Promoting Stem Cell Growth and Self-Renewal In Vitro

An engineered laminin substrate facilitates high-quality human stem cell cultures.

The Scientist Creative Services Team in collaboration with MilliporeSigma
Feb 19, 2021

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cientists grow and differentiate human embryonic and induced pluripotent stem (iPS) cells in laboratories around the world to study numerous cellular processes and develop regenerative medicines. To faithfully culture generations of these sensitive cells and drive them toward desired cell types, scientists add several components to their media and perform many steps, all while following strict protocols.

To continuously grow precious stem cells in vitro, researchers must preserve their “stemness.” They often accomplish this by using feeder cells, such as mouse or human fibroblasts, that deliver unknown factors that promote stem cell self-renewal. To grow in vitro, feeder cells require animal components such as fetal bovine serum (FBS). These ingredients do not comply with the Standard for Biological Ingredients, which regulates source materials derived from humans or animals. Additionally, feeder cells often carry viruses that infect stem cells. As an alternative, some scientists now use feeder-free cultures to avoid viruses and animal products in their cultures, and to reduce the labor involved with growing feeder cells. Cellular interactions within feeder-free media promote stem cell self-renewal.

Stem cells naturally interact with diverse extracellular matrix (ECM) proteins in their environment. The basement membrane alone is composed of numerous ECM proteins, which stem cells bind via integrin proteins. These interactions regulate stem cell fates. To replicate this cellular environment in the laboratory, scientists coat in vitro culture plates with ECM proteins and adhesion molecules to facilitate integrin binding. Some of the largest basement membrane proteins are laminins, which are heterotrimeric proteins composed of alpha, beta, and gamma chains.

Human stem cells express the α6β1 form of integrin, which binds to the laminin-511 isoform. The interaction of this integrin with human recombinant laminin-511 in cell culture promotes repeated stem cell renewal without feeder cells.However, laminin-511 is an 800 kDa heterotrimeric protein, making it unsuitable for large-scale production and distribution.

To solve this problem, researchers produced a 150 kDa recombinant fragment of human laminin-511, ECMatrix™-511 E8, that retained full integrin binding activity.2,3 The scientists maintained several human embryonic and iPS cell lines in media containing the fragment. These cells retained high levels of pluripotency marker expression and could differentiate into all three germ layers. Because of its small size, ECMatrix™-511 E8 is simple to produce at large-scale.

Compared to traditional basement membrane extracts, ECMatrix-511 E8 is more consistent lot-to-lot, promotes better cell adhesion and high proliferation rates, reduces cell clumping, and creates consistent microenvironments. The laminin fragment is ideal for CRISPR editing or clonal isolation applications because it allows for single cell passaging without the Rho-associated protein kinase (ROCK) inhibitor to prevent dissociation-induced apoptosis. Additionally, researchers can add the fragment directly to defined animal- and xeno-free media when passaging cells without pre-coating their cultureware. With ECMatrix-511 E8, scientists cultivate high quality feeder-free cultures to reliably propagate stem cells for regenerative medicine and beyond.

References

  1. S. Rodin et al., “Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511,” Nat Biotechnol, 28:611-15, 2010.
  2. T. Miyazaki et al., “Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells,” Nat Comm, 3:1236, 2012.
  3. M. Nakagawa et al., “A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells,” Sci Rep, 4:3594, 2014.