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Stem Cell Adhesion and Cell Culture Substrates

Human iPSC colony on Vitronectin XF™ defined, xeno-free substrateHuman pluripotent stem cells were originally derived from the inner cell mass of blastocyst embryos [1], and more recently, by reprogramming of adult somatic cells [2, 3]. Human pluripotent stem cells, or hPSCs, are often cultivated on a feeder layer of mitotically-inactivated mouse embryonic fibroblasts (MEFs), and grown in medium that is conditioned by MEFs.

To enable feeder-free cultivation of hPSCs, several media formulations have been developed that eliminate the use of fetal bovine serum, and reduce or eliminate all animal-derived components [4]- examples include mTeSR®1 and TeSR™2 (Stem Cell Technologies), the ‘E8’ formulation of Chen et al. [5], Stem EZ8 (Cellagen), StemPro® hESC SFM (Life Technologies), and NutriStem™ XF/FF Culture Medium (StemGent).

To enable propagation of hPSCs under feeder-free conditions, it is necessary to provide a cell adhesion coating supportive of propagation and maintenance of pluripotency. The first commercial matrix used for this purpose is Matrigel™ (Becton-Dickenson), a solubilized basement membrane preparation extracted from mouse Engelbreth-Holm-Swarm sarcoma. Major components of Matrigel™ include laminin, collagen IV, heparan sulfate proteoglycans, and entactin (nidogen). Matrigel™ also contains appreciable amounts of sarcoma derived growth factors such as TGF-beta, fibroblast growth factor, TPA, and insulin-like growth factor. Matrigel™ lots vary in protein and growth factor concentrations, and it is necessary to keep Matrigel™ cold at all times to prevent gelation during preparation for use.

Individual extracellular matrix (ECM) proteins have also been used, singly and in various combinations, to support propagation of hPSCs. Laminin, fibronectin, vitronectin, and collagen (in various combinations) were claimed in the US Patent 7,442,548 in which J. Thomson and T. Ludwig described the development of the mTeSR®1 medium. Recombinant laminin-511 (BioLamina) can be used for maintenance and propagation of hPSCs [6, 7]. Recombinant vitronectin is also sufficient for hPSC culture [8], and can be used as a substrate for subsequent differentiation (Vitronectin XF™, Primorigen Biosciences).

In addition to ECM components, certain cell adhesion proteins can be used for attachment and expansion of pluripotent cells. E-cadherin, for example, is a homophilic cell-cell adhesion protein expressed strongly in pluripotent cells. For hPSCs, coating polystyrene cultureware with recombinant E-Cadherin fusion protein such as StemAdhere™ (Primorigen Biosciences, exclusively available through Stem Cell Technologies) enables attachment and expansion of pluripotent cells in defined media such as mTeSR®1 and TeSR™2. Human iPS cultures have been maintained in mTeSR®1 on StemAdhere™ for over 100 passages with normal karyotype and sustained expression of the pluripotency markers Oct4, SSEA-4 and Tra1-81. Studies supporting proliferation of other pluripotent cell types [9] also have demonstrated the use of recombinant E-cadherin fusion protein for propagation of hPSCs.

Additional developments in defined culture surfaces include synthetic peptide coatings such as Synthemax™ (Corning). To enable a fully defined, xenobiotic-free culture system capable of clinical use, it is important to examine the sources of all components used in routine hPSC culture, including reagents used for lifting cells during passaging, and reagents used for cryopreservation. For practicality, cost also is a major variable. To date, Primorigen’s StemAdhere™ and Vitronectin XF™ appear to be the only matrices made under defined conditions that offer the same economic advantages as Matrigel™, along with the added advantage of being xenobiotic-free.

1. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
4. Ludwig, T.E., et al., Feeder-independent culture of human embryonic stem cells. Nature methods, 2006. 3(8): p. 637-46.
5. Chen, G., et al., Chemically defined conditions for human iPSC derivation and culture. Nature methods, 2011. 8(5): p. 424-9.
6. Rodin, S., et al., Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nature biotechnology, 2010. 28(6): p. 611-5.
7. Domogatskaya, A., et al., Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells, 2008. 26(11): p. 2800-9.
8. Braam, S.R., et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells, 2008. 26(9): p. 2257-65.
9. Nagaoka, M., et al., Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. BMC Developmental Biology, 2010. 10(60).