Direct and indirect regulation of ?-glucocerebrosidase by the transcription factors USF2 and ONECUT2
Mutations in GBA1 encoding the lysosomal enzyme ?-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson’s disease (PD),with 10–30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance,we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library,we uncovered 11 TFs as regulators of GCase activity. Among these,activation of MITF and TFEC increased lysosomal GCase activity in live cells,while activation of ONECUT2 and USF2 decreased it. While MITF,TFEC,and USF2 affected GBA1 transcription,ONECUT2 might control GCase trafficking. The effects of MITF,TFEC,and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase.
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Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.
Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes,both in vitro and in vivo. Beyond uses in cell replacement therapy,patient-specific cardiomyocytes may find applications in drug testing,drug discovery,and disease modeling. Recently,approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs),adult heart-derived cardiac progenitor cells (CPCs),and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts,highlighting potential applications and future challenges.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
Borowiak M et al. (APR 2009)
Cell stem cell 4 4 348--58
Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.
An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives,including lung,liver,and pancreas,are of interest for regenerative medicine,but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds,two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm,a higher efficiency than that achieved by Activin A or Nodal,commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers,can participate in normal development when injected into developing embryos,and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.
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产品类型：
产品号#：
72314
72512
72514
产品名：
(-) -Indolactam V（吲哚内酰胺 V)
IDE1
IDE1
Staerk J et al. ( 2011)
Angewandte Chemie (International ed. in English) 50 25 5734--5736
Pan-Src family kinase inhibitors replace Sox2 during the direct reprogramming of somatic cells.
Bhinge A et al. (JUN 2014)
EMBO Journal 33 11 1271--1283
MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-$\$/BMP signaling.
Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.
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Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b.
A recent study has suggested that fibroblasts can be converted into mouse-induced neural stem cells (miNSCs) through the expression of defined factors. However,successful generation of human iNSCs (hiNSCs) has proven challenging to achieve. Here,using microRNA (miRNA) expression profile analyses,we showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2,a let-7-targeting gene,enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs. Interestingly,HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2,whereas other combinations or SOX2 alone showed a limited conversion ability. Taken together,these findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal,providing a strategy for the clinical treatment of neurological diseases.
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产品类型：
产品号#：
05750
05752
产品名：
NeuroCult™ NS-A 基础培养基（人）
NeuroCult™ NS-A 分化试剂盒 (人)
Sun AX et al. (AUG 2016)
Cell reports 16 7 1942--1953
Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells.
Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.
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Hu W et al. (AUG 2015)
Cell stem cell 17 2 204--12
Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.
Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however,the introduction of ectopic genes limits the therapeutic applications of such induced neurons (iNs). Here,we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules,bypassing a neural progenitor stage. These human chemical-induced neuronal cells (hciNs) resembled hiPSC-derived neurons and human iNs (hiNs) with respect to morphology,gene expression profiles,and electrophysiological properties. This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together,our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine.
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