Biology subjects

Toprakhisar, B.

Publications and source records attributed to Toprakhisar, B..

4 recordsLinked to original sources

bioRxiv2025

Organobodies: A robust and size-controllable system for generating scalable hiPSC-derived liver organoids for drug toxicity screening

Background and aimHepatic organoids generated from pluripotent and adult stem cells faithfully mimic the architecture and function of in vivo organs making them powerful tools for preclinical applications such as disease modelling and drug screening. However, developing robust and scalable models capable of preserving liver-specific functions long-term remains technically challenging. MethodsUsing a defined self-assembling peptide, we established a robust and reproducible method to generate hepatic organoids from human induced pluripotent stem cells (hiPSCs), termed hepatic organobodies (OBs), which remained stable in culture for several weeks. The hepatic identity and maturation of OBs were characterized by qPCR, histology, immunofluorescence, and bulk RNA sequencing. Their functional competence was evaluated by ELISA, CYP3A4 induction assays, and assessment of drug biotransformation capacity using HPLC-MS/MS, in parallel with three-dimensional human primary hepatocyte (3D PHH) cultures. Finally, the model was applied to predict the hepatotoxicity of a selected panel of known compounds and benchmarked against HepG2 cells and 3D PHH microtissues. ResultshiPSC-derived hepatocyte-like cells (HLCs) cultured in OBs acquired characteristic hepatic morphology and expressed several key hepatocyte-specific genes, some at levels comparable to those in freshly isolated primary human hepatocytes (PHHs). OBs secreted significantly higher amounts of albumin and 1-antitrypsin (A1AT) compared with parallel 2D-cultured HLCs. Bulk RNA sequencing revealed higher relative expression of major drug metabolism genes, including CYP3A4, CYP2C9, and CYP1A2, as well as enhanced maturation, evidenced by upregulation of the PPAR signalling and fatty acid {beta}-oxidation pathways in OBs relative to 2D HLCs. Moreover, OBs demonstrated CYP450 enzyme activity, with CYP3A4 and CYP2C9 activities comparable to those observed in 3D PHH microtissues. Finally, we show that our 3D model accurately predicted the hepatotoxicity of more than 10 tested chemical compounds. ConclusionHere, we demonstrate a novel, defined, robust and remarkably advanced 3D liver model as a valuable platform for scalable toxicity prediction and drug screening for personalised medicine.

bioengineering↗

bioRxiv2021

Engineering large-scale perfused tissues via synthetic 3D soft microfluidics

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

bioengineering↗

bioRxiv2020

A fully defined pluripotent stem cell derived multi-liver-cell model for steatohepatitis and fibrosis

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC)-derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGF{beta}-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

bioengineering↗

bioRxiv2020

PU.1 drives specification of pluripotent stem cell-derived endothelial cells to LSEC-like cells

To date there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell-derived) endothelial cells (ECs). Furthermore, the transcriptional programs driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC transcription factor ETV2) in human pluripotent stem cells (PSCs) induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers such as CD32B and MRC1 as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

developmental biology↗