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BIOLAMININ 521 and/or BIOLAMININ 111


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PROTOCOL

A scalable and GMP-ready hepatocyte differentiation protocol

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells


VIEW ON DEMAND

Defined generation of hPSC-derived hepatocyte-like cells on Biolaminin

Watch webinar on efficient differentiation and functional organization of hPSC-derived hepatocyte on hr laminin substrates.


APPLICATION NOTE

Get the hepatocyte differentiation application note here

 


CUSTOMER TESTIMONIALS 

"The use of recombinant laminins in conjunction with our differentiation systems have significantly advanced stem cell derived hepatocyte performance and polarity. Importantly, those processes are now fully defined, and GMP ready, offering exciting prospect for human regenerative medicine”

                                             Dr. David Hay, University of Edinburgh, UK

         
More Biolaminin product testimonials here

 


REFERENCES

Find a selection of hepatic articles here

HOW TO CULTURE HEPATOCYTES ON BIOLAMININ SUBSTRATES

hPSc-derived liver cell and hepatocytes cultured on human recombinant laminin-521 and laminin-111 cell culture reagents Laminins play a vital role for 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 injury, laminin accumulate around the progenitors and the laminin-liver progenitor cell interactions are a 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, alpha-5 laminin matrix promotes HPC attachment, migration and maintenance (Kallis, 2012; Klaas, 2016). 

Biolaminin 521 promotes quiescence in isolated hepatic stellate cells

Hepatic stellate cells (HSCs) are liver-resident mesenchymal stem cells which 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 show 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).

Laminin substrates support effective hepatocyte differentiation and significantly advance hepatic maturation

In a publication by Cameron et al., the authors show that 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 high ratio of hepatocyte-like cells positively stained for ALB, CYP2D6 and CYP3A and the cells exhibit significantly 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 hESC derived hepatocytes on laminin express MRP1  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 previous 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 show that human recombinant Biolaminin 521 can be used as stage specific matrix to guide the hepatic specification of pluripotent stem cells (Kanninen, 2016). Moreover, the expression of laminin 521-specific integrins increase 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 generation of definitive endoderm cells. A screening of the acellular matrix produced by the popular hepatic tumor cell line HepaRG show 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 an recent article from a research group in 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 show 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 have also been shown to promote attachment of human primary hepatocytes and can effectively be used as culture substrate for these cells (Watanabe, 2016).

BIOLAMININ KEY ADVANTAGES

  • Hepatocyte differentiation process under defined conditions

  • Scalable and GMP-ready protocols

  • Biolaminin 521 improves and stabilizes the hepatocyte phenotype

  • Biolaminin 521 improves hepatocyte function, phenotype and homogeneity

  • Biolaminin 521 significantly increase in P450 enzyme metabolic activity

  • Polarized and functional expression of MRP1 and MRP2 in hESC-derived hepatocytes cultured on Biolaminin 521

  • Highly organized hepatocytes with lobule-like structures reminiscent of regenerating liver in plates coated with Biolaminin 521

  • Efficient expansion and maintenance of hPSC-derived hepatoblasts for >15 passages on Biolaminin 111

  • Efficient clonal expansion of hepatoblast on Biolaminin 111

  • Biolaminin 521 promotes quiescence in isolated hepatic stellate cells

  • Biolaminin 411 and 511 promote cholangiocyte differentiation of hiPSC

  • Biolaminin substrates promote attachment of human primary hepatocytes

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 CTG

Biolaminin 521 CTG cell therapy grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal component-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Mesenchymal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 MX

Biolaminin 521 MX research grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal component-free and biologically relevant cell culture system for better cell models.

  • x
  • Expansion of human PSC

  • Mesenchymal stem cells

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Animal stem cells

  • Endothelial cells

  • Pancreatic cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Biolaminin 521 LN

Biolaminin 521 LN research grade cell culture matrix makes pluripotent stem cell culture easy. A defined, animal component-free and biologically relevant cell culture system for better cell models.