Biology subjects

Florentino, R. M.

Publications and source records attributed to Florentino, R. M..

6 recordsLinked to original sources

bioRxiv2026

Liver Disease Reveals KIF12 as a Critical Regulator of Mitochondria, Lysosome and Cilia Localization

Pathogenic variants in kinesin family member 12 (KIF12) cause pediatric liver disease, yet the cellular mechanisms underlying this phenotype remain unknown. Here we show that KIF12 expression in the healthy human liver is primarily detected in biliary epithelial cells, as revealed by single cell RNA-sequencing data. To investigate its role in biliary pathology, we introduced a homozygous KIF12 p.Arg219* mutation into induced pluripotent stem cells (iPSCs), which were differentiated into 2D cholangiocyte-like cells (iCCs) and 3D biliary organoids. Pioneering single-molecule fluorescence microscopy in live iCCs, we observed wildtype KIF12 co-localizing with microtubules, consistent with its predicted role as a microtubule-associated motor protein. Our data reveals that KIF12 dysfunction causes abnormal perinuclear clustering of mitochondria and lysosomes, and mislocalization of primary cilia in cholangiocytes. Restoration of wildtype KIF12 expression in mutant KIF12 iCCs rescued organelle positioning and normalized GGT activity. This study uncovers a novel link between KIF12 dysfunction and organelle dynamics in human cholangiocytes, extending our understanding of kinesin roles beyond their established functions in neuronal systems. Our findings provide new insights into the pathogenesis of KIF12-related cholestatic liver disease and lay the groundwork for developing targeted genetic therapies.

genetics↗

bioRxiv2024

Stress-free Bioprinting of Human Primary and iPSC-derived Islets with Retained Functionality

Currently, Type 1 diabetes (T1D) can be treated through implantation of allogenic islets, which replenish the beta cell population, however this method requires an extensive post-implantation immunosuppressant regimen. Personalized cellular therapy can address this through implantation of an autologous cell population, induced pluripotent stem cells (iPSCs). Cellular therapy, however, requires an encapsulation device for implantation, and so to achieve this uniformly with cells in a clinical setting, bioprinting is a useful option. Bioprinting is dependent on having a bioink that is printable, retains structural fidelity after printing, and is supportive of cell type and function. While bioprinting of pancreatic islets has been demonstrated previously, success in maintaining islet function post-printing has been varied. The objective of this study is to investigate the feasibility of printing functional islets by determining the appropriate combination of bioink, printing parameters, and cell configuration. Here, we detail the successful bioprinting of both primary human islets and iPSC-derived islets embedded in an alginate/methylcellulose bioink, with functionality sustained within the construct for both cell lineages. Sc-RNAseq analysis also revealed that printing did not adversely affect the genetic expression and metabolic functionality of the iPSC-derived islets. Importantly, the iPSC-derived islets displayed comparable functionality to the primary islets, indicating the potential to act as a cell source alternative for T1D implantation.

bioengineering↗

bioRxiv2024

A rat model of cirrhosis with well-differentiated hepatocellular carcinoma induced by thioacetamide

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths, and commonly associated with hepatic fibrosis or cirrhosis. This study aims to establish a rat model mimicking the progression from liver fibrosis to cirrhosis and subsequently to HCC using thioacetamide (TAA). We utilized male Lewis rats, treating them with intra-peritoneal injections of TAA. These rats received bi-weekly injections of either 200 mg/kg TAA or saline (as a control) over a period of 34 weeks. The development of cirrhosis and hepatocarcinogenesis was monitored through histopathological examinations, biochemical markers, and immunohistochemical analyses. Our results demonstrated that chronic TAA administration induced cirrhosis and well-differentiated HCC, characterized by increased fibrosis, altered liver architecture, and enhanced hepatocyte proliferation. Biochemical analyses revealed significant alterations in liver function markers, including elevated alpha-fetoprotein (AFP) levels, without affecting kidney function or causing significant weight loss or mortality in rats. This TAA-induced cirrhosis and HCC rat model successfully replicates the clinical progression of human HCC, including liver function impairment and early-stage liver cancer characteristics. It presents a valuable tool for future research on the mechanisms of antitumor drugs in tumor initiation and development.

