Electrophysiological measures from human iPSC-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance.

Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRSs) for schizophrenia (SCZ) and a clinical diagnosis of SCZ, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures in the patient-derived neurons that implicated altered Na+ channel function, action potential interspike interval, and gamma-aminobutyric acid-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these SCZ patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.

Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence.

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor-derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non-bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel-running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs.

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.

Circadian mPer1 gene expression in mesencephalic trigeminal nucleus cultures.

The circadian timing system includes the major circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and less well characterized circadian pacemakers in the brain and peripheral tissues throughout the body. The coupling between these discrete circadian clocks is not well understood, although individual neurons of the SCN are considered competent circadian pacemakers that interact to produce rhythms in the SCN and in its afferents. Because the SCN is a complex assemblage of small neurons of several phenotypes, we sought a simpler circadian brain nucleus with larger neurons that might provide insight into circadian timing not easily obtained from the SCN. Using bioluminescence imaging of brain tissue explants from transgenic mice containing the firefly luciferase gene luc controlled by the mPer1 promoter, we discovered elevated transgene expression throughout the mesencephalic trigeminal nucleus (Me5) of the brain stem. Large sensory neurons of the Me5 receive proprioceptive signals from periodontal ligaments and masseter muscle spindles. The Me5 cells displayed circadian rhythms with elevated expression in culture corresponding with the dark portion of the prior light cycle. Because of known interactions between the Me5 and the tuberomammillary nucleus and because of the role of both nuclei in satiety, it is possible that a circadian clock in the Me5 serves in regulating daily feeding behavior. This newly identified circadian pacemaker in the Me5 may prove useful for single-cell analyses of circadian gene expression in clock cells and for comparison with the SCN.

Reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures.

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells. After the first 10 d of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantified using this protocol. Reprogramming retinal neurons using this protocol will take 56 d, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 d. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantifies eye field specification, optic cup formation and retinal differentiation in 3D cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol.

Imaging gene expression in live transgenic mice after providing luciferin in drinking water.

Mice expressing the firefly luciferase gene luc under the control of various gene promoters are used to image long-term changes in tumor growth, infection, development, and circadian rhythms. This novel approach enables ongoing regulation of gene expression to be visualized through repeated imaging of luciferase bioluminescence. Typically, luciferin, the luciferase substrate, is injected into mice before they are anaesthetized for imaging. To avoid the effects of handling and stress from injection on expression of the transgene, oral luciferin delivery methods were tested as an alternative to current methods. For unobscured imaging, a transgenic mouse line containing luc controlled by the enhancer and promoter for the major immediate-early gene of human cytomegalovirus (CMV) was crossed with a hairless albino mouse stock (HRS/J), resulting in the Hr-CMV line. Mice given food and water ad libitum readily drank 1-5 mM luciferin in water or apple juice and could be imaged repeatedly on subsequent days without any apparent adverse effects. Oral and injected luciferin produced similar patterns of luminescence in the body areas examined: abdomen, tail vertebrae, gonads, hind leg, foreleg and others, although the tail showed a slightly brighter relative luminescence after oral luciferin. These results show that luciferin is not appreciably degraded in the digestive tract and can be easily administered orally to avoid injection and any concomitant effects on behavior that could alter gene expression.