One microgram of total RNA was reverse transcribed to complementary DNA (cDNA) using the RevertAid H Minus First Strand cNDA Synthesis Kit (Fermentas, Thermo Fisher Scientific) according to the manufacturer’s instructions. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGF1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore , the results demonstrated the benefit of hUCS for hPSCs culture system. == 1 . Introduction == Human pluripotent stem cells (hPSCs) can be generated from isolation of the pluripotent cells using preimplantation embryos, called human embryonic stem cells (hESCs) [1] or the reprogramming of somatic cells using exogenous genes, resulting in human induced pluripotent stem cells (hiPSCs) [2]. Both hESCs and hiPSCs share phenotypic and molecular genetic similarities [3]. hPSCs can be used as a model for the study of developmental biology, toxicity, and cell-based therapy [4]. Conventionally, hPSCs are derived and propagated through coculture with supportive feeder cells isolated from mice [1, 2]. Feeder cells play an important role in supporting the pluripotency of hPSCs by producing a complex microenvironment for the growth of hPSCs. Feeder cells secrete growth factors, such as FGF2, TGF-1, or Activin A, involving the specific pathway controlling the pluripotency of hPSCs. Moreover, extracellular matrices, such as laminin, fibronectin, or collagen, produced from the feeders, are necessary for cell interaction, cell migration, or cell proliferation [5]. For therapeutic purposes, avoiding the cross-contamination of animal pathogens to hPSCs through the replacement of mouse feeder cells with the feeder cells derived from human tissue should be considered. Human feeder cells, such as human foreskin fibroblasts (HFFs), human mesenchymal stem cells (hMSCs), human amniotic epithelial cells (hAECs), or human fallopian tube-derived cells, were used as supportive feeder cells for the culture of hPSCs [6]. Moreover, previous studies have demonstrated the possibility to generate genetically modified human feeder cells for the long-term support of hPSCs [7, 8]. For routine BUN60856 culture, human cells were cultured in medium supplemented with fetal bovine serum (FBS). Although FBS promotes human cell proliferation, human cells bare the risk of contamination of bovine viruses or other pathogens. For clinical purposes, the use of animal products in human cell culture should be avoided. Since human serum (HS) BUN60856 shows BUN60856 a positive effect on culture of mesenchymal stem cells, it appears to be a promising candidate for FBS replacement. The previous studies demonstrated that HS can be used for culturing the supportive feeder cells of hESCs [9, 10] and hMSCs showed greater proliferation in the HS-containing medium compared with FBS-containing medium [11, 12]. Nonetheless, HS not only promoted hMSC cell proliferation but also enhanced osteogenic differentiation [13]. Due to the progressive research for the therapeutic application of hPSCs, it is necessary to develop xeno-free culture conditions for the maintenance of hPSCs. Recent studies have shown that both feeder cells and hPSCS can be generated and cultured under the good manufacturing practice (GMP) [14, 15] as a useful step forward for the application of hPSCs in the field of regenerative medicine. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells [16, 17]. Therefore , it is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. The objective of the present study was Elf1 to compare (i) growth, proliferation, and karyotypic stability of HFFs when cultured in a medium containing hUCS (HFF-hUCS) versus FBS (HFF-FBS), (ii) the characteristics of inactivated HFF-hUCS and inactivated HFF-FBS, and (iii) the pluripotent characteristics of hPSCs after having been cocultured for a long period with inactivated HFF-hUCS and inactivated HFF-FBS. == 2 . Results == == 2 . 1 . Effect of Serum Supplementation on the Morphology and Proliferation of Human Foreskin Fibroblasts == The HFFs were cultured in medium containing either hUCS or FBS, and the morphology and growth were subsequently observed. We first observed the difference of cell attachment between the two culture media. At 5 hours after dissociation, nearly all dissociated HFF-FBS were attached to the surface of the culture dish..