Hepatocytes and hepatoblasts

Several laminins play a vital role in liver progenitor cell-mediated regeneration

The liver contains several different laminin (LN) isoforms that play important roles in development, liver tissue homeostasis, organization, and regeneration. Hepatic progenitor cells (HPCs) are always closely associated with a laminin-rich basement membrane. Several Biolaminin substrates have been shown to promote attachment and to maintain the cell viability of human primary hepatocytes in culture (Watanabe, 2016). In addition to laminin 521 and 111 being relevant isoforms in the liver, laminin-411 was identified as a key niche protein, advancing iPS-derived hepatocytes toward functional significance and prolonging the survival of hepatic progenitor cells (Ong, 2018). 

After an injury, laminin accumulates around the progenitors and the laminin-liver progenitor cell interactions are critical for hepatic progenitor cell-mediated regeneration (Carvalho, 2008; Lorenzini, 2010). Murine regeneration models show that the alpha-1, alpha-2, alpha-4, and alpha-5 laminin chains increase in association with HPC activation and that laminin alpha-5 containing matrix (laminin 511 and laminin 521) is deposited around HPCs during regeneration. Moreover, the alpha-5 laminin matrix promotes HPC attachment, migration, and maintenance (Kallis, 2012; Klaas, 2016). 

Biolaminin 521 promotes quiescence in isolated hepatic stellate cells

Laminin proteins are critically involved in hepatic stellate cell (Whisks) function. The hepatic stellate cells (HSCs) are liver-resident mesenchymal stem cells that are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases. The HSCs reside in a quiescent state in the space of Dissé on a basement membrane-like structure that contains a5 laminins. Biolaminin 521 supports the quiescent state of the HSCs (Rohn, 2018). In the study, Biolaminin 521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC cultured on uncoated polystyrene plates or plates coated with non-relevant laminin isoforms developed into myofibroblasts-like cells. In addition, laminin-functionalized, gold-nanostructured glass surfaces further enhanced the expression of quiescence-associated genes in the HSCs.

The culture of hepatic stellate cells (HSCs) on nanostructured surfaces functionalized with Biolaminin 521 was shown to enhance hepatocyte growth factor (HGF) expression in HSC, demonstrating that laminin proteins are critically involved in HSC function (Rohn, 2020). HGF is an essential inductor of liver regeneration which is impaired during aging. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. The findings suggest that impaired mechanosensing via integrin α5β1 in HSC contributes to the age-related reduction of ECM and HGF release that could affect liver regeneration.

Biolaminin substrates support effective, GMP-ready, hepatocyte differentiation

In a publication by Cameron et al., the authors show that the culture of human ES cells (hESC) on human recombinant Biolaminin 521 and Biolaminin 111 substrates significantly improved hepatocyte differentiation, maturation, function, and stabilization of phenotype compared to Matrigel cultured cells (Cameron, 2015). The authors published a JoVE protocol that describes the scalable and GMP-ready differentiation process (Wang, 2017). The results presented in the paper represent a significant advance compared to any previously published data, especially regarding metabolic activity and functional cell organization. 

Biolaminin 521 was also used as a stage-specific matrix to effectively guide the hepatic specification of pluripotent stem cells (Kanninen et al. 2016). The expression of laminin 521-specific integrins increases during definitive endoderm and hepatic specification. hPSC-derived hepatic cells differentiated on Biolaminin 521 show up-regulation of liver-specific protein markers, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. A screening of the acellular matrix produced by the popular hepatic tumor cell line HepaRG shows that laminin isoforms 521 and 511 and fibronectin are highly expressed, however, fibronectin was not a vital matrix protein for the generation of definitive endoderm cells.

Hepatic progenitor cells can be effectively expanded and maintained on Biolaminin 111 in vitro

Isolated and hESC-derived hepatic progenitor cells can be effectively expanded with a maintained phenotype on Biolaminin 111. The group of Mizuguchi shows that Biolaminin 111 is optimal for maintenance and clonal expansion of homogenous populations of hESC and hiPSC derived hepatoblasts (Takayama, 2013). The hepatoblasts were efficiently expanded on Biolaminin 111 for more than 15 passages with maintained phenotype and could thereafter be further differentiated into both hepatic and biliary lineages. Moreover, since pluripotent stem cells cannot survive and self-renew on Biolaminin 111, residual, undifferentiated cells are effectively eliminated from the differentiated hepatoblast population by the matrix itself, an important mechanism from a therapeutic aspect. When transplanted in CCl4-treated immunodeficient mice, the laminin-cultured hepatoblasts successfully engrafted and albumin-positive cells were observed in the liver of the transplanted mice.