Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs

Dittmann, Nicole; Torabi, Pouria; Watson, Adrianne Eve Scovil; Yuzwa, Scott A.; Voronova, Anastassia

doi:10.1007/s12015-022-10492-z

Cited by 4 publications

(19 citation statements)

References 115 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells from embryonic cortex or postnatal SVZ were mechanically triturated and cultured at clonal density (10 cells/μl) as described in [ 67 ]. Briefly, cells were cultured in a 6-well polystyrene microplate (08–772-1B; Fisher) for 6 days in vitro (DIV) in 2 ml/well of serum-free media (SFM: equal parts Dulbecco’s modified Eagle medium low glucose (DMEM, 10567–014, (Sigma), 5 mM HEPES (Gibco), 0.1125% sodium bicarbonate (NaHCO3, Gibco), 1% penicillin–streptomycin (Gibco), and 100 μg/mL L-glutamine (Gibco), supplemented with 10 ng/mL FGF (fibroblast growth factor; Peprotech), 2% B27 (Gibco), 20 ng/mL EGF (epidermal growth factor; Peprotech), and 2 μg/mL heparin sodium salt (H3149-50KU; Sigma) [ 38 , 67 , 68 ].…”

Section: Methodsmentioning

confidence: 99%

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Goodkey,

Wischmeijer,

Perrin

et al. 2024

BMC Med

Self Cite

View full text Add to dashboard Cite

ANKRD11 (ankyrin repeat domain 11) is a chromatin regulator and the only gene associated with KBG syndrome, a rare neurodevelopmental disorder. We have previously shown that Ankrd11 regulates murine embryonic cortical neurogenesis. Here, we show a novel olfactory bulb phenotype in a KBG syndrome mouse model and two diagnosed patients. Conditional knockout of Ankrd11 in murine embryonic neural stem cells leads to aberrant postnatal olfactory bulb development and reduced size due to reduction of the olfactory bulb granule cell layer. We further show that the rostral migratory stream has incomplete migration of neuroblasts, reduced cell proliferation as well as aberrant differentiation of neurons. This leads to reduced neuroblasts and neurons in the olfactory bulb granule cell layer. In vitro, Ankrd11-deficient neural stem cells from the postnatal subventricular zone display reduced migration, proliferation, and neurogenesis. Finally, we describe two clinically and molecularly confirmed KBG syndrome patients with anosmia and olfactory bulb and groove hypo-dysgenesis/agenesis. Our report provides evidence that Ankrd11 is a novel regulator of olfactory bulb development and neuroblast migration. Moreover, our study highlights a novel clinical sign of KBG syndrome linked to ANKRD11 perturbations in mice and humans.

show abstract

Section: Methodsmentioning

confidence: 99%

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Goodkey,

Wischmeijer,

Perrin

et al. 2024

BMC Med

Self Cite

View full text Add to dashboard Cite

show abstract

“…We confirmed the loss of Ankrd11 mRNA via qRT-PCR from cultured primary neurospheres, which were generated from cells micro dissected from E15.5 Ankrd11 nscKO and Ankrd11 Contol neocortices 24 hours after TMX injection. This culture protocol allows NPCs to proliferate as neurospheres and does not support the survival of neurons, astrocytes, oligodendrocytes, or microglia (Dittmann et al 2023). Fig.…”

Section: Loss Of Ankrd11 In Npcs Results In Aberrant Postnatal Ob Dev...mentioning

confidence: 99%

“…and 100 μg/mL L-glutamine [Gibco], supplemented with 10 ng/mL FGF (fibroblast growth factor; Peprotech), 2% B27 (Gibco), 20 ng/mL EGF (epidermal growth factor; Peprotech), and 2 μg/mL heparin sodium salt (H3149-50KU; Sigma). Cells were cultured in a 6-well polystyrene microplate (08-772-1B; Fisher) with 2 ml of supplemented SFM per well for 6 days in vitro (DIV) as described in (Storer et al 2018;Gallagher et al 2015;Dittmann et al 2023). Spheres were then centrifuged at 465 g for 7 min at room temperature and the cell pellet was collected for total RNA purification according to manufacturer's instructions (Omega Bio-Tek E.Z.N.A kit).…”

