Abnormal fetal brain development is linked to a myriad of neurological and psychiatric diseases. Human fetal gestation is a particularly sensitive stage of brain development, during which aberrant genetic programs or external factors (viruses, pollutants, toxins, and other agents) exert the most devastating impact [43,44]. The etiopathogenesis of mental illnesses, such as autism and schizophrenia, is not exclusively determined by genetic variation, but rather by the interaction of genes with the environment [45,46]. Variations in the duration and amplitude of Ca2+ transients and their time of occurrence affect neuronal development and plasticity , but these physiological aspects of human neurodifferentiation are largely absent in clinical and laboratory research. Unsurmountable ethical and technical difficulties preclude experiments on human fetuses. One possibility for studying the physiology of young human neurons is to use hESC and iPSC technologies and mimic human neurodifferentiation in vitro [6,11,47,48].
In this experimental project, we have applied embryonic and pluriopotent stem cell technology to study physiological activity and gene expression during the earliest stages of human neurodifferentiation. Using multisite Ca2+ imaging and qPCR, we have compared time courses of spontaneous physiological Ca2+ activity and gene expression in two human cell lines (hESC-H9 and iPSC-15), as these cells transitioned from undifferentiated stem cells to postmitotic neurons. The iPSC-15 cell line was derived from a patient with schizo-affective disorder with a deletion on 22q11.2 that results in haploinsufficiency of ~ 40 genes, which is one of the most common causes of psychotic disorders [49–51]. We did not observe any significant differences in electrical properties, action potential frequencies, and the expression of genes important for neural development between the two cell lines, demonstrating the presence of intact cellular machinery for basic neuronal function .
4.1. Correlating spontaneous electrical activity with gene expression
Interestingly, as human cells differentiated from DIV-7 to DIV-21, there was a strong positive correlation between the number of cells exhibiting repetitive spontaneous Ca2+ transients and the expression levels of GAD1 and ERBB4 (p < 0.001; Table 3, Row 2). Also, there was a strong positive correlation (p < 0.001) between the number of narrow events and the expression levels of either GAD1 or ERBB4 (Table 3, Row 3). These parallel trends of ERBB4 and GAD1 are not surprising as GABAergic interneurons have been suggested to employ ERBB4 receptor signaling early in the development of the cerebral cortex . In accordance with our present observations, a previous study employing patch clamp recording followed by single-cell PCR revealed that individual neurons expressing GAD1 and ERBB4 are more likely to engage in spontaneous electrical activity than their counterparts which do not express these two genes . As human cells differentiated into neurons and matured, the expression of GAD1 and ERBB4 increased at the same rate as the number of cells showing spontaneous Ca2+ activity (Table 3, Row 1) or the number of cells showing repetitive Ca2+ transients (Figure 6C). The regulation of the GABAergic neuronal phenotype (GAD1 expression and production of GABA) appears to occur via an activity-dependent mechanism and is Ca2+-mediated, as shown by removing extracellular Ca2+ or stimulating cultured neurons with different frequencies of Ca2+ spikes [30,54].
Narrow calcium events (Ca2+ transients with duration < 8 s) fit the description of AP-induced Ca2+ signals in postmitotic human neurons obtained by simultaneous patch clamp and optical recordings , and as such these short-duration Ca2+ signals mark the arrival of newborn neurons in stages IV and V of the current neurodifferentiation protocol (Figure 3F). We, therefore, explored the possibility that the expression of all the other genes that correlated with repetitive activity also had a significant correlation to the number of narrow events. We found that except for Cx36, which is significantly correlated (p < 0.001) to percent repetitive activity, but not to the number of narrow events, the rest of the genes (BLBP, Cx45, ERBB4, GAD1, PAX6, PNX1, and vGLUT1) had a significant positive correlation to both repetitive activity and number of narrow events (Table 3, Rows 2 and 3). Cx36 has been reported to be expressed during the development of retinal ganglion cells. It is thought that Cx36 is capable of influencing spontaneous firing patterns, but is not required for spontaneous retinal wave generation .
