Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro

Nadadhur, Aishwarya G.; Leferink, Prisca S.; Holmes, Dwayne; Hinz, Lisa D.; Cornelissen-Steijger, Paulien; Gasparotto, Lisa; Heine, Vivi M.

doi:10.1016/j.scr.2018.08.017

Cited by 31 publications

(37 citation statements)

References 72 publications

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“…15 Primary antibodies targeted glial fibrillary acidic protein (GFAP; Sigma, St Louis, MO; G3893, 1:1,000), GFAP (Dako, Carpinteria, CA; Z0334, 1:1,000), nestin (BD Biosciences, Franklin Lakes, NJ; 611658, 1:500), S100β (Proteintech Group, Rosemont, IL; 15146-1-AP, 1:1,000), Olig2 ( Bromodeoxyuridine (BrdU) assay was performed (2 independent differentiations of 4 human astrocyte lines per genotype) by 2-hour incubation of BrdU at 37 C and stained with anti-BrdU antibody (Abcam) according to the manufacturer's protocol. 15 Primary antibodies targeted glial fibrillary acidic protein (GFAP; Sigma, St Louis, MO; G3893, 1:1,000), GFAP (Dako, Carpinteria, CA; Z0334, 1:1,000), nestin (BD Biosciences, Franklin Lakes, NJ; 611658, 1:500), S100β (Proteintech Group, Rosemont, IL; 15146-1-AP, 1:1,000), Olig2 ( Bromodeoxyuridine (BrdU) assay was performed (2 independent differentiations of 4 human astrocyte lines per genotype) by 2-hour incubation of BrdU at 37 C and stained with anti-BrdU antibody (Abcam) according to the manufacturer's protocol.…”

Section: Discussionmentioning

confidence: 99%

“…15 Primary antibodies targeted glial fibrillary acidic protein (GFAP; Sigma, St Louis, MO; G3893, 1:1,000), GFAP (Dako, Carpinteria, CA; Z0334, 1:1,000), nestin (BD Biosciences, Franklin Lakes, NJ; 611658, 1:500), S100β (Proteintech Group, Rosemont, IL; 15146-1-AP, 1:1,000), Olig2 ( Bromodeoxyuridine (BrdU) assay was performed (2 independent differentiations of 4 human astrocyte lines per genotype) by 2-hour incubation of BrdU at 37 C and stained with anti-BrdU antibody (Abcam) according to the manufacturer's protocol. 15 Calcium imaging of glutamate uptake in human astrocytes was performed and analyzed as described previously. RNA isolation, cDNA synthesis, and PCR analysis were performed as described previously (see Supplementary Table 1 for primer sequences).…”

Section: Discussionmentioning

confidence: 99%

“…RNA was collected with TRIzol (Invitrogen, Carlsbad, CA). 15 For reactivity assay, human astrocytes were treated with either 10μg/ml polyinosinic:polycytidylic acid (Tocris Bioscience, Bristol, UK) or dH 2 O for 21 hours, after which RNA was isolated and analyzed as described previously. 15 Calcium imaging of glutamate uptake in human astrocytes was performed and analyzed as described previously.…”

Section: Discussionmentioning

confidence: 99%

“…14 Cells were maintained and characterized as previously described, 15 by alkaline phosphatase staining, immunocytochemistry, real-time polymerase chain reaction (RT-PCR) analysis, RNAseq PluriTest, and germ layer embryoid body (EB) differentiation assay, and karyotyped or tested on the Infinium Global Screening Array for DNA abnormalities. 14 Cells were maintained and characterized as previously described, 15 by alkaline phosphatase staining, immunocytochemistry, real-time polymerase chain reaction (RT-PCR) analysis, RNAseq PluriTest, and germ layer embryoid body (EB) differentiation assay, and karyotyped or tested on the Infinium Global Screening Array for DNA abnormalities.…”

Section: Materials and Methods Hipsc Differentiation Toward Astrocytesmentioning

confidence: 99%

“…15 We characterized the human astrocytes for expression of the astrocyte-associated markers GFAP, nestin, Id3, CD44, and SOX9 by immunocytochemistry (Fig 3), and NESTIN, BLBP, S100β, ALDOC, AQP4, GLAST, GFAP, and CD44 by RT-PCR (data not shown). To generate astrocytes, we differentiated control and VWM hiPSCs toward astrocytes according to protocols described earlier.…”

Section: Vwm Patient Ipsc-based Models Confirm Intrinsic Defects In Vmentioning

confidence: 99%

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Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

