Utilization of Human Amniotic Mesenchymal Cells as Feeder Layers to Sustain Propagation of Human Embryonic Stem Cells in the Undifferentiated State

Abstract

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Developmental and Functional Nature of Human iPSC Derived Motoneurons

“…Alternatively, hESCs can be cultured permanently on feeder cells from human tissues like human foreskin fibroblasts [127,147], human bone marrow stromal cells or human amniotic mesenchymal cells (hAMCs) [227]. Feeder cells support the survival and the preservation of an undifferentiated state via supply of some critical factors either to promote selfrenewal or to suppress differentiation [214,217].…”

Feeder Cell Sources and Feeder-Free Methods for Human iPS Cell Culture

“…Zhang et al [48] reported that human amniotic MSCs can be used as feeder cells for effective growth of human ESCs. Human amniotic epithelial cells (AECs) can be isolated from the surface membrane of fresh placentas, and they express many growth factors including EGF, bFGF, TGF-β, and BMP-4 [49,59,60] in addition to stem cell markers such as Oct-4 and Nanog [50].…”

A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

“…Under more circumstances, they are plated directly on feeder layers originated from MEFs (Thomson et al 1998;Yu et al 2009;Hu et al 2011), STO cells (Park et al 2004;Takahashi and Yamanaka 2006), human embryonic fibroblasts (Kibschull et al 2011), human placental fibroblasts (Genbacev et al 2005), human foreskin fibroblasts (Unger et al 2009;Skottman 2010;Strom et al 2010), human skin fibroblasts (Richards et al 2003;Tecirlioglu et al 2010), amniotic mesenchymal cells (Zhang et al 2011), etc. Human embryonic stem cells (hESCs) can even be cultured on feeder layers derived from themselves (Xu et al 2004;Stojkovic et al 2005;Chen et al 2009).…”