Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging.

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

Cytogenetics and genomics of acute myeloid leukemia.

The diversity of genetic and genomic abnormalities observed in acute myeloid leukemia (AML) reflects the complexity of these hematologic neoplasms. The detection of cytogenetic and molecular alterations is fundamental to diagnosis, risk stratification and treatment of AML. Chromosome rearrangements are well established in the diagnostic classification of AML, as are some gene mutations, in several international classification systems. Additionally, the detection of new mutational profiles at relapse and identification of mutations in the pre- and post-transplant settings are illuminating in understanding disease evolution and are relevant to the risk assessment of AML patients. In this review, we discuss recurrent cytogenetic abnormalities, as well as the detection of recurrent mutations, within the context of a normal karyotype, and in the setting of chromosome abnormalities. Two new classification schemes from the WHO and ICC are described, comparing these classifications in terms of diagnostic criteria and entity definition in AML. Finally, we discuss ways in which genomic sequencing can condense the detection of gene mutations and chromosome abnormalities into a single assay.

RAD21 mutations in acute myeloid leukemia.

The cohesin complex is a ring-shaped protein structure involved in DNA repair and chromosomal segregation. Studies have showed that genomic alterations in the cohesin complex members are among the initial occurrences in the development of acute myeloid leukemia (AML). STAG2 is the most commonly mutated and best-studied member of the cohesin complex in AML and mutations in this gene have been associated with adverse outcomes and are diagnostically relevant. However, the exact role of mutations in other members of the cohesin complex in the development of myeloid neoplasia is controversial. In this single institution study, we retrospectively reviewed data from the molecular profiles of 1,381 AML patients and identified 14 patients with mutations in RAD21, another member of the cohesin complex. We evaluated the frequency, mutational profile, clinico-pathologic features, and prognostic impact of RAD21 in this cohort. This study showed that RAD21-mutated AML often associates with monocytic differentiation, CD7 expression, co-existing mutations in epigenetic regulators, a normal karyotype, and poor prognosis. Our findings provide additional insights into the morphologic, immunophenotypic, and genomic profile of RAD21 mutation-positive AML and suggest that RAD21 mutations should be evaluated for independent prognostic significance in AML.

Continuous Low-Intensity Ultrasound Improves Cartilage Repair in Rabbit Model of Subchondral Injury.

Subchondral drilling (SD), a bone marrow stimulation technique, is used to repair cartilage lesions that lack regenerative potential. Cartilage repair outcomes upon SD are typically fibrocartilaginous in nature with inferior functionality. The lack of cues to foster the chondrogenic differentiation of egressed mesenchymal stromal cells upon SD can be attributed for the poor outcomes. Continuous low-intensity ultrasound (cLIUS) at 3.8 MHz is proposed as a treatment modality for improving cartilage repair outcomes upon marrow stimulation. Bilateral defects were created by SD on the femoral medial condyle of female New Zealand white rabbits (n = 12), and the left joint received cLIUS treatment (3.8 MHz, 3.5 Vpp, 8 min/application/day) and the contralateral right joint served as the control. On day 7 postsurgery, synovial fluid was aspirated, and the cytokine levels were assessed by Quantibody™ assay. Rabbits were euthanized at 8 weeks and outcomes were assessed macroscopically and histologically. Defect areas in the right joints exhibited boundaries, incomplete fill, irregular cartilage surfaces, loss of glycosaminoglycan (GAG), and absence of chondrocytes. In contrast, the repaired defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte morphology, COL2A1 staining, and columnar organization. Synovial fluid collected from cLIUS-treated left knee joints had lower levels of IL1, TNFα, and IFNγ when compared to untreated right knee joints, alluding to the potential of cLIUS to mitigate early inflammation. Further at 8 weeks, left knee joints (n = 12) consistently scored higher on the O'Driscoll scale, with a higher percent hyaline cartilage score. No adverse impact on bone or change in the joint space was noted. Upon a single exposure of cLIUS to TNFα-treated cells, nuclear localization of pNFκB and SOX9 was visualized by double immunofluorescence and the expression of markers associated with the NFκB pathway was assayed by quantitative real-time polymerase chain reaction. cLIUS extends its chondroprotective effects by titrating pNFκB levels, preventing its nuclear translocation, while maintaining the expression of SOX9, the collagen II transcription factor. Our combined results demonstrate that healing of chondral defects treated with marrow stimulation by SD can be accelerated by employing cLIUS regimen that possesses chondroinductive and chondroprotective properties. Impact statement Repair of cartilage represents an unsolved biomedical burden. In vitro, continuous low-intensity ultrasound (cLIUS) has been demonstrated to possess chondroinductive and chondroprotective potential. To our best knowledge, the use of cLIUS to improve cartilage repair outcomes upon marrow stimulation, in vivo, has not been reported and our work reported here fills that gap. Our results demonstrated enhanced cartilage repair outcomes under cLIUS (3.8 MHz) in a rabbit model of subchondral injury by subchondral drilling. Enhanced repair stemmed from mesenchymal stem cell differentiation in vivo and the subsequent synthesis of articular cartilage-specific matrix.