AUG 30, 2016 08:00 AM PDT
Improved differentiation of human pluripotent stem cell-derived neurons through reduction of progenitor proliferation: impact on downstream applications
SPONSORED BY: Thermo Fisher Scientific
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Speakers:
  • Associate Director and Group Leader, Thermo Fisher Scientific
    Biography
      Dr. Kuninger leads research, development and commercialization of next generation culture 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 15 new products spanning stem cell culture & cryopreservation, differentiation (endo-, ecto- and meso-dermal lineages) and neurobiology, leading to more than 25M in revenue over the last 3 years. David is a seasoned scientist and manager, experienced in media formulation & optimization as well as with sophisticated experimental design and data modeling tools applied to cell imaging and functional analysis endpoints as well as 2D and 3D culture models. Expertise in GLP/GMP compliance, technical transfer and scale up, verification and validation processes. Prior to starting at Thermo Fisher Scientific (legacy Invitrogen) in 2007 as Staff Scientist, he joined Oregon Health Sciences University (OHSU) as a Postdoctoral Fellow investigating the actions of insulin-like growth factors (IGFs) 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, Galveston, Texas in the laboratory of Dr. John Papaconstatinou and has a B.S. in Chemistry from the University of Oregon in Eugene OR.

    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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