We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.
At a glance
- Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro . Nat. Biotechnol. 18, 399–404 (2000). , , , &
- Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001). et al.
- A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18, 1404–1409 (2003). et al.
- Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid. Proc. Natl. Acad. Sci. USA 102, 12819–12824 (2005). , , , &
- A simplified laminin nomenclature. Matrix Biol. 24, 326–332 (2005). et al.
- Laminin functions in tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 20, 255–284 (2004). &
- Expression and biological role of laminin-1. Matrix Biol. 22, 35–47 (2003). , &
- A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J. Cell Biol. 119, 679–693 (1992). et al.
- Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain. J. Biol. Chem. 272, 27862–27868 (1997). et al.
- Molecular cloning of a novel laminin chain, alpha 5, and widespread expression in adult mouse tissues. J. Biol. Chem. 270, 28523–28526 (1995). , &
- Subunits of laminin are differentially synthesized in mouse eggs and early embryos. Dev. Biol. 96, 467–471 (1983). &
- Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol. 111, 372–382 (1985). &
- Human embryonic stem cells derived without feeder cells. Lancet 365, 1636–1641 (2005). et al.
- Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006). et al.
- Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells 26, 2257–2265 (2008). et al.
- Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375, 27–32 (2008). et al.
- The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners. Proc. Natl. Acad. Sci. USA 94, 10189–10194 (1997). et al.
- Recombinant human laminin-10 (α5β1γ1). Production, purification, and migration-promoting activity on vascular endothelial cells. J. Biol. Chem. 277, 12741–12748 (2002). et al.
- Recombinant laminin-8 (α4β1γ1). Production, purification, and interactions with integrins. J. Biol. Chem. 275, 14853–14859 (2000). , &
- Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells 26, 2800–2809 (2008). , , &
- Differences between human and mouse embryonic stem cells. Dev. Biol. 269, 360–380 (2004). et al.
- Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. Stem Cells 22, 522–530 (2004). et al.
- Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (α2β1γ1), 8 (α4β1γ1), 10 (α5β1γ1). Matrix Biol. 25, 89–93 (2006). et al.
- Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum. Reprod. 22, 3051–3058 (2007). et al.
- Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly. Stem Cells Dev. 18, 919–928 (2009). et al.
- A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008). et al.
- Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo. Dev. Biol. 239, 161–175 (2001). et al.
- Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum. Mol. Genet. 17, R48–R53 (2008). , , , &
- Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23, 544–549 (2005). et al.
- Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008). et al.
- Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol. Hum. Reprod. 10, 461–466 (2004). et al.
- Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006). et al.
- Supplementary Text and Figures (3M)
Supplementary Figs. 1–7 and Supplementary Tables 1,2