Fast and Efficient Differentiation of Mouse Embryonic Stem Cells Into ATP-Responsive Astrocytes

Abstract

Astrocytes are multifunctional cells in the CNS, involved in the regulation of neurovascular coupling, the modulation of electrolytes, and the cycling of neurotransmitters at synapses. Induction of astrocytes from stem cells remains a largely underdeveloped area, as current protocols are time consuming, lack granularity in astrocytic subtype generation, and often are not as efficient as neural induction methods. In this paper we present an efficient method to differentiate astrocytes from mouse embryonic stem cells. Our technique uses a cell suspension protocol to produce embryoid bodies (EBs) that are neurally inducted and seeded onto laminin coated surfaces. Plated EBs attach to the surface and release migrating cells to their surrounding environment, which are further inducted into the astrocytic lineage, through an optimized, heparin-based media. Characterization and functional assessment of the cells consists of immunofluorescent labeling for specific astrocytic proteins and sensitivity to adenosine triphosphate (ATP) stimulation. Our experimental results show that even at the earliest stages of the protocol, cells are positive for astrocytic markers (GFAP, ALDH1L1, S100β, and GLAST) with variant expression patterns and purinergic receptors (P2Y). Generated astrocytes also exhibit differential Ca 2+ transients upon stimulation with ATP, which evolve over the differentiation period. Metabotropic purinoceptors P2Y 1 R are expressed and we offer preliminary evidence that metabotropic purinoceptors contribute to Ca 2+ transients. Our protocol is simple, efficient and fast, facilitating its use in multiple investigations, particularly in vitro studies of engineered neural networks.

Generation of Multipotential NG2 Progenitors From Mouse Embryonic Stem Cell-Derived Neural Stem Cells

“…Following this treatment, NSC converted to flattened GFAP + cells similar to those observed in primary cultures of mouse astrocytes ( Schildge et al, 2013 ; Figure 7A ). In contrast, NOP differentiated into GFAP + cells displaying a mix of morphologies including flattened GFAP + cells, and cells with multiple thickened GFAP + processes typical of ESC derived astrocytes ( Juneja et al, 2020 ; Figure 7B ). Consistent with previous reports ( Suzuki et al, 2017 ), we also detected GFAP + cells when cultures of pOPC were treated with BMP4 and LIF (data not shown).…”

“…The NOP/OL population downregulates these NSC genes during differentiation in OL medium; thus, it would be interesting to compare their astroglial and neuronal potential with the more immature NOP cells. Regarding the identify of NOP-derived GFAP+ cells, BMP4 stimulation, as used here, is recognized to drive the differentiation of NPC toward an astroglial fate ( Gross et al, 1996 ); thus, the GFAP+ cells, which had morphologies expected of cultured mouse astrocytes ( Schildge et al, 2013 ; Juneja et al, 2020 ), are likely to represent astroglial lineage cells. Nevertheless, GFAP is also expressed by NPC/radial glia populations; thus, in the absence of other lineage markers, we term these cells astroglial-like.…”

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

“…When the exogenous BRN4 remains expressed, the astrocyte differentiation is severely compromised. Astrocytes can be induced from mESCs by embryoid body (EB) formation followed by retinoic acid treatment with similar efficiency to our SrC switch [ 66 ]. Although the current version of the SrC switch using BRN4 does not necessarily improve the astrocyte differentiation system, the SrC switch is easily adaptable for further improvement of astrocyte induction; for example, by manipulating the expression pattern of BRN4, by altering BRN4 functions via mutagenesis, or by using an alternate transcription factor.…”

Localisation of oestrogen receptors in stem cells and in stem cell‐derived neurons of the mouse

“…39 In this study, we committed mouse embryonic stem cells to neural lineages, by fine tuning a mass suspension protocol by Wichterle and Peljto 40 to generate embryoid bodies (EBs) within 5 days. This fast and efficient adaptation has produced both astrocytes 41 and neurons. 42,43 Hence, the objectives of this study were to examine the subcellular localisation and colocalisation of endogenously expressed ERα, GPER-1 and ERα-36 in neurons differentiated from pluripotent mouse stem cells.…”