Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques.

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

Self-assembling human skeletal organoids for disease modeling and drug testing.

Skeletal conditions represent a considerable challenge to health systems globally. Barriers to effective therapeutic development include a lack of accurate preclinical tissue and disease models. Most recently, work was attempted to present a novel whole organ approach to modeling human bone and cartilage tissues. These self-assembling skeletal organoids mimic the cellular milieu and extracellular organization present in native tissues. Bone organoids demonstrated osteogenesis and micro vessel formation, and cartilage organoids showed evidence of cartilage development and maturation. Skeletal organoids derived from both bone and cartilage tissues yielded spontaneous polarization of their cartilaginous and bone components. Using these hybrid skeletal organoids, we successfully generated "mini joint" cultures, which we used to model inflammatory disease and test Adenosine (A2A ) receptor agonists as a therapeutic agent. The work and respective results indicated that skeletal organoids can be an effective biological model for tissue development and disease as well as to test therapeutic agents.

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

Tissue resident (TR) immune cells play important roles in facilitating tissue homeostasis, coordinating immune responses against infections and tumors, and maintaining immunological memory. While studies have shown these cells are distinct phenotypically and functionally from cells found in the peripheral blood (PB), the clonal relationship between these populations across tissues has not been comprehensively studied in primates or humans. We utilized autologous transplantation of rhesus macaque hematopoietic stem and progenitor cells containing high diversity barcodes to track the clonal distribution of T, B, myeloid and natural killer (NK) cell populations across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract, in comparison with PB longitudinally post-transplantation, in particular we focused on NK cells which do not contain endogenous clonal markers and have not been previously studied in this context. T cells demonstrated tissue-specific clonal expansions as expected, both overlapping and distinct from blood T cells. In contrast, B and myeloid cells showed a much more homogeneous clonal pattern across various tissues and the blood. The clonal distribution of TR NK was more heterogenous between individual animals. In some animals, as we have previously reported, we observed large PB clonal expansions in mature CD56-CD16+ NK cells. Notably, we found a separate set of highly expanded PB clones in CD16-CD56- (DN) NK subset that were also contributing to TR NK cells in all tissues examined, both in TR CD56-CD16+ and DN populations but absent in CD56+16- TR NK across all tissues analyzed. Additionally, we observed sets of TR NK clones specific to individual tissues such as lung or GI tract and sets of TR NK clones shared across liver and spleen, distinct from other tissues. Combined with prior functional data that suggests NK memory is restricted to liver or other TR NK cells, these clonally expanded TR NK cells may be of interest for future investigation into NK cell tissue immunological memory, with implications for development of NK based immunotherapies and an understanding of NK memory.

Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids.

How stem cells self-organize to form structured tissues is an unsolved problem. Intestinal organoids offer a model of self-organization as they generate stem cell zones (SCZs) of typical size even without a spatially structured environment. Here we examine processes governing the size of SCZs. We improve the viability and homogeneity of intestinal organoid cultures to enable long-term time-lapse imaging of multiple organoids in parallel. We find that SCZs are shaped by fission events under strong control of ion channel-mediated inflation and mechanosensitive Piezo-family channels. Fission occurs through stereotyped modes of dynamic behavior that differ in their coordination of budding and differentiation. Imaging and single-cell transcriptomics show that inflation drives acute stem cell differentiation and induces a stretch-responsive cell state characterized by large transcriptional changes, including upregulation of Piezo1. Our results reveal an intrinsic capacity of the intestinal epithelium to self-organize by modulating and then responding to its mechanical state.

Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining.

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.