Publications

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 the growth of pluripotent stem cells as monolayers even on glass, which is especially suitable for live imaging.

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

Alan Tin-Lun Lam, Jian Li, Allen Kuan-Liang Chen, William R Birch, Shaul Reuveny, Steve Kah-Weng Oh. Biores Open Access, 2015

The authors used different bioreactors up to 2.5 L in scale, and successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks where they use a short period of intense agitation in the presence of enzymes such that the cells are detached without being damaged. Here, the same approach has been effective for 15 mL ambr^TMbioreactors, 100 mL spinner flasks and 250 mL Dasgip bioreactors.  Two types of microcarrier were used, with (laminin-521) and without surface coatings, four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. Qasim Rafiq presented this data at BioLamina symposium 2015. It was found that human recombinant laminin LN-521 and recombinant fibronectin were the optimal ECM proteins for microcarrier coating. A serum-free expansion, harvest and xeno, and DMSO-free cryopreservation process, using LN-521 coated microcarriers and FREEZEstem^TM cryopreservation medium was then developed which demonstrated > 5 fold increase in hMSC yield.

Collaboration of 3D Context and Extracellular Matrix in the Development of Glioma Stemness in a 3D Model

Ma N.K.L., Kai Lim J., Fatt Leong M., Sandanaraj E., Ti Ang B., Tang C., Wan A.C.A. Biomaterials, 2015

This work illustrates that different laminin isoforms have specific effects in promoting the stemness of glioma cells, in collaboration with a 3D context. U251 glioblastoma cells were cultured on electrospun polystyrene (ESPS) scaffolds coated with 7 different laminin isoforms (LAMscreen kit) to provide a 3D model for stem cell-related genes and proteins expression studies. The results indicate the influence of 3D (versus 2D) context on stemness markers and integrin expression, specifically, the upregulation of the laminin-binding integrins a6b4. By a colony-forming assay, we showed enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins 411, 421, 511 and 521. The present results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness.

Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

Lecht S., Stabler C.T., Rylander A.L., Chiaverelli R., Schulman E.S., Marcinkiewicz C., Lelkes P.I.Biomaterials, 2014

Pre-treatment of the decellularized lung with defined ECM proteins, to evaluate the efficacy of reseeding of mESC. All current decellularization methodologies result in significant alterations of the contents, and ratios of ECM proteins, though the exact degree of the loss of ECM glycoproteins, such as laminins. Following ECMs were tested: bovine collagen type IV; human collagen type IV; human pro-collagen type I; human collagen type I; rat collagen type I; human plasma-purified fibronectin; human thrombospondin-1; VCAM; vitronectin; bovine elastin; Matrigel-purified laminin 111; and human recombinant laminins 111, 211, 332, 411, 421, 511, 521. mESCs lack major integrin receptors for collagens but express high levels of functional receptors for LM and FN. Laminin next to FN appears to be the major pro-adhesive ECM protein for mESCs. The mESCs were found to adhere differentially in an isoform-dependent manner with the following order of potencies 511 = 521 >332 >421 >211 >111 >411. These findings further support the notion that a3b1 and a6b1 integrin receptors expressed on the mES cell surface are involved in the specific binding to LM in general and to LM 511 and 521. LM and FN interact more efficiently with collagen type I than with collagen IV or elastin 90% of the available Coll I in the decellularized lung matrix forms a complex with LM. in vitro that cell-derived FN and LM interact with Coll I with much higher efficiencies than commercial FN or LM This finding may be explained by a possible partial loss of activity as a result of the purification procedures in contrast to the unprocessed CM.

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks

Sorkioa A., Kochb L., Koivusaloa L., Deiwickb A., Miettinena S., Chichkovb B., Skottman H.
Biomaterials, 2018

In this study, the authors produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell-derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue-derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The authors used two previously established LaBP setups based on laser-induced forward transfer, with different laser wavelengths and appropriate absorption layers. Recombinant human laminin and human-sourced collagen I served as the bases for the functional bioinks. For hESC-LESCs, bioink containing human recombinant laminin-521 was chosen, as laminin is a major component in LESC basement membrane in the native cornea. Three different types of corneal structures was printed: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression. The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and the successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.

Expression and biological role of laminin-1

Ekblom P., Lonai P., Talts J.F'
Matrix Biol., 2003

Engineered Human Stem Cell Microenvironment

Jordahl J.H., Villa-Diaz L., Krebsbach P.H., Lahann J.Stem Cells and Nanotechnologies, 2016

Review of engineered stem cell niches. Laminin (from various sources) is throughout the paper pointed out is the single most important factor for culturing both hPSCs but also many other stem cells.

Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

Wang H., Luo X. Leighton J.Biochemistry Insights, 2015

Here, the author summarizes the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuro-ectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. 

Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics

Kevin G. Chen K.G., Mallon B.S., McKay R.D.G., Robey P.GCell stem cell, 2014

In this review, the authors look at different large-scale hPSC culture growth components by comparing cell culture methods (matrices, media, etc.) and identifying the advantage and disadvantages and pitfalls associated with each one. Since laminin-521 was not available when the review was written, they only mention laminin-511 where the cells are seeded as clumps (Rodin et al 2010). The only disadvantage mentioned regarding laminin-511 is that the use of laminin for maintenance and expansion will be to thigh due to the price of the laminins. 

Human embryonic stem cells

Damdimopoulou P., Rodin S., Stenfelt S., Antonsson L., and Tryggvason K., Hovatta O. Best Practice & Research Clinical Obstetrics & Gynaecology, 2015

A short review on the establishment of hESC lines on LN-521. Authors state that they easily can establish and expand hESC lines in fully chemically defined animal substance-free conditions. hESC lines can be derived from single biopsied cells of embryos that need not be destroyed during the process. The genetic stability and differentiation capacity can be studied. These cell lines can today be safely expanded almost without limitations.