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Clonal Culture and Genome Editing

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.


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 

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.


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.


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

Here, the authors demonstrate a direct correlation between medium acidification linked to culture density and the occurrence of DNA damage and genomic alterations in hESC grown on feeder layers. This, in turn, results in an increase of cells in G1 and a stalling of the S phase, without an increase in cell death or a loss of pluripotency. The DNA effects are rapid and occur in the short time span of a single passage. However, culture density has no effect on the level of apoptosis. Increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. hESC grown on laminin-521 show a decreased proneness to acquiring DNA damage during suboptimal culture conditions, such as medium acidification during high culture density.


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 the 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 a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

Kim S-J., Habib O., Kim J-S., Han H-W., Koo S.K., Kim J-H.
Stem Cell Research, 2017

Here, the authors generate a homozygous Nrf2 knockout human embryonic stem cell (hESC) line, H9Nrf2KO-A13, using the CRISPR/ Cas9 genome editing method. The human embryonic stem cell H9 line was grown in Essential 8 Medium on laminin-521 coated plates. Human ESCs were dissociated into single, resuspended in Nucleofection solution and electroporated with 30 μg Cas9 and 40 μg of in vitro-transcribed sgRNA using the Amaxa P3 Primary Cell 4D–Nucleofector Kit (Lonza). After 4 days, cells were replated as single cells at a very low density on laminin 521-coated plates in Essential 8 Medium supplemented with a Rho kinase (ROCK) inhibitor (Stemgent). Individual colonies were picked and expanded. Genomic DNA was then extracted using QuickExtract (Epicentre). The target region was amplified and subjected to paired-end read sequencing using Illumina MiSeq at LAS.


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.