Uhlin E., Marin Navarro A., Rönnholm H., Day K., Kele M., Falk A.
J Vis Exp. 2017
Here the authors describe a consistent, highly reproducible and easy-to-use protocol, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. This protocol has been extensively tested and used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described.
Warren L., Manos P.D., Ahfeldt T., Loh Y-H., Li H., Lau F., Ebina W., Mandal P., Smith Z.D., Meissner A., Daley g.Q., Brack A.S., Collins J.J, Cowan C., Schlaeger T.M, Rossi D.J.
Cell Stem Cell., 2010
Here the authors describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. Laminin-521 is used as matrix. They show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols and that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells.
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 by passing bioinformatics assay PluriTest and by embryonic body assay.
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 sigthly improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in Ecadherin-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 grown and differentiated avoiding the formation of cell aggregates.
Rodin S., Antonsson L., Hovatta O., Tryggvason K.
Nature Protocols, 2014 (b)
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.
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 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 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.
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.
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.
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 is confirmed.
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%.
Lu H.F., Chai C., Lim T.C., Leong M.F., Lim J.K., Gao S., Lim K.L., Wan A.C.
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.
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.
Matsumura T., Tatsumi K., Noda Y., Nakanishi N., Okonogi A., Hirano K., Li L., Osumi T., Tada T., Kotera H.
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.
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.
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, the authors 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.
Sellgren C.M., Sheridan S.D., Gracias J., Xuan D., Fu T., Perlis R.H.
Molecular Psychiatry, 2017
Human fibroblasts were reprogrammed and the resulting iPSC colonies stabilized and expanded under xeno-free conditions by Stemiotics. Colonies were bulk passaged from the most productive well to establish passage 1 (P1) iPSC cultures on Laminin-521 in Nutristem XF media and expanded in the same culture system until at least passage 3 before being frozen down for storage. The iPSC where then used for neural differentiation.
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.
Miyazaki T., Nakatsuji N., Suemori H.
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.