a colon cancer prognostic factor.

The Lineage-Specific Transcription Factor CDX2 Navigates Dynamic Chromatin to Control Distinct Stages of Intestine Development

Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic-loss of CDX2 triggers homeotic transformation of intestinal fate, while adult-onset Cdx2-loss compromises critical physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program, but rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or maturity. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

A Method for Cryogenic Preservation of Human Biopsy Specimens and Subsequent Organoid Culture

Human tissue–derived gastrointestinal (GI) organoids have revolutionized the study of human biology, and are powerful tools for studying human physiology and disease; however, generation of organoids is limited by access to human tissue and a short window of viability for human samples, putting a hard limit on the time and place in which a patient sample can be used for research. These restraints mean that a laboratory must be relatively geographically close to the source of collection to use the sample within the window of viability. Patient-derived organoids also are being used for drug development, stem cell therapies, and personalized medicine; however, it is not always feasible to prospectively develop organoid line efforts given the time and labor involved. To overcome these limitations, we sought to develop a practical method to cryopreserve live human biopsy tissue, which then could be stored or shipped frozen and later thawed to generate new cultures of GI epithelium-only organoids (also referred to as enteroids/colonoids). Here, we describe a simple and robust method to cryopreserve human biopsy specimens that subsequently could be

Integrated growth factor signaling promotes lung epithelial progenitor cell expansion and maintenance in mice and humans

During lung branching morphogenesis, a multipotent progenitor population resides at the tips of bifurcating epithelial tubes that give rise to all lung epithelial cell types (1). Previously (2,3), we demonstrated that human pluripotent stem cells (hPSCs) could differentiate into human lung organoids (HLOs), which possessed airway-like structures and alveolar cells, but it was unclear if HLOs possessed a bona fide tip-progenitor population. The goal of the current study was to understand how tip-progenitors are regulated such that this population could be induced in HLO cultures. Using isolated embryonic mouse and human fetal tip-progenitors, we identified factors that promoted long-term growth and maintenance of this population in vitro. Our results showed significant functional and expression differences between mouse and human tip-progenitors, and demonstrated de novo induction of a robust population of budding, SOX9+ progenitor-like cells in HLOs. Abbreviations BMP Bone Morphogenic Protein FGF Fibroblast Growth Factor RA All-Trans Retinoic Acid HLO Human Lung OrganoidThe bud tip epithelium of the branching mouse and human lung contains multipotent progenitors that are able to self-renew and give rise to all mature lung epithelial cell types. The current study aimed to understand the developmental signaling cues that regulate bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified that FGF7, CHIR-99021 and RA maintained isolated human fetal lung bud tip progenitor cells in an undifferentiated state in vitro, and led to the induction of a 3-dimensional lung-like epithelium from hPSCs. 3-dimensional hPSC-derived lung tissue was initially patterned, with airway-like interior domains and bud tip-like progenitor domains at the periphery. Bud tip-like domains could be isolated, expanded and maintained as a nearly homogeneous population by serial passaging. Comparisons between human fetal lung bud tip cells and hPSC-derived bud tip-like cells were carried out using immunostaining, in situ hybridization and transcriptome-wide analysis, and revealed that in vitro derived tissue was highly similar to native lung. hPSC- derived bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed and maintained multi-lineage potential. Furthermore, hPSC- derived bud tip progenitors had successful short-term engrafment into the proximal airways of injured immunocompromised NSG mouse lungs, where they began to express markers of proximal airway cells.