STEMdiff™星形细胞无血清成熟试剂盒

从人造血干细胞来源的星形胶质细胞前体生成成熟的皮质型星形胶质细胞的无血清成熟试剂盒

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产品号 #（选择产品）

产品号 #100-1666_C

从人造血干细胞来源的星形胶质细胞前体生成成熟的皮质型星形胶质细胞的无血清成熟试剂盒

产品优势

优化，无血清配方一致和稳健的性能
支持高效生成功能成熟的星形胶质细胞
使来自多种人类胚胎干细胞和iPS细胞系的皮质型星形细胞前体可再生成熟

产品组分包括

STEMdiff™星形胶质细胞无血清成熟基础培养基，70 mL
STEMdiff™星形细胞成熟补充剂A， 20ml
STEMdiff™星形胶质细胞无血清成熟补充剂，10ml

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概述

使用STEMdiff™星形细胞无血清成熟试剂盒，快速有效地从星形细胞前体中成熟皮质型星形细胞。与the连用STEMdiff™星形细胞分化试剂盒使用该试剂盒，人类多能干细胞（hPSCs）在成熟为星形胶质细胞之前产生星形细胞前体细胞。使用这种完全无血清的系统获得高度纯净的星形胶质细胞群(平均> 70%的s100b阳性星形胶质细胞和> 60%的gmap阳性星形胶质细胞；< 15%的双皮质素阳性神经元)在短短7周内从人乳头状细胞中分离出来，并选择长期培养。该试剂盒在不同细胞系的标记表达和谷氨酸摄取方面显示出更大的一致性。使用这些产品衍生的细胞是模拟人类神经发育和疾病、药物筛选和毒性测试的通用工具。

Data Figures

Figure 1. Schematic for STEMdiff™ Astrocyte Serum-Free Kit Using the Embryoid Body Protocol for Neural Induction

Astrocyte precursors can be generated from hPSC-derived NPCs in 20 days after selecting neural rosettes from replated embryoid bodies. The resulting cells can then be matured using STEMdiff™ Astrocyte Serum-Free Maturation Kit. Snowflakes indicate optional cryopreservation timepoints. hPSC = human pluripotent stem cell; NPC = neural progenitor cell; EB = embryoid body.

Figure 2. Schematic for STEMdiff™ Astrocyte Serum-Free Kit Using the Monolayer Protocol for Neural Induction

Astrocyte precursors can be generated from hPSC-derived NPC monolayers in 21 days after three single-cell passages. The resulting cells can then be matured using STEMdiff™ Astrocyte Serum-Free Maturation Kit. Snowflakes indicate optional cryopreservation timepoints. hPSC = human pluripotent stem cell; NPC = neural progenitor cell.

Figure 3. Pure Populations of Astrocytes Are Generated Using STEMdiff™ Astrocyte Differentiation Kit and Matured Using STEMdiff™ Astrocyte Serum-Free Maturation Kit or STEMdiff™ Astrocyte Maturation Kit

NPCs were generated from a variety of hPSC cell lines maintained in mTeSR™ Plus using STEMdiff™ SMADi Neural Induction Kit. Using the embryoid body protocol, the NPCs were differentiated using STEMdiff™ Astrocyte Differentiation Kit for 3 weeks and matured using STEMdiff™ Astrocyte Maturation Kit or STEMdiff™ Astrocyte Serum-Free Maturation Kit for an additional 3 weeks. (A) Representative images display astrocytes derived from the iPSC line SCTi003-A. Nuclei are labeled with Hoechst (gray). The resulting cultures contain a highly pure population of astrocytes expressing astrocyte identity marker GFAP (magenta) and mature astrocyte marker S100B (cyan). (B) The percentage expression of GFAP, S100B, and DCX in the resulting cultures, derived from 3 ESC (H1, H7, and H9) and 4 iPSC (WLS-1C, STiPS-R038, STiPS-M001, and SCTi003-A) lines, were quantified after culture in either STEMdiff™ Astrocyte Maturation Kit (‘Serum-Containing’) or STEMdiff™ Astrocyte Serum-Free Maturation Kit (‘Serum-Free’). Each point is a biological replicate (averaged across the technical replicates). Different symbols represent different experiment setups. Numbers are % positive of total Hoechst-positive cells. Normality was determined by Anderson-Darling test and statistics were calculated by the unpaired t-test or Mann-Whitney test. * p ≤ 0.05 and *** p ≤ 0.001. NPC = neural progenitor cell; hPSC = human pluripotent stem cell; ESC = embryonic stem cell; iPSC = induced pluripotent stem cell.

