Biology subjects

Rodriguez-Fraticelli, A.

Publications and source records attributed to Rodriguez-Fraticelli, A..

3 recordsLinked to original sources

bioRxiv2024

Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing

Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to [~]70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.

cell biology↗

bioRxiv2024

Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan

Current approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.

genomics↗

bioRxiv2019

An engineered CRISPR/Cas9 mouse line for simultaneous readout of lineage histories and gene expression profiles in single cells

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Particularly in the context of stem cell biology, analysis of single cell lineages in their native state has elucidated novel fates and highlighted heterogeneity of function. Coupling of such ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CARLIN (for CRISPR Array Repair LINeage tracing) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures based on in vivo stem cell function without a need for markers or cell sorting.

developmental biology↗