Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
Extracellular matrix microarrays to study inductive signaling for endoderm specification
Braga Malta D.F., Reticker-Flynn N.E., da Silva C.L., Cabral J.M.S, Fleming H.E., Zaret K.S., Bhatia S.N., Underhill G.H. Acta Biomaterialia, 2016
The authors implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high throughput manner. Seeded definitive endoderm (DE) cells onto the arrays and evaluated cell adhesion and hepatic and pancreatic differentiation. When comparing the ECM conditions that best supported hepatic and pancreatic differentiation, we noted that two combinations (fibronectin + merosin (laminin α2) and laminin (α1) + superfibronectin) are among the most robust domains for both hepatic and pancreatic differentiation.
Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytesCell Stem Cell Reports, 2013 Oct
Takayama K., Morisaki Y., Kuno S., Nagamoto Y., Harada K., Furukawa N., Ohtaka M., Nishimura K., Imagawa K., Sakurai F., Tachibana M., Sumazaki R., Noguchi E., Nakanishi M., Hirata K., Kawabata K., Mizuguchi H. PNAS 2014
The authors had previously developed a method to maintain and proliferate PSC-derived hepatoblasts on LN-111 (Cell Stem Cell Reports, 2013 Oct). In this publication, they examine and find evidence for the increased efficiency and homogeneity of hepatocyte differentiation when the LN-111 cultivated and purified hepatoblasts are further differentiated on LN-111 to hepatocytes.
CCAAT/enhancer binding protein-mediated regulation of TGFβ receptor 2 expression determines the hepatoblast fate decision
Takayama K., Kawabata K., Nagamoto Y., Inamura M., Ohashi K., Okuno H., Yamaguchi T., Tashiro K., Sakurai F., Hayakawa T., Okano T., Furue M.K., and Mizuguchi H. Development, 2014
Examined the function of TGFBR2 in the hepatoblast fate decision using hESC-derived HBC. hESC-derived HBCs purified and maintained (HBCs passaged more than three times) on human laminin 111 (LN111)-coated dishes were used. The HBC population was nearly homogeneous and expressed human hepatoblast markers such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 19 (CK19) and EPCAM, and most of the colonies observed on human LN111-coated plates were ALB and CK19 double positive. The HBCs were capable of repopulating the liver of carbon tetrachloride (CCl4)-treated immunodeficient mice. This study reveals a molecular mechanism underlying the lineage commitment of human hepatoblasts (hepatocyte and biliary differentiation) controlled by a gradient of TGFβ signaling. It provides the first evidence of c/EBP-mediated regulation of TGFBR2 expression in the human hepatoblast fate decision.
Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
Watanabe M., Zemack H., Johansson H., Hagbard L., Jorns C., Li M., Ellis E.PLOS ONE, 2016
In this study, the authors determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. α1 and α5 expression could be detected on RNA level but not on protein level in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521, matrigel or collagen type IV. Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double-stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte-specific function in vitro.
Characterization of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver
Lorenzini S., Bird T.G., Boulter L., Bellamy C., Samuel K., Aucott R., Clayton E., Andreone P., Bernardi M., Golding M., Alison M.R., Iredale J.P., Forbes S.J.Gut, 2010
In this article, the composition and formation of stem progenitor cell niches were examined. The progenitor cell response in liver injury is intimately surrounded by myofibroblasts and macrophages, and to a lesser extent by endothelial cells. Hepatic progenitor cells are not of bone marrow origin; however, bone marrow-derived cells associate intimately with these cells and are macrophages. Laminin always surrounds the progenitor cells. In vitro studies showed that laminin aids the maintenance of progenitor and biliary cell phenotype and promotes their gene expression (Dlk1, Aquaporin 1, gammaGT) while inhibiting hepatocyte differentiation and gene expression (CEPB/alpha). During liver damage in rodents and humans, a stereotypical cellular and laminin niche forms around hepatic progenitor cells. Laminin helps the maintenance of undifferentiated progenitor cells. The niche links the intrahepatic progenitor cells with bone marrow-derived cells and links tissue damage with progenitor cell-mediated tissue repair.
