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

Yu Wang Y., Alhaque S., Cameron K., Meseguer-Ripolles J., Lucendo-Villarin B., Rashidi H., Hay D.C.
JoVE, 2017

Here, the authors developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. 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 modelling 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 cultured on human recombinant laminin-521 and laminin-111 show efficient hepatocyte specification, maturation, function and stabilization of phenotype. The results presented in the paper represents a significant advance compared to any previous 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.


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 of efficiently expanding and purifying this liver progenitor, the matrix provides an important safety mechanism as LN-111 selectively only maintain hepatoblasts while eliminating residual undifferentiated cells that cannot survive and self-renew on laminin-111.


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 the 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 HepaRG-derived acellular matrix.


Characterisation 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 was 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 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 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.


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 lavel 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.


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 were 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.


Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

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 heaptoblasts 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.


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.


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

Takayama K., Mitani S., Nagamoto Y., Sakurai F., Tachibana M., Taniguchi Y., Sekiguchi K., Mizuguchi H.
Biochem Biophys Res Commun. 2016

Here 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. Differentiated human iPSC to cholangiocyte-like cells via hepatoblasts-like cells (derived according to previous papers). The 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 the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and g-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix.


Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response 

Kallis Y.N., Robson A.J., Fallowfield J.A., Thomas H.C., Alison M.R., Wright N.A., Goldin R.D., Iredale J.P., Forbes S.J.
Gut, 2011

In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Here, the authors test the relationship between collagen degradation, laminin deposition, and the HPC response. Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.


Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats

Carvalho S., Cortez E., Stumbo A.C., Thole A., Caetano C., Marques R., Pelajo-Machado M., Porto L.C., Carvalho L.
Cell Biology International, 2008

The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation (injected into recently hepatectomyzed rats via the portal vein). Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids (endothelia) expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.