Brain-imaging studies have identified structural changes in SCZD brains, but cannot resolve which neuronal cell types are affected in SCZD. Pharmacological studies have identified a role for DA and GLU; however, chronic antipsychotic treatment alters brain structure and neural activity, confounding studies of human patients affected with SCZD. GWAS studies have yet to account for most of the heritable variance of SCZD. Though animal models have recapitulated aspects of the behavioral and cellular phenotypes of SCZD, they lack the ability to define the complex interacting genetic factors that contribute to disease. hiPSC-based studies will complement brain-imaging, pathological, pharmacological, genetic and animal studies of SCZD.
It will be critical that researchers carefully consider which specific subtypes of neurons should be compared using hiPSC neurons studies. We propose that the field begin to characterize cell-autonomous defects in midbrain DA (mDA) and cortical glutamatergic (cGLU) and GABAergic (cGABA) SCZD hiPSC neurons.
An efficient protocol can now differentiate pluripotent stem cells to populations consisting of approximately 20% DA neurons 98
by recapitulating developmental cues found in the ventral midline when SHH, FGF8 and WNT1 initiate DA differentiation; immature mDA neurons express NURR1, EN1/2 and LMX1A/B, whereas mature mDA neurons also express tyrosine hydoxylase (TH) and aromatic L-amino acid decarboxylase (AADC) 99
. Studies using mouse ESCs have shown that NODAL antagonists (LEFTYA) induce expression of the forebrain marker Brain-factor 1 (BF1, FOXG1) and subsequent treatment with WNT antagonists causes regional specification towards cortical fate 100
. Genes such as EMX1, FEZF2, and FEZ, are expressed in immature cGLU neurons 101–103
, while mature cGLU neurons express CTIP2 and OTX1. 101, 104, 105
. Subsequent culture with FGF2 has been shown to increase GABAergic differentiation 106
; GABA neurons can be identified by expression of key GABAergic markers, such as Glutamate decarboxylase (GAD65/67), DARPP32, ARPP21, CALBINDIN, or CALRETININ 107
, although few if any regional markers of basal ganglia identity have been identified.
Future molecular studies of hiPSC neurons should incorporate SNP, CNV and gene expression data. Studies of quantitative trait loci (eQTLs) will determine how genetic lesions affect gene expression in SCZD neurons. Current eQTL studies can only compare a limited supply of heterogeneous post-mortem brain tissue confounded by variables such as patient treatment history, drug/alcohol abuse and poverty. Ideally, hiPSC-based eQTL studies would produce a renewable supply of more homogeneous cell populations. As hiPSC generation, neuronal differentiation and subtype purification are streamlined and made more efficient, it will become possible to generate any defined neuronal subtypes from hiPSCs generated from hundreds of patients with known genetic backgrounds.
Currently, laborious single cell electrophysiological analysis is the best method to establish the maturity and assess synaptic function of the SCZD hiPSC neurons. Electrophysiological characterization can verify that hiPSC neurons have membrane potentials, undergo induced action potentials and show evidence of spontaneous synaptic activity. However, in order to study functional synaptic activity defects contributing to SCZD, and detect significant effects in heterogeneous patient populations, it will be necessary to increase both the number of patients and neurons that can be studied, We believe that developing more efficient and higher throughput synaptic analyses needs to become a priority of the field.
Beyond neurons, hiPSCs can of course also be differentiated to any other cell type implicated in SCZD, particularly oligodendrocytes 108, 109
. Given that consider data suggests that myelin dysfunction contributes to SCZD, comparisons of co-cultures of control and SCZD neurons and oligodendrocytes should reveal whether reduced myelination in SCZD neurons is a cell autonomous effect. Should studies of hiPSC-derived cells reveal aberrant oligodendrocyte activity in SCZD, this may indicate a new point of therapeutic intervention in SCZD.
Moving forward, hiPSC studies must make several critical advances. Future studies should focus on defined neuronal subtypes. More efficient hiPSC generation and neuronal differentiation will ultimately permit eQTL studies of SCZD. More scalable assays of synaptic function will allow characterization of increased numbers of control and patient neurons. New studies will need to recruit better-characterized patient populations with well-defined clinical endophenotypes, pharmacological responses and/or genetic lesions.