Clonal Density Research Articles

Patterns of molecular evolution in a parthenogenic terrestrial isopod (Trichoniscus pusillus).

The "paradox of sex" refers to the question of why sexual reproduction is maintained in the wild, despite how costly it is compared to asexual reproduction. Because of these costs, one might expect nature to select for asexual reproduction, yet sex seems to be continually selected for. Multiple hypotheses have been proposed to explain this incongruence, including the niche differentiation hypothesis, the Red Queen hypothesis, and accumulation of harmful mutations in asexual species due to inefficient purifying selection. This study focuses on the accumulation of mutations in two terrestrial isopods, Trichoniscus pusillus, which has sexual diploid and parthenogenic triploid forms, and Hyloniscus riparius, an obligately sexual relative. We surveyed sex ratios of both species in an upstate New York population and obtained RNA-seq data from wild-caught individuals of both species to examine within- and between-species patterns of molecular evolution in protein-coding genes. The sex ratio and RNA-seq data together provide strong evidence that this T. pusillus population is entirely asexual and triploid, while the H. riparius population is sexual and diploid. Although all the wild T. pusillus individuals used for sequencing shared identical genotypes at nearly all SNPs, supporting a clonal origin, heterozygosity and SNP density were much higher in T. pusillus than in the sexually reproducing H. riparius. This observation suggests this parthenogenic lineage may have arisen via mating between two divergent diploid lineages. Between-species sequence comparisons showed no evidence of ineffective purifying selection in the asexual T. pusillus lineage, as measured by the ratio of nonsynonymous to synonymous substitutions (dN/dS ratios). Likewise, there was no difference between T. pusillus and H. riparius in the ratios of nonsynonymous to synonymous SNPs overall (pN/pS). However, pN/pS ratios in T. pusillus were significantly higher when considering only SNPs that may have arisen via recent mutation after the transition to parthenogenesis. Thus, these recent SNPs are consistent with the hypothesis that purifying selection is less effective against new mutations in asexual lineages, but only over long time scales. This system provides a useful model for future studies on the evolutionary tradeoffs between sexual and asexual reproduction in nature.

Cue-driven microbial cooperation and communication: evolving quorum sensing with honest signaling

BackgroundQuorum sensing (QS) is the ability of microorganisms to assess local clonal density by measuring the extracellular concentration of signal molecules that they produce and excrete. QS is also the only known way of bacterial communication that supports the coordination of within-clone cooperative actions requiring a certain threshold density of cooperating cells. Cooperation aided by QS communication is sensitive to cheating in two different ways: laggards may benefit from not investing in cooperation but enjoying the benefit provided by their cooperating neighbors, whereas Liars explicitly promise cooperation but fail to do so, thereby convincing potential cooperating neighbors to help them, for almost free. Given this double vulnerability to cheats, it is not trivial why QS-supported cooperation is so widespread among prokaryotes.ResultsWe investigated the evolutionary dynamics of QS in populations of cooperators for whom the QS signal is an inevitable side effect of producing the public good itself (cue-based QS). Using spatially explicit agent-based lattice simulations of QS-aided threshold cooperation (whereby cooperation is effective only above a critical cumulative level of contributions) and three different (analytical and numerical) approximations of the lattice model, we explored the dynamics of QS-aided threshold cooperation under a feasible range of parameter values. We demonstrate three major advantages of cue-driven cooperation. First, laggards cannot wipe out cooperation under a wide range of reasonable environmental conditions, in spite of an unconstrained possibility to mutate to cheating; in fact, cooperators may even exclude laggards at high cooperation thresholds. Second, lying almost never pays off, if the signal is an inevitable byproduct (i.e., the cue) of cooperation; even very cheap fake signals are selected against. And thirdly, QS is most useful if local cooperator densities are the least predictable, i.e., if their lattice-wise mean is close to the cooperation threshold with a substantial variance.ConclusionsComparing the results of the four different modeling approaches indicates that cue-driven threshold cooperation may be a viable evolutionary strategy for microbes that cannot keep track of past behavior of their potential cooperating partners, in spatially viscous and in well-mixed environments alike. Our model can be seen as a version of the famous greenbeard effect, where greenbeards coexist with defectors in a evolutionarily stable polymorphism. Such polymorphism is maintained by the condition-dependent trade-offs of signal production which are characteristic of cue-based QS.

