The laminin-521 matrix recreates the natural stem cell niche in the dish

In the developing embryo, laminins containing the alpha-1 and alpha-5 subunits are the first extracellular proteins to be expressed. Laminin-111 (LN-111) is mostly expressed in Reichert’s membrane which supports the outer, extra-embryonic layer of trophoblasts. LN-111 is widely expressed during embryogenesis, important for the development into the different lineages. Contrary, laminin-521 (LN-521) and laminin-511 (LN-511) are expressed around the stem cells in the inner cell mass and support survival and self-renewal of the pluripotent stem cells (Domogatskaya et al., 2012; Ekblom et al., 2003; Miner & Yurchenco, 2004). The alpha-5 laminins are also produced endogenously by pluripotent stem cells cultured in vitro and are critical autocrine and paracrine factors that regulates their survival and self-renewal (Laperle et al., 2015). Knockdown and disruption of the gene encoding the α-5 laminin subunit dramatically reduce hPSC self-renewal and increased apoptosis. However, the cell viability can be restored to wild-type levels by culturing the deficient cells on LN-521 (Laperle et al., 2015). LN-521 has strong interaction to the α6β1 integrin which have shown to have a pivotal role for hPSCs.

Laminin binding induces the PI3K/Akt signaling pathway, promoting hPSC survival and stimulating cell proliferation over differentiation (Rodin et al., 2010; Miyazaki et al., 2008; Rodin et al., 2014). Activation of integrin α6β1 by α-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 et al., 2016). 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 (Rodin et al., 2014).


LN-521 is an optimal matrix for culture of pluripotent stem cells

The LN-521 matrix successfully recreates the natural nice for pluripotent stem cells when cultured in vitro. The defined and xeno-free LN-521 cell culture system making handling of hPSC easy, reliable and standardized. LN-521 supports high survival and robust, long-term expansion of hPSC cells with maintained pluripotent phenotype. We have successfully and efficiently grown hESC and iPSC for more than 100 passages with maintained pluripotency and genetic integrity. The cells grow in a homogeneous monolayer without any need for manual removal of differentiated cell areas. Motility of hES cells on LN-521 is higher than that of the cells on other matrices which correlates to the survival of the cells (Rodin et al., 2014).



LN-521 provides a robust and reliable hPSC culture system for standardized 
experiments

Compared to LN-521, LN-111-based culture matrices, such as tissue isolates or extracts of the Engelbreth-Holm-Swarm (EHS) tumor (Engel et al., 1981), cannot provide the correct biorelevant signals to support pluripotent stem cells which often results in loss of pluripotency and genetically drift in vitro. It has been reported that feeder-free substrates, such as recombinant vitronectin, fibronectin and a LN-511 fragment, can serve as feeder-free alternatives to the EHS-based matrices for culturing of human pluripotent cells. However, none of them are optimal substrates for high quality pluripotent stem cell maintenance. Many of them are unable to maintain an undifferentiated, genetically stable hPSC population. For example, collagens and fibronectin are not able to support an undifferentiated hPSC population (Laperle et al., 2015). The cells are often recommended to be passaged as colonies which significantly induces the risk of spontaneous differentiation. Moreover, none of these feeder-free cell culture matrices allows clonal survival of human embryonic stem cells without the use of apoptosis inhibitors or chemically undefined substances (Rodin et al., 2014).

Contrary to many other feeder-free matrices on the market, LN-521 provides the natural environment for human pluripotent stem cells. The cells can reliably be cultured as single cells and even seeded clonally without addition of inhibitors of anoikis (Domogatskaya et al., 2012; Rodin et al., 2014). LN-521 support maintained PSCs pluripotent phenotype and genetic stability and the cells grow faster compared to other feeder-free substrates (Rodin et al., 2014; Laperle et al., 2015).

Pluripotency 

Growth

Left figure: Cells on LN-521 grow faster compared to cells cultured on other matrices.
Growth rate of a hESC (HS181) and an iPS (C3) cell line on different feeder-free coating matrices in mTeSR1 medium and according to manufacturer’s instructions. Experiments are done in duplicates for each cell line.


