Biology subjects

Duffy, K. R.

Publications and source records attributed to Duffy, K. R..

5 recordsLinked to original sources

bioRxiv2026

Intravital microscopy and statistical image analysis of early engraftment of haematopoietic stem cells reveal dynamic and evolving cellular behaviours linked to progressive differentiation.

Haematopoietic stem cells (HSCs) have long been used in the clinic for bone marrow transplantation applications, critical for the survival of an increasingly wide range of patients with haematological, oncological and immunological pathologies. Despite this, little is known about the mechanisms through which relatively few stem cells are able to regenerate the entire haematopoietic tissue of transplant recipients. To gain insights on this biological process, we used intravital microscopy of calvarium bone marrow, collecting tissue-wide images and a total of 850 hours of tracks of engrafting haematopoietic stem and progenitor cells from 24 hours to 8 days following injection in lethally irradiated recipients. Analysis of the data revealed that regenerating HSCs and their immediate progeny are highly dynamic, migrating through the parenchyma at both the microscopic and near-macroscopic scales, i.e. within and in-and-out of fields of view. HSC-derived cell clusters, expanded locally between day 2 and day 4 post-transplant, leading to patches of densely populated bone marrow by day 8. Single cell level analysis highlighted increasingly heterogenous cellular behaviours over time. Track clustering based on migration and niche interaction parameters combined with post-tracking whole mount immunostaining revealed that persistence in the vicinity of nestin-GFP perivascular cells and relatively slow movement correlated with stemness, while more heterogeneous niche interactions coupled with slowest or faster migration correlated with differentiation, validated through flow cytometry analysis and functional studies. The findings presented here shed light on the fundamental principles driving haematopoietic regeneration in transplantation settings.

developmental biology↗

bioRxiv2024

The DivisionCounter, a method for counting large ranges of cell divisions in vivo, reveals cell dynamics of leukemic cell killing via CAR-T therapy

Cell division drives multicellular growth and its dysregulation can cause disease. While approximately 44 divisions are needed to produce without death all 1013 cells in the human body, current methods are limited to count 10 cell divisions in vivo across diverse mammalian cell types. Here we introduce the DivisionCounter, a method to count cell division in vivo over large division ranges ([~]70 divisions) using an easy fluorescence readout. We demonstrate that the DivisionCounter accurately measures the average cell division count of cells both in vitro and in vivo. Its use revealed that leukemia tumor division rates are independent of the organs specific microenvironment and CAR-T cell treatment, providing an estimate of tumor death rates in vivo. The DivisionCounter method holds unique potential for quantifying contributions of cell division, death, and migration to the growth of healthy and pathological mammalian tissues.

immunology↗

bioRxiv2022

The Impact of Brief Monocular Retinal Inactivation on the Central Visual System During Postnatal Development

During a critical period of postnatal life, monocular deprivation (MD) of kittens by eyelid closure reduces the size of neurons in layers of the dorsal lateral geniculate nucleus (dLGN) connected to the deprived eye, and shifts cortical ocular dominance in favor of the non-deprived eye, modeling deprivation amblyopia in humans. Following long-term MD, temporary retinal inactivation of the non-deprived eye with microinjection of tetrodotoxin can promote superior recovery from MD, and at older ages, in comparison to conventional occlusion therapy. This suggests that monocular inactivation (MI) is a more potent approach to producing neural plasticity than occlusion. In the current study we assessed the modification of neuron size in the dLGN as a means of measuring the impact of a brief period of MI imposed at different ages during postnatal development. The biggest impact of inactivation was observed when it occurred at the peak of the critical period for ocular dominance plasticity. The effect of MI was evident in both the binocular and monocular segments of the dLGN, distinguishing it from MD that produces changes only within the binocular segment. With increasing age, the capacity for inactivation to alter postsynaptic cell size diminished but was still significant beyond the classic critical period. In comparison to MD, inactivation consistently produced effects that were about double in magnitude, and inactivation exhibited efficacy to produce neural modifications at older ages than MD. Notwithstanding the large neural alterations precipitated by inactivation, its anatomical effects were remediated with a short period of binocular visual experience, and vision through the previously inactivated eye fully recovered after washout of TTX. Our results demonstrate that MI is a potent means of modifying the visual pathway, and does so beyond the age at which occlusion is effective. The magnitude and longevity of inactivation to evoke neural modification highlights its potential to ameliorate disorders of the visual system such as amblyopia.

neuroscience↗

bioRxiv2021

Correction of amblyopia in cats and mice after the critical period

Monocular deprivation early in development causes amblyopia, a severe visual impairment. Prognosis is poor if therapy is initiated after an early critical period. However, clinical observations have shown that recovery from amblyopia can occur later in life when the non-deprived (fellow) eye is removed. The traditional interpretation of this finding is that vision is improved by relieving interocular suppression in primary visual cortex. However, an alternative explanation is that elimination of activity in the fellow eye establishes conditions in visual cortex that enable the weak connections from the amblyopic eye to gain strength. Here we show in cats and mice that temporary inactivation of the fellow eye is sufficient to promote a full and enduring recovery from amblyopia at ages when conventional treatments fail. Thus, connections serving the amblyopic eye are capable of substantial plasticity beyond the critical period, and this potential is unleashed by reversibly silencing the fellow eye.

neuroscience↗

bioRxiv2019

Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

The advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.

immunology↗