High-throughput clonal analysis of neural stem cells in microarrayed artificial niches.
To better understand the extrinsic signals that control neural stem cell (NSC) fate, here we applied a microwell array platform which allows high-throughput clonal analyses of NSCs, cultured either as neurospheres or as adherent clones, exposed to poly(ethylene glycol) (PEG) hydrogel substrates functionalized with selected signaling molecules. We analyzed by time-lapse microscopy and retrospective immunostaining the role of integrin and Notch ligands, two key NSC niche components, in altering the behavior of several hundred single stem cells isolated from a previously described Hes5::GFP reporter mouse. NSC self-renewal was increased by 1.5-fold upon exposure to covalently tethered Laminin-1 and fibronectin fragment 9-10 (FN(9-10)), where 60-65% of single cells proliferated extensively and remained Nestin positive. Tethering of the Notch ligand Jagged-1 induced activation of Notch signaling. While Jagged-1 alone increased cell survival and proliferation, no further increase in the clonogenic potential of Hes5::GFP cells was observed upon co-stimulation with Laminin-1 and Jagged-1. We believe that the bioengineering of such in vitro niche analogues is a powerful approach to elucidate single stem cell fate regulation in a well-controlled fashion.
School of Life Sciences, Institute of Bioengineering and Laboratory of Stem Cell Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland.
Marta
M
Roccio
Samy
S
Gobaa
Matthias P
MP
Lutolf
D000071916:AC133 Antigen; D000818:Animals; D000831:Animals, Newborn; D015703:Antigens, CD / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology* / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006023:Glycoproteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D020100:Hydrogels / Q000737:chemistry; D039503:Integrin alpha6 / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000378:metabolism; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D051880:Receptors, Notch / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D055153:Stem Cell Niche; D059008:Time-Lapse Imaging
D000071916:AC133 Antigen; D015703:Antigens, CD; D051792:Basic Helix-Loop-Helix Proteins; D002135:Calcium-Binding Proteins; D066246:ErbB Receptors; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D020100:Hydrogels; D039503:Integrin alpha6; D036341:Intercellular Signaling Peptides and Proteins; D007381:Intermediate Filament Proteins; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D064231:Nestin; D010455:Peptides; D011092:Polyethylene Glycols; D051880:Receptors, Notch; D012097:Repressor Proteins; D000072099:Serrate-Jagged Proteins; C489685:Hes5 protein, mouse; C000606274:Jag1 protein, mouse; C577485:Nes protein, mouse; C541485:EGFR protein, mouse