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AUG 30, 2016 8:00 AM PDT

Improved differentiation of human pluripotent stem cell-derived neurons through reduction of progenitor proliferation: impact on downstream applications

Speaker
  • Director and Group Leader, Thermo Fisher Scientific
    Biography
      Dr. Kuninger leads research, development and commercialization of media systems for pluripotent stem cell culture & differentiation, neurobiology, and (non-hepatic) primary cell biology at Thermo Fisher Scientific in the Cell Biology business based in Frederick MD. His teams support numerous portfolios and have launched over 25 new products spanning stem cell culture & cryopreservation, differentiation (endo-, ecto- and meso-dermal lineages) and neurobiology over the past 3 years. David is a seasoned scientist and manager, experienced in media formulation & optimization, assay design and implementation, and troubleshooting. Expertise in GLP/GMP compliance, tech transfer and scale up, as well as verification and validation processes. Prior to starting at Thermo Fisher Scientific (legacy Invitrogen) in 2007 as Staff Scientist, he joined Oregon Health Sciences University as a Postdoctoral Fellow investigating the actions of insulin-like growth factors in the lab of Dr. Peter Rotwein, subsequently joining the faculty in the Department of Biochemistry at OHSU as a Research Instructor. He completed is PhD in Biochemistry and Genetics University of Texas Medical Branch in the laboratory of Dr. John Papaconstatinou and has a B.S. in Chemistry from the University of Oregon.

    Abstract
    Neurons derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are excellent resources for disease modeling and drug screening. Human PSCs derived neural stem cells (NSCs) can be expanded in culture and further differentiated into mature neurons for various applications, however, these often contain mixed population of both differentiated neurons and undifferentiated NSCs. Due to the continuing proliferation of undifferentiated NSCs, very high cell densities and cell aggregation are usually observed during the differentiation of hPSC-derived NSCs which increase over time, posing challenges for long-term maintenance and downstream analysis.  Here we demonstrate the use of a new supplement which can reduce the proliferation of undifferentiated NSCs without negatively impacting the rate or extent of differentiation for hPSC-derived NSCs.  The overall effect increases the relative population of differentiated neurons in culture.  Typically under these conditions by 2-3 weeks, differentiated neurons with extensive neurite networks are seen that are evenly distributed across the culture surface, with very little clumping or aggregation observed.  Further, this more uniform morphology and enriched neuron population greatly facilitates quantitative image analysis, as demonstrated by high content analysis using automated a CellInsight™ CX5 imaging platform which showed differentiated neurons expressing mature neuronal markers MAP2 or HuC&D without the contamination of undifferentiated Nestin positive NSCs, as seen in control samples.  Multielectrode array (MEA) analysis demonstrates that differentiated neurons fired spontaneous action potentials, indicating functional neurons. In addition, use of this new supplement enabled differentiated neurons to be maintained for longer time in culture than untreated control cells.

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