Abstract
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, TCF3, 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.
Footnotes
* Author List Footnotes: Corresponding Author and lead contact: Vanessa Jane Hall
Email addresses: Yong Liu: yong.liu{at}ki.se, Tobias Bergmann: tobi{at}sund.ku.dk, Leo Mogus: rnh644{at}alumni.ku.dk, Julie Lee: julie.lee{at}sund.ku.dk, Ulrich Pfisterer: ulrich.pfisterer{at}med.lu.se, Louis-Francois Handfield: lh20{at}sanger.ac.uk, Andrea Asenjo-Martinez: andrea.asenjo{at}bric.ku.dk, Irene Lisa-Vargas: irene.vargas{at}sund.ku.dk, Stefan E Seemann: sse{at}sund.ku.dk, Jimmy Tsz Hang Lee: tl7{at}sanger.ac.uk, Nikolaos Patikas: np13{at}sanger.ac.uk, Birgitte Rahbek Kornum: kornum{at}sund.ku.dk, Mark Denham: mden{at}dandrite.au.dk, Poul Hyttel: poh{at}sund.ku.dk, Menno P Witter: menno.witter{at}ntnu.no, Jan Gorodkin: gorodkin{at}sund.ku.dk, Tune H Pers: tune.pers{at}sund.ku.dk, Martin Hemberg: mhemberg{at}bwh.harvard.edu, Konstantin Khodosevich: konstantin.khodosevich{at}bric.ku.dk, Vanessa Jane Hall: vh{at}sund.ku.dk
This version of the manuscript includes a proof-of-concept study where we produce stellate cell-like cells from human induced pluripotent stem cells using an overexpression viral delivery of six identified transcription factors upregulated in one of the REELIN+ stellate cell populations identified from the scRNA-seq data.
Abbreviations
- 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