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Genetic manipulation of Fezf2 in human embryonic stem cells and its application in studying central nervous system development and repair


One of the major goals in the stem cell biology field is to understand the mechanisms involved in directing cells towards a particular fate. This is an especially complex problem when studying the central nervous system, due to the variety of neuronal cells that comprise the brain and spinal cord. To study any specific lineage or cell type, it is necessary to have tools that can identify the cell type of interest. One such method is to generate a reporter cell line to fluorescently label cells that differentiate towards the lineage of interest. To study the mechanisms involved in differentiation and specification of corticospinal motor neurons, the neuron type damaged in spinal cord injury and amyotrophic lateral sclerosis, we chose to study Fezf2, a gene that has been shown to play an essential role in corticospinal motor neuron development in mice. To track cells expressing Fezf2, I developed a fluorescent reporter under the control of the endogenous Fezf2 promoter via homologous recombination. This method is generally applicable to target all genes, regardless of their expression. Analysis of the targeted cell line confirmed functional reporter expression, with fluorescence being detected only after differentiation, consistent with the fact that Fezf2 is not expressed in ESCs. To study the role of Fezf2 in human cells, we developed a directed differentiation protocol to generate Fezf2+ cells. Analysis of pluripotency and neural markers confirmed our culture system efficiently induces a neural identity. At later time points, Fezf2 expression is detected in a subset of cells. Fezf2+ cells also express [Beta]-tubulin, and a subset of Fezf2+ cells co-express CTIP2, a transcription factor downstream of Fezf2. Gene expression analysis of purified Fezf2+ cells revealed conservation of expression of a number of corticospinal markers identified in mice. Together, this data suggests there is some conservation between mouse and human corticospinal development, indicating Fezf2 may be an ideal gene for studying human corticospinal development. This Fezf2 reporter line is a valuable tool that will facilitate further studies into mechanisms involved in human corticospinal development, including in vivo studies looking at development as well as repair strategies for neurological conditions

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