Liver organoids: characteristics and future perspectives

da | Giu 15, 2021 | Biologia Molecolare, Liver, Organoids

Abstract

Liver bud organoids offer an alternative to traditional 2D cell cultures, but in order to exploit all their potential it is necessary to understand how cell communication affects differentiation. Camp and collaborators [1] explored differentiation in 3D organoids with single-cell RNA sequencing (scRNA seq). The results indicated that 3D organoids diverge from 2D cell culture and express genes characteristic of hepatic fetal cells. Furthermore, analysis on signaling mechanisms suggest that VEGF-KDR interactions promote organoid development. This work defined new insights on liver bud cell communication, providing basis for future interventions.

Organoids are 3D in vitro structures that recapitulate morphological and functional features of in vivo tissues. Liver organoids, in particular, could be applied in several fields, such as translational research, drug development and regenerative medicine [2]. Despite recent progress, it is still unclear how cell communication can affect differentiation of each cell type, dampening the importance of three-dimensional organoid over two-dimensional cell cultures. To better understand interactions occurring during liver bud (LB) formation, Camp et al. used single-cell RNA sequencing (scRNA seq) which provides deeper insight into cellular complexity within the same tissue [1].

Liver organoids were obtained by co-culturing induced pluripotent stem cell (IPSc)-derived human hepatic endoderm (HE), endothelial cells (EC) and mesenchymal cells (MC). 465 single-cell transcriptomes from the organoid were compared with those from input cells isolated from 2D cultures, for a total of 890 cells analyzed by scRNA seq. Principal component analysis (PCA) was then performed to identify the most informative genes of cell subpopulations. The result revealed that the three cell populations within the organoid, identified through single nucleotide polymorphisms (SNPs), differ from those in 2D cell culture, suggesting that each lineage undergoes transcriptional changes upon LB development. In addition, hepatic endoderm cells in the organoid (HE-LB) were at an intermediate maturation stage relative to hepatic endoderm and immature hepatoblast.

Using self-organizing maps (SOMs), the authors identified 14 overexpressed gene signatures. Some of these signatures were in common with input cells, while other were specific to LB cells. Hepatic endoderm cells in the liver bud overexpressed genes involved in hypoxia, epithelial cell shape and migration, phagocytosis, GTPase activation, and lipoprotein metabolism. A single-cell network, based on Pearson’s correlation, was built by selecting genes based on PCA analysis, in order to visualize the relationships between cells within the organoid and in the 2D cell cultures. By using this approach, the authors observed that HE-LB were actually separated from the hepatic endoderm and immature hepatoblast. This finding was confirmed by using t-distributed stochastic neighbor embedding (tSNE) where it was observed that organoid’s cells did actually cluster separately from 2D cells. Furthermore, some hepatic endoderm cells in the liver bud expressed genes involved in in vivo hepatoblast migration such as PROX1 and ONECUT2. These data suggest that HE-LB begin a differentiation program distinct from cells in homotypic 2D cultures.

In contrast, LB endothelial cells overexpressed genes involved in hypoxia, endoplasmic reticulum stress and are characterized by lower levels of proliferation. The LB endothelial signatures correlated with different vasculogenic profiles which are typical of endothelial cells during liver regeneration after tissue damage. Mesenchymal cells, instead, overexpressed genes related to hypoxia, inflammatory response, TNF and NF-kB signalling. In addition, these cells expressed various collagens and other extracellular matrix components, presumably to remodel extracellular matrix and enhance tissue permeability. The authors also found evidence that hypoxic stress was resolved after 15 days from transplantation, concluding that hypoxia, inflammation and matrix remodelling in the liver bud microenvironment might contribute to vascularization.