Section: Primary Neurosphere Culturementioning

confidence: 99%

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Goodkey,

Wischmeijer,

Perrin

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

ANKRD11 (Ankyrin Repeat Domain 11) is a chromatin regulator and the only gene associated with KBG syndrome, a rare neurodevelopmental disorder. We have previously shown that Ankrd11 regulates murine embryonic cortical neurogenesis. Here, we show a novel olfactory bulb phenotype in a KBG syndrome mouse model and two diagnosed patients. Conditional knockout of Ankrd11 in murine embryonic neural stem cells leads to aberrant postnatal olfactory bulb development and reduced size. We further show relatively normal formation of olfactory bulb outer neuronal layers with a reduced density of progenitors, neuroblasts, and neurons in the olfactory bulb core. Finally, we demonstrate incomplete migration of neuroblasts along the postnatal rostral migratory stream, which results in a decreased cell density in the olfactory bulb core. We also describe two clinically and molecularly confirmed KBG syndrome patients with anosmia and olfactory bulb and groove hypo-dysgenesis/agenesis. Our report provides evidence that Ankrd11 is a novel regulator of olfactory bulb development and neuroblast migration. Moreover, our study highlights a novel clinical sign of KBG syndrome linked to ANKRD11 perturbations in mice and humans.

show abstract

“…With the wide adoption of single-cell RNA sequencing and "omics" techniques, it was recently shown that both NPCs and OPCs express genes that are typically attributed to immune cells (Falcão et al, 2018;Kirby et al, 2019;Kirby and Castelo-Branco, 2020;Nicaise et al, 2020;Watson et al, 2020;Belenguer et al, 2021;Willis et al, 2022;Dittmann et al, 2023). For example, NPCs and OPCs express MHC (major histocompatibility complex) class I antigen presentation category genes (Falcão et al, 2018;Kirby et al, 2019;Lin et al, 2021;Dittmann et al, 2023). Subsequently, OPCs were shown to present antigens and activate T-cells in rodent models of multiple sclerosis (Falcão et al, 2018;Kirby et al, 2019;Fernandez-Castaneda et al, 2020).…”

Section: "Immune" Roles Of Npcs In Cns Development and Regenerationmentioning

confidence: 99%

“…Dcx + (Doublecortin) neuroblasts, which are derived from NPCs and represent committed proliferative progenitors that are destined to differentiate into neurons, phagocytose dead neurons, whereas OPCs can phagocytose myelin debris and prune synapses; properties typically attributed to microglia and macrophages (Lu et al, 2011;Falcão et al, 2018;Auguste et al, 2022). NPCs and OPCs also directly react to frontiersin.org de Almeida et al 10.3389/fncel.2023.1130205 neuroinflammatory environment through chemokine and cytokine receptors that they express, and actively participate in modulating the inflammatory environment by secreting chemokines and cytokines (Watson et al, 2020(Watson et al, , 2021Belenguer et al, 2021;Shariq et al, 2021;Willis et al, 2022;Dittmann et al, 2023). Finally, an elegant study by Marteyn et al (2016) found that transplanting human NPCs, but not human OPCs, into the brain of Pelizaeus-Merzbacher (hypomyelinating leukodystrophy) mouse model leads to increased survival rates.…”

Section: "Immune" Roles Of Npcs In Cns Development and Regenerationmentioning

confidence: 99%

Regulation of microglia function by neural stem cells

Almeida

Goodkey

Voronova

2023

Front. Cell. Neurosci.

Self Cite

View full text Add to dashboard Cite

Neural stem and precursor cells (NPCs) build and regenerate the central nervous system (CNS) by maintaining their pool (self-renewal) and differentiating into neurons, astrocytes, and oligodendrocytes (multipotency) throughout life. This has inspired research into pro-regenerative therapies that utilize transplantation of exogenous NPCs or recruitment of endogenous adult NPCs for CNS regeneration and repair. Recent advances in single-cell RNA sequencing and other “omics” have revealed that NPCs express not just traditional progenitor-related genes, but also genes involved in immune function. Here, we review how NPCs exert immunomodulatory function by regulating the biology of microglia, immune cells that are present in NPC niches and throughout the CNS. We discuss the role of transplanted and endogenous NPCs in regulating microglia fates, such as survival, proliferation, migration, phagocytosis and activation, in the developing, injured and degenerating CNS. We also provide a literature review on NPC-specific mediators that are responsible for modulating microglia biology. Our review highlights the immunomodulatory properties of NPCs and the significance of these findings in the context of designing pro-regenerative therapies for degenerating and diseased CNS.

show abstract

Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs

Cited by 4 publications

References 115 publications

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Olfactory bulb anomalies in KBG syndrome mouse model and patients

Regulation of microglia function by neural stem cells

Contact Info

Product

Resources

About