Scientific Resources

$$-Thalassemia ($$-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific $$-Thal-induced pluripotent stem cells (iPSCs),correction of the disease-causing mutations in those cells,and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here,we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin $$ (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in $$-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction,we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally,whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore,the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in $$-Thal iPSCs. View Publication

Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here,human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes,and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay,the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay,phenotypic changes in mitochondrial membrane potential,calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes,albumin/urea secretion,and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition,the cell viability of SC-iHeps and p-Heps was increased by ketoconazole,a CYP3A4 inhibitor. Collectively,SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore,SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells. View Publication

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling,we examined textgreater 680 colonies from 3 different preparations of cells over 5 days each,generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies,correlation of colony characteristics with Oct4 expression,and identification of rare events. View Publication

In functional cytometric studies,cultured cells are exposed to effectors (e.g. drugs),and the heterogeneity of cell responses are studied using cytometry techniques (e.g. image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity,(ii) the presence of a subpopulation with an elevated cytoprotective capacity and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies,such as measurements of population heterogeneity of cytotoxicity,chemosensitivity and radiosensitivity. This article is protected by copyright. All rights reserved. View Publication

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression,together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1,marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC,transcriptional activators of the HK2 and LDHA genes,decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death,indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes,whose levels are unchanged compared to NPCs,revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth,indicating an unknown mechanism linking mitochondrial biogenesis to cell size. View Publication

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly,techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization,mtDNA carryover was reduced to textless2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease,but it may not guarantee prevention. View Publication

Oct4 and Sox2 are pluripotent stem cell factors but the interplay between them in tumorigenesis is unclear. The aim of the present study was to investigate the roles of Oct4 and Sox2 in the reprogramming of oral cancer stem cells. One or both Oct4 and Sox2 were overexpressed in immortalized oral epithelial (hTERT+-OME) cells by lentivirus transduction. In addition,Oct4 and Sox2 proteins in two oral squamous cell carcinoma cell (OSCC) lines (Cal27 and primary cultured OSCC from a T2N2M0 patient) were individually or combinedly knocked down by shRNA. The results showed that the doubly transduced (Oct4+Sox2+) cells could trigger neoplasms in immunodeficient mice after lentivirus transduction,but single transduced (Oct4+ or Sox2+) cells had no tumor formation ability. The knockdown Sox2low and knockdown Oct4lowSox2low cells resulted in decreased tumor size in the immunodeficient mice but the single knockdown Oct4low cancer cells acquired more aggressive xenografts. Our findings suggest that Oct4+Sox2+ cells may be reprogrammed cancer stem cells inducing oral carcinogenesis. View Publication

Th2-eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2-eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis,Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically,this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore,we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together,these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis. View Publication

CD8(+) T cells develop increased sensitivity following Ag experience,and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that textgreater50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation,compared with textless20% in central memory T cells (TCM). Immediately proximal to Lck signaling,we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore,we observed superior cytotoxic effector function in TEM compared with TCM,and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase,and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM,as well as increased cytotoxic effector function in TCM Collectively,this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity,and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. View Publication

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific,induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells,which develop in germinal centres and then home to the bone marrow,IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly,their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However,these IgM plasma cells are probably not antigen-selected,as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally,antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge. View Publication

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis,we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro,we generated neurons harboring disease-causing mutations from patient-specific,induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons,reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro,including oxidative stress vulnerability and altered neuronal activity. Notably,PD neurons show that elevated RBFOX1,a gene previously linked to neurodevelopmental diseases,underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes. View Publication