期刊
JOURNAL OF NEUROSCIENCE METHODS
卷 294, 期 -, 页码 91-101出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2017.07.019
关键词
Cell culture; Astroglia; Human induced pluripotent stem cell; Differentiation; hiPSC derived-neurons
资金
- University of California, Irvine California Institute for Regenerative Medicine Postdoctoral Fellowship [TG201152]
- California State University, Long Beach California Institute for Regenerative Medicine Grant (PF, ATP) [TB1-01182]
- National Institutes of Health [NS083009, R01]
Background: Human induced pluripotent stem cell (hiPSC)-derived neuronal cultures are a useful tool for studying the mechanisms of neurological disorders and developing novel therapeutics. While plating hiPSC-derived neuronal progenitors onto glial feeder layers prepared from rodent cortex has been reported to promote functional differentiation of neuronal networks, this has not been examined in detail. New method: Here we describe a method of using cryopreserved cells from primary cultures for generation of mouse astrocyte-enriched, neuron-free feeder layers that grow from 10% to 100% confluence in 1 week. Results: Electrophysiological analysis demonstrated that compared to biochemical substrates alone, astrocyte-enriched feeder layers support more rapid differentiation of hiPSC-derived progenitors into excitable neurons that form spontaneously active networks in culture. There was a positive correlation between the degree of astroglial confluence at the time of progenitor plating and the average frequency of postsynaptic currents 3 weeks after plating. One disadvantage to plating on 100% confluent feeder layers was a high incidence of the astroglial layer with the overlying neurons detaching from the coverslips during transfer to the recording chamber. Comparison with existing method(s): Prevailing methods using primary glial feeder layers can result in possible contamination with rodent neurons and an unpredictable rate of growth. We provide a reliable method of generating mouse astroglial feeder layers from cryopreserved primary cultures to support differentiation of hiPSC-derived neurons. Conclusions: The ability to make astrocyte-enriched feeder layers of defined confluence from cryopreserved primary cultures will facilitate the use of human stem cell derived neuronal cultures for disease modeling. (C) 2017 Elsevier B.V. All rights reserved.
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