Analyzing mouse neural stem cell and progenitor cell proliferation using EdU incorporation and multicolor flow cytometry.

This protocol describes an ex vivo approach to identify and quantify the proportions of proliferating neural stem cells and progenitors of the mouse subventricular zone. It uses ethynyl deoxyuridine (EdU) incorporation to identify dividing cells, combined with multicolor flow cytometry for 4 cell surface antigens to distinguish between 8 phenotypically distinct mouse neural progenitors and stem cells. It has been optimized for wild-type neonatal mice but can be used on mice of any postnatal age. For complete details on the use and execution of this profile, please refer to Kumari et al. (2020).

Developmental IL-6 Exposure Favors Production of PDGF-Responsive Multipotential Progenitors at the Expense of Neural Stem Cells and Other Progenitors.

Interleukin-6 (IL-6) is increased in maternal serum and amniotic fluid of children subsequently diagnosed with autism spectrum disorders. However, it is not clear how increased IL-6 alters brain development. Here, we show that IL-6 increases the prevalence of a specific platelet-derived growth factor (PDGF)-responsive multipotent progenitor, with opposite effects on neural stem cells and on subsets of bipotential glial progenitors. Acutely, increasing circulating IL-6 levels 2-fold above baseline in neonatal mice specifically stimulated the proliferation of a PDGF-responsive multipotential progenitor accompanied by increased phosphorylated STAT3, increased Fbxo15 expression, and decreased Dnmt1 and Tlx expression. Fate mapping studies using a Nestin-CreERT2 driver revealed decreased astrogliogenesis in the frontal cortex. IL-6-treated mice were hyposmic; however, olfactory bulb neuronogenesis was unaffected. Altogether, these studies provide important insights into how inflammation alters neural stem cells and progenitors and provide new insights into the molecular and cellular underpinnings of neurodevelopmental disorders associated with maternal infections.

Mechanisms of mouse neural precursor expansion after neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia (H-I) is the leading cause of brain damage resulting from birth complications. Studies in neonatal rats have shown that H-I acutely expands the numbers of neural precursors (NPs) within the subventricular zone (SVZ). The aim of these studies was to establish which NPs expand after H-I and to determine how leukemia inhibitory factor (LIF) insufficiency affects their response. During recovery from H-I, the number of Ki67(+) cells in the medial SVZ of the injured hemisphere increased. Similarly, the number and size of primary neurospheres produced from the injured SVZ increased approximately twofold versus controls, and, upon differentiation, more than twice as many neurospheres from the damaged brain were tripotential, suggesting an increase in neural stem cells (NSCs). However, multimarker flow cytometry for CD133/LeX/NG2/CD140a combined with EdU incorporation revealed that NSC frequency diminished after H-I, whereas that of two multipotential progenitors and three unique glial-restricted precursors expanded, attributable to changes in their proliferation. By quantitative PCR, interleukin-6, LIF, and CNTF mRNA increased but with significantly different time courses, with LIF expression correlating best with NP expansion. Therefore, we evaluated the NP response to H-I in LIF-haplodeficient mice. Flow cytometry revealed that one subset of multipotential and bipotential intermediate progenitors did not increase after H-I, whereas another subset was amplified. Altogether, our studies demonstrate that neonatal H-I alters the composition of the SVZ and that LIF is a key regulator for a subset of intermediate progenitors that expand during acute recovery from neonatal H-I.

Leukemia inhibitory factor is essential for subventricular zone neural stem cell and progenitor homeostasis as revealed by a novel flow cytometric analysis.

Stem cells rely on extracellular signals produced by the niche, which dictate their ability to self-renew, expand and differentiate. It is essential to have sensitive and reproducible methods of either quantifying or isolating these stem cells and progenitors to understand their intrinsic properties and how extrinsic signals regulate their development. However, stem cells are difficult to distinguish from multipotential progenitors, which may look and act like them. Here we define a 4-color flow cytometry panel using CD133, LeX, CD140a, NG2 to define a neural stem cell (NSC) as well as 4 classes of multipotential progenitors and 3 classes of bipotential progenitors, several of which have not been described previously. We performed gain and loss of function studies for leukemia inhibitory factor (LIF) and showed a depletion of NSCs, a subset of multipotential neural precursors and immature oligodendrocytes in LIF null mice. Gain of function studies showed that LIF increased the abundance of these precursors. Our studies also show that these NPs have differential requirements for LIF and ciliary neurotrophic factor (CNTF) and for epidermal growth factor (EGF), fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) for their propagation in vitro. Surprisingly, the related cytokine, CNTF, was less potent than LIF in increasing the NSCs and more potent than LIF in increasing the PDGF responsive multipotential precursors. Finally, we show that LIF increases the expression of the core transcription factors: Klf4, Fbx15, Nanog, Sox2 and c-Myc. Altogether our FACS (fluorescence-activated cell sorter) analyses reveal that the neonatal subventricular zone is far more heterogeneous than previously suspected and our studies provide new insights into the signals and mechanisms that regulate their self-renewal and proliferation.

Opposite effect of inflammation on subventricular zone versus hippocampal precursors in brain injury.

OBJECTIVE: Inflammation promotes epidermal wound healing but is considered detrimental to recovery from central nervous system injury. Sick infants have increased levels of cytokines in their cerebrospinal fluid that correlate with poor neurological outcome. In this study, we investigated the role of neuroinflammation and more specifically interleukin 6 (IL-6) in the amplification of subventricular zone (SVZ) and subgranular zone (SGZ) neural precursors after neonatal brain injury. METHODS: Neonatal hypoxia/ischemia (H/I) was induced in P6 rat pups, and IL-6 was quantified with or without indomethacin administration. Neural precursor responses were evaluated by neurosphere assays as well as by stereological analyses. Studies were performed to determine how IL-6 and leukemia-inhibiting factor (LIF) affect SVZ cell expansion, proliferation, and self-renewal. RESULTS: Consistent with earlier studies, medially situated SVZ cells expanded after H/I. Contrary to our expectations, indomethacin significantly decreased both the initial reactive increase in these precursors and their ability to self-renew. By contrast, indomethacin increased proliferation in the SGZ and lateral SVZ. Indomethacin diminished the accumulation of microglia/macrophages and IL-6 production after H/I. In vitro IL-6 enhanced neurosphere growth, self-renewal, and tripotentiality and was more effective than LIF in promoting self-renewal. Enhanced precursor self-renewal also was obtained using prostaglandin E2, which is downstream of cyclooxygenase 2 and a target of indomethacin. INTERPRETATION: These data implicate neuroinflammation and in particular IL-6 as a positive effector of primitive neural precursor expansion after neonatal brain injury. These findings have important clinical implications, as indomethacin and other anti-inflammatory agents are administered to premature infants for a variety of reasons.

The canonical Notch/RBP-J signaling pathway controls the balance of cell lineages in mammary epithelium during pregnancy.

Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation.