Science Room

iPSC Reprogramming and Expansion

PRODUCT RECOMMENDATIONS

LAMININ-521
 
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PROTOCOLS


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Integration Free Derivation of Human iPS Cells Using Laminin 521 Matrix

Protocol for efficient iPSC derivation and expansion on laminin 521

 

mRNA reprogramming - high-throughput, xeno-free, feeder-free, footprint-free iPSC derivation

mRNA reprogramming on laminin-521


APPLICATION NOTES


CUSTOMER TESTIMONIALS 

"We at the iPS core at Karolinska Institutet use laminin-521 for derivation and maintenance of our iPS cells. It is reliable, consistent and user-friendly for us and for our customers."

                               Malin Kele, iPS Core at Karolinska Institutet, Sweden


"We have discovered L521 is far superior to all other alternative ECMs we have tried for hPSC culture, and have switched all our hiPSC and hESC lines to L521 for maintenance. As a result, our hPSC lines have become more stable, yields increased and survival improved dramatically on passage. We can now perform more experiments with each flask, and do so with greater confidence of high quality results due to the morphological uniformity of pluripotent cells grown on L521."

                               Dr. Jonathan Niclis, Univ. of Copenhagen, Denmark

         
MORE TESTIMONIALS HERE


ARTICLES

hPSC CULTURE ARTICLES


MORE INFORMATION

EFFICIENT REPROGRAMMING AND EXPANSION OF HUMAN IPS CELLS ON LAMININ SUBSTRATES

Robust and reliable human ES and iPS cell expansion on defined and xeno-free human recombinant laminin-521 cell culture reagentsHigh-efficiency iPSC reprogramming and expansion on laminin-521

Human recombinant laminin-521 is a chemically defined and xeno-free system for robust, clinically compliant reprogramming and expansion of human induced pluripotent stem cells (Uhlin, 2017a, Uhlin, 2017b, Tano, 2014; Lu, 2014). It successfully replicates the genuine human stem cell niche in vitro and thereby provides a highly reliable and reproducible culture system to create sufficient populations of undifferentiated cells that, in turn, can be terminally differentiated. 

Reprogramming on laminin-521 has successfully been performed using mRNA (Warren, 2010; Sellgren, 2016) and Sendai-virus methods (Uhlin 2017a; Uhlin 2017b). Cells can be passaged in very low cell densities and can be cultured to high confluence without phenotypic alterations (Rodin, 2014a; Rodin, 2014b). Moreover, laminin-521 supported direct hiPSC lineage-specific differentiation (Lu et al., 2013). A publication by Matsumura and co-workers show how laminin-521 increased survival and propagation of single-cell hiPSC clones in a microfluidic device by conditioning the medium (Matsumura, 2014).

Robust, long-term expansion of human pluripotent stem cells on laminin-521 

Once the cell line is established, laminin-521 supports robust, long-term, single-cell expansion of human pluripotent stem cells. Pluripotent stem cells grow as a monolayer on laminin-521 providing you with a homogenous, pluripotent cell culture without need to dispose of unwanted differentiated cells (Rodin, 2014a; Rodin, 2014b). The cells migrate quickly on laminin-521, which correlates with the high survival of the cells (Rodin, 2014a). The natural cell-matrix interaction triggers authentic biorelevant signals and gives homogenous and genetically stable PSC cultures with a more uniformed gene expression. Importantly, culture on laminin-521 primes the hPSCs and gives more efficient differentiation and enhances cell maturation, polarization and organization (Cameron, 2015; Penton, 2016). Laminin-521 also support high post-thawed survival of cryopreserved pluripotent stem cells (Miyazaki, 2013). Cells amplify faster compared to other matrices, with about 10-fold expansion in 4 days.

A flexible culture system that is easy to control

Due to the authentic biorelevant signals provided by the culture matrice, laminin-521 is a flexible culture system. Laminin-521 works with most commercial medias and hPSCs can routinely be cultured as single cells without need to add apoptosis inhibitors, such as ROCKi. Since laminin efficiently buffers endogenously produced growth factors laminin-521 also performs well together with growth-factor reduced medias (Rodin, 2014a). The biorelevant support provided by the laminin-521 matrix also have a protective effect against DNA damage caused by suboptimal culture conditions, such as high-density culture and pH changes (Jacobs, 2016). This allows for weekend-free feeding, drastically reducing work load, time and cost for growing your cells. A detailed step-by-step protocol for secure transfer from feeders, true clone generation and expansion of hPSCs and freezing and thawing as single cells on laminin-521 has been published (Rodin et al., 2014b). There are also critical steps and reagents included for easier handling of more difficult lines and a useful trouble shooting guide (Rodin et al., 2014b).

