In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro.

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFNα) treatment to "pre-instruct" HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs' exit from dormancy via IFNα was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.

Emerging Mechanisms by which EMT Programs Control Stemness.

Tissue regeneration relies on adult stem cells (SCs) that possess the ability to self-renew and produce differentiating progeny. In an analogous manner, the development of certain cancers depends on a subset of tumor cells, called cancer stem cells (CSCs), with SC-like properties. In addition to being responsible for tumorigenesis, CSCs exhibit elevated resistance to therapy and thus drive tumor relapse post-treatment. The epithelial-mesenchymal transition (EMT) programs promote SC and CSC stemness in many epithelial tissues. Here, we provide an overview of the mechanisms underlying the relationship between stemness and EMT programs, which may represent therapeutic vulnerabilities for the treatment of cancers.

Pitfalls and requirements in quantifying asymmetric mitotic segregation.

The asymmetric inheritance of NUMB during mitosis determines future daughter cell fates in multiple model organisms. NUMB asymmetric inheritance has also been postulated for hematopoietic stem cell (HSC) divisions but remained controversial until recently. To reconcile conflicting reports, we revisited the evidence for asymmetric inheritance of NUMB during HSC divisions. We demonstrate that previously used strategies to identify dividing cells in fixed samples suffer from multiple systematic errors. Nonmitotic cells in close proximity are frequently mistaken as dividing cells, while mitotic cells are not detected. Furthermore, microtubule depolymerization by either nocodazole or low temperatures prevents the reliable detection of mitosis and introduces mitotic artifacts. Without artificial microtubule depolymerization and by the use of reliable mitotic markers, we find NUMB differences in daughter cells to be reduced and restricted to cells with low NUMB expression and thus low signal over background. This bias fits the expected random distribution of simulated noise data, suggesting that the putative asymmetric inheritance of NUMB in HSCs could be merely technical noise. We conclude that functionally relevant asymmetric inheritance of NUMB and other factors in mitotic HSCs and other cells cannot be conclusively demonstrated using snapshot data and requires alternative approaches, such as continuous quantitative single-cell analysis.

Modulation of asymmetric cell division as a mechanism to boost CD8+ T cell memory.

Asymmetric partitioning of fate determinants is a mechanism that contributes to T cell differentiation. However, it remains unclear whether the ability of T cells to divide asymmetrically is influenced by their differentiation state, as well as whether enforcing asymmetric cell division (ACD) rates would have an impact on T cell differentiation and memory formation. Using the murine LCMV infection model, we established a correlation between cell stemness and the ability of CD8+ T cells to undergo ACD. Transient mTOR inhibition was proven to increase ACD rates in naïve and memory cells and to install this ability in exhausted CD8+ T cells. Functionally, enforced ACD correlated with increased memory potential, leading to more efficient recall response and viral control upon acute or chronic LCMV infection. Moreover, transient mTOR inhibition also increased ACD rates in human CD8+ T cells. Transcriptional profiling revealed that progenies emerging from enforced ACD exhibited more pronounced early memory signatures, which functionally endowed these cells with better survival in the absence of antigen exposure and more robust homing to secondary lymphoid organs, providing critical access to survival niches. Our data provide important insights into how ACD can improve long-term survival and function of T cells and open new perspectives for vaccination and adoptive T cell transfer therapies.

Side population cells in anaplastic thyroid cancer and normal thyroid.

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated. One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12. This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.

Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis.

Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation. Lgl1 conditional knockout OPC progeny retain NG2 and show reduced OL differentiation, while undergoing more symmetric self-renewing divisions at the expense of asymmetric divisions. Moreover, Lgl1 and hemizygous Ink4a/Arf knockouts in OPC synergistically induce gliomagenesis. Time lapse and total internal reflection microscopy reveals a critical role for Lgl1 in NG2 endocytic routing and links aberrant NG2 recycling to failed differentiation. These data establish Lgl1 as a suppressor of gliomagenesis and positive regulator of asymmetric division and differentiation in the healthy and demyelinated murine brain.

CDK5 Inhibition Resolves PKA/cAMP-Independent Activation of CREB1 Signaling in Glioma Stem Cells.

Cancer stem cells promote neoplastic growth, in part by deregulating asymmetric cell division and enhancing self-renewal. To uncover mechanisms and potential therapeutic targets in glioma stem cell (GSC) self-renewal, we performed a genetic suppressor screen for kinases to reverse the tumor phenotype of our Drosophila brain tumor model and identified dCdk5 as a critical regulator. CDK5, the human ortholog of dCdk5 (79% identity), is aberrantly activated in GBMs and tightly aligned with both chromosome 7 gains and stem cell markers affecting tumor-propagation. Our investigation revealed that pharmaceutical inhibition of CDK5 prevents GSC self-renewal in vitro and in xenografted tumors, at least partially by suppressing CREB1 activation independently of PKA/cAMP. Finally, our TCGA GBM data analysis revealed that CDK5, stem cell, and asymmetric cell division markers segregate within non-mesenchymal patient clusters, which may indicate preferential dependence on CDK5 signaling and sensitivity to its inhibition in this group.

