Identification of Potential Pluripotency Determinants for Human Embryonic Stem Cells Following Proteomic Analysis of Human and Mouse Fibroblast Conditioned Media

The unique pluripotential characteristic of human embryonic stem cells heralds their use in fields such
as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current
availability of sufficient quantities of embryonic stem cells for such applications is limited, and generating
sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder
layers or their conditioned media, human embryonic stem cells readily differentiate to form embryoid
bodies, indicating that trophic factors secreted by the feeder layers are required for long-term
proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the factors
required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of
mouse or human origin) to effectively support human embryonic stem cells. Currently, the deficiency
of knowledge concerning the exact identity of factors within the pathways for self-renewal illustrates
that a number of factors may be required to support pluripotent, undifferentiated growth of human
embryonic stem cells. This study utilized a proteomic analysis (multidimensional chromatography
coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of
three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse embryonic
fibroblasts) used to support the undifferentiated growth of human embryonic stem cells. One-hundred
seventy-five unique proteins were identified between the three cell lines using a ≤1% false positive
rate of identification. These proteins were organized into 17 categories. The differentiation and growth
factor and extracellular matrix and remodeling categories contained proteins from many of the key
pathways already implicated in the maintenance of human embryonic stem cell pluripotency including
the Wnt, BMP/TGF-β1, Activin/Inhibin, and insulin-like growth factor-1 pathways. The conditioned media
of fibroblast feeder layers is a complex system, and this study assists in narrowing potential candidates
responsible for the support of undifferentiated human embryonic stem cells.
Keywords: fibroblast conditioned media • proteomic analysis • human embryonic stem cells • pluripotency •
transforming growth factor beta • undifferentiated