Abstract
BackgroundTo maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns, we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium.ResultsCells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features, including an ability to differentiate into multiple cell lineages in teratoma assays. We, therefore, present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions.ConclusionsWe propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP), which will be necessary for the clinical use of pluripotent stem cells and their derivatives.
Highlights
To maintain pluripotency of human embryonic stem cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells
We have previously reported that mouse embryonic stem cells can be successfully maintained on surfaces coated with a fusion protein consisting of the Ecadherin extracellular domain and the IgG Fc domain (Ecad-Fc) [19]
Maintenance of human embryonic stem (huES) cells on an hE-cad-Fc-coated surface under standard culture conditions We initiated our studies by examining the purity of hEcad-Fc produced as described in the methods section
Summary
To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns, we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. Such contaminations raise serious safety issues if pluripotent stem cells are to be used for autologous cell therapy
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