Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons From Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.

Comparison of the Gene Expression Profiles of Human Fetal Cortical Astrocytes with Pluripotent Stem Cell Derived Neural Stem Cells Identifies Human Astrocyte Markers and Signaling Pathways and Transcription Factors Active in Human Astrocytes

“…Gene expression data from previous microarray experiments indicated that NSC lines derived by either the EB/rosette or direct induction method have very similar gene expression profiles [20]. Prior to microarray hybridization the NSCs were examined for expression of the NSC marker Nestin and nearly all PSC-derived NSCs were Nestin+ (Figure 1A).…”

“…These datasets were from additional NSC lines [20], three astrocyte line differentiated from iPSC-derived NSCs (one from this study and two described in ref. 18) as well as cortical neurons differentiated from iPSC-derived NSCs (described ref 21).…”

Induced pluripotent stem cells as a discovery tool for Alzheimer׳s disease

“…Through modulation of patterning pathways, differentiation can be directed to generate cultures of more dorsal, glutamatergic fates (Boisvert et al, 2013; Vazin et al, 2014; Zeng et al, 2010) or else of more ventral, GABAergic fates (Cunningham et al, 2014; Nicholas et al, 2013; Vazin et al, 2014; Yan et al, 2013). It was recently shown that iPSC-derived GABAergic neurons are less susceptible to Aβ induced cell death (Vazin et al, 2014), thus demonstrating the differential phenotypes that can be understood by specifying cell fates.…”

Stem Cells and Neuronal Differentiation

“…Distinct maturation properties were attained depending on the protocol used, and functional maturation was achieved the best when cultured along with primary neuronal culture. hiPSCs have been efficiently differentiated to region and transmitter-specific neuronal cells including glutamatergic, GABAergic, cholinergic, dopaminergic, and motor neurons as well as astrocytes and oligodendrocytes [95,96]. The neural progenitors derived from hiPSCs upon transplantation into the fetal mouse brain migrated into various brain regions and showed in vivo differentiation into glutamatergic, GABAergic, and dopaminergic subtypes.…”