Stem cells of animal origin

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 the extracellular matrix and influence the behavior 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 have been used for the defined and reliable cultures of many animal origin pluripotent stem cells, such as a mouse, rabbit, and monkey.

Culture of mouse pluripotent stem cells

Only laminin-511 is expressed in the early mouse embryo instead of the human PSC-specific isoform laminin-521. Drs. Austin Smith and Jennifer Nichols, in an article published in Nature Cell Biology, studied ESCs in mouse embryos and showed that laminin-511 is crucial for the 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 the 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 a 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 proteins for mESCs. The mESCs were found to adhere with the 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).

Culture of monkey pluripotent stem cells

Laminin-521 and laminin-511 are two of the first extracellular 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 origin-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.

Protocol:

Maintaining pluripotent mouse ES cells in vitro without the addition of leukemia inhibitory factor