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Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail,namely VCR (V,VPA,an inhibitor of HDACs; C,CHIR99021,an inhibitor of GSK-3 kinases and R,Repsox,an inhibitor of TGF-β pathways),under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs re- garding their proliferative and self-renewing abilities,gene expression profiles,and multipotency for different neu- roectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation,glycogen synthase kinase,and TGF-β pathways show similar efficacies for ciNPC induction. Moreover,ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemi- cal cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. View Publication

Phospho flow is a powerful approach to detect cell signaling aberrations,identify biomarkers and assess pharmacodynamics,and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here,B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions,fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes. View Publication

Asymmetry of cell fate is one fundamental property of stem cells,in which one daughter cell self-renews,whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms,such as plants,fungi,and mammals,has already been shown. However,before this current work,asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs),and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing,differentiating human and mouse ESCs. Moreover,we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3),indicating a molecular mechanism that regulates this phenomenon. Furthermore,our data support the hypothesis that retention of chromatids with the old" template DNA preserves the epigenetic memory of cell fate� View Publication

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues,but not in neural lineages,a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here,we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging,we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs,but not in NPCs. We postulate this aging" discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC� View Publication

Inhibition of metabolic features which distinguish cancer cells from their non-malignant counterparts is a promising approach to cancer treatment. Energy support for drug extrusion in multidrug resistance (MDR) is a potential target for metabolic inhibition. Two major sources of ATP-based metabolic energy are partial (glycolysis) and complete (mitochondrial oxidative phosphorylation) oxidation of metabolic fuels. In cancer cells,the balance between them tends to be shifted toward glycolysis; this shift is considered to be characteristic of the cancer metabolic phenotype. Numerous earlier studies,conducted with cells cultured in a monolayer (2-D model),suggested inhibition of glycolytic ATP production as an efficient tool to suppress MDR in cancer cells. Yet,more recent work challenged the appropriateness of the 2-D model for such studies and suggested that a more clinically relevant approach would utilize a more advanced cellular model such as a 3-D model. Here,we show that the transition from the 2-D model (cultured monolayer) to a 3-D model (cultured spheroids) introduces essential changes into the concept of energetic suppression of MDR. The 3-D cell organization leads to the formation of a discrete cell subpopulation (not formed in the 2-D model) with elevated MDR transport capacity. This subpopulation has a specific metabolic phenotype (mixed glycolytic/oxidative MDR support) different from that of cells cultured in the 2-D model. Finally,the shift to the oxidative phenotype becomes greater when the spheroids are grown under conditions of lactic acidosis that are typical for solid tumors. The potential clinical significance of these findings is discussed. View Publication

In spite of the recent improvements,the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer,a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here,we report on a component of a traditional Chinese medicine,evodiamine,which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase,it did not hold CSLC in a specific cell cycle phase but instead,selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly,evodiamine selectively activated p53 and p21 and decreased inactive Rb,the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. View Publication

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient,strain-dependent,and requires expert skills. Over recent years,several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment,which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin,which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary,our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%. View Publication

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors,the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast,and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-$$B (NF-$$B) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-$$B was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover,both canonical and non-canonical NF-$$B signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact,canonical and non-canonical NF-$$B signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-$$B promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1$$ and interleukin-6. The results suggest the use of NF-$$B inhibitors for clinical therapy of certain breast cancers. View Publication

Tumor cells have the ability to invade and form small clusters that protrude into adjacent tissues,a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro,p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction. Using epithelial cells transfected with inducible p60AmotL2,we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo,drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models,both in 2D and in 3D,and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells. We identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly,these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models,as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells. BET inhibitors specifically target p60AmotL2-expressing invasive cancer cells,likely by exploiting differences in chromatin accessibility,leading to cell death. Additionally,our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells. The online version contains supplementary material available at 10.1186/s13046-024-03031-w. View Publication