In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Alteration of mechanical stresses in the murine brain by age and hemorrhagic stroke.

Residual mechanical stresses, also known as solid stresses, emerge during rapid differential growth or remodeling of tissues, as observed in morphogenesis and tumor growth. While residual stresses typically dissipate in most healthy adult organs, as the growth rate decreases, high residual stresses have been reported in mature, healthy brains. However, the origins and consequences of residual mechanical stresses in the brain across health, aging, and disease remain poorly understood. Here, we utilized and validated a previously developed method to map residual mechanical stresses in the brains of mice across three age groups: 5-7 days, 8-12 weeks, and 22 months. We found that residual solid stress rapidly increases from 5-7 days to 8-12 weeks and remains high in mature 22 months mice brains. Three-dimensional mapping revealed unevenly distributed residual stresses from the anterior to posterior coronal brain sections. Since the brain is rich in negatively charged hyaluronic acid, we evaluated the contribution of charged extracellular matrix (ECM) constituents in maintaining solid stress levels. We found that lower ionic strength leads to elevated solid stresses, consistent with its unshielding effect and the subsequent expansion of charged ECM components. Lastly, we demonstrated that hemorrhagic stroke, accompanied by loss of cellular density, resulted in decreased residual stress in the murine brain. Our findings contribute to a better understanding of spatiotemporal alterations of residual solid stresses in healthy and diseased brains, a crucial step toward uncovering the biological and immunological consequences of this understudied mechanical phenotype in the brain.

CD300e: Emerging role and mechanism as an immune-activating receptor.

As a transmembrane protein, CD300e is primarily expressed in myeloid cells. It belongs to the CD300 glycoprotein family, functioning as an immune-activating receptor. Dysfunction of CD300e has been suggested in many diseases, such as infections, immune disorders, obesity, and diabetes, signifying its potential as a key biomarker for disease diagnosis and treatment. This review is aimed to explore the roles and potential mechanisms of CD300e in regulating oxidative stress, immune cell activation, tissue damage and repair, and lipid metabolism, shedding light on its role as a diagnostic marker or a therapeutic target, particularly for infections and autoimmune disorders.

Emergence of nanoscale viscoelasticity from single cancer cells to established tumors.

Tumors are complex materials whose physical properties dictate growth and treatment outcomes. Recent evidence suggests time-dependent physical properties, such as viscoelasticity, are crucial, distinct mechanical regulators of cancer progression and malignancy, yet the genesis and consequences of tumor viscoelasticity are poorly understood. Here, using Wide-bandwidth AFM-based ViscoElastic Spectroscopy (WAVES) coupled with mathematical modeling, we probe the origins of tumor viscoelasticity. From single carcinoma cells to increasingly sized carcinoma spheroids to established tumors, we describe a stepwise evolution of dynamic mechanical properties that create a nanorheological signature of established tumors: increased stiffness, decreased rate-dependent stiffening, and reduced energy dissipation. We dissect this evolution of viscoelasticity by scale, and show established tumors use fluid-solid interactions as the dominant mechanism of mechanical energy dissipation as opposed to fluid-independent intrinsic viscoelasticity. Additionally, we demonstrate the energy dissipation mechanism in spheroids and established tumors is negatively correlated with the cellular density, and this relationship strongly depends on an intact actin cytoskeleton. These findings define an emergent and targetable signature of the physical tumor microenvironment, with potential for deeper understanding of tumor pathophysiology and treatment strategies.

Multiscale elasticity mapping of biological samples in 3D at optical resolution.

The mechanical properties of biological tissues have emerged as an integral determinant of tissue function in health and disease. Nonetheless, characterizing the elasticity of biological samples in 3D and at high resolution remains challenging. Here, we present a µElastography platform: a scalable elastography system that maps the elastic properties of tissues from cellular to organ scales. The platform leverages the use of a biocompatible, thermo-responsive hydrogel to deliver compressive stress to a biological sample and track its resulting deformation. By surrounding the specimen with a reference hydrogel of known Young's modulus, we are able to map the absolute values of elastic properties in biological samples. We validate the experimental and computational components of the platform using a hydrogel phantom and verify the system's ability to detect internal mechanical heterogeneities. We then apply the platform to map the elasticity of multicellular spheroids and the murine lymph node. With these applications, we demonstrate the platform's ability to map tissue elasticity at internal planes of interest, as well as capture mechanical heterogeneities neglected by most macroscale characterization techniques. The µElastography platform, designed to be implementable in any biology lab with access to 3D microscopy (e.g., confocal, multiphoton, or optical coherence microscopy), will provide the capability to characterize the mechanical properties of biological samples to labs across the large community of biological sciences by eliminating the need of specialized instruments such as atomic force microscopy. STATEMENT OF SIGNIFICANCE: Understanding the elasticity of biological tissues is of great importance, but characterizing these properties typically requires highly specialized equipment. Utilizing stimulus-responsive hydrogels, we present a scalable, hydrogel-based elastography method that uses readily available reagents and imaging modalities to generate resolved maps of internal elasticity within biomaterials and biological samples at optical resolution. This new approach is capable of detecting internal stiffness heterogeneities within the 3D bulk of samples and is highly scalable across both imaging modalities and biological length scales. Thus, it will have significant impact on the measurement capabilities of labs studying engineered biomaterials, mechanobiology, disease progression, and tissue engineering and development.