pathology↗

bioRxiv2023

Human Induced Pluripotent Stem Cell based Hepatic-Modeling of Lipid metabolism associated TM6SF2 E167K variant

BACKGROUND AND AIMSTM6SF2 rs58542926 (E167K) is associated with an increase in the prevalence of Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD). Despite all the investigation related to the role of this variant in lipid metabolism, conflicting results in mouse studies underscore the importance of creating a human model for understanding the TM6SF2 mechanism. Therefore, the aim of this study is to generate a reliable human in vitro model that mimic the effects of the TM6SF2 E167K mutation and can be used for future mechanism studies. APPROACH AND RESULTSWe performed gene editing on human-induced pluripotent stem cells (iPSC) derived from a healthy individual to obtain the cells carrying the TM6SF2 E167K mutation. After hepatic differentiation, a decrease in TM6SF2 protein expression was observed in the mutated-induced hepatocyte. An increase in intracellular lipid droplets and a decrease in the efflux of cholesterol and ApoB100 were also observed. Transcriptomics analysis showed up-regulation of genes related to the transport, flux, and oxidation of lipids, fatty acids, and cholesterol in TM6SF2 E167K cells. Additionally, signs of cellular stress were observed in the ER and mitochondria. CONCLUSIONSOur findings indicate that induced hepatocytes generated from iPSC carrying the TM6SF2 E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to help in the identification of potential clinical targets and therapies and to understand the mechanism by which the TM6SF2 E167K variant leads to vulnerability to MASLD.

cell biology↗

bioRxiv2023

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/537186v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@8ce1bdorg.highwire.dtl.DTLVardef@1385103org.highwire.dtl.DTLVardef@4ec496org.highwire.dtl.DTLVardef@14c9295_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIComplementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration. C_LIO_LIVEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis. C_LIO_LIIdentification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin. C_LIO_LIKDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered. C_LI

pathology↗

bioRxiv2021

Alkaloid and acetogenin-rich fraction from Annona crassiflora fruit peel inhibits proliferation and migration of human liver cancer HepG2 cells

Plant species from Annonaceae are commonly used in traditional medicine to treat various cancer types. This study aimed to investigate the antiproliferative potential of an alkaloid and acetogenin-rich fraction from the fruit peel of Annona crassiflora in HepG2 cells. A liquid-liquid fractionation was carried out on the ethanol extract of A. crassiflora fruit peel in order to obtain an alkaloid and acetogenin-rich fraction (AF-Ac). Cytotoxicity, proliferation and migration were evaluated in the HepG2 cells, as well as the proliferating cell nuclear antigen (PCNA), vinculin and epidermal growth factor receptor (EGFR) expression. In addition, intracellular Ca2+ was determined using Fluo4-AM and fluorescence microscopy. First, 9 aporphine alkaloids and 4 acetogenins that had not yet been identified in the fruit peel of A. crassiflora were found in AF-Ac. The treatment with 50 {micro}g/mL AF-Ac reduced HepG2 cell viability, proliferation and migration (p < 0.001), which is in accordance with the reduced expression of PCNA and EGFR levels (p < 0.05). Furthermore, AF-Ac increased intracellular Ca2+ in the HepG2 cells, mobilizing intracellular calcium stores, which might be involved in the anti-migration and anti-proliferation capacities of AF-Ac. Our results support the growth-inhibitory potential of AF-Ac on HepG2 cells and suggest that this effect is triggered, at least in part, by PCNA and EGFR modulation and mobilization of intracellular Ca2+. This study showed biological activities not yet described for A. crassiflora fruit peel, which provide new possibilities for further in vivo studies to assess the antitumoral potential of A. crassiflora, especially its fruit peel.

biochemistry↗