Right figure: LN-521 cultured cells remain pluripotent without spontaneous differentiation. 
hESC (HS181) cells cultured on different feeder-free coating matrices in mTeSR1 medium and according to manufacturer’s instructions. Immunostaining for pluripotency markers Nanog, Oct4, SSEA4 and Tra-1-81. DAPI for nuclear staining. LN-521 cultured cells remain pluripotent whereas on Matrigel and Vitronectin some cells undergo spontaneous differentiation with loss of pluripotent marker expression (white arrows).

KEY ADVANTAGES

  • No feeding required Saturdays or Sundays – Work-free weekends!
  • No strict passaging schedule
  • The cells maintain high pluripotency expression and proliferation rate
  • Easy, efficient and reliable single cell passaging enables standardized experiments
  • Defined and xeno-free long-term propagation of hPSC
  • Protocol validated with a low bFGF concentration medium

 

  

 

Figure 2. Weekend-free cultured cells maintain characteristic, pluripotent cell morphology and protein expression
 
Expression of pluripotent stem cell marker OCT-4 (red staining) was high after 6 passages for both 
the human ES cells (HS181 and HS980) and iPS cells (C3) maintained in Nutristem using standard or weekend-free feeding protocols. DAPI staining (blue) was used as control. The cells also consitently maintained undifferentiated cell morphology (bright-field pictures).

LN-521 facilitates long-term self-renewal of human pluripotent stem cells without weekend feeding

Most established human pluripotent stem cells (hPSC) culture protocols require daily medium replacement, demanding researchers to routinely work weekends. Based on the biorelevant support from LN-521, we developed a protocol that eliminates weekend feeding. The protocol is flexible - just feed your cells daily during weekdays and perform a simple LN-521 single cell passage on Thursday or Friday. Splitting frequency will be cell line dependent, therefor simply adjust the seeding density, optimal for your cell line and work schedule. The only important guideline is that the cells must be passaged late in the week (e.g. Thursday or Friday). Your hPSC cultures will maintain characteristic cell morphology, high pluripotent cell marker expression and high expansion rates. 


We validated the weekend-free feeding protocol (feeding only weekdays) for support of long-term maintenance of human ES and iPS cells by com- paring it to standard, every day feeding protocol (feeding 7 days/week). In both cases, cells were routinely passaged as single cells twice/week but always with a passage at the end of the week (Thursday or Friday). For more information about the easy and reliable LN-521 single-cell passage protocol, see INSTRUCTIONS FOR USE BL003. Experiments were performed using two hESC lines (HS181 and HS980) and one iPS cell line (C3), maintained in Nutristem XF medium on LN-521 stem cell matrix for 6 passages. Both culturing protocols were performed in parallel with 4 independent wells (n=4) for each cell line and protocol.


Compared to the standard protocol, all three cell lines cultured with the weekend-free culture protocol maintained the same high, long-term expansion rate for 6 passages (Figure 1). Independent of culture protocol, all three cell lines grew as a homogenous monolayer with characteristic hES and iPS cell morphology with prominent nucleoli and a high nuclear to cytoplasmic ratio (Figure 2, bright-field pictures). The cells also maintained the features of undifferentiated human ES and iPS cells as illustrated by high expression of the pluripotent stem cell marker OCT-4 (Figure 2, red staining).

 

 

Figure 1. Cell expansion rates are just as high under weekend-free feeding condition
Human ES cells (HS181 and HS980) or iPS cells (C3) were maintained in Nutristem under standard, every day-feeding (filled lines) or weekend-free feeding conditions (dashed lines) for 6 passages. Fold expansion at each passage was determined by comparing the number of cells generated to the amount seeded.

 

 

REFERENCES

  • Functional diversity of laminins. Domatskaya et al. Annu Rev Cell Dev Biol., 2012
  • Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. Engel et al. Journal of Molecular Biology, 1981
  • α-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal. Laperle et al. Stem Cell Reports, 2015
  • Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Miyazaki et al. Biochem Biophys Res Commun, 2008
  • Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Rodin et al. Nat Biotechnol, 2010
  • Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Rodin et al. Nat Commun, 2014
  • Inhibition of FAK Signaling by Integrin α6β1 Supports Human Pluripotent Stem Cell Self‐Renewal. Villa-Diaz et al. Stem Cells, 2016