Here is a selection of publications where different laminin isoforms were used to create more authentic cell culture systems.
Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions
Börger A-K., Eicke D., Wolf C., Gras C., Aufderbeck S., Schulze K., Engels L., Eiz-Vesper B., Schambach A., Guzman C.A., Lachmann N., Moritz T., Martin U., Blasczyk R., Figueiredo C. Molecular Medicine, 2016
Here, the authors have established a protocol for the generation of megakaryocytes (MK) and platelets (PLTs) from an HLA-universal and virtually unlimited cell source (iPSCs) under xeno-free and defined conditions to facilitate their future translation into clinical application. HLA-universal iPSC was generated and cultured on laminin-521. The expression of HLA class I was silenced (shRNA) by up to 82% and remained stable during iPSC cultivation. The generation of MK and PLTs from an HLA-universal iPSC were conducted on laminin-521. On d19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion.
Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform
Sivalingam J., Lam A.T., Chen H.Y., Yang B.X., Chen A.K., Reuveny S., Loh Y.H, Oh S.K. Tissue Eng Part C Methods, 2016
Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or co-culture with xenogeneic cell lines. In this study, the authors describe the development of a scalable, serum-free, xeno-free, and chemically defined microcarrier-based platform using human recombinant laminin-521 as an extracellular matrix (ECM) for hPSC expansion, EB formation, and subsequently hematopoietic differentiation of hPSC to red blood cells (RBS). Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in an 80-fold improvement in the yield of RBS generation compared to a conventional EB-based differentiation method. In addition, they show efficient terminal maturation and generation of mature enucleated RBCs using a co-culture system that comprised primary human mesenchymal stromal cells.
Large-scale production of megakaryocytes in microcarrier supported stirred suspension bioreactors
Eicke D., Baigger A., Schulze K., Latham S.L., Halloin C., Zweigerdt R., Guzman C.A., Blasczyk R., Figueiredo C.Scientific reports, 2018
Although extensive research has been dedicated to developing processes for differentiating PLTs from MKs, no sustainable process for large-scale MK production is available. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 Å~ 107 MKs per 50 ml medium. Biolaminin 521-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion.
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.
Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors
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 were then used for neural differentiation.
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, the authors used the CloneStem kit that contains 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. Analyzed pluripotency, G-banding karyotype analysis, SNP genotyping, differentiation capacity (EB). The authors 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.
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.
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.
Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA
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
In this article, the authors describe a simple, non-integrating strategy for reprogramming cell fate based on the administration of synthetic mRNA modified to overcome innate antiviral responses. Laminin-521 is used as a 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.
Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix
Uhlin E., Marin Navarro A., Rönnholm H., Day K., Kele M., Falk A. J Vis Exp. 2017
In this video publication, 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.