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 trouble shooting 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 generation and culture of human pluripotent stem cells. Clonal derivation and single-cell expansion of hPSCs on laminin-521.This article provides scientific evidence that LN-521 is the optimal matrix for 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.

 

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 was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Synthemax 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 show how much better laminin-521 performs compared to other competitor matrices.

 

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 occurs 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.

 

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

Newly identified pathway in hPSCs contribute to a better understanding of how laminin-521 maintain 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 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. Knockdown of laminin α5 resulted in 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.

 

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.

 

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.

 

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 demonstrates Laminin-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 Laminin-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 Laminin-521, allowing for efficient and easy handling of cells and bulk storage of high-quality hPSCs.

 

Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures

Lam A.T., Li J., Chen A.K., Birch W.R., Reuveny S., Oh S.K.
BioResearch Open Access, 2015

The authors show that LN-521 enables efficient cell attachment and spreading of hPSCs that results in high cell yields (~3.5×10^6 cells/mL) within 7 days in agitated plate and scalable spinner cultures. This offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN-521 on microcarriers also enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10^8 cells.

 

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 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.

 

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 protocol, 2016

Here, the authors developed an 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 (LNLN-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.

 

North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13

Small K.W., DeLuca A.P, Whitmore S.S, Rosenberg T., Silva-Garcia R., Udar N., Puech b., Garcia C.A., Rice T.A., Fishman G.A, Héon E., Folk J.C, Streb L.M., Haas C.M., Wiley L.A., Scheetz T.E., Fingert J.H., Mullins R.F., Tucker B.A., Stone E.M.
American Academy of Ophtalmology, 2015

Genome sequencing of patients to identified rare mutations involved in macular dystrophy. iPSCs were maintained in Essential 8 media on 521-To-Go plates and then differentiated by a 3D differentiation protocol to retinal tissues.

 

Control of ground-state pluripotency by allelic regulation of Nanog

Miyanari Y., Torres-Padilla M.E.
Nature, 2012 

In this article laminin-511 used to coat glass-bottomed dishes.

 

Live visualization of chromatin dynamics with fluorescent TALEs

Miyanari Y., Ziegler-Birling C., Torres-Padilla M.E.
Nature structural & molecular biology, 2013 

The authors of these two Nature publications show that laminin can be coated directly on glass, which many other substrates and proteins can not. This enables growth of pluripotent stem cells as monolayeres even on glass, which is especially suitable for live imaging.

  

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.

 

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, and Matrigel, gelatin and poly-D-lysine are compared as substrata maintaining pluripotent mouse ES cells in vitro without 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. 

 

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 was created with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated to 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 significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation.

 

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 purpose. 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-521The 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 is confirmed.

 

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 were 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.

 

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 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.

 

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 culture and on a new microfluidic device. By conditioning the medium they show drastically increased efficiency and represents an additional protocol to the LN-521/E-cadherin method presented in Rodin et al., 2014 for clonal growth.

 

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 Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,- 15,-18,-21 Embryo

Aparecida Siqueira Fonseca S., Montero Costas R., Morato-Marques M., Costa S., Roberto Alegretti J., Rosenberg C., Leme E., da Motta A., Serafini P.C., Pereira L.V.
PLOS ONE, 2015

Aneuploid embryos (Array-CGH analysis) cultured until day-5 or 6 after in vitro fertilization. To derive new hESC lines under defined xeno-free culture condition, we used the CloneStem kit that contain recombinant laminin-521 and E-cadherin as matrix and E8 medium supplemented with 10% human albumin serum for 48h. The first passage was made mechanically after 15 days and the fragments were transferred to laminin-521 and e-cadherin, in E8 medium supplemented with 5 uM of Rock Inhibitor. The next three passages were made mechanically and the fragments were transferred to plates coated only with Laminin-521 in E8 medium, in a split ratio of 1:2. After passage five, the hESCs were maintained in Geltrex and E8 medium. Analysed pluripotency, G-banding karyotype analysis, SNP genotyping, differentiation capacity (EB). The autors show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo ́s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the trophectoderm lineage. Two embryos out of ten embryos attached to the culture plate and presented cell growth. From these only one embryo gave rise to a new line of hESC. This rate of derivation is relatively low and is similar to those obtained on Matrigel with euploid embryos.