Correct interactions with extracellular matrix are essential to human pluripotent stem

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during culture. on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture SC-144 and differentiation. In addition this SC-144 matrix is ideal for the efficient generation of new hiPSC lines. Introduction Human pluripotent stem cells (hPSC including both human embryonic stem cells hESC and induced pluripotent stem cells hiPSC) require either a feeder cell layer or an extracellular matrix (ECM) coating to support their self-renewal suggesting that signals originating from the ECM have a significant role in hPSC regulation. Consequently there has been a growing interest in the extracellular milieu (or niche) of hPSCs. hPSCs are predominantly cultured on either mouse embryonic fibroblasts (mEF) or Matrigel an extracellular matrix preparation isolated from mouse sarcoma [1-4]. However undefined ECM preparations based on various animal glycoproteins and growth factors are not ideal for hPSC cultures as they may have unexpected Rabbit Polyclonal to OR52E5. and poorly controllable biological effects on the cells and furthermore they cannot be used in eventual clinical applications. A specialized extracellular matrix structure basement membrane underlies epithelial and endothelial cells creating boundaries between different tissue types in a body [5 6 Basement membranes consist of diverse protein and carbohydrate macromolecules that are secreted in cell type specific manner. Importantly it has been shown that basement membranes not only provide mechanical support for tissues but SC-144 also maintain tissue homeostasis [7 8 The most important group of biologically active signaling proteins in basement membranes is laminins (lm). Laminins are composed of one alpha (α) one beta (β) and one gamma (γ) chain that are twisted together to form either a cruciform or a T-shaped structure. Currently at least 15 different combinations (αβγ) of laminins are known [9-11]. We have previously shown that laminins-511 (α5β1γ1) and -111 (α1β1γ1) the two laminin isoforms expressed in early mouse embryos SC-144 are also synthesized by the hPSC cultures [12]. Our study also demonstrated that hPSCs utilize specific cell surface receptors when they adhere to the laminin isoforms. Crucially we showed that undifferentiated hPSCs could be maintained on purified human lm-511 in defined culture medium. Various human recombinant proteins including lm-511 vitronectin fibronectin and their combinations have been shown to support hPSC maintenance [13-15]. However large-scale purification or production of biologically functional human laminins by recombinant technologies is laborious and expensive. Therefore here we have developed a feeder-free cost-effective and user-friendly hPSC culture system that is based on the matrix secreted by human choriocarcinoma cell line JAR producing high quantities of lm-511 and -111. Hereafter the matrix is called JAR matrix. Materials and Methods Ethics Statement The generation of human ES lines and their use in these studies was approved by the Ethics Committee of the Helsinki University Central Hospital (statement nr. 143/E8/01 on December 18 2003 Donors provided their written informed consent for participation. The procedure generation and use of human iPS cells were approved by the Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District (statement nr. 423/13/03/00/08) on April 9 2009 The National Animal Experiment Board ( authorized the SC-144 use of mice in the teratoma assays. The animals were anesthetized by a mixture of Ketamine and Xylatsine and Carprofen was used as painkiller during the operation and day after. The animals were housed under controlled humidity temperature and light regimen and care was consistent with institutional and National Institute of Health guidelines. Teratoma growth was followed.