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Report No. 002 Recombinant Human Laminin-511 E8 Fragment, iMatrix-511
Report No. Recombinant Human Laminin-511 E8 Fragment, iMatrix-511
002
Laminin is a family of large glycoproteins that is a component of the extracellular matrix existing in basement membranes of animals, and is involved in cell adhesion, migration, and proliferation. Nippi has been manufacturing and selling recombinant human laminin-511 E8 fragment, which is about one fifth of the size of the molecule.

Extracellular matrix

Estimates of the number of cells in the human body vary depending on the method used, but one example is that there are approximately 36 trillion cells in a 70 kg man, 28 trillion cells in a 60 kg woman, and 17 trillion cells in a 32 kg 10 year old child (Ref. 1). The cell size varies greatly depending on the type, but is usually around 10 μm. The cells of the human body cannot form the complex structure of an animal body simply by gathering together. The structure that plays an important role is called the extracellular matrix. As shown in Figure 1, ECM forms physical structures between cells, acts as a backing for single or multilayered cell sheets, separates cells of different types, and plays a role in creating an environment in which the various types of cells that make up the human body can perform their appropriate functions in the appropriate locations. The main components of ECM is collagen protein family, which is said to be the most abundant protein in the human body. The ECM not only functions as a mechanical support for organ tissue, but also has the ability to transmit various biological signals to cells, such as cell survival, proliferation, movement, and what molecules to synthesize. The ECM contains proteins such as collagen, laminin, fibronectin, and proteoglycans with sugar chains, as well as polysaccharides (glycosaminoglycans) such as hyaluronic acid, and these are intricately combined and exist in all parts of the body.

Figure 1. Schematic diagram showing the relationship between the ECM and cells

Recombinant Human Laminin-511 E8 (The product name: iMatrix-511)

Laminin exists in the thin sheets of basement membrane together with other ECM components such as type IV collagen, perlecan, and nidogen. Laminin molecules are composed of three polypeptide chains: α, β, and γ chains, and more than 16 isoforms of laminin have been reported, depending on the combination of alpha, beta and gamma chains (Fig. 2, Ref. 2). iMatrix-511 is a recombinant protein made from parts of the human α5, β1 and γ1 chains. Some cells synthesize receptor proteins called integrins that bind to extracellular matrix components, and through these receptors, they receive various signals related to cell adhesion, expansion and proliferation. Integrins are membrane proteins composed of two chains, an alpha chain and a beta chain. The integrin receptors that mainly interact with laminin 511 are the α6β1 and α3β1 integrins. The globular domains LG1-LG3 in the α5 chain of laminin and the carboxyl terminus of the gamma 1 chain of laminin are the binding sites for integrin (Refs. 3-5), and the laminin-511 E8 fragment contains a binding site for α6β1 integrin (Ref. 5).


Figure 2. Schematic diagram of laminin-511E8

The use of Laminin-511 E8

The laminin-511 E8 fragment can be used as a coating material on a substrate for culturing pluripotent stem cells, such as iPS cells and ES cells, as well as other cell types such as epidermal cells and nerve cells. In cell culture, the fragment, diluted in a solvent such as PBS, can be coated onto a culture substrate. Furthermore, as described later, even if the laminin-511 E8 is not coated on the culture substrate, pluripotent stem cells can be cultured while maintaining their undifferentiated state by simply suspending them with the cells during seeding and pouring them onto the culture substrate. Figure 3 shows images of epidermal cells and vascular endothelial cells cultured on iMatrix-511. Epidermal cells hardly adhere on uncoated plates, but many cells adhere and spread on iMatrix-511-coated plates. Vascular endothelial cells adhere even on uncoated dishes, but many are observed to remain in a rounded shape without spreading. On the other hand, when cultured on iMatrix-511, most cells spread well.


Figure 3. Adhesion and spreading of human skin epidermal cells and vascular endothelial cells cultured on iMatrix-511

More than a decade ago, it was known that pluripotent stem cells, such as iPS cells and ES cells, were extremely difficult to handle. For example, in order to increase the number of cells while maintaining their pluripotency, it was necessary to passage the proliferated cell mass by dividing it into appropriate sizes, but if the division operation was not appropriate, the pluripotency could be lost. In addition, mouse-derived feeder cells and mouse tumor-derived basement membrane extracts had been used as culture substrates for these cells, but due to the risk of contamination with foreign components, this was considered to be one of the problems in medical applications. Joint research by researchers at Osaka University and Kyoto University revealed that the laminin-511 E8 fragment is effective for culturing iPS cells and ES cells (References 6-7). Nippi manufactures this product under license for this patent. On the laminin-511 E8 fragment, even if iPS cell masses are broken up into individual cells, many of them can be maintained and efficiently proliferated. In addition to laminin-511, Nippi also manufactures and sells the E8 regions of laminin isoforms 111, 221, 332, and 411. The integrin proteins expressed on the cell membrane differ depending on the organ and tissue from which the cells are derived. By using the laminin isoform corresponding to the integrin, appropriate signals are sent to the cells during cell culture. It has been shown that the laminin-511 E8 fragment can maintain undifferentiated properties even if it is seeded on the substrate together with the cells, without coating the culture substrate (Reference 8).

References

1. Hatton IA et al. The human cell count and size distribution., Proc Natl Acad Sci USA 120: e2303077120 (2023)
2. Aumailley M et al. A simplified laminin nomenclature. Matrix Biol. 24: 326-332 (2005)
3. Ido H et al. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins. J Biol Chem. 282: 11144-11154 (2007)
4. Takizawa M et al. Mechanistic basis for the recognition of laminin-511 by α6β1 integrin. Sci Adv. 3: e1701497 (2017)
5. Arimori et al. Structural mechanism of laminin recognition by integrin. Nat Commun. 12: 4012 (2021)
6. Miyazaki T et al. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat Commun. 3: 1236 (2012)
7. Nakagawa et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 4: 3594 (2014)
8. Miyazaki T et al. Efficient adhesion culture of human pluripotent stem cells using laminin fragments in an uncoated manner. Sci Rep. 7: 41165 (2017)

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