Kruppel-like factor 4 suppresses neuroblastoma growth by promoting smooth-muscle differentiation

Poster Board Number: 2105Neuroblastoma (NB) is an embryonic tumor and possesses a unique propensity to exhibit either a spontaneous regression or an unrestrained growth. Growing evidence suggests that NB comprises heterogeneous populations of improperly differentiated neural crest cells and a small subset of NB cells behaves as stem cells. Commitment of NB stem cells to the fibromuscular lineage may give a favorable outcome, while to the neuronal lineage results in a malignant tumor progression. Kruppel like factor 4 (KLF4) is one of the key reprogramming factors. Intriguingly, it also possesses paradoxical functions in cancers, either as an oncogene or tumor suppressor dependent of cell context. In this study, we elucidated the roles of KLF4 in the lineage determination of NB stem cells and tumor progression. Quantitative RT-PCR showed that loss of KLF4 expression ...published_or_final_versio

Greater variability in lipid measurements associated with kidney diseases in patients with type 2 diabetes mellitus in a 10-year diabetes cohort study.

This study aimed to evaluate the associations between variability of lipid parameters and the risk of kidney disease in patients with type 2 diabetes mellitus. Low-density lipoprotein-cholesterol, total cholesterol to high-density lipoprotein-cholesterol ratio and triglyceride were specifically addressed in this study. This retrospective cohort study included 105,552 patients aged 45-84 with type 2 diabetes mellitus and normal kidney function who were managed under Hong Kong public primary care clinics during 2008-2012. Those with kidney disease (estimated glomerular filtration rate < 60 mL/min/1.73 m2 or urine albumin to creatinine ratio ≥ 3 mg/mmol) were excluded. Variabilities of low-density lipoprotein-cholesterol, total cholesterol to high-density lipoprotein-cholesterol ratio and triglyceride were determined using the standard deviation of the respective parameter obtained from a mixed effects model to minimize regression dilution bias. The associations between lipid variability and renal outcomes including incident kidney disease, renal function decline defined as ≥ 30% reduction in estimated glomerular filtration rate since baseline, and end-stage renal disease (estimated glomerular filtration rate < 15 mL/min/1.73 m2) were evaluated by multivariable Cox regression. After a median follow-up of 66.5 months (0.5 million person-years in total), 49,653 kidney disease, 29,358 renal function decline, and 1765 end-stage renal disease cases were recorded. Positive linear associations between low-density lipoprotein-cholesterol and total cholesterol to high-density lipoprotein-cholesterol ratio variabilities and the risk of all renal outcomes were demonstrated. However, no association between triglyceride variability and any outcome was found. Each mmol/L increase in low-density lipoprotein-cholesterol variability was associated with 20% (Hazard ratio 1.20 [95% CI 1.15-1.25]), 38% (Hazard ratio 1.37 [95% CI 1.30-1.45]), and 108% (Hazard ratio 2.08 [95% CI 1.74-2.50]) higher risk in incident kidney disease, renal function decline and end-stage renal disease respectively. Similarly, each unit increase in total cholesterol to high-density lipoprotein-cholesterol ratio variability was associated with 35% (Hazard ratio 1.15 [95% CI 1.10-1.20]), 33% (Hazard ratio 1.33 [95% CI 1.26-1.40]), and 75% (Hazard ratio 1.75 [95% CI 1.46-2.09]) heightened risk in incident kidney disease, renal function decline and end-stage renal disease respectively. Cholesterol variability may potentially be a useful predictor of kidney diseases in patients with type 2 diabetes mellitus. Attention should be drawn to cholesterol variability when managing diabetic patients and further research is warranted to investigate the modifiable risk factors for lipid variability

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function

ACKGROUND & AIMS: Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. METHODS: We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/- and RET-/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. RESULTS: ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. CONCLUSIONS: We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.postprin

