Decoding the porcine developing spatial processing system and production of human entorhinal stellate cell-like cells by a direct programming approach

Classic studies investigating how and when the entorhinal cortex (component of the memory processing system of the brain) develops have been based on traditional thymidine autoradiography and histological techniques. In this study, we take advantage of modern technologies to trace at a high resolution, the cellular complexity of the developing porcine medial entorhinal cortex by using single-cell profiling. The postnatal medial entorhinal cortex comprises 4 interneuron, 3 pyramidal neuron and 2 stellate cell populations which emerge from intermediate progenitor and immature neuron populations. We discover four MGE-derived interneurons and one CGE-derived interneuron population as well as several IN progenitors. We also identify two oligodendrocyte progenitor populations and three populations of oligodendrocytes. We perform a proof-of-concept experiment demonstrating that porcine scRNA-seq data can be used to develop novel protocols for producing human entorhinal cells in-vitro. We identified six transcription factors ( RUNX1A1, SOX5, FOXP1, MEF2C, TCF4, EYA2 ) important in neurodevelopment and differentiation from one RELN + stellate cell population. Using a lentiviral vector approach, we reprogrammed human induced pluripotent stem cells into stellate cell-like cells which expressed RELN, SATB2, LEF1 and BCL11B. Our findings contribute to the understanding of the formation of the brain’s cognitive memory and spatial processing system and provides proof-of-concept for the production of entorhinal cells from human pluripotent stem cells in-vitro. ### Competing Interest Statement The authors have declared no competing interest. * BMP : bone morphogenetic protein CHIR : GSK-3α/β inhibitor CNS : central nervous system D : Day DMEM : dulbecco’s modified eagle’s medium E : Embryonic day EC : entorhinal cortex EDTA : Ethylenediaminetetraacetic acid gDNA : genomic DNA GOI : gene of interest h : hours iPSC : induced pluripotent stem cells IN : interneurons IP : intermediate progenitor L : layer LV : lentivirus LEC : lateral entorhinal cortex MEC : medial entorhinal cortex MOI : multiplicity of infection OPC : oligodendrocyte progenitor cell P : postnatal day PBS : phosphate-buffered saline PCR : polymerase chain reaction PYR : pyramidal neuron qPCR : quantitative real time PCR RELN : Reelin gene RT : room temperature scRNA-seq : Single-cell RNA sequencing SC : stellate cell SD : standard deviation SPE : speed cell SST : somatostatin TF : transcription factor U : units of viral particles UMI : unique molecular identifier VPD : volume of produced virus