Abstract
Extracellular ATP and trophic factors released by exocytosis modulate in vivo proliferation, migration, and differentiation in multipotent stem cells (MpSC); however, the purinoceptors mediating this signaling remain uncharacterized in stem cells derived from the human olfactory epithelium (hOE). Our aim was to determine the purinergic pathway in isolated human olfactory neuronal precursor cells (hONPC) that exhibit MpSC features. Cloning by limiting dilution from a hOE heterogeneous primary culture was performed to obtain a culture predominantly constituted by hONPC. Effectiveness of cloning to isolate MpSC-like precursors was corroborated through immunodetection of specific protein markers and by functional criteria such as self-renewal, proliferation capability, and excitability of differentiated progeny. P2 receptor expression in hONPC was determined by Western blot, and the role of these purinoceptors in the ATP-induced exocytosis and changes in cytosolic Ca2+ ([Ca2+]i) were evaluated using the fluorescent indicators FM1-43 and Fura-2 AM, respectively. The clonal culture was enriched with SOX2 and OCT3/4 transcription factors; additionally, the proportion of nestin-immunopositive cells, the proliferation capability, and functionality of differentiated progeny remained unaltered through the long-term clonal culture. hONPC expressed P2X receptor subtypes 1, 3-5, and 7, as well as P2Y2, 4, 6, and 11; ATP induced both exocytosis and a transient [Ca2+]i increase predominantly by activation of metabotropic P2Y receptors. Results demonstrated for the first time that ex vivo-expressed functional P2 receptors in MpSC-like hONPC regulate exocytosis and Ca2+ signaling. This purinergic-triggered release of biochemical messengers to the extracellular milieu might be involved in the paracrine signaling among hOE cells.
Highlights
In many species including humans, neurogenic process in the central nervous system (CNS) and the olfactory epithelium (OE) prevails in adulthood [1, 2]
Since key neurogenic phenomena are modulated by purinoceptors, our aim was to characterize the P2 receptors’ expression in ex vivo human olfactory neuronal precursor cells with multipotent stem cells (MpSC)-like features and in addition, by evaluating exocytosis, to explore whether this Ca2+-dependent purinergic signaling pathway might play a role in paracrine communication between cells from the human olfactory epithelium (hOE)
To corroborate that human olfactory neuronal precursor cells (hONPC) with MpSC-like traits were effectively isolated from the primary culture and predominated in the clonal culture, we looked for protein markers expressed by pluri- and multipotent precursors with an antibody array
Summary
In many species including humans, neurogenic process in the central nervous system (CNS) and the olfactory epithelium (OE) prevails in adulthood [1, 2] This allows the replacement of dead cells [3] to preserve structural and functional features of both the CNS and OE by the integration of de novo specialized cells in preestablished neuronal circuits [4, 5]. This process requires the proliferation and migration of multipotent stem cells (MpSC) that are capable of self-renewal, and their progeny differentiates into neuronal or glial lineages [6]. The effectiveness of cloning is usually corroborated through functional criteria and detection of specific protein markers expressed by these stem cells [9,10,11]
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