STEMdiff™运动神经元分化试剂盒

用于从hPSCs生成运动神经元的分化试剂盒

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产品号 #100-0871_C

用于从hPSCs生成运动神经元的分化试剂盒

产品优势

14天内从人类诱导多能干细胞（iPSCs）分化为运动神经元
简化运动神经元培养过程，提供简单且可扩展的工作流程
使用集成的 BrainPhys™ 神经元培养基，支持神经元活动和成熟，产生生理相关的结果
通过与兼容的分化试剂盒配对用于共培养应用，模拟细胞间相互作用的复杂性

产品组分包括

STEMdiff™ 运动神经元分化基础培养基，100 mL
STEMdiff™ 运动神经元分化补充剂 A，120 μL
STEMdiff™ 运动神经元分化补充剂 B，200 μL
STEMdiff™ 运动神经元分化补充剂 C，120 μL

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总览

STEMdiff™ 运动神经元分化试剂盒基于 Maury 等人（Nature Biotechnology, 2015）已建立的方案，您可以生成高纯度的脊髓运动神经元群，其中平均 III 类 β-微管蛋白阳性率 >80%，运动神经元标志物 ISL1 阳性率 >50%，运动神经元标志物 HB9 阳性率 >60%。这些运动神经元还高表达HOXA5 。所得运动神经元培养物可用于多种应用，例如模拟人类神经系统疾病或毒性测试。这些用途广泛的培养物适用于与使用MyoCult™ 分化试剂盒（货号 #05965）生成的肌管进行神经肌肉共培养，与使用STEMdiff™ 小胶质细胞分化试剂盒（货号 #100-0019）生成的小胶质细胞共培养，或与AggreWell™400（货号 #34460）共培养以提高产量。

为了使该试剂盒生成的运动神经元进一步成熟，可与STEMdiff™ 运动神经元成熟试剂盒配对使用，以在额外的 14 天内产生 >40% 的 ChAT 细胞。

Data Figures

Experimental Protocol for STEMdiff™ Motor Neuron Differentiation and Maturation Kits.

Figure 1. Schematic for the STEMdiff™ Motor Neuron Culture System Protocol

Motor neurons can be generated from human pluripotent stem cells (hPSCs) in 14 days. The resulting cells can then be matured for an additional 14 or more days using STEMdiff™ Motor Neuron Maturation Kit.

Motor Neuron Progenitors With >60% OLIG2+ Expression Created Using the STEMdiff™ Motor Neuron Differentiation Kit

Figure 2. Pure Population of Motor Neuron Progenitors Are Generated Using the STEMdiff™ Motor Neuron Differentiation Kit

hPSCs from a variety of cell lines were differentiated to motor neuron progenitors using the STEMdiff™ Motor Neuron Differentiation Kit in an ultra-low attachment or an AggreWell™400 plate. (A) hPSCs maintained in mTeSR™1 or mTeSR™ Plus were aggregated to EBs with the STEMdiff™ Motor Neuron Differentiation Kit. On Day 9, EBs were dissociated into single cells and replated for adherent culture.The resulting cultures contain a population of cells expressing motor neuron progenitor marker OLIG2 (red) and are negative for spinal interneuron marker NKX2.2 (green). Nuclei are labeled with Hoechst (blue). (B) The percentage expression of OLIG2 and negative controls NKX2.2, FOXG1, OTX1, and OTX2 in the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (WLS-1C and STiPS-M001) cell lines, were quantified. This differentiation generated OLIG2+ motor neuron progenitors (83.20% ± 2.890%, mean ± SEM; n=4 cell lines, 2 replicates per condition). Numbers are % positive of total Hoechst-positive cells. hPSCs = human pluripotent stem cells; EB = embryoid body

Post-Mitotic Motor Neurons Exhibit βIII-TUB, HB9 & ISL1 Expression Generated Using STEMdiff™ Motor Neuron Differentiation Kit

Figure 3. Pure Population of Post-Mitotic Motor Neurons Are Generated Using the STEMdiff™ Motor Neuron Differentiation Kit

Motor neuron progenitors derived from a variety of cell lines were transitioned to post-mitotic motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit. (A) Post-mitotic motor neurons were generated by Day 14 after hPSCs were differentiated with the STEMdiff™ Motor Neuron Differentiation Kit. The resulting cultures contain a population of cells expressing neuronal identity marker βIII-TUB (green), mature motor neuron marker HB9 (red), and (B) ISL1 (red). Nuclei are labeled with Hoechst (blue). (C) The percentage expression of βIII-TUB, ISL1, and HB9 in the resulting cultures, derived from 3 hES (H1, H7, and H9) and 3 hiPS (WLS-1C, STiPS-M001, and STiPS-R038) cell lines, were quantified. This differentiation generated βIII-TUB+ (92.59% ± 1.079%, mean ± SEM; n=6 cell lines, 2 replicates per condition), ISL1+ (55.99% ± 3.723%, mean ± SEM), and HB9+ (65.17% ± 3.514%, mean ± SEM) motor neurons. Numbers are % positive of total Hoechst-positive cells. hPSC = human pluripotent stem cell

