Topographical analysis of the subependymal zone neurogenic niche

The emerging model for the adult subependymal zone (SEZ) cell population indicates that neuronal diversity is not generated from a uniform pool of stem cells but rather from diverse and spatially confined stem cell populations. Hence, when analysing SEZ proliferation, the topography along the anterior-posterior and dorsal-ventral axes must be taken into account. However, to date, no studies have assessed SEZ proliferation according to topographical specificities and, additionally, SEZ studies in animal models of neurological/psychiatric disorders often fail to clearly specify the SEZ coordinates. This may render difficult the comparison between studies and yield contradictory results. More so, by focusing in a single spatial dimension of the SEZ, relevant findings might pass unnoticed. In this study we characterized the neural stem cell/progenitor population and its proliferation rates throughout the rat SEZ anterior-posterior and dorsal-ventral axes. We found that SEZ proliferation decreases along the anterior-posterior axis and that proliferative rates vary considerably according to the position in the dorsal-ventral axis. These were associated with relevant gradients in the neuroblasts and in the neural stem cell populations throughout the dorsal-ventral axis. In addition, we observed spatially dependent differences in BrdU/Ki67 ratios that suggest a high variability in the proliferation rate and cell cycle length throughout the SEZ; in accordance, estimation of the cell cycle length of the neuroblasts revealed shorter cell cycles at the dorsolateral SEZ. These findings highlight the need to establish standardized procedures of SEZ analysis. Herein we propose an anatomical division of the SEZ that should be considered in future studies addressing proliferation in this neural stem cell niche.Fundação para a Ciência e a Tecnologia (FCT

Ovine Carotid Artery-Derived Cells as an Optimized Supportive Cell Layer in 2-D Capillary Network Assays

BACKGROUND: Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs) and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. METHODS AND RESULTS: Human umbilical artery SMCs (HUASMCs) and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs). Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab), had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. CONCLUSIONS: Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast tissue cells. The use of ovine carotid artery-derived cells simplifies the endothelial co-culture assay with respect to testing large amounts of pro- and anti-angiogenic factors

Biofunctionalized Microfiber-Assisted Formation of Intrinsic Three-Dimensional Capillary-Like Structures

Objectives: A vascular supply network is essential in engineered tissues >100–200-μm thickness. To control vascular network formation in vitro, we hypothesize that capillarization can be achieved locally by using fibers to position and guide vessel-forming endothelial cells within a three-dimensional (3D) matrix. Materials and Methods: Biofunctionalization of poly-(L-lactic acid) (PLLA) fibers was performed by amino-functionalization and covalent binding of RGD peptides. Human foreskin fibroblasts (HFFs) and human umbilical vein endothelial cells (HUVECs) were seeded on the fibers in a mould and subsequently embedded in fibrin gel. After 9–21 days of coculture, constructs were fixed and immunostained (PECAM-1). Capillary-like structures with lumen in the 3D fibrin matrix were verified and quantified using two-photon microscopy and image analysis software. Results: Capillary-like networks with lumen formed adjacent to the PLLA fibers. Increased cell numbers were observed to attach to RGD-functionalized fibers, resulting in enhanced formation of capillary-like structures. Cocultivation of HFFs sufficiently supported HUVECs in the formation of capillary-like structures, which persisted for at least 21 days of coculture. Conclusions: The guidance of vessel growth within tissue-engineered constructs can be achieved using biofunctionalized PLLA microfibers. Further methods are warranted to perform specified spatial positioning of fibers within 3D formative scaffolds to enhance the applicability of the concept

of capillary-like structures in the co-culture system.

<p>(A) Pre-processing of the obtained mosaix images for analysis in Angioquant, (B) explanation of length, junctions, area and complex as defined in Angioquant, CD31 staining (endothelial cells, red)</p

Correlation of VEGFR-2 with VE-Cadherin and capillary-like structures.

<p>(A) Confluent and sparse endothelial seeding in HUVEC/cartid artery-derived cell co-culture systems; CD31 endothelial cell staining; (B) Immunoprecipitated VEGFR2 blotted with anti-phosphotyrosine (PY 20) for detection of phosphorylated levels of VEGFR2; whole cell lysates were blotted with α-Tubulin to show equal loading.</p

The effect of Bevacizumab on the formation of capillary-like structures.

<p>The effect of Bevacizumab on 17.500 cells/ well (each cell type) and 40+10 ng/ml VEGF+PDGF-BB; (A) without Bevacizumab and (B) with Bevacizumab; CD31 staining (endothelial cells, red)</p

Evaluation of cell number of both supportive and vessel-forming cells in the co-culture assay.

<p>(A) 10,000 cells/well, (B) 17,500 cells/well and (C) 20,000 cells/well for each cell type; CD31 staining (endothelial cells, red)</p

Characterization of supportive cells.

<p>Immunhistological staining of (A–C) human umbilical artery smooth muscle cells and (D–F) ovine carotid artery smooth muscle cells for (A, B) α-SMA (red), (C, D) smoothelin (red) and (E, F) CD31, (DAPI staining in blue)</p

Effect of PDGF-BB and VEGF on HUVEC donor 1.

<p>Addition of PDGF-BB and/or VEGF to (A+B) 10.000 and (C+D) 17.500 cells/well of each cell type in the co-culture assay (HUVEC donor 1); amount of added growth factors: (A+C) 10 ng/ml PDGF-BB and (B+D) 40 ng/ml VEGF +10 ng/ml PDGF-BB; CD31 staining (endothelial cells, red)</p