Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.
Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APNnull mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/ or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.lung cancer | melanoma | proteolytic activity | shRNA | tumorigenesis A minopeptidase N (APN, CD13; EC 3.4.11.2) is a widely expressed type II membrane-bound metalloprotease (1, 2). It functions in the enzymatic cleavage of peptides, in endocytosis, and as a signaling molecule and has been implicated in the regulation of complex and diverse processes, including cell migration, cell survival, viral uptake, and angiogenesis (3). APN has also been linked specifically to cancer, having been identified as a cellsurface marker for malignant myeloid cells (4-7) and reaching high levels of expression in association with the progression of tumors, including breast, ovarian, and prostate cancer (8-14). Indeed, vascular endothelial growth factor (VEGF), a key angiogenesis regulator, induces the expression of APN at an early stage of tumor growth (15), again highlighting the role of this enzyme in angiogenesis, a process crucial for sustained growth of most solid tumors (16). Studies of bestatin, a CD13 inhibitor and antiangiogenic agent, also suggest that APN enzymatic activity is relevant for tumorigenesis (17,18). Nevertheless, the substrates of APN in the context of angiogenesis are still unknown. The only well-defined substrate is angiotensin III in the renin-angiotensin pathway, in which APN cleaves the NH 2 -terminal arginine residue of angiotensin III to form angiotensin IV. Consistent with several lines of evidence, we have previously identified APN as a target for inhibition of tumor vascularization and growth (18)(19)(20).Tumor growth relies on a complex microenvironment in which malignant cells cooperate with various other cell types: endothelial cells of the blood and lymphatic circulation, mesenchymal stromal cells/cancer-associated fibroblasts, and a variety of bone marrow-derived cells such as myeloid-derived suppressor cells and lymphocytes (21, 22). Some of these cell populations c...
Globally, over 33.2 million people who mostly live in developing countries with limited access to the appropriate medical care suffer from the human immunodeficiency virus (HIV) infection. We developed an on-chip HIV capture and imaging method using quantum dots (Qdots) from fingerprick volume (10 μl) of unprocessed HIV-infected patient whole blood in anti-gp120 antibodyimmobilized microfluidic chip. Two-color Qdots (Qdot525 and Qdot655 streptavidin conjugates) were used to identify the captured HIV by simultaneous labeling the envelope gp120 glycoprotein and its high-mannose glycans. This dual-stain imaging technique using Qdots provides a new and effective tool for accurate identification of HIV particles from patient whole blood without any preprocessing. This on-chip HIV capture and imaging platform creates new avenues for point-of-care diagnostics and monitoring applications of infectious diseases.
Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.
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