Investigation of the hepatic development in the coculture of hiPSCs-derived LSECs and HLCs in a fluidic microenvironment

Abstract

Interactions between the different liver cell types are critical to the maintenance or induction of their function in vitro . In this work, human-induced Pluripotent Stem Cells (hiPSCs)-derived Liver Sinusoidal Endothelial Cells (LSECs) and Hepatocytes-Like Cells (HLCs) were cultured and matured in a microfluidic environment. Both cell populations were differentiated in Petri dishes, detached, and inoculated in microfluidic biochips. In cocultures of both cell types, the tissue has exhibited a higher production of albumin (3.19 vs 5.31 μ g/mL/10 6 cells in monocultures and cocultures) as well as a higher inducibility CYP450 over monocultures of HLCs. Tubular-like structures composed of LSECs and positive for the endothelial marker PECAM1, as well as a tissue more largely expressing Stabilin-2 were detected in cocultures only. In contrast, monocultures exhibited no network and less specific endothelial markers. The transcriptomic analysis did not reveal a marked difference between the profiles of both culture conditions. Nevertheless, the analysis allowed us to highlight different upstream regulators in cocultures (SP1, EBF1, and GATA3) and monocultures (PML, MECP2, and NRF1). In cocultures, the multi-omics dataset after 14 days of maturation in biochips has shown the activation of signaling related to hepatic maturation, angiogenesis, and tissue repair. In this condition, inflammatory signaling was also found to be reduced when compared to monocultures as illustrated by the activation of NFKB and by the detection of several cytokines involved in tissue injury in the latter. Finally, the extracted biological processes were discussed regarding the future development of a new generation of human in vitro hepatic models.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation‐like profiles in human‐induced pluripotent stem cell‐derived liver sinusoidal endothelial cells and carboxypeptidase M‐positive liver progenitor cells cocultured in a fluidic microenvironment

“…The hiPSC‐derived LSECs were prepared using 454E2 clones (Riken BioResource Research Center) as previously described 27 . The cells were stored in liquid nitrogen and proliferated for a total of 9 days as previously described 25 before further use in the biochip.…”

“…To partially solve these remaining issues, the work described in this manuscript demonstrated the coculture of hiPSC-derived CPM-positive LPCs with hiPSC-derived LSECs within a single microfluidic biochip. The choice of cellular model was set on these populations due to their excellent prospects in terms of maturation and the fact that those cells can be proliferated and cryopreserved, [23][24][25] which allows for relatively easier handling than their primary counterparts or other hiPSCderived models. In those biochips, the resulting tissue, both inlets and outlets, were analyzed separately, and a comparison was carried out at the transcriptome and proteome levels.…”

Microfluidic devices: The application in TME modeling and the potential in immunotherapy optimization

“…Different from traditional 2D cell culture models, organ chips with microchannels allowed the fluids to flow across the cell chambers, which enabled the recapitulation for in vivo physical conditions such as vascular perfusion, air-liquid interfaces, shear stresses as well as the physical and chemical gradients (Murugesan et al, 2017;Liu et al, 2021). For instance, microfluidic chips were used to culture the human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) (Danoy et al, 2021). And the multi-omics analysis of the chip demonstrated a typical signature of a liver regenerative process, which provided an original overview of the sophisticated mechanisms of liver regeneration by the use of microfluidic ii) The schematic views of this platform.…”

“…Frontiers in Genetics frontiersin.org technology (Danoy et al, 2021). In addition, some sophisticated organ chips even succeed in modeling organ-relevant mechanical activity by manipulating the organotypic tissue interfaces with the designed hollow side chambers (Choi et al, 2015;Hassell et al, 2017).…”

Improvements in Maturity and Stability of 3D iPSC-Derived Hepatocyte-like Cell Cultures

“…The 3D models provide structural organization with more cell-cell and cell-extracellular matrix (ECM) contacts, thus promoting liver functions. Various 3D culture systems can be utilized, from bio-printed structures to microfluidic chips or spheroid culturing, either as monocultures of hepatocytes or co-cultures with NPCs [3][4][5]. Primary human hepatocytes (PHHs) represent the current gold standard cell type in liver modeling, but they have a tendency to rapidly lose their functionality in vitro [6,7].…”