Biology subjects

Chandrakanthan, V.

Publications and source records attributed to Chandrakanthan, V..

7 recordsLinked to original sources

bioRxiv2021

Defined microenvironments trigger in vitro gastrulation in human pluripotent stem cells

Embryogenesis is orchestrated through local morphogen gradients and endometrial constraints that give rise to the three germ layers in a well-defined assembly. In vitro models of embryogenesis have been demonstrated by treating pluripotent stem cells in adherent or suspension culture with soluble morphogens and small molecules, which leads to tri-lineage differentiation. However, treatment with exogenous agents override the subtle spatiotemporal changes observed in vivo that ultimately underly the human body plan. Here we demonstrate how microconfinement of pluripotent stem cells on hydrogel substrates catalyses gastrulation-like events without the need for supplements. Within six hours of initial seeding, cells at the boundary show elevated cytoskeletal tension and yes-associated protein (YAP) activity, which leads to changes in cell and nuclear morphology, epithelial to mesenchymal transition, and emergence of defined patterns of primitive streak containing SRY-Box Transcription Factor 17 (SOX17)+ T/BRACHYURY+ cells. Immunofluorescence staining, transcript analysis, and the use of pharmacological modulators reveal a role for mechanotransduction-coupled non-canonical wingless-type (WNT) signalling in promoting epithelial to mesenchymal transition and multilayered organization within the colonies. These microscale gastruloids were removed from the substrate and encapsulated in 3D hydrogels, where biomaterials properties correspond to maintenance and spatial positioning of the primitive streak. Together, this approach demonstrates how materials alone can nurture embryonic gastrulation, thereby providing an in vitro model of early development.

bioengineering↗

bioRxiv2021

Self-renewing tissue-resident endothelial-macrophage progenitor cells originate from yolk sac and are a local source of inflammation and neovascularization in postnatal aorta

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and forming perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally.

cell biology↗

bioRxiv2021

Mesoderm-Derived PDGFRA+ Cells Regulate the Emergence of Hematopoietic Stem Cells in the Dorsal Aorta

Mouse hematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5) on the ventral surface of the dorsal aorta, by endothelial-to-hematopoietic transition (EHT). We investigated whether cells with mesenchymal stem cell-like activity, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta- gonad-mesonephros (AGM) and contribute to the structural development of the dorsal aorta and EHT. Using transgenic mice, we demonstrate a lineage hierarchy for AGM stromal cells and traced the E10.5/E11.5 aortic endothelium and HSCs to mesoderm derived (Mesp1) PDGFRA+ stromal cells (Mesp1der PSCs). Mesp1der PSCs dominate the sub-endothelial and ventral stroma in the E10.5-E11.5 AGM but by E13.5 were replaced by neural crest (Wnt1) derived PDGFRA+ stromal cells (Wnt1der PSCs). Co-aggregating non-hemogenic embryonic and adult endothelial cells with Mesp1der PSCs but not with Wnt1der PSCs resulted in activation of a hematopoietic transcriptional program in endothelial cells accompanied by EHT and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA signalling or BMP, WNT, NOTCH signalling interrupted this reprogramming event. This partnership between endothelial cells and AGM Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

developmental biology↗

bioRxiv2020

Label-free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes using Inertial Microfluidics

To advance our understanding of cardiomyocyte identity and function, we need appropriate tools to isolate pure primary cardiomyocytes. We have developed a label-free method to purify viable cardiomyocytes from mouse neonatal hearts using a simple inertial microfluidics biochip. Cardiomyocytes were sorted from neonatal hearts and isolated to >90% purity and their physico-mechanical properties were evaluated using real time deformability cytometry. Purified cardiomyocytes were viable and retained their identity and function as depicted by expression of cardiac specific markers and contractility. Furthermore, we showed that cardiomyocytes have a distinct physico-mechanical phenotype that could be used as an intrinsic biophysical marker to distinguish these cells from other cell types within the heart. Taken together, this cardiomyocyte isolation and phenotyping method could serve as a valuable tool to progress our understanding of cardiomyocyte identity and function, which will ultimately benefit many diagnostic development and cardiac treatment studies.

bioengineering↗

bioRxiv2020

Capturing Neonatal Cardiomyocyte - Endothelial interactions on-a-Chip

The neonatal heart has been the focus of numerous investigations due to its inherent regenerative potential. However, the interactions between neonatal cardiomyocytes (CMs) and endothelial cells (ECs) have been difficult to model and study due to the lack of an appropriate device. Here, we developed a method to culture primary neonatal CMs and ECs in a microchip and characterise their behavioural properties over a 14-day period. By implementing cell migration analyses coupled with immunostaining and confocal microscopy, we were able to identify and quantify sub-populations of migratory and non-migratory ECs. In CM-EC co-cultures, migrating ECs were found to move in higher numbers and longer distances compared to migrating CMs. In the presence of CMs, non-migrating ECs established connexin gap junctions and formed CM-EC cell aggregates, which were likely a priming event for endothelial organoid formation. This microfluidic device also enabled us to visualise the temporal sequence organoid formation and phenomena such as collective cell migration, CM-EC trans-differentiation and synchronisation of CM beating. This microchip based culture system has potential applications for tissue engineering and drug discovery.

bioengineering↗

bioRxiv2020

Induction of Muscle Regenerative Multipotent Stem Cells from Human Adipocytes by PDGF-AB and 5-Azacytidine.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-Azacytidine into multipotent stem cells with stromal cell characteristics. To generate a product that is amenable for therapeutic application, we have modified and optimised culture conditions to reprogram human adipocytes into induced multipotent stem cells (iMS) and expand them in vitro. The basal transcriptomes of adipocyte-derived iMS cells and matched adipose-tissue-derived mesenchymal stem cells were remarkably similar. However, there were distinct changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to new muscle, bone, cartilage and blood vessels with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibres without ectopic tissue formation. Taken together, human adipocyte derived iMS cells regenerate tissues in a context dependent manner without ectopic or neoplastic growth.

cell biology↗

bioRxiv2017

PDGFRα signaling in cardiac stem and stromal cells modulates quiescence, metabolism and self-renewal, and promotes anatomical and functional repair

The interstitial and perivascular spaces of the mammalian heart contain a highly interactive tissue community essential for cardiac homeostasis, repair and regeneration. Mesenchymal cells (fibroblasts) are one of the most abundant cell types, playing key roles as sentinels, tissue architects, paracrine signaling hubs and lineage precursors, and are linked to heart disease through their roles in inflammation and fibrosis. Platelet-derived growth factors (PDGFs) are secreted by several cell types involved in cardiac injury and repair, and are recognized mitogens for cardiac fibroblasts and mesenchymal stem cells. However, their roles are complex and investigations of their impact on heart repair have produced contrasting outcomes, leaving therapeutic potential uncertain. Here, we use new approaches and tools, including single cell RNA sequencing, to explore cardiac fibroblast heterogeneity and how PDGF receptor (PDGFR) signaling impacts fibroblasts during heart repair. Short-term systemic delivery of PDGF-AB to mice from the time of myocardial infarction (MI) led to enhanced anatomical and functional recovery. Underpinning these benefits was a priming effect, in which PDGF-AB accelerated exit of fibroblasts from quiescence and induced a higher translational biosynthetic capacity in both fibroblasts and macrophages without triggering fibrosis. Our study highlights the significant biosynthetic heterogeneity and plasticity in cardiac fibroblast populations, and suggests a rationale for a novel therapeutic approach to cardiac injury involving controlled stimulation of fibroblast activation.

cell biology↗