Abstract
Understanding how autocrine/paracrine factors regulate neural stem cell (NSC) survival and growth is fundamental to the utilization of these cells for therapeutic applications and as cellular models for the brain. In vitro, NSCs can be propagated along with neural progenitors (NPs) as neurospheres (nsphs). The nsph conditioned medium (nsph-CM) contains cell-secreted factors that can regulate NSC behavior. However, the identity and exact function of these factors within the nsph-CM has remained elusive. We analyzed the nsph-CM by mass spectrometry and identified DSD-1-proteoglycan, a chondroitin sulfate proteoglycan (CSPG), apolipoprotein E (ApoE) and cystatin C as components of the nsph-CM. Using clonal assays we show that CSPG and ApoE are responsible for the ability of the nsph-CM to stimulate nsph formation whereas cystatin C is not involved. Clonal nsphs generated in the presence of CSPG show more than four-fold increase in NSCs. Thus CSPG specifically enhances the survival of NSCs. CSPG also stimulates the survival of embryonic stem cell (ESC)-derived NSCs, and thus may be involved in the developmental transition of ESCs to NSCs. In addition to its role in NSC survival, CSPG maintains the three dimensional structure of nsphs. Lastly, CSPG's effects on NSC survival may be mediated by enhanced signaling via EGFR, JAK/STAT3 and PI3K/Akt pathways.
Highlights
In vivo, neural stem cells (NSCs) reside in tightly regulated microenvironments known as the stem cell niche
We fractionated the nsph conditioned medium (nsph-CM) into fraction A (.30 kDa) and fraction B (,30 kDa; Fig. 1B) and found that in low density cultures fraction A has nsph stimulatory activity approximating that of whole nsph-CM, and fraction B stimulated nsph formation by 1.5-fold (Fig. 1C)
DSD-1proteoglycan, apolipoprotein E (ApoE) and cystatin C were identified as unique factors present in the nsph-CM (Fig. 1D)
Summary
Neural stem cells (NSCs) reside in tightly regulated microenvironments known as the stem cell niche. This niche consists of supporting niche cells, extrinsic signals, membranebound molecules and the extracellular matrix (ECM). The niche protects stem cells from differentiation signals, apoptotic stimuli and excessive proliferation which can result in cancer [1]. NSCs can be propagated along with lineage restricted neural progenitors (NPs) as neurospheres (nsphs) or adherent cultures. The nsph conditioned medium (nsph-CM) contains cell-secreted factors that have been shown to increase NSC/NP survival and proliferation [2,3]. Nsph-CM regulates cell fate decisions [4,5]. Proteoglycans within the ECM are known to act as reservoirs for growth factors [6]
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