Abstract
Next to embryonic stem cell research, adult stem cell research is providing a promising alternative for enhanced tissue regeneration and transplantation. The key biochemical networks controlling the differentiation processes regulating stem cell biology remain largely disputed and or undefined, contributing to a lack of knowledge of the principle phosphoregulatory events propagating signal transduction. To effectively monitor these events relative to adipocyte differentiation, this study utilized a high throughput reverse phase protein microarray platform and characterized adult adipose-derived stem cell (ASC) differentiation through the monitoring of ∼100 phosphospecific endpoints with 33 distinct time points examined across 14 days. This kinetic-based analysis showed time ordered signal transduction ultimately implicating pathways correlated with adipogenic differentiation. To further validate the causal significance of these network activations, pharmacological targeting was implemented to include the chemical inhibitors MAPK inhibitor PD169316, rapamycin, and HNMPA-(AM)3 yielding partial or complete disruption of adipocytic differentiation, as noted by a decrease or lack of lipid formation within the mature adipocytes. Based on this analysis, v-crk sarcoma virus CT10 oncogene homolog (CRKII) and c-abl oncogene 1, non-receptor tyrosine kinase (c-ABL) were implicated as novel key regulators of adipocyte differentiation, with v-akt murine thymoma viral oncogene (AKT), mammalian target of rapamycin (mTOR), and SMAD family member (SMAD) pathways being implicated as secondary regulators. This dynamic molecular profiling provides a novel insight into the signaling architecture of mesenchymal stem cell differentiation and may be useful in the development of therapeutic modulators for clinical applications; in addition to advancing the collective understanding of key cellular processes, ultimately contributing to more confident stem cell manipulation.
Highlights
Recent breakthroughs in adult stem cell research have increased industry focus on the potency and availability of these cells
Multilineage Time Course (Study Set#1)—Dynamic time course reverse phase protein microarray (RPMA) data from each lineage was visualized via unsupervised hierarchical clustering, which facilitated end point clustering and established time windows used for histogram based analysis
This portrayed kinetic activation portraits that were cleanly reproducible across all three samples, including even subtle and time-dependent changes (Fig. 2B and 2C) and interesting oscillations in phosphorylation levels seen for akt murine thymoma viral oncogene (AKT) at the later time points (Fig. 2B)
Summary
Recent breakthroughs in adult stem cell research have increased industry focus on the potency and availability of these cells. Adipose-Derived Stem Cell Differentiation Pathway Mapping teractions leading to an array of cellular events from differentiation to gene expression, encompassing signal transduction Characterization of this broad-scale signaling architecture is necessary to provide a more finite depiction of the complex signaling events directing a given cellular phenotype and aid in the understanding of the repercussions of alterations in regulation [13, 14]. Lineage signal transduction profiles were established through the monitoring of protein network activation during the course of differentiation into adipocyte, osteoblast, and chondrocyte lineages This multilineage kinetic experimentation allowed for global examination of signal transduction through the monitoring of ϳ100 phosphospecific endpoints, across 33 consecutive time points that spanned a 14 day period to reach terminal differentiation demonstrating timespecific and lineage-specific signaling. This experimental design allowed for isolation of a subset of time-specific endpoints unique to adipogenesis relative to the other lineages that could be further tested for causal significance using pharmacologic knock-out analysis
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