Angiogenic factors stimulate growth of adult neural stem cells

Abstract

Background: The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools.

Methodology/principal findings: Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes.

Conclusions/significance: We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.

Figure 1. Efficient culture of adult NSCs by angiogenic factors.

(A–E) Treatments increase rat and monkey adult neural precursor expansion in vitro (Rat, 5-d expansion; Monkey, 10-d). (F) Diagram of the areas dissected for the rat cultures. (G) Precursor and differentiation marker expression in the expansion and differentiation (by 2-weeks mitogen withdrawal) stages from rat lateral forebrain. (H) Precursor and differentiation marker expression in the expansion and differentiation (by a 10-day mitogen withdrawal) stages from rat spinal cord. (I) Precursor and differentiation marker expression in the expansion and differentiation (by 2-weeks mitogen withdrawal) stages from the adult monkey SVZ. [Size bars: 20 µm].

Figure 2. Hes3 is a marker of normal and cancer human stem cells.

(A–C) Hes3⁺ cell in the striatum of non-cancerous adult human brain tissue (blood vessels identified by RECA-1 expression), human hemangioblastoma (HBM) biopsy (HBM Hes3⁺ megakaryocytes shown), human glioblastoma multiforme (GBM) biopsy (Hes3 co-expressed with prominin). (D) Fetal cortical cells sorted for prominin express Sox2 and Hes3. (E) Enrichment for Sox2+ and Hes3+ cells by magnetic sorting using an anti-prominin antibody. (Size bar, 50 µm).

Figure 3. Increased vascular coverage and neuronal projections by angiogenic factors.

(A, B) CT treatment of organotypic slice cultures (every 4 days for 2 weeks) retains the vasculature (confocal projection for the pan-endothelial marker RECA-1 and TH), (C) increases the thickness of the striatal portion of the slice, (D,E) promotes the sprouting of TH⁺ fibers from the S. Nigra section to the striatal section (2-weeks after control (BSA) and CT treatment). [Size bars: 20 µm].