A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

Isolation of XO Subclones from XY Murine Embryonic Stem Cells.

This is a simple procedure to isolate XO subclones from XY murine embryonic stem cells in situations that require transmission of a mutation through the female germline-for example, if the mutation adversely affects spermatogenesis. XY cells are plated at clonal density, and resulting colonies are genotyped by polymerase chain reaction for a Y-specific probe to identify clones that have spontaneously lost the Y chromosome.

Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our exvivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Abstract 1638: Discovery and preclinical characterization of anti-Siglec-15 antibodies that rescue T cells from macrophage-mediated immune suppression

Abstract Background: Siglec-15 is an immunosuppressive sialic acid-binding Ig-like lectin expressed by myeloid cells, tumor associated macrophages (TAMs) and some human tumors. Siglec-15 expressed by TAMs inhibits anti-tumor immune responses by engaging unknown immune checkpoint(s) on T cells. Interactions between Siglec-15 on TAMs and sialy Tn (sTn) antigen found on tumor cells contributes to the immunosuppressive tumor microenvironment. Notably, the mutually exclusive expression of Siglec-15 and the checkpoint ligand PD-L1 by cancer cells emphasizes Siglec-15 as an attractive target for cancer immunotherapy. Methods: Anti-Siglec-15 antibodies were cloned from B cells derived from rabbits immunized with human Siglec-15. Cells were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to human, murine and cynomolgus Siglec-15. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant rabbit human IgG1 Fc chimeras. Anti-Siglec-15 chimeric antibodies were evaluated in a panel of functional and phenotypic assays using primary human macrophages and T cells, and then prioritized for evaluation in murine tumor models. Results: Thirty-one rabbit anti-Siglec-15 clones were expressed as rabbit-human IgG1 chimeras based on binding to recombinant human, cynomolgus and murine Siglec-15 proteins, binding to Siglec-15 expressing cell lines, and lack of binding to other Siglec family members. A subset of these clones inhibited the binding of Siglec-15 to sTn. The top 5 clones were identified in functional screens modeling Siglec-15 mediated immune suppression. The clones rescued the NFAT promotor activity of a T cell reporter cell line as well as the proliferative and IFN-γ response of anti-CD3 activated human T cells from the inhibitory activity of recombinant Siglec-15-Fc protein. Selected clones also relieved M2c-macrophage-mediated immune suppression in M2c/T cell coculture assays by restoring T-cell proliferation and IFN-γ secretion. The prioritized clones are currently under evaluation in in vivo tumor models for PK and efficacy. Conclusions: We identified novel anti-Siglec-15 antibodies that restore T cell effector function following suppression by recombinant and cell-expressed Siglec-15. These data support further development of these anti-Siglec 15 antibodies as an anti-cancer immunotherapy. Citation Format: Myriam Bouchlaka, Huyen Dinh, Sam Lam, Valerie Wall, Darbie Whitman, Ramya Chandrasekaran, Johanna Harshman, Texia Loh, Lauren Loh, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of anti-Siglec-15 antibodies that rescue T cells from macrophage-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1638.

Abstract 1719: OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages

Abstract Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1719.

Genomic Profiling Reveals Spatial Intra-Tumor Heterogeneity in Follicular Lymphoma