To further explore differences between 3D and 2D cell culture systems, genes identified through PCA were used to perform a lineage reconstruction (Fig.2). This analysis revealed that the 2D and 3D lineages bifurcated shortly after LB generation and continued to diverge during hepatic maturation. For a better comprehension, scRNA sequencing was applied on fetal and adult human liver samples. Fetal and adult hepatocytes clustered separately, but interestingly LB-hepatic cells were much more similar to fetal hepatocytes rather than adult or 2D hepatic cells. Likewise, mesenchymal and endothelial cells derived from the organoid were more similar to the fetal counterparts. All this information leads to the conclusion that signals in the organoid promote transcriptional states comparable to those of fetal hepatic tissues.

In the second part of the study, the authors focused on the analysis of the potential signaling mechanisms involved in the liver bud maturation process. Initially, an in silico screening was performed based on the presence of a complementary receptor or ligand among cells. Results showed that MCs had the most potential interactions, and HE cells tended to interact more with MC and EC rather than with other HE cells. The same approach was used by Zhou and collaborators to identify growth factors involved in fibroblast-macrophage communication [3] (inserire link review Zagaria-Ghinato)

Figure 2 – lineage reconstruction using genes identified by PCA reveals a bifurcation in 2D and 3D trajectories (Camp et al, 2017)

Many pathways were identified, such as TNF, FGF, JAK/STAT, NF-κB, HIF, and VEGF signalling. To confirm these prediction, two strategies were carried out. First, small interfering RNAs (siRNAs) were used to knockdown the receptor TIE1 or its ligand EDN1 in EC cells, which are known to be involved in angiogenesis and in the HE/EC interaction [4]. Results indicated that hepatic differentiation was impaired in both conditions, meaning that EC activity is important in HE differentiation. Second, a high-throughput imaging approach based on miniaturized organoids was adopted to test the effect of pathway inhibitors on LB development and differentiation. The authors assayed 70 chemical inhibitors, at two different concentrations, targeting previously reported pathways. By measuring the ratio of hepatic to endothelial cells, it was possible to identify that NF-kB, FGFR, IGF1R, JAK, and VEGFR pathways had an effect on LB development. Recent study showed that cell interactions are important to modulate cellular composition within a system and to reach homeostasis [3]. In particular, VEGFA resulted to be expressed by all the organoid cell types and that inhibition of the KDR receptor (VEGFR2), expressed only by ECs, had effects on endothelial cells sprouting and hepatoblasts differentiation. This suggests that heterotypic VEGFA–KDR interaction promotes hepatic differentiation specifically within LBs.

In conclusion, scRNA sequencing of 2D and 3D cell cultures revealed that both systems recapitulate certain transcriptomic characteristics of human hepatogenesis. However, only cells in 3D cultures showed gene expression profiles that can be traced back to fetal liver cells. This difference may be a consequence of heterotypic interaction occurring during liver bud development. Camp et al. provided great evidences on cellular communication within the organoid, but still preliminary. Therefore, in the future, it will be necessary to further investigate these mechanisms. Since cells within the organoid are highly similar to hepatic fetal cells, the next step in research could be to develop organoids already differentiated in adult tissues. Finally, it might be interesting to analyse the limiting factors of this three-cell system in the same way as Zhou et al. did[3].

References

  1. Camp, J., Sekine, K., Gerber, T. et al. Multilineage communication regulates human liver bud development from pluripotency. Nature 546, 533–538 (2017). https://doi.org/10.1038/nature22796
  2. Nuciforo S, Heim MH. Organoids to model liver disease. JHEP Rep. 3(1):100198 (2020). https://doi:10.1016/j.jhepr.2020.100198
  3. Zhou, X. et al. Circuit Design Features of a Stable Two-Cell System. Cell 172, 744–757 (2018). https://doi.org/10.1016/j.cell.2018.01.015
  4. D’Amico, G. et al. Tie1 deletion inhibits tumor growth and improves angiopoietin antagonist therapy. J. Clin. Invest. 124, 824–834 (2014). https://doi.org/10.1172/JCI68897

Giuseppe Panarello

Master Industrial Biotechnology student

Sabrina Dezzani

Master Industrial Biotechnology student