Abstract
Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.
Highlights
Human pluripotent stem cells, including both human embryonic stem cells and human induced pluripotent stem cells, have the unlimited self-renewal capacityPLOS ONE | DOI:10.1371/journal.pone.0149023 February 16, 2016Xeno-Free Culture System for Human iPS Cells
We found about sixty percent of human induced pluripotent stem cells (hiPSCs) colonies spontaneously differentiated after passage onto the Human umbilical cords (hUCs)-Mesenchymal stem cells (MSCs) feeder from mouse embryonic fibroblast (MEF) feeder, the hiPSCs adapted to the new xeno-free feeder as they gradually decreased the number of differentiated colonies
Flow cytometric analysis revealed that the hUC-MSCs expressed a set of cluster of differentiation (CD) MSC markers (CD73, CD90, and CD105), they did not express hematopoietic markers (CD34 and CD45) or human leukocyte antigen (HLA-DR) (Fig 1C)
Summary
Isolation and culture of hUC-MSCs. Human umbilical cords (hUCs) were collected from West China Women’s and Children’s Hospital without any complications of pregnancy or parturition, and the collected hUCs were transferred in sterile boxes that contained cold Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, USA). The remaining pieces were chopped into 0.2cm size. These explants were transferred in the CELLstartTM CTSTM Substrate (Life Technologies, USA)-coated 100 mm plates (Nunc, Denmark). A few StemPro MSC SFM XenoFree medium (Life Technologies, USA) with 1% penicillinstreptomycin (Life Technologies, USA) was added to the plates, and the explants were cultured at 37°C in a 5% CO2 incubator and left undisturbed to allow the cells to migrate from the explants. The cells were passaged into another plates and further split 1:4 by 0.05% Trypsin-EDTA (Life Technologies, USA) once the cells reached 80% confluence
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