Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Relations between Speed-Accuracy Trade-Off and Muscle Synergy in Isometric Contraction Tasks.

Muscle synergy is the theory that movements are controlled by a module of coordinated combined muscles. This theory is thought to solve the degrees-of-freedom problem in the musculoskeletal system. Previous studies have investigated the robustness of muscle synergies under conditions such as varying speeds and required degrees of accuracy. One of the principles of human movement is that when movement becomes faster, spatial accuracy is reduced. This is called the "speed-accuracy trade-off" (SAT), and many models have been proposed to explain this phenomenon. Studies on muscle synergies have shown that muscle synergy modules are robust against changes in speed; however, the relationship between SAT and motor control by muscle synergies remains unclear. Therefore, we investigated the relationship between changes in spatial accuracy and changes in speed and muscle synergies from measured behavioral data and surface electromyography. This was achieved by performing an isometric contraction task in which subjects exerted a horizontal force with various movement speeds. The results showed that the module structures of muscle synergies were robust against speed changes, and that the neural commands to muscle synergies changed in response to speed changes. In addition, changes in spatial accuracy with variations in speed tended to increase when movement was performed with a single muscle synergy. These results suggest that the number of muscle synergies used for movement may affect movement accuracy.Clinical Relevance-The results of this study suggest that the number of muscle synergies used for movement affects spatial accuracy.