Effect of neurosphere size on the growth rate of human neural stem/progenitor cells.
Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 micro m and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 micro m in diameter quickly by culturing them in plates with U-bottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of gamma-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs.
Mori
Hideki
H
Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
Ninomiya
Kazuaki
K
Kino-oka
Masahiro
M
Shofuda
Tomoko
T
Islam
Mohammed Omedul
MO
Yamasaki
Mami
M
Okano
Hideyuki
H
Taya
Masahito
M
Kanemura
Yonehiro
Y
D053829:Amyloid Precursor Protein Secretases / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000196:embryology; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D048429:Cell Size; D002470:Cell Survival; D005333:Fetus; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling; D007211:Indoles; D007381:Intermediate Filament Proteins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D018874:Spheroids, Cellular / Q000502:physiology*; D013234:Stem Cells / Q000502:physiology*; D000072056:Transcription Factor HES-1
D053829:Amyloid Precursor Protein Secretases; D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D007211:Indoles; D007381:Intermediate Filament Proteins; D019394:Ki-67 Antigen; D009419:Nerve Tissue Proteins; D064231:Nestin; D012333:RNA, Messenger; D012097:Repressor Proteins; D000072056:Transcription Factor HES-1; C577484:NES protein, human; C077478:HES5 protein, human; C078493:HES1 protein, human; C007293:DAPI