Laminin-511 and laminin-521 based matrices for efficient hepatic specification of human pluripotent stem cells
Kanninen L.K., Harjumäki R., Peltoniemi P., Bogacheva M.S., Salmi T., Porola P., Niklander J., Smutny T., Urtti A., Yliperttula M.L., Lou Y-R. Biomaterials, 2016
In this study, the authors used laminin-511, laminin-521, and fibronectin, as culture matrices for hPSC-derived definitive endoderm cells. By screening the acellular matrix produced by HepaRG cells and found that laminin-511 (LN-511), laminin-521 (LN-521), and fibronectin were highly expressed. The authors observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification. They did not observe any improvement in cell differentiation efficacy with fibronectin. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Use of recombinant matrix proteins is faster, more consistent, more efficient, and more scalable compared to the HepaRG-derived acellular matrix.
Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on Human Laminin 111-Coated Dishes
Takayama K., Nagamoto Y., Mimura N., Tashiro K., Sakurai F., Tachibana M., Hayakawa T., Kawabata K., Mizuguchi H. Cell Stem Cell Reports, 2013
The authors of this important study demonstrated that laminin-111 is the optimal matrix for hepatoblasts. In addition to solving the need to efficiently expanding and purifying this liver progenitor, the matrix provides an important safety mechanism as LN-111 selectively only maintains hepatoblasts while eliminating residual undifferentiated cells that cannot survive and self-renew on laminin-111.
Protocol: Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells
Wang Y., Alhaque S., Cameron K., Meseguer-Ripolles J., Lucendo-Villarin B., Rashidi H., Hay D.C. JoVE, 2017
Here, the authors show in a video how they developed a defined optimized system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. The protocol shows the procedures for culturing hPSCs on LN521 and differentiating them on either LN521 or a blend of LN521 and LN111 (Biolaminin 521 and 111). Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines promising advances in cell-based modeling and therapies.
Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes
Cameron K., Tan R., Schmidt-Heck W., Campos G., Lyall M.J, Wang Y., Lucendo-Villarin B., Szkolnicka D., Bates N., Kimber S.J., Hengstler J.G., Godoy P., Forbes S.J., Hay D.C. Stem Cell Reports, 2015
Human ES cells are cultured on human recombinant laminin-521 and laminin-111 shows efficient hepatocyte specification, maturation, function and stabilization of phenotype. The results presented in the paper represent a significant advance compared to any previously published data especially regarding metabolic activity and functional organization. Cells cultured on the laminin matrices exhibited significantly increased metabolic function relative to cells on Matrigel and human primary hepatocytes. The laminin cultured hepatocyte-like cells were arranged in lobule like structures, reminiscent of regenerating liver, with positive staining for MRP1 and MRP2 and were capable of biliary efflux.
Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications
Ong J., Paola Serra M., Segal J., Cujba A.-M., Seng Ng S., Butler R., Millar V., Hatch S., Zimri S., Koike H., Chan K., Bonham A., Walk M., Voss T., Heaton N., Mitry R., Dhawan A., Ebner D., Danovi D., Nakauchi H., Rashid S.T.Stem Cell Reports, 2018
Here, the authors show that extracellular niche factors likely play a critical role in bridging this gap in functional differences between hepatocytes derived from induced pluripotent stem cells (i-Heps) and primary cells. They defined a profile of healthy, freshly isolated primary hepatocytes (Hepatocyte Likeness Index; HLI) that cells of interest can be compared against for high-throughput screening. They applied this platform to screen hepatocyte niche factors for their ability to drive i-Heps closer to that target and validated their findings in a pharma-like screening environment. The HLI was applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Results from the screen performed highlighted the important role played by laminins where laminin 411 was identified as a key niche protein. Laminin 411 is a component of the hepatic niche. Laminin 411 advanced i-Heps toward functional significance and prolonged survival of hepatic progenitor cells, contributing to better i-Hep-based drug-screening applications. This paper underscores the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.