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%.
Generation of human iPS cell line CTL07-II from human fibroblasts, under defined and xeno-free conditions
Kele M., Day K., Rönnholm H., Schuster J., Dahl N., Falk A.Stem cell research, 2016
CTL07-II is a healthy feeder-free and characterized human induced pluripotent stem (iPS) cell line cultured under xeno-free and defined conditions. iPS cell coating during derivation and expansion was human recombinant Laminin-521. The line is generated from healthy human fibroblasts with non-integrating Sendai virus vectors encoding the four Yamanaka factors, OCT4, SOX2, KLF4, and cMYC. The generated iPS cells are free from reprogramming vectors and their purity, karyotypic stability and pluripotent capacity are confirmed.
Derivation of human iPS cell lines from monozygotic twins in defined and xeno-free conditions
Uhlin E., Rönnholm H., Day K., Kele M., Tammimies K., Bölte S., Falk A.Stem Cell Res., 2017
Human-induced pluripotent stem (hiPS) cell lines CTRL-9-II and CTRL-10-I were derived from healthy monozygotic twin donors using non-integrating RNA based Sendai virus reprogramming and cultured in a xeno-free chemically defined condition on the laminin-521 cell culture substrate. The established hiPS cell lines, CTRL-9-II and CTRL-10-I, are karyotypically normal, free from reprogramming vectors, display endogenously expression of pluripotency factors at levels similar to embryonic stem cells. The generated iPS cell lines demonstrate pluripotency bypassing bioinformatics assay PluriTest and by embryonic body assay.
The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines
Canham M.A, Van Deusen A., Brison D.R., De Sousa P.A., Downie J., Devito L., Hewitt Z.A., Ilic D., Kimber S.J., Moore H.D., Murray H., Kunath T.Scientific Reports, 2015
It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. In this study, they evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. The hESC lines were all cultured on laminin-521.
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.
Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability
Jacobs K., Zambelli F., Mertzanidou A., Smolders I., Geens M., Nguyen H.T., Barbé L., Sermon K., Spits C. Stem Cell Reports, 2016
In this study, the authors elucidate a direct relationship between culture density and the emergence of DNA damage and genomic alterations in human embryonic stem cells (hESC). Compared with culture on feeder cells, hESCs cultured on laminin-521 (LN521) exhibit reduced susceptibility to DNA damage under suboptimal culture conditions, such as medium acidification during high-density culture. This phenomenon leads to an accumulation of cells in the G1 phase and a halt in the S phase, without concomitant cell death or loss of pluripotency. Remarkably, these DNA effects manifest rapidly, occurring within a single passage.
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.
Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment
Rodin S., Antonsson L., Niaudet C., Simonson O.E., Salmela E., Hansson E.M., Domogatskaya A., Xiao Z., Damdimopoulou P., Sheikhi M., Inzunza J., Nilsson A.S., Baker D., Kuiper R., Sun Y., Blennow E., Nordenskjöld M., Grinnemo K.H., Kere J., Betsholtz C., Hovatta O., Tryggvason K.Nature Communications 2014 (a)
This article provides scientific evidence that LN-521 is the optimal matrix for the generation and culture of human pluripotent stem cells. Laminin-521 successfully recreates the biologically relevant hPSC milieu in vitro and via integrin binding, laminin-521 induces the PI3K/Akt signaling pathway, promoting survival and robust self-renewal of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Clonal derivation and single-cell expansion of hPSCs on laminin-521.This article provides scientific evidence that LN-521 is the optimal matrix for the generation and culture of human pluripotent stem cells. It is described in detail how this physiologically relevant laminin establishes genetically stable hESC lines in an efficient, defined, xeno-free and feeder-free procedure, suitable for stem cell banking and regenerative medicine applications. It is even possible to derive embryonic stem cells from a single blastomere, thereby avoiding the ethical dilemma associated with the destruction of donated embryos. LN-511 binds the same integrin but the α6β1 integrin mediating effects of LN-521 is much stronger than that of LN-511 which results in a more robust PSC expansion on LN-521.
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.
Laminin 521 stabilizes the pluripotency expression pattern of human embryonic stem cells initially derived on feeder cells
Albalushi H., Kurek M., Karlsson L., Landreh L., Rós Kjartansdóttir K., Söder O., Hovatta O., Stukenborg J-B.Stem Cell International, 2017
In this article, the authors tested the effect of the laminin-521 substrate on cultured hES cells. Five male hES cell lines, originally derived on human foreskin fibroblasts (hFFs), were cultured on hFF, Matrigel, laminin-521 or laminin-121 for nine passages. Variations in gene expression related to pluripotency, stemness, and male germ and somatic gonadal cells at different passages and different culture methods were evaluated. All cell lines expressed pluripotency markers at protein and gene level and were able to differentiate into cell types of the three germ layers after being cultured on laminin-521 for nine passages. Laminin 521 had no obvious effect on the expression of genes related to male germ cells and somatic gonadal cells when used as a matrix for hES cell cultures. Importantly, reduction in variation of pluripotency marker expression was observed after culturing the cells on laminin-521 compared to culture on hFFs, and could be reduced further with an increased culture period on laminin-521. Changing the cell culture medium of hES cells on hFFs from Dulbecco's Modified Eagle Medium (DMEM) to NutriStem, which is commonly used for culturing the cells on matrices, did not affect the variation of pluripotency genes and genes related to stemness expression. hES cells cultured on laminin-521 were more homogenous, attached better and grew faster compared to hES cells cultured on matrigel. The results show that laminin-521 provides optimal culture conditions for adaptation to feeder and xeno-free conditions of hES cells derived and cultured on feeder cells. Moreover, laminin-521 has a positive effect on stabilizing and homogenizing pluripotent gene expression profiles between hES cell lines provides the first step towards more controllable and robust culture conditions for hES cells.