An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

Abstract

The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometrybased method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Human embryonic stem cells (hESCs)1 are non-transformed cell lines that can proliferate indefinitely in culture, although maintaining the potential to form all primary human cell types (pluripotency) (1, 2). These cells, which originate from the inner cell mass of pre-implantation blastocysts, represent a unique source of human cells for cell replacement therapies and for creating model human systems for understanding disease and development (3). Like other mammalian ESCs, hESCs were originally derived and propagated on replication-deficient mouse embryonic fibroblast (MEF) feeder cells in serum (2, 4), with varying efficiencies (5). At the heart of this variability is a lack of understanding of the regulatory pathways and growth factors that govern hESC self-renewal and pluripotency (6). This ambiguity restricts the application of hESCs in both research and therapeutic applications.We hypothesize that under optimal hESC culture conditions, there exist autocrine and paracrine growth factors, produced both by the feeder cells and the hESCs themselves, that establish the complex microenvironment required to retain hESC potential in culture. Previous genomic-based studies suggested the presence of such networks of hESC transcriptional regulation (7); however, these networks were not correlated to the extracellular microenvironment that ultimately controls hESC fate. Moreover, prior attempts to identify proteins within the hESC microenvironment using MSbased approaches produced few potential candidate regulators and provided little new insight or tangible improvements upon hESC line derivation and culture (6, 8 -11).From the ‡Don Rix Protein Identification Facility,

A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells

“…As the total energy budget was greater in hESCs than in NSCs, this indicated a faster macromolecule synthesis rate in hESCs; however, in the absence of increased cell division, these extra macromolecules must ultimately be degraded or secreted. hESCs have been shown to secrete a diverse range of chemokines, cytokines, growth factors and matrix remodelling enzymes, along with cytosolic and cytoskeletal components (Bendall et al, 2009;LaFramboise et al, 2010). We found that hESCs had morphological indications of goblet-cell-like secretion and secreted significantly more protein into the extracellular environment than did NSCs.…”

Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

“…5C). Several of these have previously been found in proteomic screens of MEF and hESC-conditioned medium (29). However, their detection in conditioned Matrigel™ suggests that the ECM is playing a role in their activity.…”

“…The total number of overlapping identifications can be found at the top of the bar in text. The rightmost bars in the top graphs of C and D represent total identifications found in Matrigel™ (11) and the H9 hESC secretome (29).…”

“…Secretome Analysis of the hESC and hiPSC Microenvironment-To further validate and enrich for candidates in the conditioned Matrigel™ data, a comparison was performed with a previously published proteomics screen of the H9 hESC-secretome obtained from MS analysis of hESC-conditioned medium (29). We performed a re-analysis of the H9 hESC-secretome raw dataset with the same software (PEAKS 5.3) used to process the conditioned Matrigel™ data to facilitate comparison and minimize variability from protein assignments or identifiers.…”

Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

“…Some of the factors presented in the ESCM such as MIF, PAI-1, sICAM-1, IL-6 and IL-8 have been detected in conditioned medium from undifferentiated human H1/H9 (Bendall et al 2009) and Hsf1 ES cell lines (LaFramboise et al 2010). Interestingly, undifferentiated human H1 and H9 ES cell lines could produce bFGF and TGFb1 which have been identified as growth factor for MSCs proliferation.…”