Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells

Abstract

Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

“…However, the basic culture conditions for growing neurons in vitro have not been updated to reflect fundamental principles of brain physiology. Currently, most human neuronal cultures are grown in media based on DMEM/F12 (4,5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), Neurobasal (25)(26)(27)(28)(29)(30), or a mixture of DMEM and Neurobasal (DN) (31)(32)(33)(34). To promote neuronal differentiation and survival, a variety of supplements, such as serum, growth factors, hormones, proteins, and antioxidants, are typically added to these basal media.…”

Potential of GABA-ergic cell therapy for schizophrenia, neuropathic pain, and Alzheimer׳s and Parkinson׳s diseases

“…Hence, generation of MGE-and LGE-like progenitors from hESCs and hiPSCs through directed differentiation approaches is critical. The methods to generate MGE-like cells from hESCs and hiPSCs are already available Maroof et al, 2013;Liu et al, 2013aLiu et al, , 2013bNicholas et al, 2013). Fig.…”

“…The various steps involved in the generation of gamma-amino butyric acid positive (GABAergic) progenitors from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and the utility of these GABA-ergic progenitors for treating conditions such as schizophrenia, Parkinson's disease, Alzheimer's disease and neuropathic pain. As described by Liu et al (2013aLiu et al ( , 2013b, the pluripotent stem cells (PSCs) are first expanded as embryoid bodies in a suspension culture using a chemically defined medium (A, B). The embryoid bodies are next treated with neural induction medium to obtain neural rossettes, which are then lifted and grown in suspension cultures with high doses of sonic hedgehog (Shh) to obtain neurospheres of cells expressing markers of medial ganglionic eminence progenitor cells (NK×2.1+ cells).…”

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells

“…During embryogenesis, these cells are not generated from the same niche as pyramidal neurons, but rather from two ventral telencephalic regions, namely the medial (MGE) and caudal (CGE) ganglionic eminences (DeFelipe et al, 2013;Sur and Rubenstein, 2005;Wilson and Rubenstein, 2000). Recapitulating in vivo development, the specification of ventral telencephalic cells from both human and mouse PSCs requires inductive input from the Shh morphogen, alone or together with inhibition of the dorsal morphogen Wnt (Danjo et al, 2011;Germain et al, 2013;Li et al, 2009;Liu et al, 2013;Maroof et al, 2010Maroof et al, , 2013Nicholas et al, 2013;Watanabe et al, 2005). The concentration and timing of exposure to Shh lead to different types of ventral progenitors and hence to distinct subtypes of neurons, including the hypothalamic/striatal projection neurons and cortical/striatal interneurons (Danjo et al, 2011;Germain et al, 2013;Ma et al, 2012), which is thus reminiscent of the time dependence of Shh signaling in in vivo contexts (Briscoe and Thérond, 2013).…”