Publications
Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System
Tano K., Yasuda S., Kuroda T., Saito H., Umezawa A., Sato Y. PLoS One, 2014
In the article, the authors use LN-521 for a safety step for iPS cells going for therapeutic purposes. This group is responsible for dictating the safety aspects of future regen med in Japan. Tano and colleagues show a novel approach based on LN-521 for direct and sensitive detection of trace amounts of residual undifferentiated hPSCs for cell therapy products. The presence of contaminating hPSCs in cell therapy products is a major quality concern associated with tumorigenicity and this first in vitro assay is direct, simple and cost-effective. The highly efficient culture system using LN-521 detected colony-forming hPSCs spiked into primary human MSCs or neurons at a ratio as low as 0.001%–0.01%.
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.
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.
Monolayer culturing and cloning of human pluripotent stem cells on laminin-521 based matrices under xeno-free and chemically defined conditions
Rodin S., Antonsson L., Hovatta O., Tryggvason K. Nature Protocols, 2014
Detailed step-by-step protocols for transfer, expansion and clonal growth of hPSCs on laminin-521. Here the authors describe predictable monolayer, xeno-free and defined culturing of hPSCs on LN-521. In the article, there is an important assembly of protocols for LN-521 based hPSC bulk expansion, true clone generation, the secure transfer step-by-step from feeders to LN-521, freezing and thawing as single cells using FREEZEstem. There are also critical steps and reagents included for easier handling of more difficult lines and a useful troubleshooting guide for solving problems faster.
a-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal
Laperle A., Hsiao C., Lampe M., Mortier J., Saha K., Palecek S.P., and Masters K.S. Stem Cell Reports, 2015
The authors study the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. They identify a-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hESC and hiPSC cultured on defined substrates. The cells also produced collagen I but no vitronectin or fibronectin. Knockdown and disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Self-renewal and survival were restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Systemax or Vitronectin could not restore survival. Treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous a-5 laminin promotes hPSC survival and self-renewal in an autocrine and paracrine manner. A good publication that also shows how much better laminin-521 performs compared to other competitor matrices.
Inhibition of FAK Signaling by Integrin a6B1 Supports Human Pluripotent Stem Cell Self‐Renewal
Villa-Diaz L.G., Kim J.K., Laperle A., Palecek S.P., Krebsbach P.H. Stem Cells, 2016
A newly identified pathway in hPSCs contributes to a better understanding of how laminin-521 maintains pluripotency and self‐renewal. In hPSCs, α6β1 is the dominant integrin of which laminin-521 is a strong inducer. Here the authors describe a signaling pathway in hPSCs linking self‐renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. During differentiation, integrin α6 levels diminish and FAK is phosphorylated and activated. Integrin α6 functions in the inactivation of integrin B1 and FAK signaling and prevention of hPSC differentiation. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. The knockdown of laminin α5 resulted in a reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self‐renewal.
Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling
Atefeh Namipashaki, Kealan Pugsley, Xiaodong Liu, Kirra Abrehart, Sue Mei Lim, Guizhi Sun, Marco J. Herold, Jose M. Polo, Mark A. Bellgrove, and Ziarih Hawi. Stem Cell Reports, 2023
This article presents an improved method for generating genetically homogeneous induced pluripotent stem cell (iPSC) clones following CRISPR-Cas9 genome editing. The greatest single-cell clone survival rate, 70 %, was noted when the human full-length laminin-521 (Biolaminin 521, LN521) surface matrix was applied, with a substantial increase from the 36% survival rate observed when vitronectin was utilized. The method employs ribonucleoproteins and lipid transfection. Validation of the approach across three iPSC lines consistently showed high editing efficiencies. The resulting cloned iPSC lines retained pluripotency, differentiation potential, and genomic stability.
Protocol for the derivation, culturing, and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro
Javier Calvo-Garrido, Dania Winn, Camilla Maffezzini, Anna Wedell, Christoph Freyer, Anna Falk, Anna Wredenberg. STAR Protocols, 2021
This protocol describes the derivation of neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into neurons and glia. The PSC culture and NES differentiation were done on plates coated with Biolaminin 521 (laminin-521). To avoid clonal selection of isolated NES cells, it is recommended to follow the culture conditions described. The protocol is expected to result in highly proliferate NES cells providing a good source of cells of a neuronal cell lineage. Glial cells are formed after approximately 45 days of differentiation.
CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development
Pia Annette Johansson, Anita Adami, Johan Jakobsson. STAR Protocols, 2022
This protocol describes using CRISPRi-mediated transcriptional silencing in human induced pluripotent stem cells. In addition, it contains an efficient protocol for neural progenitor differentiation. The method is directly applicable to loss-of-function studies in brain development research. Biolaminin 521 (laminin-521) is applied as the cell culture matrix for PSCs and Biolaminin 111 (laminin-111) for hiPSC differentiation into forebrain neural progenitor cells. The protocol is expected to achieve high transduction and silencing efficiency. After two weeks of differentiation, the authors detected no reduction in the percentage of positive cells, and the cerebral organoids had stable transcriptional silencing even after 4 months.
Generation of a CHIP isogenic human iPSC-derived cortical neuron model for functional proteomics
Catarina Dias, Erisa Nita. Jakub Faktor, Lenka Hernychova, Tilo Kunath, Kathryn L. Ball. STAR Protocols, 2022
This protocol describes the production of gene-edited cells using CRISPR-Cas9 and a patient-derived induced pluripotent stem cell (iPSC) line. Biolaminin 521 was used as the matrix to improve single cell survival and pluripotency. The resulting panel of iPSC lines was differentiated into cortical neurons with the Biolaminin 111 culture matrix. The overall aim was to identify protein and pathway targets for the neuroprotective E3-ubiquitin ligase CHIP, which is important in healthy brain aging. The protocol can be adapted to other proteins and pluripotent stem cell lines.
