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. 2017 Aug 15;26(16):1199-1213.
doi: 10.1089/scd.2016.0350. Epub 2017 Jul 20.

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage

Courtney M Dumont  1   2 , Jennifer M Piselli  1   2 , Nadeem Kazi  1   2 , Evan Bowman  2 , Guoyun Li  2   3 , Robert J Linhardt  2   3 , Sally Temple  4 , Guohao Dai  1   2 , Deanna M Thompson  1   2
Affiliations

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage

Courtney M Dumont et al. Stem Cells Dev. .

Abstract

The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures. Endothelial factors generated under dynamic conditions significantly increased neuronal differentiation, while those released under static conditions stimulated oligodendrocyte differentiation. Flow increases EC release of neurogenic factors and of heparin sulfate glycosaminoglycans that increase their bioactivity, likely underlying the enhanced neuronal differentiation. Additionally, endothelial factors, especially from static conditions, promoted adherent growth. Together, our data suggest that blood flow may impact proliferation, adhesion, and the neuron-glial fate choice of adult NPCs, with implications for diseases and aging that reduce flow.

Keywords: neural stem cells; neurogenesis; oligodendrocytes; shear stress; vascular niche.

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Conflict of interest statement

The authors declare no competing interests.

Figures

<b>FIG. 1.</b>
FIG. 1.
EC-conditioned medium reduces neurosphere formation in favor of adherent NPC cultures. Schematic representation of the experimental design (A) for cells isolated from the adult SVZ and expanded in neurosphere cultures using a base medium of DMEM (control) (B1-3), static EC-conditioned (C1-3), or dynamic EC-conditioned (D1-3) medium for 14 days. Neurospheres developed in control and dynamic EC-conditioned medium-treated cultures; however, the static EC-conditioned medium formed only adherent cultures. Dynamic EC-conditioned medium also supported adherent cultures. Control medium supports greater neurosphere numbers (E) compared with dynamic EC-conditioned medium. The average neurosphere size (F) was not significantly different for cells expanded in either the control or dynamic EC-conditioned medium. (G) Evaluation of the total cell number shows that control medium generated significantly (*P < 0.05, n = 6) more cells than those cultured in EC-conditioned medium. Mixed cultures in dynamic EC-conditioned medium generated significantly more cells than those expanded as adherent cells in static EC-conditioned medium (*P < 0.05, n = 6). (H) Cells expanded for 14 days in experimental and control conditions were dissociated and subsequently cultured for an additional 21 days in an NCFC assay to assess potency. Cells preconditioned in EC-conditioned medium (dynamic and static, P < 0.05, n = 6) generate larger and more numerous colonies. Colonies greater than 2 mm are considered to be indicative of NSC colonies, rather than NPC colonies. Only SVZ cells preconditioned with EC-conditioned medium collected under dynamic culture generated the largest >2-mm neurospheres at the end of 21 days; however, this result was not statistically significant as so few colonies were detected. Data are represented as mean ± standard deviation. Scale bar = 0.5 mm. EC, endothelial cell; NCFC, neural colony-forming cell; NPC, neural progenitor cell; SVZ, subventricular zone.
<b>FIG. 2.</b>
FIG. 2.
NPCs expanded in control or EC-conditioned medium maintain SVZ precursor phenotypes. NPCs cultured in (A) control, (B) static EC-conditioned, or (C) dynamic EC-conditioned medium were stained for Mash1+ (type C cells, magenta, indicated by *), EGFR (type C cells, red), or PSA-NCAM+ (type A neuroblasts, indicated by ^). Similarly, NPCs cultured in (D) control, (E) static EC-conditioned, or (F) dynamic EC-conditioned medium were stained for GFAP+ (yellow, indicated by caret [<]), EGFR+ (red), or CD133+ (green). Cells immunoreactive for GFAP+CD133+ (quiescent type B cells) were not detected in any culture conditions. (G) No statistically significant (n = 4) differences were observed in phenotype across any of the control or EC-conditioned media. Data are represented as mean ± standard deviation. Scale bar = 50 μm.
<b>FIG. 3.</b>
FIG. 3.
EC-conditioned medium enhances neuronal and oligodendrocyte differentiation. Isolated SVZ cells were cultured in either the control (nonconditioned) or EC-conditioned (static or dynamic) medium, dissociated, and subsequently differentiated in 1% FBS for 3 weeks. At 3 weeks, cells initially cultured in control (A, B), static EC-conditioned (C, D), or dynamic EC-conditioned (E, F) medium were stained either for astrocytes (GFAP; red) and neurons (NeuN; green) (A, C, E) or oligodendrocytes (O4, green) and oligodendrocyte precursor cells (OPCs: NG2; red) (B, D, F). All cells were visualized with DAPI (blue). (G) Cells expanded in EC-conditioned medium from static cultures resulted in O4+ oligodendrocyte populations that were not present in dynamic EC-conditioned or control medium. Expansion in dynamic EC-conditioned medium resulted in a significant increase (*P < 0.05, n = 4) in NeuN+ cells compared with the static and control-expanded populations. Nestin+ precursor cells were not detected after 3 weeks. See Supplementary Fig. S3 for individual channels of this figure. Scale bar = 50 μm. Data are represented as mean ± standard deviation.
<b>FIG. 4.</b>
FIG. 4.
Increased cytokine production and stabilization are observed in dynamic EC-conditioned medium. EC-produced FGF2 (A) and EGF (B) increase in response to dynamic culture (10 dynes/cm2), resulting in a significant 2.8-fold and 8.7-fold increase over 24 h relative to statically cultured ECs. (C) Heparan sulfate (HS) and chondroitin sulfate (CS) GAGs are present in both the static and dynamic EC-conditioned media. There is a significant 2.6-fold increase in HS within the medium collected from dynamic culture compared with the static EC-conditioned medium, while no statistically significant difference was detected in CS content. GAG content is known to stabilize growth factors and the buffering capacity of the EC-conditioned medium for growth factor stabilization was evaluated. (D) Degradation of FGF2 in EC-conditioned medium (dynamic or static) or in control medium supplemented with 100 pg/mL mouse FGF2 at 37°C was evaluated using ELISA. Model HBGF factor (FGF2) degrades rapidly for all culture media over the first 12 h. In control and static EC-conditioned media, FGF2 continues to degrade below ELISA detection limits (8 pg/mL) by 72 h, while FGF2 levels plateau in dynamic EC-conditioned medium between 6 and 72 h at 40% of the original FGF2 concentration. * indicates dynamic is significantly (P < 0.05, n = 4) higher than control; + indicates dynamic is significantly (P < 0.05, n = 4) higher than static medium. Data are represented as mean ± standard deviation. GAG, glycosaminoglycan.
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