Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells Siqin Wu a , Jan Johansson a, b, c , Pauliina Damdimopoulou d , Mansoureh Shahsavani e , Anna Falk e , Outi Hovatta d , Anna Rising a, b, * a Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Novum 5th floor, 141 86 Stockholm, Sweden b Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, 751 23 Uppsala, Sweden c Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 25,101 20 Tallinn, Estonia d Department of Clinical Sciences, Intervention and Technology, Division of Obstetrics and Gynecology, Karolinska Institutet and Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden e Department of Neuroscience, Karolinska Institutet, Retzius v. 8, 171 77 Stockholm, Sweden article info Article history: Received 24 April 2014 Accepted 20 June 2014 Available online 17 July 2014 Keywords: Biomaterial Chemically defined Human embryonic stem cells Human induced pluripotent stem cells Functionalized materials Scaffold abstract Human pluripotent stem cells (hPSCs) can undergo unlimited self-renewal and have the capacity to differentiate into all somatic cell types, and are therefore an ideal source for the generation of cells and tissues for research and therapy. To realize this potential, defined cell culture systems that allow expansion of hPSCs and subsequent controlled differentiation, ideally in an implantable three- dimensional (3D) matrix, are required. Here we mimic spider silk e Nature's high performance mate- rial e for the design of chemically defined 2D and 3D matrices for cell culture. The silk matrices do not only allow xeno-free long-term expansion of hPSCs but also differentiation in both 2D and 3D. These results show that biomimetic spider silk matrices enable hPSC culture in a manner that can be applied for experimental and clinical purposes. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Human pluripotent stem cells (hPSCs), including human em- bryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the unique ability to form any specialized tissue in the human body. These cells therefore represent powerful re- sources for applications in regenerative medicine and pharma- ceutical development. However, several technical challenges must be addressed before hPSCs can be used routinely for clinical ther- apeutic applications and generation of tissues or organs [1]. First, in order to generate sufficient number of cells, culture systems that are cheap, easy-to handle and chemically defined are needed. Second, mechanically robust 3D matrices that are adaptable, well tolerated by the host and able to regulate stem cell fate commit- ments have to be developed. Recently, several groups have devel- oped matrices for long-term xeno-free expansion of hPSCs [2e7]. In these reports, hPSCs are maintained on recombinant extracellular matrix (ECM) proteins or synthetic peptides derived from ECM proteins but none of these substrates have been reported to generate 3D scaffolds that support proliferation and differentiation of hPSCs, cf below under 3.4. for further details. Spider silk is an ideal biomaterial, since it is strong, extendible and is well tolerated and degraded when implanted in living tissues [8,9]. However, spiders are difficult to farm and therefore native spider silk is practically impossible to obtain at large scale. Pro- duction in heterologous hosts may be an alternative route to in- dustrial production of spider silk, but this strategy is associated with problems since the spider silk proteins are large and prone to aggregate. Spider silk proteins are composed of an extensive re- petitive region flanked by small folded terminal domains that regulate silk assembly [10,11]. The low complexity of the about 3000 amino acid residue long repetitive segment likely contributes both to the impressive mechanical properties and presumed low immunogenicity of spider silk. Despite the technical problems, recent progress has resulted in cost-efficient methods to produce artificial spider silk in heterologous hosts [12]. We have found that a miniature spider silk protein, referred to as 4RepCT, is easily produced in Escherichia coli, can be purified to homogeneity and * Corresponding author. Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Novum 5th floor, 141 86 Stockholm, Sweden. Fax: þ46 8 58583610. E-mail addresses: anna.rising@ki.se, anna.rising@slu.se (A. Rising). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials http://dx.doi.org/10.1016/j.biomaterials.2014.06.039 0142-9612/© 2014 Elsevier Ltd. All rights reserved. Biomaterials 35 (2014) 8496e8502