Background and Aims: Follicular lymphoma (FL) is an incurable B cell malignancy characterized by advanced stage disease and a heterogeneous clinical course. Despite recent genomic studies focused on profiling single FL biopsies over several time-points, little is known about the spatial heterogeneity as multi-site sampling is rarely conducted in lymphomas. However, in solid cancers, this approach has demonstrated profound spatial intra-tumor heterogeneity (ITH), impacting mechanisms of drug resistance and compromising precision-based strategies. As the majority of FL patients manifest disseminated tumor involvement, we sought to characterize the extent and clinical importance of spatial genetic heterogeneity in FL.Methods: Our study cohort comprised 9 FL patients (SP1-9) from which 22 synchronously removed spatially-separated samples from several tissue sources (lymph node (LN), bone marrow (BM), skin (SK), pleural effusion (PE), ascites (AS), spleen (SP)) were collected. For patients SP3 and SP4, both spatial and longitudinal samples at 2 time-points; at FL and histological transformation, were included. FL B-cells and normal T-cells were enriched by fluorescence activated cell sorting (FACS) in most cases. Each sample was subjected to whole exome sequencing and we defined somatic variants, copy number alterations (CNAs) and mutation signatures using established bioinformatic tools. We employed a statistical clustering model (mclust) for clonal density estimation and together with ultra-deep targeted sequencing, reconstructed each tumor's subclonal architecture.Results: Whilst spatially-separated FL tumors shared identical BCL2 - IGH breakpoints, we observed variable degrees of ITH. On average, 82% (range 50%-99%) of variants were shared between sites with two patterns of spatial ITH observed: high (SP1, SP8, SP9) and low (SP5, SP6, SP7). Copy number analyses showed a high level of concordance in most cases, except for SP4, SP7 and SP8. The overall distribution of known mutational signatures between spatially shared and discordant variants was different. Shared variants were predominantly enriched for mutational signature 1, representing age-related changes. In contrast signatures 6 and 24, both suggestive of defective DNA mismatch repair, were more pronounced in the spatially-discordant variants (χ2 test, p <0.0001), indicating that distinct mutagenic processes operate at different times during a tumor's evolution. Clonal architecture analyses revealed that some tumors comprised multiple competing subclones, with distinct clusters of mutations demonstrating differential expansions within spatially-separated sites, exemplified in SP2 (Figure 1). Importantly, for cases where spatial tumors were examined at two time-points, the degree of heterogeneity markedly increased with transformation. Detailed mutation analysis showed that apart from CREBBP mutations, that were clonally maintained throughout spatially separated biopsies, there were several instances of site-specific alterations in genes encoding proteins known to activate signaling pathways amenable to targeted therapies such as, TNFAIP3 (SP3 and SP9), TNFRSF14 (SP1), PIK3CD (SP4) and copy number loss of PTEN (SP8). In depth characterization of these variants using deep-sequencing techniques confirmed their discordant nature, suggesting ongoing genetic diversification driving evolution after widespread tumor dissemination.Conclusion: Collectively, our results demonstrate that spatial ITH is prevalent in FL. The existence of site-specific aberrations suggests a single biopsy insufficiently captures the full genomic complexity present and these spatial variations need to be considered in biomarker-led clinical studies.Figure 1: Dot plot demonstrating variants allele frequencies (VAFs) of all non-synonymous variants in SP2. Three distinct subclones circled in purple, orange and green can be inferred. The purple cluster includes ATP 6V1B2 and KMT2D variants classified as clonal in the BM and subclonal in the LN. The orange cluster includes different variants in KMT2D classified as subclonal in the BM and clonal in the LN. The green cluster includes variants in EZH2 and BCL2 that have a higher VAF in the LN compared to the BM. VAFs for selected variants in each of these putative subclones are shown in the horizontal bar graphs. [Display omitted] DisclosuresAuer:Janssen: Consultancy. Gribben:Kite: Honoraria; Karyopharm: Honoraria; Pharmacyclics: Honoraria; TG Therapeutics: Honoraria; Celgene: Honoraria; Acerta: Honoraria; Janssen: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

SummaryAdult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Identification of mesenchymal stromal cell survival responses to antimicrobial silver ion concentrations released from orthopaedic implants

