Abstract

Making Clinical Quality Cells From hES Cells Using Laminin-Based Cell Culture Substrata

Karl Tryggvason, Professor, Duke-NUS Graduate Medical School & Karolinska Institutet, Stockholm

Human pluripotent stem cells (hPSCs) constitute an attractive cell source for regenerative medicine and recent work has demonstrated the possibility to generate from hPSCs several cell types that may become applicable for cell therapy. Induced pluripotent stem cells (iPSCs) and stem cells made using nuclear transfer (NTSCs) are possible cell sources, but human embryonic stem cells (hESCs) are still considered the gold standard as a stem cell source. A major ethical concern with hESCs is that their derivation has required destruction of a human embryo used for hESC line derivation. From a regulatory and patient’s safety standpoint, it is also important that hPSCs used for differentiation of cells are devoid of infectious agents or immunogens and therefore there is a need to be able to use fully chemically defined and xeno-free systems for both derivation and expansion of hPSCs as well as in the differentiation protocols themselves. Furthermore, hPSC propensity to genetic mutations of in vitro expanded hPSCs and potential tumorigenicity are a challenge for HPSC-based therapies. We have shown that by deriving new hESC lines on and expanding them on certain stem cell niche adhesion proteins, laminins (LN), one can develop fully chemically defined and animal reagent free cell culture systems. These LNs (LN-511 and LN-521) are made as recombinant human proteins that are thus not immunogenic. Importantly, the new hESCs can be derived clonally without a need to destroy a human IVF embryo from which they are derived. hESCs derived on LN-521 can be expanded in cell cultures from single cell suspension and they grow homogeneously as a monolayer. There is no need for use of Rho kinase inhibitors which are potentially dangerous as they inhibit abnormal cells from going into apoptosis. We have also developed an efficient differentiation protocol for generating hESC-derived cardiomyocyte progenitors using LN-521 and LN-221 the most abundant laminin of striated muscle fibers