STEMdiff™ Motor Neuron System Generates Motor Neurons Patterned to the Cervical Axis Expressing HOXA5

Figure 4. STEMdiff™ Motor Neuron System Generates Motor Neurons Patterned to the Cervical Axis

hPSCs from a variety of cell lines were differentiated to motor neuron progenitors using the STEMdiff™ Motor Neuron Differentiation Kit. Expression levels of the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (STiPS-M001 and STiPS-R038) cell lines, were measured by qPCR and hPSC-derived forebrain progenitor cells were used as a control. Analysis of Day 9 motor neuron progenitors showed high expression of cervical AP axis HOXA5 (n = 4) relative to hPSC. hPSCs = human pluripotent stem cells; qPCR = quantitative polymerase chain reaction; AP = anteroposterior

hPSC-Derived Motor Neurons Matured in STEMdiff™ Motor Neuron Maturation Medium Express Expected Markers

Figure 5. hPSC-Derived Motor Neurons Can Be Matured in STEMdiff™ Motor Neuron Maturation Medium

Motor neuron progenitors derived from a variety of cell lines were matured using the STEMdiff™ Motor Neuron Maturation Kit. (A) Mature motor neurons were generated after hPSCs were cultured with the STEMdiff™ Motor Neuron Differentiation Kit for 14 days and then the STEMdiff™ Motor Neuron Maturation Kit for an additional 14 days. The resulting cultures contain a population of cells expressing neuronal identity marker βIII-TUB (green), mature motor neuron markers HB9 (red), (B) SYNAPSIN (red), and MAP2 (green), as well as (C) cholinergic neuron marker ChAT (green). Nuclei are labeled with Hoechst (blue). (D) The percentage expression of ChAT, HB9, and βIII-TUB in the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (STiPS-R038 and STiPS-M001) cell lines, were quantified. This differentiation generated ChAT+ (65.16% ± 3.737%, mean ± SEM; n=4 cell lines, 2 replicates per condition), HB9+ (79.58% ± 2.570%, mean ± SEM), and βIII-TUB+ (86.56% ± 2.331%, mean ± SEM) motor neurons. Numbers are % positive of total Hoechst-positive cells. hPSC = human pluripotent stem cell

qPCR Data Shows hPSC-Derived Motor Neurons Matured in STEMdiff™ Motor Neuron Maturation Medium Produce Cholinergic Neurons

Figure 6. hPSC-Derived Motor Neurons Matured in STEMdiff™ Motor Neuron Maturation Medium Produces Cholinergic Neurons

hPSCs from a variety of cell lines were differentiated to motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit for 14 days and then the STEMdiff™ Motor Neuron Maturation Kit for an additional 14 days prior to analysis. Expression levels of the resulting cultures, derived from 3 hES (H1, H7, and H9) and 3 hiPS (WLS-1C, STiPS-M001, and STiPS-R038) cell lines, were measured by qPCR and hPSC-derived forebrain neurons were used as a control. Analysis of Day 28 mature motor neurons showed higher expression of ChAT (n = 5, P<.0001, Two-way ANOVA with Sidak's multiple comparisons test). hPSCs = human pluripotent stem cells; qPCR = quantitative polymerase chain reaction; ChAT = choline acetyltransferase

Imaging Showing hPSC-Derived Motor Neurons Co-Cultured with hPSC-Derived Myotubes for In Vitro Modeling

Figure 7. hPSC-Derived Motor Neurons Can Be Co-Cultured with hPSC-Derived Myotubes for In Vitro Modeling

hPSCs were differentiated to motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit and Maturation Kit. hPSC-derived myogenic progenitors were generated using the STEMdiff™ Myogenic Progenitor Kit and then induced to differentiate into myotubes using MyoCult™ Differentiation Medium. The resulting co-culture contains a population of cells expressing neuronal identity marker βIII-TUB (red), mature motor neuron marker ISL1 (blue), and AChR (green). Arrows indicate neuromuscular junctions. hPSC = human pluripotent stem cell; AChR = acetylcholine receptor.

Bulk RNA-seq heatmap showing that STEMdiff™ Motor Neurons express expected markers at various time points.

Figure 8. STEMdiff™ Motor Neurons Express Expected Motor Neuron Markers

Motor neurons were generated from iPS cell lines STiPS-R038, STiPS-M001, SCTi003-A (all maintained in mTeSR™ Plus), and SCTi003-A (maintained in mTeSR™1). hPSCs were then differentiated with the STEMdiff™ Motor Neuron culture system. RNA from Day 9, Day 14, and mature (in maturation media for 14 days) motor neurons was harvested and subsequently assayed using bulk RNA-seq. Their transcriptomic profiles were also compared to a parent hPSC control. The heat map displays expression levels for select genes associated with hPSCs, motor neuron progenitors, and motor neurons. iPS = induced pluripotent stem; hPSC = human pluripotent stem cell

Protocols and Documentation

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

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