Optimization of Neurite Tracing and Further Characterization of Human Monocyte-Derived-Neuronal-like Cells

Abstract

Deficits in neuronal structure are consistently associated with neurodevelopmental illnesses such as autism and schizophrenia. Nonetheless, the inability to access neurons from clinical patients has limited the study of early neurostructural changes directly in patients’ cells. This obstacle has been circumvented by differentiating stem cells into neurons, although the most used methodologies are time consuming. Therefore, we recently developed a relatively rapid (~20 days) protocol for transdifferentiating human circulating monocytes into neuronal-like cells. These monocyte-derived-neuronal-like cells (MDNCs) express several genes and proteins considered neuronal markers, such as MAP-2 and PSD-95. In addition, these cells conduct electrical activity. We have also previously shown that the structure of MDNCs is comparable with that of human developing neurons (HDNs) after 5 days in culture. Moreover, the neurostructure of MDNCs responds similarly to that of HDNs when exposed to colchicine and dopamine. In this manuscript, we expanded our characterization of MDNCs to include the expression of 12 neuronal genes, including tau. Following, we compared three different tracing approaches (two semi-automated and one automated) that enable tracing using photographs of live cells. This comparison is imperative for determining which neurite tracing method is more efficient in extracting neurostructural data from MDNCs and thus allowing researchers to take advantage of the faster yield provided by these neuronal-like cells. Surprisingly, it was one of the semi-automated methods that was the fastest, consisting of tracing only the longest primary and the longest secondary neurite. This tracing technique also detected more structural deficits. The only automated method tested, Volocity, detected MDNCs but failed to trace the entire neuritic length. Other advantages and disadvantages of the three tracing approaches are also presented and discussed.

Modulation of human endogenous retroviruses and cytokines expression in peripheral blood mononuclear cells from autistic children and their parents

“…In addition, this is an in vitro study on PBMCs, given the inability of accessing human neurons. A protocol for transdifferentiating human circulating monocytes into neuron-like cells, expressing several genes and proteins associated with neuronal structure, could circumvent this obstacle [ 85 ].…”

Emerging Translational Research in Neurological and Psychiatric Diseases: From In Vitro to In Vivo Models

“…Bellon et al investigate the optimization of neurite tracing and the further characterization of neuronal-like cells derived from human monocytes [32]. Revealing the mechanisms underlying the differentiation of human circulating monocytes into neuronal-like cells is crucial for identifying novel therapeutic strategies for neurological conditions, as demonstrated by this study.…”

Comparing stem cells, transdifferentiation and brain organoids as tools for psychiatric research

“…Neuronal-like cells derived from pluripotent monocytes are the most affordable approach and among the fastest methods to deliver differentiated cells (Table 1 ). In addition, access to pluripotent monocytes is non-invasive as it only requires a standard blood sample [ 114 , 137 ]. The downside is that these neuronal-like cells are also among the least characterized and their differentiation efficiency with the protocols currently available is low [ 114 , 126 , 127 , 134 ].…”

“…MDNCs decrease and in some instances completely abolish its expression of CD14 [ 114 ]. Other proteins present in monocytes such as CD11B and CCR2 are also absent in MDNCs [ 137 ]. Instead, these cells express a variety of neuronal markers.…”

“…Instead, these cells express a variety of neuronal markers. We have studied, through flow cytometry, immunofluorescence, RT-PCR and single cell mRNA sequencing either in combination or each technique individually (Table 2 ), the expression of 43 neuronal markers involved in synaptic functioning, neuronal structure [ 137 ] or considered as markers of specific neuronal types such as glutamatergic, GABAergic, dopaminergic, serotoninergic, cholinergic or motor neurons [ 114 ]. Of the 43 neuronal markers searched, MDNCs express 28 (Table 2 ).…”