Here is a selection of publications where different laminin isoforms are being used to create more authentic cell culture systems
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.
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.