論文

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo–grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5β1 to α2β1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Primary hepatocytes are widely recognized for their ability to accurately represent the in vivo hepatocyte phenotype. However, traditional avascular primary hepatocyte culture models are limited by inadequate mass transfer, which leads to a rapid decline in hepatocyte function and survival. To address these challenges, vascularization of hepatic spheroids is crucial for enhancing oxygen and nutrient supply, thereby enabling the construction of larger and more complex hepatic tissues in vitro. In this study, we achieved vascularization of hepatic spheroids containing freshly isolated primary hepatocytes by incorporating fibroblasts as a source of paracrine factors to induce angiogenesis. Multicellular spheroids composed of primary hepatocytes and fibroblasts were formed in non-adhesive concave wells, and one of the spheroids was subsequently embedded in a fibrin-collagen hydrogel within a microfluidic device. Endothelial cells were then seeded onto adjacent microfluidic channels. They formed microvascular networks that extended toward and penetrated the hepatic spheroid. The vascularized hepatic spheroid closely mimicked hepatic sinusoids, with hepatocytes in close contact with microvessels. Moreover, the vascularized spheroid exhibited significantly enhanced hepatic function, specifically albumin secretion and urea synthesis. Our findings provide insights into the establishment of highly vascularized hepatic spheroids in vitro, which is crucial for constructing scalable hepatic tissues in the context of biofabrication.

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic and adult skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of skin, including nucleated/enucleated epithelium, mesenchyme, and hair follicle, as well as the role of collagens in maintaining its integrity. Furthermore, quantitative analysis comparing stiffness distributions in live tissue samples with those in preserved tissues, including formalin-fixed and cryopreserved tissue samples, unveiled the distinct impacts of preservation processes on tissue stiffness patterns. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples to accurately capture the true spatiotemporal variations in mechanical properties. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. Statement of significance: Stiffness, a simple mechanical parameter, has drawn attention in understanding the mechanobiological principles underlying the homeostasis and pathology of living tissues. To explore tissue-scale mechanobiology, we propose a technique integrating an upright structured illumination microscope and an atomic force microscope. This technique enables live measurements of stiffness distribution and fluorescent observation of thick living tissue slices. Experiments revealed the highly heterogeneous mechanical nature of mouse embryonic and adult skin in three dimensions and the previously unnoticed influences of preservation techniques on the mechanical properties of tissue at microscopic resolution. This study provides a new technical platform for live stiffness measurement and biomolecular observation of tissue-scale samples with micron-scale resolution, thus contributing to future studies of tissue- and organ-scale mechanobiology.