Figure 4. Astrocytes Maintained in STEMdiff™ Astrocyte Serum-Free Maturation Kit and STEMdiff™ Astrocyte Maturation Kit Are Transcriptionally Similar

Bulk RNA-seq data from hPSCs maintained in mTeSR™ Plus; neural progenitor cells, and hPSC-derived forebrain neuron precursors maintained for 7 days in STEMdiff™ Forebrain Neuron Differentiation Kit; hPSC-derived forebrain neurons after 14 days in STEMdiff™ Forebrain Neuron Maturation Kit; hPSC-derived astrocyte precursor cells after 3 weeks in STEMdiff™ Astrocyte Differentiation Kit; hPSC-derived astrocytes matured in STEMdiff™ Astrocyte Maturation Kit or in STEMdiff™ Astrocyte Serum-Free Maturation Kit. Principal component analysis was performed on this data. The astrocytes maintained in both STEMdiff™ Astrocyte Serum-Free Maturation Kit and STEMdiff™ Astrocyte Maturation Kit cluster together, demonstrating the similar global gene expression patterns between the resulting astrocytes after maturation with either of these two kits when generated from various hPSC cell lines. The spread of cell types along the PC2 y-axis and overlap of the astrocyte precursor cell group with the forebrain neuron group demonstrates that the precursor cells pass through a neurogenic fate before reaching their gliogenic gate. The top genes for the PC2 top loading are GFAP, COL14A1, and CD44. hPSCs = human pluripotent stem cells, NPCs = neural progenitor cells.

Figure 5. Astrocytes Generated with STEMdiff™ Astrocyte Kits Modulate Internal Calcium Concentration After ATP and Glutamate Treatments

Astrocytes generated from iPSC lines WLS-1C and SCTi003-A were matured in either STEMdiff™ Astrocyte Maturation Kit or STEMdiff™ Astrocyte Serum-Free Maturation Kit. The astrocytes were kept in BrainPhys™ Imaging Optimized Neuronal Medium to reduce the background fluorescence in the images and loaded with the fluorescent calcium indicator Fluo-4AM. (A) The WLS-1C-derived astrocytes were then exposed to 3 µM ATP and (B) the SCTi003-A-derived astrocytes were exposed to 25 µM glutamate. The calcium response to the treatments were captured for 1 or 2 minutes total at 500 ms intervals. Representative time-lapse image series shows the calcium response at various pre- and post-treatment timepoints. Orange arrows identify an astrocyte that increases in fluorescence after treatment and the blue arrows identify an astrocyte wherein calcium fluctuated from increased to decreased within the representative time series. iPSC = induced pluripotent stem cell.

Figure 6. hPSC-Derived Neurons, Astrocytes, and Microglia Can Be Tri-Cultured to Model Cell-Cell Interactions In Vitro

Astrocyte precursor cells from the ESC line H7 were maintained in STEMdiff™ Astrocyte Serum-Free Maturation Kit for 3 weeks and passaged weekly. Hematopoietic progenitor cells from the iPSC line WLS-1C were thawed into STEMdiff™ Microglia Differentiation Kit for 24 days. Forebrain neuron precursors from the iPSC line STiPS-R038 were thawed and maintained in STEMdiff™ Forebrain Neuron Maturation Kit for 1 week. After the neurons were matured for 1 week, the astrocytes were passaged on top of the neurons at a 1:1 seeding density and maintained overnight in the STEMdiff™ Astrocyte Serum-Free Maturation Kit. The next day, 24-day-old microglia were plated on top of the neuron and astrocyte co-culture in the tri-culture medium. For more detailed instructions on the tri-culture protocol, please refer to the full protocol. (A) 30-day-old tri-culture composed of (B) hPSC-derived class III β-tubulin-positive forebrain neurons, (C) GFAP-positive astrocytes, and (D) IBA1-positive microglia. (E) Nuclei are labeled with Hoechst. The microglia extend their processes to interact with the neurons and astrocytes. ESC = embryonic stem cell; iPSC = induced pluripotent stem cell.

Figure 7. Tri-Culture Containing hPSC-Derived Neurons, Astrocytes, and Microglia Recovers from Wound Injury

Astrocyte precursor cells from the ESC line H7 were maintained in STEMdiff™ Astrocyte Serum-Free Maturation Kit for 3 weeks and passaged weekly. Hematopoietic progenitor cells from the iPSC line WLS-1C containing a stable transfection to express GFP were thawed into STEMdiff™ Microglia Differentiation Kit for 24 days. Forebrain neuron precursors from the iPSC line STiPS-R038 were thawed and maintained in STEMdiff™ Forebrain Neuron Maturation Kit for 1 week. After the neurons were matured for 1 week, the astrocytes were passaged on top of the neurons at a 1:1 seeding density and maintained overnight in the STEMdiff™ Astrocyte Serum-Free Maturation Kit. The next day, 24-day-old microglia were plated on top of the neuron and astrocyte co-culture in the tri-culture medium. For more detailed instructions on the tri-culture protocol, please refer to the full protocol. On Day 7, the culture was scratched with the Incucyte® WoundMaker and the recovery process was captured every 12 hours with the Incucyte® SX5 instrument. (A) The culture was fixed and stained for GFAP (cyan) and βIII-TUB (magenta) after 48 hours of injury. (B) Representative phase images of the same area (see insets) within the wound pre- and post-scratch with the hours post-injury labelled on-top of the images are shown. The GFP-expressing microglia (green) are seen interacting with a cluster of forebrain neurons labelled with black arrows. The same cluster of forebrain neurons labelled with the black arrows expresses βIII-TUB and displays neurite re-growth (white arrow). ESC = embryonic stem cell; iPSC = induced pluripotent stem cell; GFP = green fluorescent protein.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.