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Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous,a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult,resulting in inaccuracies. In this study,we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts,human induced PSCs,and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time,with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2?4 and 2?3 hours after starvation,respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs,focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types,including undifferentiated PSCs,differentiated cells,and cells undergoing cellular reprogramming,and addresses critical issues,such as differences in basal metabolic levels and sensitivity to normalization,providing valuable insights into cellular energetics. View Publication

ObjectivesMutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD),a dominantly inherited,rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells,which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients,such as glucose,to the retina. Recently,metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis.MethodsQuantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3,known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways,processes,and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U–13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U–13C6] glucose and [U–13C5] glutamine isotopic tracing in SFD iRPE cells.ResultsQuantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U–13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly,[U–13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally,[U–13C5] glutamine tracing found evidence of altered malic enzyme activity.ConclusionsThis study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases,emphasizing RPE cellular metabolism as a therapeutic target. Highlights•SFD mice display alterations in proteins associated with metabolism.•SFD RPE cells have increased glycolytic activity and glucose contribution to the TCA cycle.•Glutamine contribution to energy metabolism is unaltered in SFD RPE cells however there is reduced malic enzyme activity.•SFD RPE cells display metabolic dysfunction potentially implicating metabolism as a viable therapeutic target. View Publication

SummaryGABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs; generating embryoid bodies; and differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses.For complete details on the use and execution of this protocol,please refer to Pilotto et al.1 Graphical abstract Highlights•Steps described for generating GABAergic neurons from human iPSCs•Instructions for the enrichment of cerebellar GABAergic interneurons (cGNs)•Guide to calcium imaging of cGNs using genetically encoded calcium indicators Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. GABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human-cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs,and generating embryoid bodies,differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses. View Publication

PURPOSE Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy,temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN ZR2002 is a combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR downstream Erk1/2 phosphorylation increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line decreased EGFR Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability blood-brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM." View Publication

Dental tissue has been acknowledged as an advantaged source for high-quality dental pulp stem cell (DPSC) preparation. However,despite the accomplishment of the separation of DPSCs from permanent teeth and supernumerary teeth,the deficiency of rigorous and systematic clarification on the signatures and efficacy will hinder their prospects in regenerative medicine. In this study,we primitively isolated permanent teeth-derived DPSCs and supernumerary teeth-derived apical papillary stem cells (SCAP-Ss) with parental consent. Immunophenotype of DPSCs and SCAP-Ss was determined by a flow cytometry assay,and the cell viability was verified by multidimensional detections including cell proliferation,cell cycle,apoptosis,and senescence. The migration and clonogenic capacity were examined by a wound healing test and crystal violet staining,respectively. The multilineage differentiation potential was quantitated by utilizing Oil Red O staining and Alizarin Red staining,together with real-time PCR analysis. The efficacy on a mouse hepatic fibrosis model was evaluated by using histologic sections and liver function tests. Herein,we showed that SCAP-Ss exhibited comparable immunophenotype and adipogenic differentiation capacity as DPSCs. However,different from DPSCs,SCAP-Ss exhibited superiority in cell viability and osteogenic differentiation. Simultaneously,injection of DPSCs and SCAP-Ss significantly reduced inflammatory infiltration,enhanced liver-associated gene expression,and finally relieved symptoms of hepatic fibrosis. In conclusion,SCAP-Ss possess preferable characteristics and efficacy on hepatic fibrosis in mice. Our findings suggest that SCAP-Ss are an easily accessible postnatal stem cell source with multifaceted characteristics for regenerative medicine. View Publication

The present study demonstrates that CD4(+)CD25(+) T cells,expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART),exhibit phenotypic,molecular,and functional characteristics of regulatory T cells. The majority of peripheral CD4(+)CD25(+) T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4(+)CD25(+) T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4(+)CD25(-) T cells,CD4(+)CD25(+) T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin,cytomegalovirus (CMV),and p24 significantly increased following depletion of CD4(+)CD25(+) T cells. Furthermore,addition of increasing numbers of CD4(+)CD25(+) T cells resulted in a dose-dependent inhibition of CD4(+)CD25(-) T-cell proliferation to tuberculin and p24. CD4(+)CD25(+) T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10),thus indicating the presence of p24-specific CD4(+) T cells among the CD4(+)CD25(+) T-cell subset. Suppressive activity was not dependent on the secretion of TGF-beta or IL-10. Taken together,our results suggest that persistence of HIV antigens might trigger the expansion of CD4(+)CD25(+) regulatory T cells,which might induce a tolerance to HIV in vivo. View Publication

Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental,pathological,and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field,few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK),a metabolic fuel gauge" of the biological system. In the present study� View Publication

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar theratype"; however a standardized approach confirming efficacy in these cohorts has not been reported. Here we demonstrate that patients bearing the rare mutation: c.3700 A>G causing protein misprocessing and altered channel function-similar to ΔF508-CFTR are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor respectively this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound effective in increasing the levels of immature CFTR protein augmented the Orkambi® response. Importantly this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue will facilitate therapy development for patients with rare CF mutations. View Publication

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance,but they can also play a detrimental role by preventing antitumor responses. Here,we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution,focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently,Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall,our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival,higher migratory capacity,and selective T-effector suppressive ability. View Publication

Objectives gamma$delta$ T cells,a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens,are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation,we compared how exposure to an autoantigenic lipid,cardiolipin (CL),during co-stimulation with an amino-bisphosphonate (zoledronate,zol),affects the activation and cytokine production of SSc and healthy control (HC) gamma$delta$ T cells. Methods Expression of CD25 on Vgamma$9+,Vdelta$1+,and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs),their binding of CD1d tetramers,and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNgamma$ and IL-4 was assessed after overnight culture. Results Percentages of CD25+ among CD3+ and Vdelta$1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC,CL and zol,respectively,suppressed {\%}CD25+ Vgamma$9+ and Vdelta$1+ T cells but,when combined,CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly,Vdelta$1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers,and a CD1d-blocking mAb decreased CL-induced enhancement of {\%}SSc CD25+ Vdelta$1+ T cells in the presence of zol. {\%}IFNgamma$+ cells among Vgamma$9+ T cells of SSc was lower than HC cultured in medium,CL,zol,or CL + zol,whereas {\%}IFNgamma$+ Vdelta$1+ T cells was lower only in the presence of CL or CL + zol. {\%}IL-4+ T cells were similar in SSc and HC in all conditions,with the exception of being increased in SSc Vgamma$9+ T cells in the presence of CL. Conclusion Abnormal functional responses of gamma$delta$ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression,characteristics of this disease. View Publication

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions,where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh),whose posttranslational lipid modifications and assembly into multimers enhance its biological potency,potentially through receptor clustering. Investigations of Shh typically utilize recombinant,monomeric protein,and thus the impact of multivalency on ligand potency is unexplored. Among its many activities,Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies,compared to the monomeric Shh,increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%,respectively. Thus,multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients. View Publication

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells,retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE),and fetal RPE. The protocols include collection of RNA for analysis by microarray,and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally,cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. View Publication