Self-Renewal and Differentiation of Reactive Astrocyte-Derived Neural Stem/Progenitor Cells Isolated from the Cortical Peri-Infarct Area after Stroke

Abstract

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells, such as Glial Fibrillary Acidic Protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER™;tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 days after stroke, and cultured them in neural stem cell medium containing EGF and bFGF. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knockout mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 days after transplantation. In contrast with neurogenic post-natal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.

Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions

“…3). As mentioned, the similarities between parenchymal astrocytes and neural stem cells even extend to an intrinsic capacity of parenchymal astrocytes to generate neurons, at least when it comes to striatal astrocytes in vivo (Magnusson et al, 2014;Nato et al, 2015;Duan et al, 2015;Luzzati et al, 2014) and cortical astrocytes when isolated and cultured in vitro (Buffo et al, 2008;Shimada et al, 2012;Sirko et al, 2013). But the relatedness between the two cell types might go even further than that.…”

“…There are cells in the neocortex, including astrocytes, with an intrinsic capacity to generate neurons. This has been demonstrated by the fact that such cells can be isolated from the injured (Buffo et al, 2008;Sirko et al, 2013;Shimada et al, 2012) or even the healthy neocortex of rodents (Palmer et al, 1999;Grande et al, 2013;Sirko et al, 2013) and coaxed into producing neurons in vitro. But this, on its own, does not constitute adult neurogenesis.…”

“…Indeed, like these stem cells, some astrocytes in the brain parenchyma even have an intrinsic ability to generate neurons. Although this ability is dormant, it can be brought out by stimulating the cells with growth factors in vitro (Buffo et al, 2008;Sirko et al, 2013;Shimada et al, 2012). Recently, it has been shown that some parenchymal astrocytes in the striatum can, after certain injuries, also produce neurons in vivo (Magnusson et al, 2014;Nato et al, 2015).…”

Notch1–STAT3–ETB R signaling axis controls reactive astrocyte proliferation after brain injury

“…We isolated reactive astrocytes from peri-infarct tissues by magnetic-activated cell sorting (MACS) with antibodies against glutamate aspartate transporter-1 (GLAST) (20). At 3 d after stroke, FACS phenotyping of GLAST + reactive astrocytes from cortical tissues of C57BL/6J mice demonstrated that cellsurface Jagged1 expression was markedly up-regulated on cells from the ischemic ipsilateral side of the brain compared with cells from the contralateral side ( Fig.…”

“…Although there is a conventional method for isolating and culturing astrocytes from neonatal mice, primary adult cortical reactive astrocytes survive poorly in culture. We recently reported the isolation and expansion of self-renewing, multipotent reactive astrocyte-derived neural stem cells (Rad-NSCs) derived from the cortical peri-infarct area after stroke (20). To avoid possible differences between neonatal and adult astrocytes, we developed methods to study signaling in adult astrocytes based on redifferentiated Rad-NSCs.…”