Biology subjects

Rahbari, N.

Publications and source records attributed to Rahbari, N..

7 recordsLinked to original sources

bioRxiv2025

CD271 sorting for improved liver cell isolation: Semiautomated and simultaneous preparation of parenchymal and non-parenchymal cells from mouse and human livers

BackgroundA detailed understanding of the dynamic fate changes of hepatocytes, hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC) is critical for studying liver (patho)physiology during disease progression. Current isolation methods often focus on single cell types, limiting utility in comprehensive research. AimTo develop a novel, semi-automated protocol for the simultaneous isolation of hepatocytes and non-parenchymal cells (NPCs), including HSC, KC, and LSEC, from mouse and human, with high yield, purity, and viability from healthy and diseased livers. MethodThe protocol employs a two-step EGTA and collagenase II perfusion for tissue digestion. Hepatocytes were isolated by low-speed centrifugation and a Percoll gradient. Subsequently, magnetic-activated cell separation, using CD271 as a selective surface marker for HSC, CD11b for KC and CD146 for LSEC) was performed. Validation was achieved with immunofluorescence staining, flow cytometry, RT-PCR, and UV fluorescence, whereby yield, purity, and viability were assessed. ResultsWith our method, yield of hepatocytes, HSC, KC, and LSEC, is 33.4{+/-}5.5x10, 5.2{+/-}6.3x10, 12.4{+/-}4.8x10 and 18.2{+/-}8.9x10 cells per healthy mouse liver, respectively, with cell viabilities exceeding 89%, and purity surpassing 90%. CD271 was validated as an effective marker for purifying HSC in healthy and diseased human (n=4-6) and mouse livers. Compared to microfluidic and organ-on-a-chip approaches, with our protocol, we achieve higher yield and purity values while enabling the simultaneous isolation of multiple cell types from a single sample. ConclusionOur semi-automated protocol offers a scalable, reliable, and versatile solution for isolating main liver cell types with high yield, purity, and viability from both healthy and diseased tissues, advancing liver research and facilitating downstream investigations. Impact and implicationsO_LIBroad applicability: The CD271-based method efficiently isolates key liver cell types (Hepatocyte, HSC, KC, LSEC) simultaneously. C_LIO_LIVersatility in disease models: Effective for studying healthy, fibrotic, and damaged liver tissues. C_LIO_LIRobust across variability: Works reliably across different mouse strains, age groups, and conditions. C_LIO_LIHuman research potential: Scalable for high-purity isolation from human liver tissue, enabling translational studies. C_LIO_LIHigh-quality results: Ensures >85% viability and >90% purity, supporting reproducible liver research applications. C_LI

cell biology↗

bioRxiv2024

Modulatory Effects of M3 Muscarinic Acetylcholine Receptor on Inflammatory Profiles of Human Memory T Helper Cells

Memory T helper (Th) cells, generated after immunogenic challenge, are crucial in directing the adaptive immune response. Muscarinic ACh receptor (mAChR) subtypes expressed by immune cells can be stimulated with acetylcholine or muscarinic-selective drug oxotremorine-M. Cholinergic signaling can influence immune cells, but it is not known how cholinergic stimuli regulate memory Th cells. This study focused on the role of mAChRs, specifically the M3 muscarinic ACh receptor (M3R), in the cytokine profile and NF-{kappa}B p65 activity of primary human memory Th cells. Memory Th cells (CD3+CD4+CD45RA-CD45RO+) were isolated from healthy participants peripheral blood. Cell culture was performed with anti-CD3/anti-CD28/anti-CD2 reagent, oxotremorine-M (M1R-M5R agonist), atropine (M1R-M5R antagonist), and J104129 (M3R-selective antagonist). MR1-MR5 genes CHRM1-CHRM5 were measured with RT-qPCR. Protein expression of M3R and phosphorylated NF-{kappa}B p65 were quantified by Western blot. The secretion of IFN-{gamma}, IL-17A, and IL-4 was assessed by ELISA and intracellular cytokine staining flow cytometry. CHRM3, encoding M3R, was knocked out using CRISPR-Cas9 gene targeting. Memory Th cells expressed all five mAChR subtypes. Oxotremorine-M increased IFN-{gamma} and IL-17A while reducing IL-4 in an atropine-sensitive manner. Stimulation of mAChRs in cells with CHRM3-knockout or M3R blockade prevented increases in IFN-{gamma} and IL-17A but continued to inhibit IL-4. mAChR stimulation enhanced NF-{kappa}B p65 activity without affecting cell proliferation, viability, or M3R expression. This investigation demonstrates that muscarinic signaling increases the pro-inflammatory profile of memory Th cells, including NF-{kappa}B p65, IFN-{gamma}, and IL-17A, with a reduction in IL-4. Focusing on M3R blockers could modulate adaptive immune responses and alleviate immune-related conditions.