Antimicrobial silver (Ag+) coatings on orthopaedic implants may reduce infection rates, but should not be to the detriment of regenerative cell populations, primarily mesenchymal stem/stromal cells (MSCs). We determined intramedullary silver release profiles in vivo, which were used to test relevant Ag+ concentrations on MSC function in vitro. We measured a rapid elution of Ag+ from intramedullary pins in a rat femoral implantation model, delivering a maximum potential concentration of 7.8 µM, which was below toxic levels determined for MSCs in vitro (EC50, 33 µM). Additionally, we present in vitro data of the reduced colonisation of implants by Staphylococcus aureus. MSCs exposed to Ag+ prior to/during osteogenic differentiation were not statistically affected. Notably, at clonal density, the colony-forming capacity of MSCs was significantly reduced in the presence of 10 µM Ag+, suggesting that a subpopulation of clonal MSCs was sensitive to Ag+ exposure. At a molecular level, surviving colony-forming MSCs treated with Ag+ demonstrated a significant upregulation of components of the peroxiredoxin/thioredoxin pathway and processes involved in glutathione metabolism compared to untreated controls. Inhibition of glutathione synthesis using l-buthionine sulfoxamine eliminated MSC clonogenicity in the presence of Ag+, which was rescued by exogenous glutathione.

Shifts in coral clonality along a gradient of disturbance: insights on reproduction and dispersal of Pocillopora acuta

Pocillopora acuta, formerly synonymized with P. damicornis, is an ecologically important reef-building coral that exhibits mixed reproductive modes, geographic variation in clonality, and conflicting reports of population genetic structure. Using 16 polymorphic microsatellite loci, this study examined clonality, genetic differentiation, and connectivity of genetically identified P. acuta (n = 428) in the Bolinao–Anda Reef Complex (BARC), Philippines, characterized by varying levels of wave exposure. Estimates of clonal richness indicate that the populations are largely derived from asexual reproduction, more likely via dispersal of ameiotic larvae. Clonal richness, population density, and mean colony size vary with wave exposure, suggesting the potential influence of local-scale disturbance on clonality, reproductive mode, and population structure. Populations in low-energy environments were characterized by greater colony density, larger colonies, and a greater proportion of clones compared to high-energy environments. Despite evidence for realized clonal dispersal of P. acuta extending up to 22 km, significant genetic differentiation among BARC populations reveals restricted gene flow at small spatial scales. Moreover, genetic differentiation is more pronounced when considering the spatial distribution of clones (FST including clones = 0.059; FST excluding clones = 0.028), suggesting that (1) asexually produced propagules are likely retained locally and across-site settlement is not as common; and (2) sexually derived propagules may have broader scales of dispersal. This study reexamines the population genetics of this often-problematic coral and underlines the importance of contextualizing site and species biology in designing or enhancing management towards the maintenance of functional genetic diversity and pathways of connectivity among populations.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.

SummaryThe Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

Growth and Morphological Responses of Duckweed to Clonal Fragmentation, Nutrient Availability, and Population Density.

Connected ramets of aquatic clonal plants are susceptible to fragmentation by disturbance. Such clonal fragmentation may interact with nutrient availability and individual density to affect growth and morphology of aquatic clonal plants. We grew the widespread floating clonal plant Spirodela polyrhiza (duckweed) under three levels of population density (low, medium, or high), two levels of nutrient availability (low or high), and two levels of clonal fragmentation (with or without). Clonal fragmentation and high nutrients increased biomass and ramet number, but decreased frond width, frond length, and specific frond area of S. polyrhiza. Increasing population density decreased growth (biomass and ramet number) and frond and root size, and increased frond thickness of individual ramets of S. polyrhiza. The negative effect of population density on growth of S. polyrhiza was greater under high than under low nutrient availability. Furthermore, the negative effect of population density on total mass and frond mass of S. polyrhiza was greater with fragmentation than without. These results suggest that clonal fragmentation, nutrient availability and population density can interact to affect growth and morphology of clonal floating plants. Competition for nutrients and space, rather than light, may be the mechanisms underlying reduced growth of clonal floating plants. As clonal fragmentation can increase biomass and ramet production of S. polyrhiza, disturbance that potentially causes clonal fragmentation cannot be recommended as a measure to limit the spread of clonal floating plants.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors

It has generally proven challenging to produce functional β cells invitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. β cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.

Enriching Stem/Progenitor Cells from Dental Pulp Cells by Low-density Culturing.

