Biology subjects

Vijaykumar, A.

Publications and source records attributed to Vijaykumar, A..

5 recordsLinked to original sources

bioRxiv2023

Tissue-scale dynamic mapping of Hematopoietic Stem Cells and supportive niche cells in the fetal liver

The fetal liver (FL) plays a fundamental role in the ontogeny of the hematopoietic system, by transiently providing a fertile microenvironment for the maturation, proliferation and expansion of fetal hematopoietic progenitors, as well as definitive hematopoietic stem cells (HSCs). Nonetheless, the cellular make up and identity of HSC niches in the FL remain poorly described. Here we employed a combination of 3D quantitative microscopy, bulk mRNA-seq, flow cytometry and cell-specific reporter models, to dissect the spatiotemporal dynamics of putative niche cells and HSCs in the FL microenvironment. We find that at peak stages of FL hematopoiesis, pro-hematopoietic cytokines are promiscuously expressed by endothelial, mesenchymal cells and most prominently by hepatoblasts. These multicellular consortia of parenchymal/stromal cells are spatially predominant in the FL, thus providing unrestricted access to supportive factors throughout the entire tissue. Accordingly, in these early phases HSCs are found scattered across the parenchyma not displaying obvious spatial biases within the broad microanatomy of the FL, but exhibiting clustering behaviors. This highly conducive microenvironment is transient and gets rapidly remodeled through hepatoblast differentiation and sterile inflammatory signaling, leading to the downregulation of hematopoietic factors and the contraction of supportive niches, which temporarily coincide with the exit of HSCs towards emergent BM tissues.

developmental biology↗

bioRxiv2023

Adrenal extramedullary hematopoiesis as an inducible model of the adult hematopoietic niche

Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and stromal cells. While the BM niche forms a complex structure, evidence exists for simpler, albeit functional, extramedullary hematopoietic niches. However, the composition of what constitutes the simplest unit of an HSPC supportive microenvironment remains largely unknown. Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon splenectomy and hormonal stimulation, the adult adrenal gland can be induced to recruit and host HSPC function, including serial transplantation. Furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR) cells. Mirroring this, we found CXCL12+ cells in patient samples obtained from a local cohort of myelolipoma, a benign adrenal tumor composed of adipose and hematopoietic tissue that constitutes the most common site of extramedullary hematopoiesis specific to the adult. We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

cell biology↗

bioRxiv2021

Particle Uptake Driven Phagocytosis in Macrophages and Neutrophils Enhances Bacterial Clearance

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

immunology↗

bioRxiv2021

CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration ([~]80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.

molecular biology↗

bioRxiv2019

DETECTing Merkel cell Polyomavirus in Merkel Tumours

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post integration ([~]80% cases), or by UV mediated DNA damage. Interestingly, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this differential information of significant diagnostic and prognostic value. Also, MCV as a causative agent also provides a direct target for therapy in virus positive MCC patients. Currently, the methods used for diagnosis of MCV in tumours are often tedious, discordant and unreliable. In this study we used a guided molecular scissors based - DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme - gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC tumours, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that this sophisticated MCV DETECTR system can detect MCV integrated in Merkel tumour rapidly, specifically and with femto-molar sensitivity. This new MCV DNA detecting system is promising and we hope it can be coupled with histopathological and immunohistochemical studies to diagnose the viral status of MCC in clinics in the near future.

molecular biology↗