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Defined generation of hPSC-derived hepatocyte-like cells on laminin
=> APPLICATION NOTE
=> Customer testimonial
"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
LAMININ KEY ADVANTAGES
Highly efficient hepatocyte specification with improved function, phenotype and homogeneity
Significant effect on hepatocyte P450 enzyme metabolic activity
Cells arrange themselves in lobule like structures, express MRP1 and MRP2 and are capable of biliary efflux
Easy to scale up
Efficient expansion and maintenance of hPSC-derived hepatoblasts for >15 passages
Undifferentiated cells are effectively eliminated from the differentiated hepatoblast population
Efficient clonal expansion of hepatoblast
Promote attachment of human primary hepatocytes
Defined and xeno-free culture method for reproduceble experments
Laminins play a vital role for liver progenitor cell mediated regeneration
The liver contains several different laminin (LN) isoforms that play important roles in development, for 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 progenitorsa and the laminin-liver progenitor cell interactions are a critical for hepatic progenitor cell (HPC) mediated regeneration. Murine regeneration models show that the alpha-1, alpha-2 and alpha-5 laminin chains increase in association with HPC activation and that laminin alpha-5 containing matrix is deposited around HPCs during regeneration. Moreover, alpha-5 laminin matrix promote HPC attachment, migration and maintenance.
Laminin-521 and laminin-111 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 laminin-521 and laminin-111 substrates significanly improved hepatocyte differentiation, maturation, function and stabilization of phenotype compared to Matrigel cultured cells. 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 laminin 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 laminin-521 and laminin-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.
The positive effect of laminin-521 on hepatic differentiation is supported by a recent publication by Kanninen et al. show that human recombinant laminin-521 can be used as stage specific matrix to guide the hepatic specification of pluripotent stem cells. 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-521, laminin-511 and fibronectin are highly expressed.
Hepatic progenitor cells can be effectively be expanded and maintained on laminin-111 in vitro
These results are in accordance with in vitro studies that show that isolated and hESC-derived hepatic progenitor cells effectively can be expanded with maintained phenotype on laminin-111. The group of Mizuguchi show that human recombinant laminin-111 is optimal for maintenance and clonal expansion of homogenous populations of hESC and hiPSC derived hepatoblasts. The hepatoblasts were efficiently expanded on laminin-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 laminin-111, residual, undifferentiated cells are effectively eliminated from the differentiated hepatoblast population by the matrix itself, an important mechanism from an 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 promote attachment of human primary hepatocytes
In a publication by Watanabe et al., laminin have also been shown to promote attachment of human primary hepatocytes and can effectively be used as culture substrate for these cells.
Hepatocyte differentiation on laminin
- Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes. Cameron et al. Stem cell reports, 2015
- Laminin-511 and laminin-521 based matrices for efficient hepatic specification of human pluripotent stem cells. Kanninen et al. Biomaterials, 2016
- Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells, Wang et al. JoVE, 2017
Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on Human Laminin 111-Coated Dishes. Takayama et al. Cell Stem Cell Reports, 2013
Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response. Kallis et al. Gut, 2011
Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver. Lorenzini et al. Gut, 2010
The alterations in the extracellular matrix composition guide the repair of damaged liver tissue. Klaas et al. Scientific reports, 2016
Culture of primary hepatocytes
Maintenance of hepatic functions in primary human hepatocytes cultured on xeno-free and chemical defined human recombinant laminins. Watanabe et al. PLOS ONE, 2016
Laminin isoforms promote attachment of hepatocytes via different integrins. Forsberg et al. Exp Cell Res, 1994
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