Brief Report: Interleukin-17A-Dependent Asymmetric Stem Cell Divisions Are Increased in Human Psoriasis: A Mechanism Underlying Benign Hyperproliferation.

The balance between asymmetric and symmetric stem cell (SC) divisions is key to tissue homeostasis, and dysregulation of this balance has been shown in cancers. We hypothesized that the balance between asymmetric cell divisions (ACDs) and symmetric cell divisions (SCDs) would be dysregulated in the benign hyperproliferation of psoriasis. We found that, while SCDs were increased in squamous cell carcinoma (SCC) (human and murine), ACDs were increased in the benign hyperproliferation of psoriasis (human and murine). Furthermore, while sonic hedgehog (linked to human cancer) and pifithrinα (p53 inhibitor) promoted SCDs, interleukin (IL)-1α and amphiregulin (associated with benign epidermal hyperproliferation) promoted ACDs. While there was dysregulation of the ACD:SCD ratio, no change in SC frequency was detected in epidermis from psoriasis patients, or in human keratinocytes treated with IL-1α or amphiregulin. We investigated the mechanism whereby immune alterations of psoriasis result in ACDs. IL17 inhibitors are effective new therapies for psoriasis. We found that IL17A increased ACDs in human keratinocytes. Additionally, studies in the imiquimod-induced psoriasis-like mouse model revealed that ACDs in psoriasis are IL17A-dependent. In summary, our studies suggest an association between benign hyperproliferation and increased ACDs. This work begins to elucidate the mechanisms by which immune alteration can induce keratinocyte hyperproliferation. Altogether, this work affirms that a finely tuned balance of ACDs and SCDs is important and that manipulating this balance may constitute an effective treatment strategy for hyperproliferative diseases. Stem Cells 2017;35:2001-2007.

A miR-34a-Numb Feedforward Loop Triggered by Inflammation Regulates Asymmetric Stem Cell Division in Intestine and Colon Cancer.

Emerging evidence suggests that microRNAs can initiate asymmetric division, but whether microRNA and protein cell fate determinants coordinate with each other remains unclear. Here, we show that miR-34a directly suppresses Numb in early-stage colon cancer stem cells (CCSCs), forming an incoherent feedforward loop (IFFL) targeting Notch to separate stem and non-stem cell fates robustly. Perturbation of the IFFL leads to a new intermediate cell population with plastic and ambiguous identity. Lgr5+ mouse intestinal/colon stem cells (ISCs) predominantly undergo symmetric division but turn on asymmetric division to curb the number of ISCs when proinflammatory response causes excessive proliferation. Deletion of miR-34a inhibits asymmetric division and exacerbates Lgr5+ ISC proliferation under such stress. Collectively, our data indicate that microRNA and protein cell fate determinants coordinate to enhance robustness of cell fate decision, and they provide a safeguard mechanism against stem cell proliferation induced by inflammation or oncogenic mutation.

Inhibition of mTOR sensitizes breast cancer stem cells to radiation-induced repression of self-renewal through the regulation of MnSOD and Akt.

The sensitization of breast cancer stem cells (BrCSCs) to the inhibitive effects of radiotherapy through adjuvant therapy which targets oncogenic pathways represents a prospective strategy for improving the effect of radiation in patients with triple-negative breast cancer (TNBC). Mammalian target of rapamycin (mTOR) activation is one of the most frequent events in human malignancies, and is critical for sustaining the self­renewing ability of cancer stem cells (CSCs); inhibition by rapamycin is an effective and promising strategy in anticancer treatments. In the present study, we found that mTOR activity was closely related to the self-renewal ability of BrCSCs, and in triple negative MDA-MB-453 and MDA-MB­468 cells, rapamycin repression of mTOR phosphorylation decreased the number of mammospheres and helped to sensitize the resistant CSCs to low-dose radiation therapy. By inhibiting mTOR and mitochondrial manganese superoxide dismutase (MnSOD), we confirmed that rapamycin functioned through the mTOR/MnSOD/reactive oxygen species (ROS) signaling pathway, and the existence of Akt governed the rapamycin­induced asymmetric division (AD) of stem cells in cases of radiation­treated breast cancer. The synergic effects of rapamycin and low-dose radiation induced the AD of stem cells, which then resulted in a decrease in the number of mammospheres, and both were mediated by MnSOD. Governed by Akt, the consequent inhibition of ROS formation and oxidative stress preserved the AD mode of stem cells, which is critical for an improved radiotherapy response in clinical treatment, as the tumor group is thus easier to eliminate with radiation therapy. We posit that an in-depth understanding of the interaction of radiation with CSCs has enormous potential and will make radiation even better and more effective.

Emergence of antibiotic resistance from multinucleated bacterial filaments.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes.

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.