immunology↗

bioRxiv2024

Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumourigenesis

Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. Regulating cell identity requires gene activation and repression. While master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether unwanted plasticity is actively suppressed by lineage-specific repressors. Here, we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that Prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternate fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and acted as a tumour suppressor in multiple liver cancer models. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo, and promoted transition of hepatocellular carcinoma to cholangiocarcinoma, conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide mechanistic evidence for PROX1 as a hepatocyte-specific safeguard and support a model where individual cell type-specific repressors actively suppress plasticity throughout life to safeguard lineage choice and prevent disease.

cancer biology↗

bioRxiv2024

β2-Adrenergic Biased Agonist Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner.

IntroductionAdrenergic receptors regulate metabolic, cardiovascular and immunological functions in response to the sympathetic nervous system. The effect of {beta}2-adrenergic receptor (AR) as a high-expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional {beta}2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells highlighting its potential for IL-17A suppression via a non-canonical {beta}2AR cell-signaling pathway. MethodsThe effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with antiCD3/antiCD28/antiCD2 ImmunoCult reagent. IFN-{gamma}, IL-4, and IL-17A which are primarily derived from Th1, Th2, and Th17 cells respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated-serine133-CREB and phosphorylated-NF-{kappa}B p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. ResultsNebivolol treatment decreased IL-17A and IFN-{gamma} secretion by activated-memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the {beta}2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells towards the Th17 phenotype. IL-10 and proliferation index remained unchanged. Nebivolol-treated {beta}2-knockout memory Th cells showed significant inhibition of {beta}2AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-{kappa}B p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. ConclusionsThe findings demonstrate that nebivolol acts through a {beta}2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing phosphorylation of NF-{kappa}B. This highlights nebivolols potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

immunology↗

bioRxiv2024

A Digital Microfluidic Platform for the Microscale Production of Functional Immune Cell Therapies

Genetically engineering human immune cells has been shown to be an effective approach for developing novel cellular therapies to treat a wide range of diseases. To expand the scope of these cellular therapies while solving persistent challenges, extensive research and development is still required. Electroporation has recently emerged as one of the most popular techniques for inserting biological payloads into human immune cells to perform genetic engineering. However, several recent studies have reported that electroporation can negatively impact cell functionality. Additionally, the requirement to use large amounts of cells and expensive payloads to achieve efficient delivery can drive up the costs of development efforts. Here we use a digital microfluidic enabled electroporation system (referred to as triDrop) and compare them against two state-of-the-art commercially available systems for the engineering of human T cells. We describe the ability to use triDrop for highly viable, highly efficient transfection while using substantially fewer cells and payload. Subsequently, we perform transcriptomic analysis on cells engineered with each of the three systems and show that electroporation with triDrop lead to less dysregulation of several functionally relevant pathways. Finally, in a direct comparison of immunotherapeutic functionality, we show that T cells engineered with triDrop have an improved ability to mount an immune response when presented with tumor cells. These results show that the triDrop platform is uniquely suited to produce functionally engineered immune cells while also reducing the costs of cell engineering compared to other commercially available systems.

bioengineering↗

bioRxiv2024

Plectin-mediated cytoskeletal crosstalk as a target for inhibition of hepatocellular carcinoma growth and metastasis.

