Defining the adult hippocampal neural stem cell secretome: in vivo versus in vitro transcriptomic differences and their correlation to secreted protein levels

Abstract

Recent evidence shows that adult hippocampal neural stem and progenitor cells (NSPCs) secrete a variety of proteins that affect tissue function. Though several individual NSPC-derived proteins have been shown to impact cellular processes like neuronal maturation and stem cell maintenance, a broad characterization of NSPC-secreted factors is lacking. Secretome profiling of low abundance stem cell populations is typically achieved via proteomic characterization of in vitro, isolated cells. Here, we analyzed the in vitro NSPC secretome using conditioned media from cultured adult mouse hippocampal NSPCs and detected over 200 different bioactive proteins with an antibody array. We next assessed the NSPC secretome on a transcriptional level with RNA sequencing (RNAseq) of cultured NSPCs. This comparison revealed that quantification of gene expression did not accurately predict relative protein abundance for several factors. Furthermore, comparing our transcriptional data with previously published single cell RNA sequencing datasets of freshly isolated hippocampal NSPCs, we found key differences in gene expression of secreted proteins between cultured and acutely isolated NSPCs. Understanding the components and functions of the NSPC secretome is essential to understanding how these cells may modulate the hippocampal neurogenic niche, as well as how they can be applied therapeutically. Cumulatively, our data emphasize the importance of using proteomic analysis in conjunction with transcriptomic studies and highlights the need for better methods of global unbiased secretome profiling.

Neural stem cell expressed vascular endothelial growth factor maintains the neurogenic vascular niche of the adult mouse hippocampus

“…Sheddases are membrane-bound enzymes that cleave trans-membrane proteins, consisting of members from the ADAM, BACE and MMP protein families. Analysis of our own published single-cell RNA sequencing and liquid chromatography tandem mass spectrometry data from cultured adult DG NSCs revealed that multiple sheddases are detectable throughout the cell cycle and in quiescent NSCs, particularly Mmp15, Adam9, Adam10 and Adam12 (Figure 4B,C) (Denninger et al, 2020). Multiple sheddases were also detected in single cell RNAseq of in vivo DG RGL-NSCs from (Shin et al, 2015) (Figure 4D).…”

Online versus Cognitive Control: A Dividing Line between Physical Action and Motor Imagery

“…To begin investigating the role of EAATs in NSCs, we used cultured NSCs derived from adult male and female DG 26 . These cultures are composed primarily of quiescent and cycling NSCs when maintained in standard conditions 27 . Using our published bulk RNAseq and liquid chromatography tandem mass spectrometry data from these cells 27 , we first confirmed that cultured NSC expression of glutamate transporter and receptor genes was similar to that of in vivo NSCs, with EAAT1 being most abundant, followed by EAAT2, then lower but detectable expression levels of AMPA receptor subunits and metabotropic glutamate receptor transcripts (Fig S2A -C).…”

“…These cultures are composed primarily of quiescent and cycling NSCs when maintained in standard conditions 27 . Using our published bulk RNAseq and liquid chromatography tandem mass spectrometry data from these cells 27 , we first confirmed that cultured NSC expression of glutamate transporter and receptor genes was similar to that of in vivo NSCs, with EAAT1 being most abundant, followed by EAAT2, then lower but detectable expression levels of AMPA receptor subunits and metabotropic glutamate receptor transcripts (Fig S2A -C). We also confirmed that glutamate stimulated NSC proliferation, as revealed by incorporation of the thymidine analog ethynyl deoxyurdine (EdU) (Fig 2A ,D).…”

“…All experiments used cells between passages 4 and 20 and were replicated in two separate cell lines, one derived from 4 pooled male DG and the other from 4 pooled female DG. Both lines were mycoplasma tested and confirmed to produce neurons and glia upon growth factor withdrawal as previously described (Denninger et al, 2020). uHPLC-ECD Glutamate was quantified using uHPLC-ECD (ALEXYS Neurotransmitter Analyzer;Antec Scientific, USA).…”

“…Mice were administered carprofen prior to surgery and daily for 3 days post surgery as well as buprenorphine immediately post surgery. RNAseq library generation, sequencing, alignment, and transcript quantification Library generation was performed as previously described 27 using the Clontech SMART-Seq HT (Takara) kit. Purified library products were then submitted to HiSeq 4000 paired-end sequencing (Illumina) with a depth of 15 -20 million 2 × 150 bp clusters.…”