HOW TO CULTURE HEPATOCYTES ON BIOLAMININ SUBSTRATES

Laminins play a vital role in liver progenitor cell-mediated regeneration

The liver contains several different laminin (LN) isoforms that play important roles for development, liver tissue homeostasis, organization, and regeneration. Hepatic progenitor cells (HPCs) and are always closely associated with a laminin-rich basement membrane. After an injury, laminin accumulates around the progenitors and the laminin-liver progenitor cell interactions are critical for hepatic progenitor cell (HPC) 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 cells (HSCs) function. The 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. The fact that a5-laminins are important elements of the HSC niche was shown in an article by German researches where they show that culture on Biolaminin 521 supports the quiescent state of the HSCs. 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 (Rohn, 2018).

In a follow-up article, the authors (Rohn, 2020) provide evidence that integrin α5β1 is an important mechanosensor in hepatic stellate cells (HSC) involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with Biolaminin 521 enhances HGF expression in HSC, demonstrating that these laminin proteins are critically involved in HSC function. During aging, HSC acquires a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. These findings suggest that impaired mechanosensing via integrin α5β1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.

Laminin substrates support effective hepatocyte differentiation and significantly advance hepatic maturation

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). In addition, the authors recently published a JoVE protocol that describes the scalable and GMP-ready differentiation process (Wang, 2017). The laminin differentiation protocol generates a high ratio of hepatocyte-like cells positively stained for ALB, CYP2D6, and CYP3A and the cells exhibit a significant increase in P450 metabolic enzyme functions relative to cells on Matrigel or human primary hepatocytes. The Biolaminin cultured hESC-derived hepatocyte-like cells display a more primary hepatocyte-like appearance and are often bi-nucleate with very pronounced nuclei. Moreover, the hepatocyte-like cells arranged themselves in lobule-like structures within the laminin-coated culture dish, reminiscent of regenerating liver. hESC differentiated on Biolaminin 521 and 111 exhibit vast networks of highly organized hepatocytes which express multidrug resistance-associated protein 1 (MRP1) and 2 (MRP2) and are capable of biliary efflux. The results presented in the paper represents a significant advance compared to any previously published data, especially regarding metabolic activity and functional cell organization. 

hESC-derived hepatocytes on Biolaminin 521 and 111. Cells positively stained for HNF4a (red) and exhibit vast networks of highly organized hepatocytes which express MRP1 (green) are capable of biliary efflux.
Courtesy of Dr. Kate Cameron, University of Edinburgh.

The positive effect of laminin 521 on hepatic differentiation is supported by a recent publication by Kanninen et al. that shows that human recombinant Biolaminin 521 can be used as a stage-specific matrix to guide the hepatic specification of pluripotent stem cells (Kanninen, 2016). Moreover, the expression of laminin 521-specific integrins increases during definitive endoderm and hepatic specification. hPSC-derived hepatic cells differentiated on these laminin matrices show up-regulation of liver-specific protein markers, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Contrary, the authors show that fibronectin is not a vital matrix protein for the generation of definitive endoderm cells. 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.

The importance of extracellular niche factors for bridging the gap in functional differences between hepatocytes derived from human induced pluripotent stem cells (i-Heps) and primary cells is further discussed in a recent article from a research group in the UK. They applied a Hepatocyte Likeness Index platform to screen hepatocyte extracellular factors for their ability to drive i-Heps closer to the standard. In their study, laminin 411 was identified as a key niche protein, advancing i-Heps toward functional significance and prolonging survival of hepatic progenitor cells (Ong, 2018). 

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

The research results presented above are in accordance with studies from Japanese researchers showing that isolated and hESC-derived hepatic progenitor cells effectively can be expanded with maintained phenotype on Biolaminin 111. The group of Mizuguchi shows that human recombinant 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.

Laminin 411 and 511 promote cholangiocyte differentiation of human-induced pluripotent stem cells

In a recent article, the authors searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells and found that both laminin 411 and laminin 511 were suitable for this purpose. Hepatoblast-like cells were mixed in a collagen gel and plated and for cholangiocyte differentiation, laminin was added to the medium. The gene expression levels of cholangiocyte markers were increased by the addition of laminin 411 or laminin 511. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% (Takayama, 2016).
 

Laminins promote attachment of human primary hepatocytes

In a publication by Watanabe et al., Biolaminin has also been shown to promote attachment of human primary hepatocytes and can effectively be used as a culture substrate for these cells (Watanabe, 2016).