Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.
Ontology highlight
ABSTRACT:
SUBMITTER: Herring B
PROVIDER: S-EPMC8734644 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.
| S-EPMC8789283 | biostudies-literature
Project description:The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues of human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 1-2 mm explants and cultured on gelatin sponges for 12 days. Immunohistochemistry revealed that human PDAC explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC explants were treated with Abraxane and we observed different levels of response between patients. PDAC explants were also transfected with polymeric nanoparticles?+?Cy5-siRNA and we observed abundant cytoplasmic distribution of Cy5-siRNA throughout the PDAC explants. Overall, our novel model retains the 3D architecture of human PDAC and has advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis, and no tissue manipulation, digestion, or artificial propagation of organoids. This provides unprecedented opportunity to study PDAC biology including tumour-stromal interactions and rapidly assess therapeutic response to drive personalised treatment.
| S-EPMC7820421 | biostudies-literature
Project description:Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 ?m sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.
| S-EPMC7250419 | biostudies-literature
Project description:Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h-60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.
| S-EPMC8229413 | biostudies-literature
Project description:Recently it was discovered that tissue-resident macrophages derive from embryonic precursors, not only from peripheral blood monocytes, and maintain themselves by self-renewal. Most in-vitro studies on macrophage biology make use of in-vitro cultured human monocyte-derived macrophages. Phagocytosis of IgG-opsonized particles by tissue-resident macrophages takes place via interaction with IgG receptors, the Fc-gamma receptors (FcγRs). We investigated the FcγR expression on macrophages both in-vivo and ex-vivo from different human tissues. Upon isolation of primary human macrophages from bone marrow, spleen, liver and lung, we observed that macrophages from all studied tissues expressed high levels of FcγRIII, which was in direct contrast with the low expression on blood monocyte-derived macrophages. Expression levels of FcγRI were highly variable, with bone marrow macrophages showing the lowest and alveolar macrophages the highest expression. Kupffer cells in the liver were the only tissue-resident macrophages that expressed the inhibitory IgG receptor, FcγRIIB. This inhibitory receptor was also found to be expressed by sinusoidal endothelial cells in the liver. In sum, our immunofluorescence data combined with ex-vivo stainings of isolated macrophages indicated that tissue-resident macrophages are remarkably unique and different from monocyte-derived macrophages in their phenotypic expression of IgG receptors. Tissue macrophages show distinct tissue-specific FcγR expression patterns.
| S-EPMC6793881 | biostudies-literature
Project description:The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.
| S-EPMC7555450 | biostudies-literature
Project description:BackgroundAlthough the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear.MethodsWe transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR.ResultsWe found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4me3 mark at transcriptional start sites.ConclusionsOur investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.
| S-EPMC5846061 | biostudies-literature
Project description:MotivationFunctional relationship networks, which summarize the probability of co-functionality between any two genes in the genome, could complement the reductionist focus of modern biology for understanding diverse biological processes in an organism. One major limitation of the current networks is that they are static, while one might expect functional relationships to consistently reprogram during the differentiation of a cell lineage. To address this potential limitation, we developed a novel algorithm that leverages both differentiation stage-specific expression data and large-scale heterogeneous functional genomic data to model such dynamic changes. We then applied this algorithm to the time-course RNA-Seq data we collected for ex vivo human erythroid cell differentiation.ResultsThrough computational cross-validation and literature validation, we show that the resulting networks correctly predict the (de)-activated functional connections between genes during erythropoiesis. We identified known critical genes, such as HBD and GATA1, and functional connections during erythropoiesis using these dynamic networks, while the traditional static network was not able to provide such information. Furthermore, by comparing the static and the dynamic networks, we identified novel genes (such as OSBP2 and PDZK1IP1) that are potential drivers of erythroid cell differentiation. This novel method of modeling dynamic networks is applicable to other differentiation processes where time-course genome-scale expression data are available, and should assist in generating greater understanding of the functional dynamics at play across the genome during development.Availability and implementationThe network described in this article is available at http://guanlab.ccmb.med.umich.edu/stageSpecificNetwork.
| S-EPMC4296147 | biostudies-literature
Project description:Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4(+) T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4(+) T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4(+) T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.
| S-EPMC525063 | biostudies-literature
Project description:We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer's patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer's Disease which are characterized by abnormal iron accumulation in the brain.
| S-EPMC5150947 | biostudies-literature