Probabilistic Model-Based Cell Tracking

The study of cell behavior is of crucial importance in drug and disease research. The fields of bioinformatics and biotechnology rely on the collection, processing, and analysis of huge numbers of biocellular images, including cell features such as cell size, shape, and motility. However manual methods of inferring these values are so onerous that automated methods of cell tracking and segmentation are in high demand. In this paper, a novel model-based cell tracker is designed to locate and track individual cells. The proposed cell tracker has been successfully applied to track hematopoietic stem cells (HSCs) based on identified cell locations and probabilistic data association

Fludarabine as a cost-effective adjuvant to enhance engraftment of human normal and malignant hematopoiesis in immunodeficient mice

There is still an unmet need for xenotransplantation models that efficiently recapitulate normal and malignant human hematopoiesis. Indeed, there are a number of strategies to generate humanized mice and specific protocols, including techniques to optimize the cytokine environment of recipient mice and drug alternatives or complementary to the standard conditioning regimens, that can be significantly modulated. Unfortunately, the high costs related to the use of sophisticated mouse models may limit the application of these models to studies that require an extensive experimental design. Here, using an affordable and convenient method, we demonstrate that the administration of fludarabine (FludaraTM) promotes the extensive and rapid engraftment of human normal hematopoiesis in immunodeficient mice. Quantification of human CD45+ cells in bone marrow revealed approximately a 102-fold increase in mice conditioned with irradiation plus fludarabine. Engrafted cells in the bone marrow included hematopoietic stem cells, as well as myeloid and lymphoid cells. Moreover, this model proved to be sufficient for robust reconstitution of malignant myeloid hematopoiesis, permitting primary acute myeloid leukemia cells to engraft as early as 8 weeks after the transplant. Overall, these results present a novel and affordable model for engraftment of human normal and malignant hematopoiesis in immunodeficient mice

Segmentation, tracking and cell cycle analysis of live-cell imaging data with Cell-ACDC

Background: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. Results: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htbl concentrations decrease with replicative age. Conclusions: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis.Peer reviewe

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function

This work was funded by the CICE/FEDER (P08-CTS-3678) de la Junta de Andalucia with a grant to PM, the FIS/FEDER (PI10/00449 to PM and PI11/00119 to CB), the MICINN (Fondo Especial del Estado para Dinamizacion de la Economia y Empleo/PLE-2009-0111) with a grant to PM, and the Foundation "Spanish Association Against Cancer"/Junta Provincial de Albacete (CI110023 to PM). DRM (PFIS scholarship FI11/0511), RM and CB (CP07/0059) are supported by the ISCIII. ON-M was supported by the Health of Department of the Junta de Andalucia.The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34(-) cells.Junta de Andalucia P08-CTS-3678European Union (EU) Instituto de Salud Carlos III PI10/00449 PI11/00119MICINN (Fondo Especial del Estado para Dinamizacion de la Economia y Empleo) PLE-2009-0111Foundation "Spanish Association Against Cancer"/Junta Provincial de Albacete CI110023Instituto de Salud Carlos III FI11/0511 CP07/0059ICRE

Junctional Adhesion Molecule-C Mediates the Recruitment of Embryonic-Endothelial Progenitor Cells to the Perivascular Niche during Tumor Angiogenesis

The homing of Endothelial Progenitor Cells (EPCs) to tumor angiogenic sites has been described as a multistep process, involving adhesion, migration, incorporation and sprouting, for which the underlying molecular and cellular mechanisms are yet to be fully defined. Here, we studied the expression of Junctional Adhesion Molecule-C (JAM-C) by EPCs and its role in EPC homing to tumor angiogenic vessels. For this, we used mouse embryonic-Endothelial Progenitor Cells (e-EPCs), intravital multi-fluorescence microscopy techniques and the dorsal skin-fold chamber model. JAM-C was found to be expressed by e-EPCs and endothelial cells. Blocking JAM-C did not affect adhesion of e-EPCs to endothelial monolayers in vitro but, interestingly, it did reduce their adhesion to tumor endothelium in vivo. The most striking effect of JAM-C blocking was on tube formation on matrigel in vitro and the incorporation and sprouting of e-EPCs to tumor endothelium in vivo. Our results demonstrate that JAM-C mediates e-EPC recruitment to tumor angiogenic sites, i.e., coordinated homing of EPCs to the perivascular niche, where they cluster and interact with tumor blood vessels. This suggests that JAM-C plays a critical role in the process of vascular assembly and may represent a potential therapeutic target to control tumor angiogenesis

An improved ontological representation of dendritic cells as a paradigm for all cell types

The Cell Ontology (CL) is designed to provide a standardized representation of cell types for data annotation. Currently, the CL employs multiple is_a relations, defining cell types in terms of histological, functional, and lineage properties, and the majority of definitions are written with sufficient generality to hold across multiple species. This approach limits the CL’s utility for cross-species data integration. To address this problem, we developed a method for the ontological representation of cells and applied this method to develop a dendritic cell ontology (DC-CL). DC-CL subtypes are delineated on the basis of surface protein expression, systematically including both species-general and species-specific types and optimizing DC-CL for the analysis of flow cytometry data. This approach brings benefits in the form of increased accuracy, support for reasoning, and interoperability with other ontology resources. 104. Barry Smith, “Toward a Realistic Science of Environments”, Ecological Psychology, 2009, 21 (2), April-June, 121-130. Abstract: The perceptual psychologist J. J. Gibson embraces a radically externalistic view of mind and action. We have, for Gibson, not a Cartesian mind or soul, with its interior theater of contents and the consequent problem of explaining how this mind or soul and its psychological environment can succeed in grasping physical objects external to itself. Rather, we have a perceiving, acting organism, whose perceptions and actions are always already tuned to the parts and moments, the things and surfaces, of its external environment. We describe how on this basis Gibson sought to develop a realist science of environments which will be ‘consistent with physics, mechanics, optics, acoustics, and chemistry’

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis

EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs