This study provides transcriptomic characterization of the cells of the crista ampullaris, sensory structures at the base of the semicircular canals that are critical for vestibular function. We performed single cell RNA-seq on ampullae microdissected from E16, E18, P3 and P7 mice. Cluster analysis identified the hair cells, support cells and glia of the crista as well as dark cells and other nonsensory epithelial cells of the ampulla, mesenchymal cells, vascular cells, macrophages and melanocytes. Cluster-specific expression of genes predicted their spatially restricted domains of gene expression in the crista and ampulla. Analysis of cellular proportions across developmental time showed dynamics in cellular composition. The new cell types revealed by single cell RNA-seq could be important for understanding crista function and the markers identified in this study will enable the examination of their dynamics during development and disease.

Summary:The CD34 antigen is expressed by human hematopoietic progenitor and stem cells. These cells are capable of reconstituting marrow function after marrow-ablative chemo-radiotherapy. Several different technologies have been developed for the separation of CD34 ؉ cells from bone marrow or peripheral blood stem cell (PBSC) components. We used an immunomagnetic separation technique to enrich CD34 ؉ cells from PBSC components in anticipation of autologous transplantation for patients with B lymphoid malignancies. Twenty-nine patients enrolled on this study and received mobilization chemotherapy followed by G-CSF. Of these, 21 achieved a peripheral blood CD34 ؉ cell level of at least 2.0 ؋ 10 4 /l required by protocol for separation of the stem cell components. A median of three components per patient was collected for processing. The average CD34 ؉ cell concentration in the components after apheresis was 1.0 ؎ 1.2%. After the CD34 ؉ cell selection, the enriched components contained 0.6 ± 0.6% of the starting nucleated cells. The recovery of CD34 ؉ cells, however, averaged 58.4 ؎ 19.2% of the starting cell number, with a purity of 90.8 ؎ 6.5%. Overall depletion of CD34 ؊ cells was 99.96 ؎ 0.06%. Nineteen patients were treated with marrow-ablative conditioning regimens and received an average of 6.2 ؎ 2.0 ؋ 10 6 CD34 ؉ cells/kg body weight. These patients recovered to an ANC >0.5 ؋ 10 9 /l at a median of 11 days (range 8-14), and platelet transfusion independence at a median of 9 days (range 5-13). Four patients died of transplant-related complications or relapse before 100 days after transplantation. No patient required infusion of unseparated cells because of failure of sustained bone marrow function. These data demonstrate that peripheral blood-derived CD34 ؉ cells enriched by use of an immunomagnetic separation technique are capable of rapid engraftment after autologous transplantation.

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We conducted 2 sequential studies of donor leukocyte infusion (DLI) in 26 patients with chronic myeloid leukemia in hematologic relapse after unmodified allogeneic bone marrow transplantation. In the first study, cells for DLI were collected from 13 donors who were not treated with granulocyte colony-stimulating factor (G-CSF) (group 1). In the second study, cells were collected from 13 donors who received G-CSF before apheresis (group 2) in an attempt to avoid aplasia after DLI. Patients in group 2 received 550-fold more CD34+ cells than those in group 1. We found no significant difference in the incidence (31% versus 22%), onset time (41 vs. 48 days), or duration (15 vs. 14 days) of cytopenia after DLI in the 2 groups. G-CSF given to donors before collection of cells did not prevent aplasia. These findings support the hypothesis that the pathogenesis of aplasia after DLI is not restricted to the destruction of recipient hematopoietic cells but also involves failure of donor hematopoiesis by undefined mechanisms.