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Page 265 of 548-
Advances in Hydrogels in Organoids and Organs-on-a-Chip.
Significant advances in materials, microscale technology, and stem cell biology have enabled the construction of 3D tissues and organs, which will ultimately lead to more effective diagnostics and therapy. Organoids and organs-on-a-chip (OOC), evolved from developmental biology and bioengineering principles, have emerged as major technological breakthrough and distinct model systems to revolutionize biomedical research and drug discovery by recapitulating the key structural and functional complexity of human organs in vitro. There is growing interest in the development of functional biomaterials, especially hydrogels, for utilization in these promising systems to build more physiologically relevant 3D tissues with defined properties. The remarkable properties of defined hydrogels as proper extracellular matrix that can instruct cellular behaviors are presented. The recent trend where functional hydrogels are integrated into organoids and OOC systems for the construction of 3D tissue models is highlighted. Future opportunities and perspectives in the development of advanced hydrogels toward accelerating organoids and OOC research in biomedical applications are also discussed.
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Finnish type of sialic acid storage disease with sialuria (Salla disease): the occurrence and diagnostic significance of cytoplasmic vacuoles in blood lymphocytes.
In this study peripheral blood smears from 29 patients (17 males and 12 females; mean age 28 years, range 3-65 years) with a confirmed diagnosis of the Finnish type of sialic acid storage disease (FSASD) and 200 controls with mental retardation without any evidence of metabolic disease were examined for the presence of vacuolated lymphocytes. Urine samples were analysed by thin-layer chromatography for free sialic acid. Only 62% of the patients with FSASD had a clearly increased percentage of vacuolated lymphocytes (greater than normal mean + 2 s.d.). In thin-layer chromatography all the FSASD patients gave a positive test result. No false positive or negative results were obtained. Electronmicroscopical examination of peripheral blood lymphocytes demonstrated only non-specific changes in a few cells. Examination of peripheral lymphocytes for vacuoles is not a reliable screening test for FSASD. The screening method of choice is the analysis of free sialic acid by thin-layer chromatography.
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[Morphology and diagnosis of Zellweger syndrome. A contribution to combined cytochemical-finestructural identification of peroxisomes in autopsy material and frozen liver tissue with case report].
A female newborn, the second child of healthy non consanguineous parents, exhibited muscular hypotonia, areflexia, apathy, seizures, hepatomegaly and failure to thrive since birth. The peculiar skull shape was lacking. In the urine pipecolic acid and trihydroxycoprostanoic acid were excreted. At the age of seven weeks she died of bronchopneumonia. Lightmicroscopy revealed malformations and deficiency of myelinisation in the brain, renal cysts and fatty metamorphosis in the enlarged liver, which showed only minimal siderosis. Ultrastructurally no peroxisomes could be found in liver and kidney. No peroxisomes were detected by histochemical demonstration of catalase in frozen liver tissue which was taken immediately after death and stored for three months. Absence of peroxisomes is pathognomonic for the cerebro-hepato-renal syndrome of Zellweger and occurs in the liver irrespective of duration and degree of liver damage. It is best demonstrated by enzymehistochemical electron microscopy. With this method peroxisomes can be visualized even 30 h post mortem. In deep frozen normal liver tissue the activity of catalase remains very stable and enables the identification of peroxisomes even after a 12 months period of storage. In the cerebro-hepato-renal syndrome of Zellweger, frozen liver tissue should be stored for biochemical and diagnostic enzymehistochemical studies.
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Vacuolation of an identified peptidergic (proctolin-containing) neuron.
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Changes in perikaryal organelles during axonal regeneration in goldfish retinal ganglion cells: an analysis of protein synthesis and routing.
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USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids.
There is an emblematic clinical and genetic heterogeneity associated with inherited retinal diseases (IRDs). The most common form is retinitis pigmentosa (RP), a rod-cone dystrophy caused by pathogenic variants in over 80 different genes. Further complexifying diagnosis, different variants in individual RP genes can also alter the clinical phenotype. USH2A is the most prevalent gene for autosomal-recessive RP and one of the most challenging because of its large size and, hence, large number of variants. Moreover, USH2A variants give rise to non-syndromic and syndromic RP, known as Usher syndrome (USH) type 2, which is associated with vision and hearing loss. The lack of a clear genotype-phenotype correlation or prognostic models renders diagnosis highly challenging. We report here a long-awaited differential non-syndromic RP and USH phenotype in three human disease-specific models: fibroblasts, induced pluripotent stem cells (iPSCs), and mature iPSC-derived retinal organoids. Moreover, we identified distinct retinal phenotypes in organoids from multiple RP and USH individuals, which were validated by isogenic-corrected controls. Non-syndromic RP organoids showed compromised photoreceptor differentiation, whereas USH organoids showed a striking and unexpected cone phenotype. Furthermore, complementary clinical investigations identified macular atrophy in a high proportion of USH compared with RP individuals, further validating our observations that USH2A variants differentially affect cones. Overall, identification of distinct non-syndromic RP and USH phenotypes in multiple models provides valuable and robust readouts for testing the pathogenicity of USH2A variants as well as the efficacy of therapeutic approaches in complementary cell types.
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Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons.
BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.
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Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.
Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.
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Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge.
Monospecific antibodies against the homogeneous Ca++ dependent regulatory protein of cyclic nucleotide phosphodiesterase (CDR protein) from bovine brain were used in indirect immunofluorescence microscopy to visualize the cytoplasmic organization of this key regulatory protein in growing tissue culture cells. Although cells during interphase reveal only a weak general cytoplasmic fluorescence, a dramatic reorganization of CDR protein occurs with the onset of mitosis. Throughout the different mitotic stages CDR protein is strongly concentrated in the two polar parts of each half spindle. After completion of telophase (CDR protein appears at both cytoplasmic ends of the intercellular bridge which still connects the two daughter cells. Parallel use of monospecific antibodies against CDR protein and tubulin emphasizes the spatial restriction in the localization of CDR protein during mitosis and early G1 phase of the cell cycle.
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Patient-Derived Organoids from Multiple Sites of a Single Tumor Recapitulates Intratumoral Heterogeneity in Patients with Gastric Cancer.
BACKGROUND/AIMS: Patient-derived organoids (PDOs) are promising preclinical models that replicate critical tumor features. However, intratumoral heterogeneity challenges the clinical utility of PDOs, especially in capturing diverse tumor cell subpopulations. METHODS: Single-cell transcriptomics was used to analyze PDOs from distinct sites within a single gastric cancer tumor, aiming to assess their ability to reflect intratumoral heterogeneity. RESULTS: The PDOs displayed similarities in gene expression but also exhibited distinct profiles. Single-cell analysis of PDOs revealed upregulation of markers for neuroendocrine tumors, which was validated via immunohistochemistry staining of neuron-specific enolase in the primary tumor. Notably, heat shock proteins showed significant variability among the PDOs, impacting immune responses. Tumors with abundant heat shock proteins are reported to have increased cytotoxic T cell activity. CONCLUSIONS: Intratumoral heterogeneity poses challenges for PDO-based models, highlighting the need for comprehensive assessment. Despite their limitations, PDOs offer valuable insights into precision medicine for patients with gastric cancer, aiding in the development of therapeutic strategies.