The most common primary malignancy of the liver, hepatocellular carcinoma (HCC), is a heterogeneous tumor entity with high metastatic potential and complex pathophysiology. Increasing evidence suggests that tissue mechanics plays a critical role in tumor onset and progression. Here we show that plectin, a major cytoskeletal crosslinker protein, plays a crucial role in mechanical homeostasis and mechanosensitive oncogenic signaling that drives hepatocarcinogenesis. Our expression analyses revealed elevated plectin levels in liver tumors, which correlated with poor prognosis for HCC patients. Using autochthonous and orthotopic mouse models we demonstrated that genetic and pharmacological inactivation of plectin potently suppressed the initiation and growth of HCC. Moreover, plectin targeting potently inhibited the invasion potential of human HCC cells and reduced their metastatic outgrowth in the lung. Proteomic and phosphoproteomic profiling linked plectin-dependent disruption of cytoskeletal networks to attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signatures. Importantly, by combining cell line-based and murine HCC models, we show that plectin inhibitor plecstatin-1 (PST) is well-tolerated and potently inhibits HCC progression. In conclusion, our study demonstrates that plectin-controlled cytoarchitecture is a key determinant of HCC development and suggests that pharmacologically induced disruption of mechanical homeostasis may represent a new therapeutic strategy for HCC treatment.

cancer biology↗

bioRxiv2023

Spatial omics imaging of fresh-frozen tissue and routine FFPE histopathology on a single cancer needle core biopsy: freezing device and multimodal workflow

Complex molecular alterations underlying cancer pathophysiology are intensely studied with omics methods using bulk tissue extracts. For spatially resolved tissue diagnostics using needle biopsy cores, however, histopathological analysis using stained FFPE tissue and immuno-histochemistry (IHC) of few marker proteins is currently the main clinical focus. Today, spatial omics imaging using MSI or IRI are emerging diagnostic technologies for identification and classification of various cancer types. However, to conserve tissue-specific metabolomic states, fast, reliable and precise methods for preparation of fresh-frozen (FF) tissue sections are crucial. Such methods are often incompatible with clinical practice, since spatial metabolomics and routine histopathology of needle biopsies currently require two biopsies for FF and FFPE sampling, respectively. Therefore, we developed a device and corresponding laboratory and computational workflows for multimodal spatial omics analysis of fresh-frozen, longitudinally sectioned needle biopsies to accompany standard FFPE histopathology on the same biopsy core. As proof-of-concept, we analyzed surgical human liver cancer specimen by IRI and MSI with precise co-registration and, following FFPE processing, by sequential clinical pathology analysis on the same biopsy core. This workflow allowed spatial comparison between different spectral profiles and alterations in tissue histology, as well as direct comparison to histological diagnosis without the need of an extra biopsy. SIMPLE SUMMARYRoutine clinical approaches for cancer diagnosis demand fast, cost-efficient, and reliable methods, and their implementation within clinical settings. Currently, histopathology is the golden standard for tissue-based clinical diagnosis. Recently, spatially resolved molecular profiling techniques like mass spectrometry imaging (MSI) or infrared spectroscopy imaging (IRI) have increasingly contributed to clinical research, e.g., by differentiation of cancer subtypes using molecular fingerprints. However, adoption of the corresponding workflows in clinical routine remains challenging, especially for fresh-frozen tissue specimen. Here, we present a novel device based on 3D-printing technology, which facilitates sample preparation of needle biopsies for correlated clinical tissue analysis. It enables combination of MSI and IRI on fresh-frozen clinical samples with histopathological examination of the same needle core after formalin-fixation and paraffin-embedding (FFPE). This device and workflow can pave the way for a more profound understanding of biomolecular processes in cancer and, thus, aid more accurate diagnosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/528125v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1da99aborg.highwire.dtl.DTLVardef@9ee706org.highwire.dtl.DTLVardef@5153daorg.highwire.dtl.DTLVardef@1581594_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