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Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

ABSTRACT: Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45^dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45^dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45^dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45^dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

SUBMITTER: Ayabe T

PROVIDER: S-EPMC9510753 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Ayabe Taro T Hisasue Masaharu M Yamada Yoko Y Nitta Suguru S Kikuchi Kaoruko K Neo Sakurako S Matsumoto Yuki Y Horie Ryo R Kawamoto Kosuke K

Frontiers in veterinary science 20220912

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HS ...[more]

PMID: 36172613

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Project description:Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45lowCD271+ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r = -0.527, p < 0.0001) or flow cytometry (r = -0.307, p = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, p < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p < 0.0001, and 27%, p < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45lowCD271+ MSCs from young and old donors (n = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45lowCD271+ MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.

Project description:IntroductionThe colony forming assay (CFA) stands as a cornerstone technique for evaluating the clonal expansion ability of single cancer cells and is crucial for assessing drug efficacy. However, traditional CFAs rely on labor-intensive, endpoint manual counting, offering limited insights into the dynamic effects of treatment. To overcome these limitations, we developed an Artificial Intelligence (AI)-assisted automated CFA combining time-lapse microscopy for real-time tracking of colony formation.MethodsUsing B-acute lymphoblastic leukemia (B-ALL) cells from an E2A-PBX1 mouse model, we cultured them in a collagen-based 3D matrix with cytokines under static conditions in a low volume (60 µl) culture vessel and validated its comparability to methylcellulose-based media. No significant differences in final colony count or plating efficiency were observed. Our automated platform utilizes a deep learning and multi-object tracking approach for colony counting. Brightfield images were used to train a YOLOv8 object detection network, achieving a mAP50 score of 86% for identifying single cells, clusters, and colonies, and 97% accuracy for Z-stack colony identification with a multi-object tracking algorithm. The detection model accurately identified the majority of objects in the dataset.ResultsThis AI-assisted CFA was successfully applied for density optimization, enabling the determination of seeding densities that maximize plating efficiency (PE), and for IC50 determination, offering an efficient, less labor-intensive method for testing drug concentrations. In conclusion, our novel AI-assisted automated colony counting platform enables automated, high-throughput analysis of colony dynamics, significantly reducing labor and increasing accuracy. Furthermore, it allows detailed, long-term studies of cell-cell interactions and treatment responses using live-cell imaging and AI-assisted cell tracking.DiscussionFuture integration with a perfusion-based drug screening system promises to enhance personalized cancer therapy by optimizing broad drug screening approaches and enabling real-time evaluation of therapeutic efficacy.

Project description:Stem cells are special cells with the distinctive capability to self-renew, forming a new pool of undifferentiated stem cells. They are also able to differentiate into lineage-specific cell types that are specialized and matured. Thus, stem cells are considered as the building blocks of tissues and organs in which they reside. Among the many types of stem cells, hematopoietic stem cells (HSCs) are the most studied adult stem cells and are considered as a promising source of cells for applications in the clinical and basic sciences. Historically, research on HSCs was initiated in the 1940s, where in a groundbreaking experiment, intravenously injected bone marrow (BM) cells prevented the death of irradiated mice by restoring blood cell production. Since then, HSCs have been studied and utilized in medical therapies and research for over several decades. Over time, more sophisticated tools have been developed to evaluate the behaviour of specifically purified subsets of hematopoietic cells that have the capacity to produce blood cells. One of the established tools is the colony-forming units (CFUs) assay. This assay facilitates the identification, enumeration, and analysis of colonies formed by differentiated hematopoietic stem and progenitor cells (HSPCs) from myeloid, erythroid and lymphoid lineages. Hence, the CFUs assay is a fundamental in vitro platform that allows functional studies on the lineage potential of an individual HSPCs. The outcomes of such studies are crucial in providing critical insights into hematopoiesis. In this review, we explore the fundamental discoveries concerning the CFUs assay by covering the following aspects: (i) the historical overview of the CFUs assay for the study of clonal hematopoiesis involving multilineage potential of HSPCs, (ii) its use in various experimental models comprising humans, mice/rodents, zebrafish and induced pluripotent stem cells (iPSCs) and (iii) research gaps and future direction concerning the role of CFUs assay in clinical and basic sciences. Overall, the CFUs assay confers a transformative platform for a better understanding of HSPCs biology in governing hematopoiesis.

Dataset Information

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Publications

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

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