Identification of the gliogenic state of human neural stem cells to optimize in vitro astrocyte differentiation

Alisch, Marlen; Kerkering, Janis; Crowley, Tadhg; Rosiewicz, Kamil Sebastian; Paul, Friedemann; Siffrin, Volker

doi:10.1016/j.jneumeth.2021.109284

Cited by 7 publications

(9 citation statements)

References 57 publications

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“…2d). Finally, a recent study also corroborates our findings, Alisch et al documented that fetal neural stem cells increase their gliogenic potential by dampening neurogenic genes during successive cell division [ 43 ]. Hence, NES increase their gliogenic potential at higher passages, similarly to what has been shown for fetal neural stem cells.…”

Section: Resultssupporting

confidence: 92%

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

Voulgaris

Nikolakopoulou

Herland

2022

Stem Cell Rev and Rep

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The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine. Graphical abstract

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Section: Resultssupporting

confidence: 92%

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

Voulgaris

Nikolakopoulou

Herland

2022

Stem Cell Rev and Rep

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“…In accordance with the data of Alisch et al 55 differentiation of NSCs with increasing passage number resulted in higher percentages of NG2 positive cells (Fig. 3 C).…”

Section: Resultssupporting

confidence: 92%

“…It has been previously reported that NSCs of higher passage number have a greater tendency in fate choice towards the glial lineage compared to NSCs of lower passages 55 . This allows for the rapid generation of glial progenitors in shorter time spans and the establishment of models optimized for the investigation of glial biology.…”

Section: Resultsmentioning

confidence: 94%

Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation

Campo Garcia,

Bueno,

Salla

et al. 2024

Sci Rep

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During neuroinflammation, monocytes that infiltrate the central nervous system (CNS) may contribute to regenerative processes depending on their activation status. However, the extent and mechanisms of monocyte-induced CNS repair in patients with neuroinflammatory diseases remain largely unknown, partly due to the lack of a fully human assay platform that can recapitulate monocyte-neural stem cell interactions within the CNS microenvironment. We therefore developed a human model system to assess the impact of monocytic factors on neural stem cells, establishing a high-content compatible assay for screening monocyte-induced neural stem cell proliferation and differentiation. The model combined monocytes isolated from healthy donors and human embryonic stem cell derived neural stem cells and integrated both cell-intrinsic and -extrinsic properties. We identified CNS-mimicking culture media options that induced a monocytic phenotype resembling CNS infiltrating monocytes, while allowing adequate monocyte survival. Monocyte-induced proliferation, gliogenic fate and neurogenic fate of neural stem cells were affected by the conditions of monocytic priming and basal neural stem cell culture as extrinsic factors as well as the neural stem cell passage number as an intrinsic neural stem cell property. We developed a high-content compatible human in vitro assay for the integrated analysis of monocyte-derived factors on CNS repair.

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“…First, we analyzed astrocytes' ability to react to hypoxia. We used human embryonal stem cell (ESC; H9)‐derived induced (H9 i ) astrocytes (Alisch et al, 2021) and mouse wild type (wt) astrocytes. These cultures were generated in media containing fetal cattal serum (FCS), which was removed 3 days before starting the assays to minimize activating effects REF.…”

Section: Resultsmentioning

confidence: 99%

“…H9 cells were purchased from Gibco and were finally differentiated to astrocytes (H9 i astrocytes) by using the methods published by (Alisch et al, 2021). For experiments, previously differentiated and frozen H9 i astrocytes were thawed and plated on Geltrex coated plates and cultivated in ADM medium (DMEM, 1× GlutaMAX, 1% FCS, 20 ng/mL CNTF (Miltenyi Biotec), 1× N2 Supplement (gibco), 1% Pen/Strep).…”

Section: Methodsmentioning

confidence: 99%

HIF prolyl hydroxylase 2/3 deletion disrupts astrocytic integrity and exacerbates neuroinflammation

Rosiewicz

Muinjonov

Kunz

et al. 2023

Glia

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Astrocytes constitute the parenchymal border of the blood-brain barrier (BBB), modulate the exchange of soluble and cellular elements, and are essential for neuronal metabolic support. Thus, astrocytes critically influence neuronal network integrity. In hypoxia, astrocytes upregulate a transcriptional program that has been shown to boost neuroprotection in several models of neurological diseases. We investigated transgenic mice with astrocyte-specific activation of the hypoxia-response program by deleting the oxygen sensors, HIF prolyl-hydroxylase domains 2 and 3 (Phd2/3).We induced astrocytic Phd2/3 deletion after onset of clinical signs in experimental autoimmune encephalomyelitis (EAE) that led to an exacerbation of the disease mediated by massive immune cell infiltration. We found that Phd2/3-ko astrocytes, though expressing a neuroprotective signature, exhibited a gradual loss of gapjunctional Connexin-43 (Cx43), which was induced by vascular endothelial growth factor-alpha (Vegf-a) expression. These results provide mechanistic insights into astrocyte biology, their critical role in hypoxic states, and in chronic inflammatory CNS diseases.

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Identification of the gliogenic state of human neural stem cells to optimize in vitro astrocyte differentiation

Cited by 7 publications

References 57 publications

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation

HIF prolyl hydroxylase 2/3 deletion disrupts astrocytic integrity and exacerbates neuroinflammation

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