Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
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.
Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro
Domogatskaya A., Rodin S., Boutaud A., Tryggvason K. Stem Cells, 2008
Different laminin isoforms, LN-511, -332, -411 and -111, as well as Matrigel, gelatin and poly-D-lysine are compared as substrates maintaining pluripotent mouse ES cells in vitro without the addition of leukemia inhibitory factor. Conclusions are that only LN-511 is able to sustain self-renewal for up to 169 days of culturing and cells maintain expression of pluripotency markers and can be used for generation of chimeric mice.
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.
Self-organization of human embryonic stem cells on micropatterns
Deglincerti A., Etoc F., Guerra C.M., Martyn I., Metzger J., Ruzo A., Simunovic M., Yoney A., Brivanlou A.H. Siggia E., Warmflash A. Nature protocols, 2016
Here, the authors developed a reproducible in vitro protocols that allow the study of spatial organization associated with this developmental transition. They use a micropatterning approach in which human embryonic stem cells are confined to disk-shaped, submillimeter colonies. After 42 h of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. The protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 as extracellular matrix coating, and either conditioned or chemically defined medium (mTeSRSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The LN521 coating allows for a simpler coating protocol with robust results. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.
Directed differentiation of human iPSC into insulin-producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors
Walczak M. P., Drozd A. M., Stoczynska-Fidelus E., Rieske P., Grzela D.P.Journal of Translational Medicine, 2016
Here, the authors show that the highest efficiencies of reprogramming of fibroblasts were obtained on Laminin-511 and Laminin-521, compared to other coatings. iPSC cell lines were created with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of the doxycycline-inducible promoter. These cells were differentiated into insulin-producing cells. Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with a significantly higher hormone release level in the case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. The efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cell maturation.
High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells
Carlson-Stevermer J., Goedland M., Steyer B., Movaghar A., Lou M., Kohlenberg L., Prestil R., Saha K.Stem Cell Reports, 2016
The authors developed a genome-editing method where they utilize surface-modified multiwell plates containing one-pot transcribed single-guide RNAs for automated, live, high-content imaging and analysis. To test the speed and efficiency of the genome editing method the authors used the LAMA5 gene encoding a-5 laminin since this extracellular matrix protein is known to be an important autocrine/paracrine factor regulating survival and self-renewal of hESCs. The LAMA5 edited hESC clones exhibited decreased rates of self-renewal and increased rate of apoptosis and culture on Matrigel was insufficient to rescue the growth phenotype. However, when cultured on human recombinant laminin-521, all LAMA5 gene-edited lines were rescued with growth rate and levels of apoptosis similar to the wild-type cells. This publication is another proof of principle for the LN-521 stem cell matrix showing that a5 laminin is a critical factor for hPSC survival and self-renewal.
Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin-521 and E-cadherin-Fc
Leino M., Astrand C., Hughes-Brittain N., Robb B., McKean R., Chotteau V.J Biomed Mater Res Part B 2017
Here, the authors describe a nonaggregate culture system of human embryonic stem cells inside electrospun polycaprolactone (PCL) fiber scaffolds combined with the defined extracellular proteins laminin-521, naturally occurring in the stem cell niche. PCL fiber scaffolds coated with recombinant human laminin-521 readily supported initial stem cell attachment and growth from a single-cell suspension. The combination of recombinant E-cadherin-Fc and laminin-521 slightly improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in E-cadherin-Fc- and laminin-521-coated PCL scaffolds could differentiate into all three germ layers. Importantly, we provided a chemically defined 3-D system in which pluripotent stem cells grew and differentiated avoiding the formation of cell aggregates.
Optimization of slow cooling cryopreservation for human pluripotent stem cells
Miyazaki T., Nakatsuji N., and Suemori H. Genesis, 2013
Increased viability of hPSCs through single-cell freezing/thawing/expansion on laminin-521. This is one of the first customer publications that demonstrate Biolaminin-521 as an optimal xeno- and feeder-free matrix for pluripotent stem cells. The authors show cells should be cryopreserved as single cells for highest survival which is specifically supported by Biolaminin-521 that promotes adhesion and self-renewal of fully dissociated single cells in the absence of ROCK inhibitor. They demonstrate 80-90% survival of hPSCs post-thawing and 60% survival following subculture on Biolaminin-521, allowing for efficient and easy handling of cells and bulk storage of high-quality hPSCs.
Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture
Hongisto H., Vuoristo S., Mikhailova A., Suuronen R., Virtanen I., Otonkoski T., Skottman H. Stem Cell Res., 2012
The authors of this study show that fibroblast feeders synthesize laminin-511 and that this is the main protein responsible for the maintenance of hESC pluripotency. This strengthens the already known biological function of laminin-511 and laminin-521 as the main cell-adhesion molecules for pluripotent stem cells.
A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells
Lu H.F., Chai C., Lim T.C., Leong M.F., Lim J.K., Gao S., Lim K.L., Wan A.C. Biomaterials 2014
Reprogramming of iPSCs on LN-521 and direct differentiation to dopaminergic cells on Laminin-521. This article demonstrates LN-521 as an optimal defined, xeno- and feeder-free matrix for the reprogramming of human iPS cells. Laminin-521 achieves high-efficiency reprogramming in different media, fast and easy expansion as well as direct differentiation to dopaminergic neurons on LN-521. The authors conclude that the efficient transgene-free hiPSC derivation and expansion on LN-521 enables clinical applications useful for human patient iPSCs and derivatives for cellular therapy.