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.

Enrichment and Identification of Neural Stem Cells in Neurospheres Using Rigidity-Tunable Gels.

IMPACT STATEMENT: Neural stem cells (NCSs) are integral to establishing in vitro models and regenerative medicine. To this day, there is an unmet need to enrich these cells from a heterogeneous cell population for clinical applications without irreversible manipulation. We identified a method to propagate human NCSs via computational analysis of their mechanical signature. In this study, we report a novel analytical method for mechanical forces in three-dimensional cultures. Further, our results revealed that stemness may, in part, be mediated by physical properties of the extracellular matrix. In conclusion, our findings have potential implications in understanding stem cell mechanobiology for enrichment or differentiation.

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.

Alcohol inhibits the proliferation of Neuro2a cells via promoting the asymmetric cell division through down-regulation of the expression of centrosome protein-J.

Alcohol can decrease cell proliferation in neural cells. The proliferation of neural cells can be inhibited by the asymmetric division of neural progenitor cells. However, whether alcohol inhibits cell proliferation through inducing cell asymmetric division is not yet clear. Here, we reported that the percentage of asymmetric division was increased in alcohol-treated Neuro2a cells owing to the impaired-spindle orientation. Meanwhile, the expression of Centrosome protein-J (CPAP) which plays an important role in spindle orientation was reduced in Neuro2a cells. The overexpression of GFP-CPAP in Neuro2a cells rescued the disorder of spindle orientation and the asymmetric cell division induced by alcohol. Taken together, the results demonstrate that alcohol exposure diminished the pool of proliferative Neuro2a cells through disordering the spindle orientation and promoting the asymmetric division. And these abnormal orientation and division were due to the reduced CPAP protein level.

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.

Numb regulates somatic cell lineage commitment during early gonadogenesis in mice.

During early gonadogenesis, proliferating cells in the coelomic epithelium (CE) give rise to most of the somatic cells in both XX and XY gonads. Previous dye-labeling experiments showed that a single CE cell could give rise to additional CE cells and to both supporting and interstitial cell lineages, implying that cells in the CE domain are multipotent progenitors, and suggesting that an asymmetric division is involved in the acquisition of gonadal cell fates. We found that NUMB is asymmetrically localized in CE cells, suggesting that it might be involved. To test this hypothesis, we conditionally deleted Numb on a Numbl mutant background just prior to gonadogenesis. Mutant gonads showed a loss of cell polarity in the surface epithelial layers, large interior cell patches expressing the undifferentiated cell marker LHX9, and a loss of differentiated cells in somatic cell lineages. These results indicate that NUMB is necessary for establishing polarity in CE cells, and that asymmetric divisions resulting from CE polarity are required for commitment to differentiated somatic cell fates. Surprisingly, germ cells, which do not arise from the CE, were also affected in mutants, which may be a direct or indirect effect of loss of Numb.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

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.

TGN38 is required for the metaphase I/anaphase I transition and asymmetric cell division during mouse oocyte meiotic maturation.

The cellular functions of the trans-Golgi network protein TGN38 remain unknown. In this research, we studied the expression, localization and functions of TGN38 in the meiotic maturation of mouse oocytes. TGN38 was expressed at every stage of oocyte meiotic maturation and colocalized with γ-tubulin at metaphase I and metaphase II. The spindle microtubule disturbing agents nocodazole and taxol did not affect the colocalization of TGN38 and γ-tubulin. Depletion of TGN38 with specific siRNAs resulted in increased metaphase I arrest, accompanied with spindle assembly checkpoint activation and decreased first polar extrusion (PB1). In the oocytes that had extruded the PB1 after the depletion of TGN38, symmetric division occurred, leading to the production of 2 similarly sized cells. Moreover, the peripheral migration of metaphase I spindle and actin cap formation were impaired in TGN38-depleted oocytes. Our data suggest that TGN38 may regulate the metaphase I/anaphase I transition and asymmetric cell division in mouse oocytes.

A new loss-of-function allele 28y reveals a role of ARGONAUTE1 in limiting asymmetric division of stomatal lineage ground cell.

In Arabidopsis thaliana L., stomata are produced through a series of divisions including asymmetric and symmetric divisions. Asymmetric entry division of meristemoid mother cell produces two daughter cells, the smaller meristemoid and the larger sister cell, a stomatal lineage ground cell (SLGC). Stomatal lineage ground cells can differentiate into epidermal pavement cells but have the potential to divide asymmetrically, spacing divisions, to create satellite meristemoids. Peptide ligands and TOO MANY MOUTHS (TMM) and ERECTA family receptors regulate the initiation of stomatal lineages, activity, and orientation of spacing divisions. Here, we reported that a natural mutant 28y displayed an increased stomatal density and index. Using map-based cloning, we identified mutation in ARGONAUTE1 (AGO1) as the cause of 28y phenotypes. Time-lapse tracing of stomatal lineage cells reveals that stomatal overproduction in 28y is caused by the excessive asymmetric spacing division of SLGCs. Further genetic results demonstrated that AGO1 acts downstream of TMM and negatively regulates the SPCH transcripts, but in a brassinosteroid-independent manner. Upregulation of AGAMOUS-LIKE16 (AGL16) in 28y mutants suggests that AGO1 is required to restrict AGL16-mediated stomatal spacing divisions, an miRNA pathway in addition to ligand-receptor signaling modules.

A self-renewing division of zebrafish Müller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons.

Müller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Müller glia partially dedifferentiate, re-express neuroepithelial markers and re-enter the cell cycle. We show that the immunoglobulin superfamily adhesion molecule Alcama is a novel marker of multipotent retinal stem cells, including injury-induced Müller glia, and that each Müller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Müller glia involves interkinetic nuclear migration, but mitosis of retinal progenitors occurs in situ. Rapidly dividing retinal progenitors form neurogenic clusters tightly associated with Alcama/N-cadherin-labeled Müller glial radial processes. Genetic suppression of N-cadherin function interferes with basal migration of retinal progenitors and subsequent regeneration of HuC/D(+) inner retinal neurons.

Key events during the transition from rapid growth to quiescence in budding yeast require posttranscriptional regulators.

Yeast that naturally exhaust the glucose from their environment differentiate into three distinct cell types distinguishable by flow cytometry. Among these is a quiescent (Q) population, which is so named because of its uniform but readily reversed G1 arrest, its fortified cell walls, heat tolerance, and longevity. Daughter cells predominate in Q-cell populations and are the longest lived. The events that differentiate Q cells from nonquiescent (nonQ) cells are initiated within hours of the diauxic shift, when cells have scavenged all the glucose from the media. These include highly asymmetric cell divisions, which give rise to very small daughter cells. These daughters modify their cell walls by Sed1- and Ecm33-dependent and dithiothreitol-sensitive mechanisms that enhance Q-cell thermotolerance. Ssd1 speeds Q-cell wall assembly and enables mother cells to enter this state. Ssd1 and the related mRNA-binding protein Mpt5 play critical overlapping roles in Q-cell formation and longevity. These proteins deliver mRNAs to P-bodies, and at least one P-body component, Lsm1, also plays a unique role in Q-cell longevity. Cells lacking Lsm1 and Ssd1 or Mpt5 lose viability under these conditions and fail to enter the quiescent state. We conclude that posttranscriptional regulation of mRNAs plays a crucial role in the transition in and out of quiescence.

Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity.

The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.