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.

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21-23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4-5days. By 35-38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4-5days and in ~80% by 21-23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics.

Objectives

Clinical research is often time-consuming and difficult to conduct in busy academic institutions. Previous studies have proposed methods to integrate undergraduate students as a means to increase research productivity. The authors aimed to describe the possibility to enhance emergency department research productivity at an academic emergency department in the United States, using undergraduate students in an Emergency Medicine Research Associates Program.

Methods

The authors described the Emergency Medicine Research Associates Program curriculum and its implementation. We also conducted a retrospective study at a university-based emergency department from January 2005 to December 2014 to demonstrate the benefit of having an established Emergency Medicine Research Associates Program. The primary outcomes were number of Emergency Medicine Research Associates Program-related studies, number of enrolled patients, extramural/intramural funding, abstract presentations, and peer-reviewed publications. The authors analyzed the data using descriptive statistics.

Results

Over the 10-year period, 110 Emergency Medicine Research Associates Program-assisted research studies were conducted, with research associates enrolling 46,219 patients. These studies yielded a total of 31 peer-reviewed publications and 77 abstract presentations (13 international, 27 national, 37 state/regional). The Emergency Medicine Research Associates Program-related studies were used as pilot studies to obtain US$1,751,036 in extramural grant funding and US$31,047 in intramural grant funding.

Conclusion

The implementation of Emergency Medicine Research Associates Program can enhance emergency department clinical research productivity, and the inclusion of supplemental academic programs enhanced the undergraduate students' research experience.