STEMdiff™ Astrocyte Maturation Kit is used to rapidly and efficiently generate cortical-type astrocytes from astrocytic precursors derived from human pluripotent stem cells (hPSCs) using the STEMdiff™ Astrocyte Differentiation Kit (Catalog #100-0016). Using this system, a highly pure population of astrocytes (an average of > 70% S100B-positive and > 60% GFAP-positive astrocytes; < 15% doublecortin-positive neurons) can be generated from hPSCs in as few as 7 weeks and can be maintained long term in culture. Cells derived using these products are versatile tools for modeling human neurological development and disease, drug screening, toxicity testing, and cell therapy validation.
Area of Interest Disease Modeling, Drug Discovery and Toxicity Testing, Neuroscience
Data Figures
Figure 1. Schematic for the Embryoid Body Protocol
Cortical-type astrocytes can be generated from astrocyte precursors after 20 days in STEMdiff™ Astrocyte Differentiation Medium. For differentiation of precursors from embryonic and induced pluripotent stem cells, see the PIS.
Figure 2. Schematic for the Monolayer Protocol
Cortical-type astrocytes can be generated from astrocyte precursors after 21 days in STEMdiff™ Astrocyte Differentiation Medium. For differentiation of precursors from embryonic and induced pluripotent stem cells, see the PIS.
Figure 3. Cortical-Type Astrocytes Are Generated After Culture in STEMdiff™ Astrocyte Differentiation and Maturation Kits
NPCs generated from hPSCs in TeSR™-E8™ using the STEMdiff™ SMADi Neural Induction Kit embryoid body (EB) protocol were differentiated and matured to cortical-type astrocytes using the STEMdiff™ Astrocyte Differentiation and Maturation Kits. Cortical-type astrocytes were formed after iPS cell-derived NPCs were cultured with the STEMdiff™ Astrocyte Differentiation Kit for 3 weeks and STEMdiff™ Astrocyte Maturation Kit for 3 weeks. (A) Nuclei are labeled with DAPI (gray). The resulting cultures contain a highly pure population of astrocytes, which are (B) more than 60% GFAP-positive (green) and (C) more than 70% S100B-positive (magenta), with (D) fewer than 15% neurons (DCX-positive cells, cyan). Scale bar = 100 μm.
Figure 4. STEMdiff™ Astrocyte Kits Generate Cells Expressing Expected Levels of Genes Characteristic for Astrocytes
Embryonic stem and induced pluripotent stem cells from a variety of lines (n = 6, maintained in mTeSR™1 or TeSR™-E8™) were differentiated to NPCs using the STEMdiff™ SMADi Neural Induction Kit embryoid body protocol. Cells were then grown in STEMdiff™ Astrocyte Differentiation Kit for 3 weeks followed by STEMdiff™ Astrocyte Maturation Kit for 3 weeks prior to analysis. Expression levels were measured by quantitative PCR (qPCR) and normalized to hPSC controls relative to housekeeping genes 18S and TBP.
Figure 5. PSC-Derived Astrocytes and Neurons Can Be Co-Cultured to Model Cell-Cell Interactions In Vitro
NPCs generated from the H1 cell line were differentiated to astrocytes using STEMdiff™ Astrocyte Differentiation and Maturation Kits. H9 cell-derived NPCs were differentiated to forebrain-type neurons using STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits. For co-culture, matured astrocytes were seeded onto forebrain neurons that had been in STEMdiff™ Forebrain Neuron Maturation Medium for at least one week. Co-cultures were then switched to STEMdiff™ Forebrain Neuron Maturation Medium the following day and for the remaining co-culture. (A) Neurons cultured alone, following the co-culture feeding schedule, are labeled with DCX (green). (B) DCX-positive neurons (green) and astrocytes (GFAP, red) can be co-cultured for at least 1 - 2 weeks prior to analysis. For a detailed co-culture protocol, please see the Methods Library.
Figure 6. PSC-Derived Neurons Survive and Mature when Co-Cultured with PSC-Derived Astrocytes
NPCs generated from the STiPS-R038 cell line were differentiated to astrocytes using STEMdiff™ Astrocyte Differentiation and Maturation Kits. STiPS-M001 cell-derived NPCs were differentiated to forebrain-type neurons using STEMdiff™ Forebrain Neuron Differentiation and Maturation Kits. After co-culture for one week, neurons (A) had significantly increased neurite outgrowth as measured on MAP2-positive neurons with the NeuriteTracer plugin for ImageJ (M Pool et al. J Neurosci Methods, 2008) and (B) were more numerous than neurons cultured alone using the same feeding schedule. *, p < 0.05; **, p < 0.01.
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