Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia

Abstract

Consistent with recent reports indicating that neurons differentiated in vitro from human induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain (1, 2), gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two, independent discovery-based approaches - microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses – to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. This approach, consisting of reproducible phenotypes identified through scalable assays, can be applied to expanded cohorts of SZ patients, making it a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

“…Though NPCs appear to be morphologically similar by eye, they are a heterogeneous population comprised of cells capable of generating both glutamatergic and GABAergic neurons. While we observe remarkably consistent forebrain patterning of NPC lines between individuals using this method 1 , this is only when comparing fully validated high quality NPC lines -there are substantial differences between good and poor quality NPC lines. Additionally, viral transduction yields genetically heterogeneous cells, each with an integration of the viral vector at a unique genomic location; therefore, individual cells will vary both in the location and number of genomic integration sites.…”

“…Furthermore, because one can rapidly generate large numbers of cells and need not wait weeks or months for neuronal maturation, NPC-based assays are suitable for the study of larger patient cohorts and are more amenable to high throughput screening. We believe that hiPSC NPCs can serve as a proxy for the developmental pathways potentially contributing to disease pathogenesis, as has already been demonstrated in disorders as diverse as schizophrenia 1 and Huntington's disease 11 .…”

“…Gene expression studies of neurons differentiated in vitro from human induced pluripotent stem cells (hiPSCs) by us 1 and others 2,3 indicate that hiPSC neurons resemble fetal rather than adult brain tissue. At present, hiPSC-based models may be more appropriate for the study of predisposition to, rather than late features of, neurological disease.…”

“…We have previously reported that a significant fraction of the gene signature of schizophrenia hiPSC-derived neurons is conserved in schizophrenia hiPSC-derived neural progenitor cells (NPCs), indicating that NPCs may be a useful cell type for studying the molecular pathways contributing to schizophrenia 1 . We and others have reported aberrant migration, increased oxidative stress and reactive oxygen species, sensitivity to sub-threshold environmental stresses and impaired mitochondrial function in schizophrenia hiPSC NPCs 1,[4][5][6] , as well as decreased neuronal connectivity and synaptic function in schizophrenia hiPSC neurons 5,[7][8][9][10] . If the molecular factors contributing to aberrant migration and/or oxidative stress in schizophrenia hiPSC NPCs also underlie the reduced neuronal connectivity in schizophrenia hiPSC-derived neurons, NPCs could be a robust and highly replicative neural population with which to study the mechanisms responsible for disease.…”

Direct somatic lineage conversion

“…Direct reprogramming of somatic cells into a target cell type by forced expression of reprogramming factors without going through an intermediate iPS cell state is a very powerful tool for disease modelling because of the ability to generate specific mature human somatic cells within a short period of time. Our laboratory has demonstrated that fibroblasts could be directly converted into functional mature neuronal cells within two weeks, whereas conventional differentiation of pluripotent cells into somatic lineages (such as neural, hematopoietic, cardiac and hepatic) through exposure to morphogens and small molecules produces cells that are typically immature, resembling fetal stages [72]. Even maturation times of several months are often insufficient to generate mature, adult-like cell types from pluripotent stem cells.…”