Clonogenicity is a key feature of stem/progenitor cells. The present study aimed to enrich stem/progenitor cells from dental pulp cells by means of culturing the cells at a low clonal density with spatial separation and the evaluate differentiation potential of the surviving cells. Pulp cells derived from wisdom teeth were seeded into wells of a 96-plate at a mean density of 1 cell/well and cultured for 2 weeks. Surviving cells were harvested, pooled together and subjected to differentiation into adipocytes, osteoblasts and neurons using respective inducing conditions for 3 weeks. The former two types of cells were examined by staining with Oil Red O and Alizarin Red, respectively. Neuron-like cells were inspected for their morphology and immunostained for microtubule-associated protein 2 and β-tubulin III. Vital cells were obtained in eight wells of a 96-well plate, corresponding to a survival rate of 8%. Since fewer than two wells would be expected to contain more than four cells at seeding, the majority of surviving cells likely grew from 1-3 cells, which is a very low density. These cells differentiated into functional adipocytes and osteoblasts, and morphologically neuron-like cells. Low-density seeding with spatial separation enables statistical estimation of cell number in wells and provides an effective strategy for enriching stem/progenitor cells and for isolating clonal dental pulp cells.

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface

Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

Engagement of cellular prion protein with the co-chaperone Hsp70/90 organizing protein regulates the proliferation of glioblastoma stem-like cells

BackgroundGlioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion, and therapeutic resistance. GSCs are therefore a promising target for GBM treatment. Our group identified the cellular prion protein (PrPC) and its partner, the co-chaperone Hsp70/90 organizing protein (HOP), as potential target candidates due to their role in GBM tumorigenesis and in neural stem cell maintenance.MethodsGSCs expressing different levels of PrPC were cultured as neurospheres with growth factors, and characterized with stem cells markers and adhesion molecules markers through immunofluorescence and flow cytometry. We than evaluated GSC self-renewal and proliferation by clonal density assays and BrdU incorporation, respectively, in front of recombinant HOP treatment, combined or not with a HOP peptide which mimics the PrPC binding site. Stable silencing of HOP was also performed in parental and/or PrPC-depleted cell populations, and proliferation in vitro and tumor growth in vivo were evaluated. Migration assays were performed on laminin-1 pre-coated glass.ResultsWe observed that, when GBM cells are cultured as neurospheres, they express specific stemness markers such as CD133, CD15, Oct4, and SOX2; PrPC is upregulated compared to monolayer culture and co-localizes with CD133. PrPC silencing downregulates the expression of molecules associated with cancer stem cells, upregulates markers of cell differentiation and affects GSC self-renewal, pointing to a pivotal role for PrPC in the maintenance of GSCs. Exogenous HOP treatment increases proliferation and self-renewal of GSCs in a PrPC-dependent manner while HOP knockdown disturbs the proliferation process. In vivo, PrPC and/or HOP knockdown potently inhibits the growth of subcutaneously implanted glioblastoma cells. In addition, disruption of the PrPC-HOP complex by a HOP peptide, which mimics the PrPC binding site, affects GSC self-renewal and proliferation indicating that the HOP-PrPC complex is required for GSC stemness. Furthermore, PrPC-depleted GSCs downregulate cell adhesion-related proteins and impair cell migration indicating a putative role for PrPC in the cell surface stability of cell adhesion molecules and GBM cell invasiveness, respectively.ConclusionsIn conclusion, our results show that the modulation of HOP-PrPC engagement or the decrease of PrPC and HOP expression may represent a potential therapeutic intervention in GBM, regulating glioblastoma stem-like cell self-renewal, proliferation, and migration.

Purification of Distinct Subsets of Epithelial Cells from Normal Human Breast Tissue.

The mammary epithelium is composed of a variety of specialized cell types that function in a coordinated fashion to produce and eject milk through multiple cycles of pregnancy. The ability to identify and purify these subsets of cells in order to interrogate their growth and differentiation capacities, as well as to characterize the molecular mechanisms that regulate their behavior, is essential in identifying the processes associated with breast cancer initiation and progression. This methods chapter outlines the step-by-step methods for dissociating human breast reduction specimens to a single cell suspension of viable cells. As well, strategies for purifying four distinct subsets of epithelial cells by using fluorescence-activated cell sorting and protocols for interrogating the growth and differentiation properties of these purified cells at clonal densities in adherent culture are also described.