The addition of growth factors BDNF and GDNF to the cell culture media appeared to have an effect on the expression of ERBB4, vGLUT1, and Cx36. These three genes were present at minimal levels, until the addition of growth factors on DIV-12 caused a sharp increase in their level of expression (Figure 4). Interestingly, the three genes showing exponential rise after DIV-14 are also highly correlated with Ca2+ signals of Moderate duration of 8–20 s and amplitude greater than 5 % ΔF/F (Table 3, Row 5). Cx36 is thought to participate in neuronal gap junction formation in vivo. An increased expression of Cx36 has been demonstrated to promote neuronal differentiation by enhancing cell–cell contact between progenitors . In situ hybridization techniques have shown high levels of Cx36 mRNA in olfactory bulbs, pineal gland, inferior olive, hippocampal pyramidal neurons, and in the retina. We found a significant correlation between Cx36 and GAD1 expression patterns (r = 0.79, p < 0.01), suggesting some functional link between these two genes. Cx36 has been reported to be present on GABAergic interneurons in the neocortex and hippocampus . In Cx36-deficient mice, gap junction-coupling among neocortical inhibitory interneurons has been shown to be nearly absent. GAD1 (GAD67) is an enzyme that synthesizes GABA and is itself influenced by Ca2+ signaling [17,30]. Thus, Cx36 and GAD1 have a close relationship, which may be a part of the regulatory mechanisms for expression of GABA and/or GABAergic interneuron differentiation.
In addition to having a significant correlation to GAD1, we found that there was a significant correlation between Cx36 and the vGLUT1 expression patterns (r = 0.63, p < 0.05) over the period DIV-8 to DIV-21. The Cx36 gene has been reported to be induced at the time of the surge of the transcription factors that determine β-cell differentiation . Thus, during neuronal differentiation, Cx36 could potentially influence transcription factors that determine the terminal differentiation of neurons to either glutamatergic or GABAergic, with the Cx36 channels forming intercellular pathways for transmission of developmentally relevant molecules. Upregulation of Cx36 expression has also been previously associated with the sequential expression of specific ligand-gated responses (GABA and glutamate), thus heralding terminal differentiation of neurons, with the peak expression of Cx36 being associated with a developmental window in which the neuronal network connections are being developed .
In the present study, we found that Cx43, Cx47, and TUBB3 showed a hyperbolic pattern of expression, wherein there was an upregulation of these genes during the cell proliferation stage (in the presence of the proliferation factor bFGF) and a subsequent decrease in their expression. Both the increase and the decrease of Cx43, Cx47, and TUBB3, relative to the characteristic time periods of the differentiation protocol (e.g. replacement of proliferation media on DIV-12; appearance of first APs on DIV-14; sharp onset of Cx36 expression on DIV-17), may become useful for understanding the roles of these three genes in human neurodifferentiation.
We found that the decline of TUBB3 after DIV-14 was paralleled by an upregulation of GAD1 (putative GABAergic neurons) and vGLUT1 (putative glutamatergic neurons) during the differentiation phase (Figure 4, compare TUBB3, GAD1, and vGLUT1). Similar to our results, a decrease in the expression of the neuronal progenitor marker TUBB3 along with a simultaneous increase in GABA and glutamatergic neurons during neuronal differentiation was also observed by . We found a strong correlation between Cx43 and TUBB3 expression (r = 0.91, p < 0.001), which suggests a close relationship between Cx43 expression on astrocytes and TUBB3 expression on neuronal progenitors. Cx43 is essential for the maintenance of neural progenitors in a proliferative state [59, 60] and forms communication channels, between migrating neurons and the glial cells, during neurogenesis .
By sampling data points on consecutive days (DIV-7 to DIV-21), we observed that the expression of human GAD1 occurred before the expression of human vGLUT1 (Figure 4), as has been reported in the literature, where the formation of GABAergic synapses in the CNS preceded that of glutamatergic synapses . In summary, as cells exit from the cell cycle and undergo lineage commitment, as shown by the expression of ERBB4, GAD1, and vGLUT1, they demonstrate a changing profile of connexin expression with Cx43 and Cx47 declining, Cx45 and PNX1 increasing, and Cx36 being activated.
Although BLBP was expressed in our cell culture, GFAP was not expressed at detectable levels. Both GFAP and BLBP are expressed on radial glia during early neuronal development, but their expression has been reported to be nonoverlapping . In line with our experimental results, GFAP was observed to be expressed only after the expression of BLBP during in vitro culture of human embryonic stem cells . Thus, culturing cells for a period longer than 21 days is necessary for observing GFAP expression.
When correlation analysis was performed between Ca2+ transients with specific characteristics of duration and amplitude, and the expression of specific genes involved in neurodifferentiation, we observed that specific subclasses of Ca2+ transients are linked to specific genes. Two possible scenarios can explain the observed positive correlations between spontaneous physiological activity and gene expression. First, specific calcium transients could be driven by the expression of specific human genes. Second, one type of calcium transients (e.g., short duration and small amplitude) may selectively influence the expression of some but not all human genes during early neuronal development.
Spike stimulation mimicking the frequency of spontaneous transients has been shown to be most effective in replicating the effects of spontaneous transients on neurotransmitter expression, for example, GABA expression [30,54]. Thus, it would be quite useful to find calcium spike properties (amplitude, duration, and frequencies) that promote the expression of genes involved in early neuronal differentiation in order to improve current methods for modeling human brain development in culture.