iPSC reprogramming and expansion

High-efficiency iPSC reprogramming and expansion on Biolaminin 521

Human recombinant Biolaminin 521 LN (LN521) is a chemically defined and animal origin-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 LN521 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, LN521 supported direct hiPSC lineage-specific differentiation (Lu et al., 2013). A publication by Matsumura and co-workers shows how laminin-521 increased the 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 Biolaminin 521

Once the cell line is established, LN521 supports the robust, long-term, single-cell expansion of human pluripotent stem cells. Pluripotent stem cells grow as a monolayer on LN521 providing you with a homogenous, pluripotent cell culture without the need to dispose of unwanted differentiated cells (Rodin, 2014a; Rodin, 2014b). The cells migrate quickly on LN521, 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 more uniformed gene expression. Importantly, culture on LN521 primes the hPSCs and gives more efficient differentiation and enhances cell maturation, polarization, and organization (Cameron, 2015; Penton, 2016). LN521 also supports 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, LN521 is a flexible culture system. LN521 works with most commercial media and hPSCs can routinely be cultured as single cells without the need to add apoptosis inhibitors, such as ROCKi. Since laminin efficiently buffers endogenously produced growth factors, LN521 also performs well together with growth-factor reduced media (Rodin, 2014a). The biorelevant support provided by the LN521 matrix also has 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 workload, 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 LN521 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 troubleshooting guide (Rodin et al., 2014b).

Biolaminin 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 isoforms 521 and 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 regulate their survival and self-renewal (Laperle, 2015). 

LN521 has strong interaction with 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 has also shown 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 LN521 (Rodin, 2014a).

Protocols:

Integration free derivation of human iPS cells using LN521 matrix

Highly efficient reprogramming of human cells using synthetic modified mRNA

Defined and animal origin-free, clonal culture, and expansion of hiPSC and hESC on LN521