Laminin-521 successfully replicates the genuine human stem cell niche in vitro

In the developing embryo, the alpha-1 and alpha-5 laminins are the first extracellular matrix proteins to be expressed. Laminin-111 is mostly expressed in extra-embryonic basement membranes and stimulates cells to commit to differentiation. Contrary, laminin-521 and laminin-511 are expressed around the stem cells in the inner cell mass and support survival and self-renewal of the pluripotent stem cells (Domogatskaya, 2012; Rodin, 2014a). The alpha-5 laminins are also produced endogenously by human pluripotent stem cells cultured in vitro and are critical autocrine and paracrine factors that regulates their survival and self-renewal (Laperle, 2015). 

Laminin-521 has strong interaction to the α6β1 integrin which induces the PI3K/Akt signaling pathway. In pluripotent stem cells, this interaction promotes survival and efficient long-term self-renewal (Rodin, 2014a; Rodin, 2014b). Activation of integrin α6β1 by alpha-5 laminins have also show to regulate focal adhesion kinase (FAK) signaling in hPSCs and disruption of this pathway results in hPSC differentiation (Villa-Diaz, 2016). Laminin-511 binds the same integrin but the α6β1 integrin mediating effects of laminin-521 is much stronger than that of laminin-511 which results in more robust hPSC expansion on laminin-521 (Rodin, 2014a).

LAMININ KEY ADVANTAGES

  • Robust iPSC reprogramming and expansion on laminin-521

  • Defined and xeno-free cell culture matrix for clinical compliance

  • A biologically relevant culture environment for human ES and iPS cells 

  • No lot-to-lot variability for standardized experiments with less variation

  • Rapid cell expansion for fast scale-up

  • Homogenous and genetically stable hPSC cultures without need to remove differentiated parts

  • Laminin-521 triggers the authentic cell signaling pathways which generates hPSCs with a more uniform gene expression profile

  • Flexible culture system and easy to control: compatible with any medium, supports weekend-free feeding, single-cells passage without ROCKi, low density seeding to high confluence culture

  • Scientifically validated in high-impact journals

  • x
  • Expansion of human PSC

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Neural cells

  • Skeletal muscle cells

  • Kidney cells

  • Hepatic cells

  • Cancer cells

  • Lung cells

  • Intestinal cells

  • Pancreatic cells

  • Hematopoietic stem and progenitor cells

  • Mesenchymal stem cells

  • Animal stem cells

  • Endothelial cells

  • Normal and cancerous mammary cells

  • Epithelial cells

Laminin-521

Laminin-521 human stem cell matrix makes pluripotent stem cell culture easy in a defined, xeno-free and biologically relevant cell culture system.

  • x
  • Neural cells

  • Hepatic cells

  • Lung cells

  • Intestinal cells

  • Normal and cancerous mammary cells

  • Expansion of human PSC

  • Clonal cell culture applications

  • Eye cells

  • Cardiac cells

  • Skeletal muscle cells

  • Kidney cells

  • Cancer cells

  • Pancreatic cells

  • Hematopoietic stem and progenitor cells

  • Mesenchymal stem cells

  • Animal stem cells

  • Endothelial cells

  • Epithelial cells

Laminin-111

Laminin-111 is important during early epithelial development and differentiation of the epiblast, and is commonly used as a general attachment protein in cell culture.

  • x
  • Neural cells

  • Eye cells

  • Endothelial cells

  • Animal stem cells

  • Pancreatic cells

  • Epithelial cells

  • Cardiac cells

  • Cancer cells

  • Hepatic cells

  • Intestinal cells

  • Kidney cells

  • Lung cells

  • Skeletal muscle cells

  • Expansion of human PSC

  • Clonal cell culture applications

  • Hematopoietic stem and progenitor cells

  • Mesenchymal stem cells

  • Normal and cancerous mammary cells

LAMscreen

LAMscreen is a kit with all our currently available laminin isoforms so that you can easily screen the best laminins for different cell types.