Generation of isogenic iPSC with RET mutation using CRISPR-Cas9

Symposium celebrates 10 years of iPS Cell Technology with CiRA and ISSCRTheme: Pluripotency: from Basic Science to Therapeutic ApplicationsThe Hirschsprung (HSCR) disease is a congenital disorder that causes serve intestinal obstruction in affected patients due to absence of neuronal cells in the distal part of the gut. Although mutations in the RET-proto tyrosine kinase receptor have frequently been associated with this disease, it is still not entirely clear what is the precise underlying molecular mechanism. Evidences from genetic and animal studies suggest mutations in the RET gene can result in reduced expression level or comprised protein function that might be the cause of HSCR. To date, advancements in stem cell technology allow us to directly address individual gene function using an in vitro neuronal differentiation system that can mimics this process during embryonic development. In this study, we aim to define the role of RET signalling in human neural crest cell (NCC) development by generating an isogenic human iPS cell harbouring a RET deletion mutation using CRISPR-Cas9. By comparing the in vitro neuronal differentiation capabilities of RET mutant cells to that of isogenic wild type control cell, we found that iPS cells carrying coding sequence mutation in RET was able to generate NCCs in a comparable manner to the control. However, when these RET mutant NCCs were subjected to further neuronal differentiation, we observed reduced neuronal differentiation as compared to the control. We also found reduced cell migration and increased cell death in the RET mutant NCCs. Together, our data suggests proper RET function is essential for neuronal differentiation and migration of human NCCs

Investigating the implications of RET variant/mutation in Hirschsprung disease pathogenesis using patient-specific iPSC

Theme: Pluripotency: from Basic Science to Therapeutic ApplicationsSymposium celebrates 10 years of iPS Cell Technology with CiRA and ISSCRHirschsprung (HSCR) disease is a complex congenital disorder and is attributed to failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Genetic lesions that affect NCC development (proliferation/survival, migration and differentiation) may lead to HSCR disease. RET/GDNF signaling is crucial for ENCC development and RET is the most predominant susceptibility gene for HSCR. Genetic variant/mutations in coding (CD) and non-coding (NCD) regions of RET are respectively associated with the severe (total colonic aganglionosis, TCA) and mild (short segment, S-HSCR) forms of HSCR. In addition, many of these genetic lesions directly lead to reduced or defective RET/GDNF signaling, suggesting that these variant/mutations may impair NCC development. The goal of the studies was to investigate how these RET CD and NCD variant/mutations affect NCC development. Recently, we have successfully established and characterized two induced pluripotent stem cell (HSCR-iPSC) lines from patients who presented with S-HSCR and TCA, harboring RET NCD and CD variant/mutations respectively. Moreover, we have adapted an efficient protocol for the differentiation of human iPSCs into NCCs. We found that NCCs derived from the HSCR-iPSC line carrying RET NCD showed defects in migration and neuronal differentiation. Importantly, the expression of genes involved in migration, neuronal and glial differentiation was dysregulated, and RET expression was reduced. Besides, early NCC development defects and impaired RET expression were observed in another iPSC line from a TCA-HSCR patient carrying RET CD mutation. These results suggest that RET variant/mutations would affect both differentiation and migration of NCCs in HSCR patients

CRISPR-Cas9 mediated correction of Hirschsprung-associated mutations in human iPSC restores neural crest cell functions

Poster presentationHirschsprung (HSCR) disease is caused by the failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, thus leading to bowel obstruction. Heterozygous mutations in the coding region of the RET gene account for the severe form of HSCR (total colonic aganglionosis, TCA). However, 80% of HSCR patients have short segment HSCR (S-HSCR), whose genetic etiology remains unclear. Here, we aimed to establish a new strategy for the functional identification of novel mutation(s) in HSCR disease. Three HSCR-iPSC lines (1 TCA-HSCR patient with a RET G731del mutation and 2 S-HSCR patients without RET mutation) and two RET isogenic mutant iPSC lines (RET+/- and RET-/-) were established to generate disease-relevant ENCCs. ENCCs derived from all diseased iPSC lines exhibited defects in migration and neuronal lineage differentiation. RET mutations were functionally associated with cellular phenotypes of ENCCs in vitro. Integration of genetic and transcriptomic data further identified a novel mutation in Vinculin (VCL M209L) associated with S-HSCR. Correcting the RET and VCL mutations in HSCR-iPSCs restored the ENCC functions. Taken together, we identified VCL as a novel HSCR gene. More importantly, our results also illustrate the value of human iPSCs for defining mutations that functionally contribute to a complex disease

