Figure 1. STEMdiff™ SMADi Neural Induction Kit Supports Generation of Neural Progenitor Cells with High Levels of PAX6 and SOX1 Expression.
Neural progenitor cells (NPCs) can be generated from hPSCs cultured in mTeSR™1 or TeSR™-E8™ via embryoid body or monolayer protocol using the STEMdiff™ SMADi Neural Induction Kit. Resulting NPCs express CNS-type NPC markers PAX6 and SOX1.
Multiple human ES and iPS lines (cultured in mTeSR™1 or TeSR™-E8™) were subjected to the monolayer neural induction protocol. Cells were harvested after 7 days in culture and processed for immunostaining with PAX6, SOX1 and SOX10 antibodies. Cultures were imaged and quantified using the high content imager ImageXpress Micro, which counts positive nuclei across the entirety of the culture well. n=3 replicates per cell line. Data showed that neural progenitor cells produced using the STEMdiff™ SMADi Neural Induction Kit expressed very high levels of CNS-type markers PAX6 and SOX1, while the neural crest marker SOX10 was low to undetectable.
Figure 3. Neural Progenitor Cells Produced Using the Stemdiff™ SMADi Neural Induction Kit Support Highly Efficient Downstream Differentiation Into Neurons and Astrocytes.
Starting hPSCs were maintained in mTeSR™1 and differentiated using an embryoid body (EB) protocol. Resulting cells were differentiated using the STEMdiff™ Neuron Differentiation/Maturation Kits, STEMdiff™ Astrocyte Differentiation/Maturation Kits, and STEMdiff™ Dopaminergic Neuron Differentiation/Maturation Kits as per the respective protocols.
Figure 4. Generation of Neural Progenitor Cells from hPSCs Maintained in mTeSR™ Plus
Human ES (H9) and iPS (STiPS-M001) cells were maintained in (A) mTeSR™1 with daily feeds or (B) mTeSR™ Plus with restricted feeds and differentiated using an embryoid body (EB)-based protocol with STEMdiff™ SMADi Neural Induction Kit. Neural progenitor cells derived from hPSCs maintained in either mTeSR™1 or mTeSR™ Plus clearly display neural rosettes (arrowheads) after replating EBs.
EPHRIN-B1 Mosaicism Drives Cell Segregation in Craniofrontonasal Syndrome hiPSC-Derived Neuroepithelial Cells Niethamer TK et al. Stem Cell Reports 2017 MAR
Abstract
Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types, they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial, skeletal, and neurological anomalies. Heterozygous females are more severely affected than hemizygous males, a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation, no direct evidence for this has been demonstrated. Here, by generating hiPSCs from CFNS patients, we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells, thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients.
Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells Xu X et al. Stem Cell Reports 2017 MAR
Abstract
Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.
Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids. E. Gabriel et al. Cell stem cell 2017 JAN
Abstract
The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.
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