Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro
Anu Hyysalo, Mervi Ristola, Meeri E.-L. Mäkinen, Sergei Häyrynen, Matti Nykter, Susanna Narkilahti. Stem Cell Research, 2017
In this work, the authors compared different recombinant human laminin isoforms in the culture of hPSC derived neurons. They show that LN α5 chain is important for neuronal attachment, viability and network formation, and that neuronal functional development is enhanced by LN α5-substrates.
Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro
Hyysalo A., Ristola M., Jpki T., Honkanen M., Vippola M., Narkilahti S.Macromolecular Bioscience, 2017
Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of the spinal cord in vivo. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Clinically relevant human recombinant laminin substrates (laminin-521 and laminin-511) are compatible with PCL nanofibers and can be used for efficient culturing of hPSC-derived neurons and astrocytes on PCL nanofibers. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and the nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment.
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.
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.
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.
Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method
Hongisto H., Ilmarinen T., Vattulainen M., Mikhailova A., Skottman H. Stem cell research and therapy, 2017
Here the authors describe a robust xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient and scalable methods to derive high-quality retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs). Multiple genetically distinct hPSC lines were adapted to a robust, defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins laminin-521 and Collagen IV. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, the authors established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing on laminin-521 and Collagen IV. The simple xeno-free methods described here could be upgraded to GMP-quality for future preclinical testing and safety and functional efficacy testing of the hPSC-RPE and hPSC LESCs produced with these protocols are currently ongoing in non-human primates and rabbit models of LSCD, respectively.
Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511
Rodin S., Domogatskaya A., Ström S., Hansson E.M., Chien K.R., Inzunza J., Hovatta O., Tryggvason K. Nat Biotechnol., 2010
In this article, the authors describe, for the first time, the use of laminin-511 as a substrate for human ES and iPS cells in vitro. The culture system is defined and devoid of animal products and feeder cells. Human pluripotent cells cultured on LN-511 substrate in this way maintained self-renewal capacity and pluripotency long-term, as well as karyotypic stability. Human ES cells plated on laminin-511 grow as a monolayer, which makes cell homogeneity particularly high.
Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix
Uhlin E., Marin Navarro A., Rönnholm H., Day K., Kele M., Falk A. J Vis Exp. 2017
In this video publication, the authors describe a consistent, highly reproducible and easy-to-use protocol, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single-cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. This protocol has been extensively tested and used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described.
Single-cell cloning and expansion of human-induced pluripotent stem cells by a microfluidic culture device
Matsumura T., Tatsumi K., Noda Y., Nakanishi N., Okonogi A., Hirano K., Li L., Osumi T., Tada T., Kotera H. BBRC, 2014
Validation of increased survival of single-cell hiPSC clones on LN-521 in culture and a microfluidic chip. Here the authors describe increased survival and propagation of single hiPSC clones on LN-521 in both cultures and on a new microfluidic device. By conditioning the medium they show drastically increased efficiency and represent an additional protocol to the LN-521/E-cadherin method presented in Rodin et al., 2014 for clonal growth.
Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform
Sivalingam J., Lam A.T., Chen H.Y., Yang B.X., Chen A.K., Reuveny S., Loh Y.H, Oh S.K. Tissue Eng Part C Methods, 2016
Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or co-culture with xenogeneic cell lines. In this study, the authors describe the development of a scalable, serum-free, xeno-free, and chemically defined microcarrier-based platform using human recombinant laminin-521 as an extracellular matrix (ECM) for hPSC expansion, EB formation, and subsequently hematopoietic differentiation of hPSC to red blood cells (RBS). Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in an 80-fold improvement in the yield of RBS generation compared to a conventional EB-based differentiation method. In addition, they show efficient terminal maturation and generation of mature enucleated RBCs using a co-culture system that comprised primary human mesenchymal stromal cells.