Human iPSC-derived Neuron Maturation IF Antibody Sampler Kit #68228

The antibodies in this kit serve to characterize human iPSC-derived neurons across various maturation stages. The cytoskeleton of these cells plays an important role in generating neuronal processes. The cytoskeleton consists of three types of cytosolic fibers: actin microfilaments, intermediate filaments, and microtubules. Nestin is an intermediate filament family member protein that is structurally related to the neurofilament proteins (1). Nestin is widely accepted as a marker of neural stem/progenitor cells (2). It is highly expressed in the developing brain, where it may help to regulate cell structure and intracellular processes required for neural cell division and migration. Upon maturation, nestin expression is quickly downregulated and replaced by expression of the neurofilament proteins (1,3). Neurofilaments are the major intermediate filaments found in neurons, consisting of light (NFL), medium (NFM), and heavy (NFH) subunits (4). A heterotetrameric unit (NFL-NFM and NFL-NFH) forms a protofilament, with eight protofilaments comprising the typical 10 nm intermediate filament (5). While neurofilaments are critical for radial axon growth and determine axon caliber, microtubules are involved in axon elongation. β3-tubulin (TUBB3) is one of six β-tubulin isoforms that make up the building blocks of microtubules and is expressed highly during fetal and postnatal development (axon guidance and maturation) (6). Microtubules enriched in β3-tubulin are more dynamic than those composed of other β-tubulin isoforms (7).

Doublecortin is a microtubule-associated protein that facilitates neurite outgrowth and cell migration (8). The dual expression of doublecortin and neural cell adhesion molecule (NCAM, also known as CD56), combined with the lack of expression of mature neuronal markers, is evidence of an immature neuronal phenotype (9). NCAM mediates neuronal attachment, neurite extension, and cell-to-cell interactions through homo and heterophilic interactions. Polysialic acid (PSA) post-translational modification of NCAM disrupts cell-to-cell adhesion, promoting axonal growth, cell migration, and synaptic plasticity during neurogenesis (10-12).

Transcription factors also play a key role in immature neuron growth and differentiation. Research studies demonstrate that a set of transcription factors that includes Oct-4, Sox2, and Nanog forms a transcriptional network that maintains cells in a pluripotent state (13,14). Sox2 is highly expressed in neural stem/progenitor cells and is largely downregulated in post-mitotic neurons and glia (15). NeuroD1 is a member of the basic helix-loop-helix (BHLH) family of transcription factors. These proteins function by forming heterodimers with E-proteins and binding to the canonical E-box sequence CANNTG (7,8). NeuroD1 functions to promote cell survival and neuronal differentiation (16,17). Neuronal activity results in CaMKII-mediated phosphorylation of NeuroD1 at Ser336, which is necessary for the formation and growth of dendrites (18,19).

Several neuron-enriched markers can be used to identify mature neurons. Neuronal nuclei (NeuN), also known as Fox-3 or RBFOX3, is a nuclear protein expressed in most post-mitotic neurons of the central and peripheral nervous systems. NeuN is not detected in Purkinje cells, sympathetic ganglion cells, Cajal-Retzius cells, INL retinal cells, inferior olivary, or dentate nucleus neurons (20). Mature neurons function as cellular mediators of synaptic transmission. Postsynaptic Density protein 95 (PSD95) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. These family members consist of an amino-terminal variable segment followed by three PDZ domains, an SH3 domain, and an inactive guanylate kinase (GK) domain. PSD95 is a scaffolding protein involved in the assembly and function of the mature postsynaptic density complex (21,22).