- Proteins and Peptides
- Lysates and Cell Lines
By Jennifer Sokolowski, MD, PhD.
Human induced pluripotent stem cells (iPSCs) can be used to create models of human organ systems and are useful for a) ascertaining the mechanisms underlying pathological conditions and b) developing and testing therapeutics. For example, studies have used iPSCs from human patients with diseases like Alzheimer's and Parkinson's to test the influence of pathological isoforms of proteins as well as the efficacy of genetic rescue.1,2
Zhang et al. have optimized methods using human iPSCs to generate dorsal telencephalic neural precursor cells that can mature into forebrain cortical neurons.3 Prior protocols lacked robust, consistent differentiation of neural precursor cells; therefore, Zhang et al. set out to adapt methods to optimize production of large, homogenous populations of dorsal telencephalic neural precursor cells. For one line of work, they used human iPSC lines, confirming they expressed the key pluripotency markers OCT4, SOX2, KLF4, LIN28, and NANOG prior to neural induction.
Immunocytochemistry/Immunofluorescene: OCT4 Antibody [NB100-2379]- OCT4 staining in NTERA-2 cells detected with a Dylight 488 labeled secondary antibody.
To induce neural precursor cells, they allowed cells to form embryoid bodies, and cultured them in media containing dorsomorphin, which inhibits BMP type 1 receptors, to promote dorsal specification. For some experiments, they did double inhibition of BMP/Smad pathway, but ultimately showed that single inhibition of BMP was sufficient. They then cultured cells in the presence of FGF2, in conditions to promote formation and selection of neural rosettes. They confirmed that the human iPSC-derived neural precursor cells expressed the neural progenitor markers Nestin, PAX6, SOX1, and SOX2. Dorsal forebrain identity was further verified with the expression of the dorsal marker FOXG1, and the absence of the ventral marker DLX2.
Immunocytochemistry/Immunofluorescence: Nestin Antibody [NB300-265] - The Nestin antibody was tested in NTERA2 cells against Dylight 488 (Green). Alpha-tubulin and nuclei were counterstained against Dylight 550 (Red) and DAPI (Blue), respectively.
After further differentiating neural precursor cells into mature neurons, to further distinguish the type of neurons they derived, they immunostained for a forebrain telencephalic marker, FOXG1; a superficial cortical layer (II–IV) marker, BRN2; a deep cortical layer (V and VI) marker, TBR1; a neuronal nuclear marker, NeuN; a dendritic marker, MAP2; and astrocyte markers GFAP, SOX9, and GLT1. This staining confirmed that their neural precursor cells almost exclusively differentiated into cortical forebrain neurons.
Differentiation of h uman iPSCs into neural cells provides substrate for study of not only neurodegenerative diseases, but also epilepsy. There are many cases of epilepsy associated with specific genetic conditions that, at least in theory, could be rescued.4,5 The neurons derived by the methods described by Zhang et al. could be used in high throughput studies that test new anti-epileptic therapeutics.
Jennifer Sokolowski, MD, PhD
University of Virginia, Department of Neurosurgery
Jennifer is doing a postdoc while completing her residency in Neurosurgery and has background in basic science, specifically neuroscience, cell death, and immunology, as well as background in medicine and translational and clinical research.