title: Single-cell mRNA profiling identifies progenitor subclasses in neurospheres.
abstract: Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/SSC(high) cells generated 2.29-fold more neurospheres than FSC/SSC(low) cells at clonal density. FSC/SSC(high) cells were enriched for NSCs and Lewis-X(+ve) cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC(high) and FSC/SSC(low) cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore, Singapore.
authors: Gunaseelan
authors: G
authors: Narayanan
authors: Anuradha
authors: A
authors: Poonepalli
authors: Jinmiao
authors: J
authors: Chen
authors: Shvetha
authors: S
authors: Sankaran
authors: Srivats
authors: S
authors: Hariharan
authors: Yuan Hong
authors: YH
authors: Yu
authors: Paul
authors: P
authors: Robson
authors: Henry
authors: H
authors: Yang
authors: Sohail
authors: S
authors: Ahmed
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D002490:Central Nervous System / Q000196:embryology; D020869:Gene Expression Profiling; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis; D051379:Mice; D058953:Neural Stem Cells / Q000145:classification* / Q000166:cytology / Q000502:physiology; D063150:Polycomb Repressive Complex 1 / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051741:Kruppel-Like Transcription Factors; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D012097:Repressor Proteins; C489421:Bmi1 protein, mouse; C489685:Hes5 protein, mouse