Self-Renewal of a de Novo Population of C-Kit+ Cells Confers a Sustainable Growth of Neuroblastoma

Poster Presentation - Session: Cancer CellHigh cellular heterogeneity within neuroblastomas (NB) may account for the non-uniform response to treatment. c-KIT+ cells are frequently detected in NB, but how they influence NB behavior still remains elusive. Here, we used NB tumor initiating cells to reconstitute NB development and demonstrated that c-KIT+ cells are de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIT+ NB cells express higher levels of neural crest and stem cell markers (SLUG, SOX2, NANOG) and are endowed with high clonogenic capacity, differentiation plasticity and are refractory to drugs. With serial transplantation assays, we found that c-KIT expression is not required for tumor formation, but c-KIT+ cells are more aggressive and can induce tumors 9-fold more efficiently than c-KIT-/low cells. Intriguingly, c-KIT+ cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of secondary and tertiary tumors in mice. In addition, we showed that Prokineticin signaling and MAPK pathways are crucial for the maintenance of c-KIT+ cells in tumor to promote NB progression. Our results highlight the importance of this de novo population of NB cells in sustainable growth of NB and reveal specific signalling pathways that may provide targets leading to more effective NB therapies

Dynamic regulation of c-KIT+ population in neuroblastoma

Conference Theme: Integration and Collaboration - the Way Forwar

Dysregulation of Gli Signaling Underlies Degective Enteric Nervous System Development

Session: Intestinal / Gut CellsPoster PresentationDuring enteric nervous system (ENS) development, coordination of neurogenesis and gliogenesis require an appropriate balance between the proliferation and differentiation of enteric neural crest cells (NCCs). Defects in these processes may lead to intestinal aganglionosis, Hirschsprung (HSCR) disease in humans. We have previously shown that aberrant Hedgehog signaling interferes both proliferation and differentiation of NCCs and results in disease predisposition, but the underlying molecular and cellular events remain unclear. In this study, we further delineated the roles of the three key Hedgehog effector genes namely Gli1, Gli2 and Gli3 in ENS development. Western blot analysis with FACS-sorted NCCs revealed that Gli signaling is transiently activated during NCC differentiation. Using an in vivo reporter of Gli activity, we demonstrated that Gli activity is elevated in the early committed ENCCs. Transient high Gli signaling is required for the proper ENS development, as constitutively high Gli activity by conditional ablation of Suppressor of Fused (Sufu) in NCCs resulted in impaired neuronal differentiation, axonal fasciculation, gangliogenesis and early onset of glial lineage differentiation of NCCs. The ratio of Gli2 activator (Gli2A) and Gli3 repressor (Gli3R) was particularly critical for ENS development, as they restrained the differentiation and cell cycle progression of NCCs. Aberrantly high Gli2A to Gli3R ratio in the NCCs resulted in dysregulation of cell cycle gene expression and an increase in S and G2/M phase, accompanied by early onset of NCC differentiation. Similarly, Gli3Δ699/ Δ699 mice, where a truncated form of Gli3 that mimics Gli3R was constitutively produced, exhibited reduced neuronal and glial differentiation, suggesting that forced expression of Gli3R was deleterious to NCC differentiation. Taken together, our findings suggest that transient activation of Gli activity promotes cell cycle progression and initiates NCC differentiation by regulating multiple proliferative signaling pathways. Aberrant Gli activity interferes NCC development and may contribute to HSCR pathogenesis