Leferink

Dooves

Hillen

et al. 2019

Annals of Neurology

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Objective: Astrocytes have gained attention as important players in neurological disease. In line with their heterogeneous character, defects in specific astrocyte subtypes have been identified. Leukodystrophy vanishing white matter (VWM) shows selective vulnerability in white matter astrocytes, but the underlying mechanisms remain unclear. Induced pluripotent stem cell technology is being extensively explored in studies of pathophysiology and regenerative medicine. However, models for distinct astrocyte subtypes for VWM are lacking, thereby hampering identification of disease-specific pathways. Methods: Here, we characterize human and mouse pluripotent stem cell-derived gray and white matter astrocyte subtypes to generate an in vitro VWM model. We examined morphology and functionality, and used coculture methods, high-content microscopy, and RNA sequencing to study VWM cultures. Results: We found intrinsic vulnerability in specific astrocyte subpopulations in VWM. When comparing VWM and control cultures, white matter-like astrocytes inhibited oligodendrocyte maturation, and showed affected pathways in both human and mouse cultures, involving the immune system and extracellular matrix. Interestingly, human white matter-like astrocytes presented additional, human-specific disease mechanisms, such as neuronal and mitochondrial functioning. Interpretation: Astrocyte subtype cultures revealed disease-specific pathways in VWM. Cross-validation of human-and mouse-derived protocols identified human-specific disease aspects. This study provides new insights into VWM disease mechanisms, which helps the development of in vivo regenerative applications, and we further present strategies to study astrocyte subtype vulnerability in neurological disease.

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Section: Discussionmentioning

confidence: 99%

“…15 Primary antibodies targeted glial fibrillary acidic protein (GFAP; Sigma, St Louis, MO; G3893, 1:1,000), GFAP (Dako, Carpinteria, CA; Z0334, 1:1,000), nestin (BD Biosciences, Franklin Lakes, NJ; 611658, 1:500), S100β (Proteintech Group, Rosemont, IL; 15146-1-AP, 1:1,000), Olig2 ( Bromodeoxyuridine (BrdU) assay was performed (2 independent differentiations of 4 human astrocyte lines per genotype) by 2-hour incubation of BrdU at 37 C and stained with anti-BrdU antibody (Abcam) according to the manufacturer's protocol. 15 Calcium imaging of glutamate uptake in human astrocytes was performed and analyzed as described previously. RNA isolation, cDNA synthesis, and PCR analysis were performed as described previously (see Supplementary Table 1 for primer sequences).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Materials and Methods Hipsc Differentiation Toward Astrocytesmentioning

confidence: 99%

Section: Vwm Patient Ipsc-based Models Confirm Intrinsic Defects In Vmentioning

confidence: 99%

See 3 more Smart Citations

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

Leferink

Dooves

Hillen

et al. 2019

Annals of Neurology

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Altered lipid profile and reduced neuronal support in human induced pluripotent stem cell‐derived astrocytes from adrenoleukodystrophy patients

Ferrer,

Jaspers,

Dijkstra

et al. 2024

J of Inher Metab Disea

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X‐linked adrenoleukodystrophy (ALD) is a peroxisomal disorder resulting from pathogenic variants in the ABCD1 gene that primarily affects the nervous system and is characterized by progressive axonal degeneration in the spinal cord and peripheral nerves and leukodystrophy. Dysfunction of peroxisomal very long‐chain fatty acid (VLCFA) degradation has been implicated in ALD pathology, but the impact on astrocytes, which critically support neuronal function, remains poorly understood. Fibroblasts from four ALD patients were reprogrammed to generate human‐induced pluripotent stem cells (hiPSC). hiPSC‐derived astrocytes were generated to study the impact of ALD on astrocytic fatty acid homeostasis. Our study reveals significant changes in the lipidome of ALD hiPSC‐derived astrocytes, characterized by an enrichment of VLCFAs across multiple lipid classes, including triacylglycerols, cholesteryl esters, and phosphatidylcholines. Importantly, ALD hiPSC‐derived astrocytes not only exhibit intrinsic lipid dysregulation but also affect the dendritic tree complexity of neurons in co‐culture systems. These findings highlight the cell‐autonomous effects of pathogenic variants in the ABCD1 protein on astrocytes and their microenvironment, shed light on potential mechanisms underlying ALD neuropathology, and underscore the critical role of astrocytes in neuronal health.

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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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Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro

Cited by 31 publications

References 72 publications

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

Altered lipid profile and reduced neuronal support in human induced pluripotent stem cell‐derived astrocytes from adrenoleukodystrophy patients

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

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