(558d) Multiplexed Single Cell Analysis of Embryonic Stem Cells and Induced Pluripotent Stem Cells

Embryonic stem cells (ESCs) are derived from the inner cell mass of a developing blastocyst, propagated in in vitro culture systems, and defined by the ability to self-renew their pluripotent state.  The ability to grow ESCs in culture relies heavily on the media components in which they grow, where specific cytokines and growth factors are necessary to maintain the self-renewal of ESCs and prevent spontaneous, terminal differentiation into heterogeneous somatic cells.  Typically, human ESCs need to be grown on a layer of fibroblasts, known as feeder cells, where exogenously supplied FGF2 induces the feeders to secrete the signaling proteins TGFB1 and IGF2 to ESCs, instructing them to stably maintain their pluripotent state.  ESCs can also be grown in feeder-free cultures, on the condition that the growth media are still supplemented with FGF2, TGFB1, and IGF2.  It has been reported that in the absence of exogenously supplied feeders, a minor population of an ESC colony will differentiate into fibroblast-like cells in order to fulfill the role of feeders in establishing an in vitro niche.  While the contents of basal media and supplemental growth factors are well defined, the profile of molecules secreted by either ESCs or feeder cells are not well understood.  Thus the microenvironment, defined as the interface where ESCs within a colony exchange signals with each other as well as with feeder cells, remains to be further characterized.

While immunofluorescent labeling of ESC surface receptors can suggest a signal dependence on the cognate ligand for those receptors, it provides limited insight as to whether this pathway is autocrine or paracrine in nature.  Bulk analysis of media conditioned by ESCs and feeders have been profiled with proteomic studies, but the distinct molecules within a heterogenous mixture cannot be traced back to the secretory cell of origin.  Our platform uniquely enables the high thoroughput, quantitative profiling of candidate signaling proteins secreted by a single ESC or ESC-derivative.  Furthermore, the ability to simultaneously assay a multiplex of secreted proteins can define and classify subpopulations of cells with distinct secretomes, several of which may be characteristic of pluripotent cells.  Lastly, this novel diagnostic tool will also be useful in the field of regenerative medicine, particularly in studies related to the generation and application of induced pluripotent stem cell (iPSC) technology.  The reprogramming of a somatic cell to a pluripotent cell is highly inefficient, and the mechanism underlying this process remains largely unknown.  Probing the signaling aspects of a single cell undergoing the reprogramming process will yield novel insights into molecular mechanisms and thereby enhance the efficiency of the process.  In terms of the clinical application of patient-specific iPSCs for cell replacement therapies, the safety of reprogrammed cells must be assessed prior to transplantation.  Assaying for single cell secretomes anti-correlative to tumorigenic secretomes provides an additional parameter to ensure the safety of iPSCs used for regenerative medicine.