Abstract
Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.
Highlights
Human embryonic stem cells and induced pluripotent stem cells are attractive stem cell sources for cell therapy [1,2]
Unlike previously reported methods which typically rely on the addition of soluble factors to affect pluripotent stem cell differentiation, the present study reports an alternative approach using a biomaterial coating on a cell culture plate made of fibrillar collagen Type I to promote the derivation of mesenchymal stem cells (MSCs)-like cells
Both Human embryonic stem cells (hESCs) and hiPSCs were subjected to MSC derivation on a fibrillar type I collagen coating prepared by self-assembly from collagen solutions on non-treated tissue culture plastic which thereby reproduces physiological Type I collagen, i.e. collagen with individual fibrils 200–300 nm in diameter and several mm in length [27]
Summary
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are attractive stem cell sources for cell therapy [1,2]. Multi-potent adult stem cells, such as human bone marrow derived mesenchymal stem cells (MSCs) show promise for the treatment of large and severe skeletal defects including repair of damaged cartilage [3], but they are limited in number and quickly lose their differentiation potential during expansion [4]. Differentiating hESCs and hiPSCs into multi-potent progenitors or overtly differentiated cells prior to transplantation is one of the most promising approaches for the safe and effective use of pluripotent stem cells. Transplantation of lineage-committed cells can avert in vivo teratoma formation that is caused by the rapid growth and uncontrolled spontaneous differentiation of pluripotent stem cells [5]. MSCs have been obtained from spontaneously differentiating embryoid bodies (EBs) or aggregates in simple culture medium without complex growth factor supplements, removal of the EBs and prolonged serial passaging was required [8,9]. While promising in many aspects, these methods have intrinsic limitations including complicated cell sorting, extensive culture time and related labor cost, and, most importantly, low efficiency and yields
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