HOW TO CULTURE ANIMAL STEM CELLS ON LAMININ SUBSTRATES

The laminin protein family is highly conserved

Laminins are a large family of conserved, multi-domain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behaviour of associated cells. 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 relative similarity of each pair of laminin chains within a chain constellation is conserved in most vertebrate species (Domogatskaya et al., 2012) and human recombinant laminins has been used for defined and reliable culture of many animal origin pluripotent stem cells, such as mouse, rabbit and monkey.

Culture of Mouse Pluripotent Stem Cells

In contrast to the inner cell mass of the human embryo where both laminin-521 and laminin-511 is expressed, the only laminin isoform expressed in the early mouse embryo is laminin-511. This data is nicely presented in an article published in Nature Cell Biology by the groups of Drs. Austin Smith and Jennifer Nichols where they also show that laminin-511 is crucial for acquisition of naive pluripotency (Boroviak et al., 2014).

Since human recombinant laminin-511 provides the mouse pluripotent stem cells with their natural environment it supports long-term expansion of mouse embryonic stem cells (Domogatskaya et al., 2008). Laminin-511 alone, without the addition of leukemia inhibitory factor (LIF) or other differentiation inhibitors, is sufficient to enable self-renewal of mouse embryonic stem cells (mESCs) for up to 169 days (31 passages) with maintained expression of pluripotency markers. Cells cultured for 95 days (17 passages) on laminin-511 were used to successfully generate chimeric mice (Domogatskaya et al., 2008). This data is supported by a publication by Lecht and co-workers where they show that laminin is one of the major pro-adhesive ECM protein for mESCs. The mESCs were found to adhere with highest potencies to laminin-511 and laminin-521, most likely via specific binding to a3B1 and a6B1 integrin receptors expressed on the mES cell surface (Lecht et al., 2013).

The main benefits of Laminin-511 for mouse pluripotent stem cells are:

• Fast adherence of mESCs to laminin-511
  3 to 5-fold higher affinity than other matrices 
• Rapid cell expansion with minimal spontaneous differentiation 
  Reliable and fast scale-up where cells grow as a homogenous monolayer 
• Biorelevant matrix where no differentiation inhibitors are needed
  Self-renewal of mouse ES cells for over 5 months without the presence of LIF 
• Highly efficient single-cell mESC derivation
  Laminin-511 is crucial for acquisition of naive pluripotency 
• Chemically defined
  No lot-to-lot variation for less experimental variation
• Easy coating and single-cell split/seeding protocol
  Standardized monolayer culture system for automation
• Scientifically proven in high-impact journals
  Validated methods

Culture of Monkey Pluripotent Stem Cells

Laminin-521 and laminin-511 are two of the first extra cellular proteins expressed during development. Both laminin isoforms are present in the inner cell mass of the human embryo and expressed by the pluripotent cells (Domogatskaya et al., 2012). 

Human recombinant laminin-521 is a chemically defined and animal component-free system for robust, long-term expansion of human pluripotent stem cells. Since it successfully replicates the specific human stem cell niche in vitro, it makes it the optimal matrix for efficient generation and robust large-scale expansion of human ES and iPS cells (Rodin et al. 2014). Several researchers are successfully routinely using laminin-521 for pluripotent expansion of monkey pluripotent stem cells (unpublished data).