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.

Nasal Airway Volumes are More Asymmetric in Skeletally Mature Patients With Cleft lip and Palate Than Controls on 3-Dimensional Analysis.

BACKGROUND: This study assesses nasal airway volumes in skeletally mature patients with CLP and healthy controls and examines the relationship among nasal volumes, cleft laterality, and facial asymmetry. METHODS: Computed tomography images from patients with CLP and controls were analyzed using Mimics Version 23.0 (Materialise, Leuven, Belgium). Relationships among nasal airway volume, cleft laterality, and facial asymmetry were compared. RESULTS: The 89 patients in this study included 66 (74%) CLP and 23 (17%) controls. Nasal airway volumes in CLP were more asymmetric than controls (26.8±17.5% vs. 17.2±14.4%; P=0.015). In UCLP, the smaller nasal airway was on the cleft side 81% of the time (P<0.001). Maximum airway stenosis was on the cleft side 79% of the time (P<0.001), and maximum stenosis was on the same side as the smaller airway 89% of the time (P<0.001). There was a mild linear relationship between nasal airway asymmetry and maximum stenosis (r=0.247, P=0.023). On 3-dimensional image reconstruction, the septum often bowed convexly into the cleft-sided nasal airway with a caudal deviation towards the noncleft side. Nasal airway asymmetry was not associated with facial midline asymmetry (P>0.05). CONCLUSION: The nasal airway is more asymmetric in patients with cleft lip and palate compared with the general population, with the area of maximum stenosis usually occurring on the cleft-sided airway. In patients with unilateral cleft lip and palate, the septum often bows into the cleft side, reducing the size of that nasal airway. Nasal airway asymmetry did not correlate with facial asymmetry.

Crowdsourcing the Impact of Illustration in Cleft Surgery Education.

OBJECTIVE: This study aimed to (1) assess layperson preferences for how surgical information is presented; (2) evaluate how the format of visual information relates to layperson comfort with undergoing surgery, perceptions of surgeon character traits, and beliefs about artistic skill impacting plastic surgery practice; and (3) identify sociodemographic characteristics associated with these outcomes. DESIGN: A survey was developed in which one of five standardized sets of information depicting a unilateral cleft lip repair was presented as (1) text alone, (2) quick sketches, (3) simple drawings, (4) detailed illustrations, or (5) photographs. SETTING: Online crowdsourcing platform. PARTICIPANTS: Raters aged 18 years and older from the United States. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): After viewing the surgical information, participants answered three sets of Likert scale questions. Ratings were averaged to produce three composite scores assessing (1) comfort with undergoing surgery (2) perceptions of surgeon character traits, and (3) beliefs about plastic surgery and artistry. RESULTS: Four hundred seventy-nine participants were included. Surgeon character traits score was highest among participants who viewed detailed illustrations at 4.46 ± 0.59, followed by photographs at 4.43 ± 0.54, text alone at 4.28 ± 0.59, simple drawings at 4.17 ± 0.67, and quick sketches at 4.17 ± 0.71 (p = 0.0014). Participants who viewed detailed illustrations rated surgical comfort score and plastic surgery and artistry score highest, although differences did not achieve statistical significance. CONCLUSIONS: Viewing detailed cleft lip repair illustrations was significantly associated with positive perceptions of surgeon character traits. Our data help to contextualize methods of communication and education valued by the public when seeking cleft care.

AKT but not MYC promotes reactive oxygen species-mediated cell death in oxidative culture.

Oncogenes can create metabolic vulnerabilities in cancer cells. We tested how AKT (herein referring to AKT1) and MYC affect the ability of cells to shift between respiration and glycolysis. Using immortalized mammary epithelial cells, we discovered that constitutively active AKT, but not MYC, induced cell death in galactose culture, where cells rely on oxidative phosphorylation for energy generation. However, the negative effects of AKT were temporary, and AKT-expressing cells recommenced growth after ∼15 days in galactose. To identify the mechanisms regulating AKT-mediated cell death, we used metabolomics and found that AKT-expressing cells that were dying in galactose culture had upregulated glutathione metabolism. Proteomic profiling revealed that AKT-expressing cells dying in galactose also upregulated nonsense-mediated mRNA decay, a marker of sensitivity to oxidative stress. We therefore measured levels of reactive oxygen species (ROS) and discovered that galactose-induced ROS exclusively in cells expressing AKT. Furthermore, ROS were required for galactose-induced death of AKT-expressing cells. We then confirmed that galactose-induced ROS-mediated cell death in breast cancer cells with upregulated AKT signaling. These results demonstrate that AKT but not MYC restricts the flexibility of cancer cells to use oxidative phosphorylation.This article has an associated First Person interview with the first author of the paper.

The FDA Reclassification of Cervical Pedicle and Lateral Mass Screws: A Case Study in Regulatory History.

The classification of medical devices by the Food and Drug Administration (FDA) involves rigorous scrutiny from specialized panels that designate devices as Class I, II, or III depending on their levels of relative risk to patient health. Posterior rigid pedicle screw systems were first classified by the FDA in 1984 and have since revolutionized the treatment of many spine pathologies. Despite this early classification by the FDA, posterior cervical pedicle and lateral mass screws were not reclassified from unclassified to Class III and then to Class II until 2019, nearly 35 years after their initial classification. This reclassification process involved a decades-long interplay between the FDA, formal panels, manufacturers, academic leaders, practicing physicians, and patients. It was delayed by lawsuits and a paucity of data demonstrating the ability to improve outcomes for cervical spinal pathologies. The off-label use of thoracolumbar pedicle screw rigid fixation systems by early adopters assisted manufacturers and professional organizations in providing the necessary data for the reclassification process. This case study highlights the collaboration between physicians and professional organizations in facilitating FDA reclassification and underscores changes to the current classification process that could avoid the prolonged dichotomy between common medical practice and FDA guidelines.

Cellular reprogramming in vivo initiated by SOX4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.

ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

All-trans retinoic acid induces durable tumor immunity in IDH-mutant gliomas by rescuing transcriptional repression of the CRBP1-retinoic acid axis.

Diffuse gliomas are epigenetically dysregulated, immunologically cold, and fatal tumors characterized by mutations in isocitrate dehydrogenase (IDH). Although IDH mutations yield a uniquely immunosuppressive tumor microenvironment, the regulatory mechanisms that drive the immune landscape of IDH mutant (IDHm) gliomas remain unknown. Here, we reveal that transcriptional repression of retinoic acid (RA) pathway signaling impairs both innate and adaptive immune surveillance in IDHm glioma through epigenetic silencing of retinol binding protein 1 (RBP1) and induces a profound anti-inflammatory landscape marked by loss of inflammatory cell states and infiltration of suppressive myeloid phenotypes. Restorative retinoic acid therapy in murine glioma models promotes clonal CD4 + T cell expansion and induces tumor regression in IDHm, but not IDH wildtype (IDHwt), gliomas. Our findings provide a mechanistic rationale for RA immunotherapy in IDHm glioma and is the basis for an ongoing investigator-initiated, single-center clinical trial investigating all-trans retinoic acid (ATRA) in recurrent IDHm human subjects.

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

Digging deeper into the early steps of cancer progression.

Epigenetically mediated cell signaling is emerging as a core principle of tumor biology. Through single cell multi-omic profiling, Burdziak, Alonso-Curbelo et al. have recently uncovered key epigenetic plasticity programs associated with cellular communication, shedding light on the role of epigenetic cell states during malignant transformation of the inflamed pancreas.

Dissecting the tumor microenvironment of epigenetically driven gliomas: Opportunities for single-cell and spatial multiomics.

Malignant gliomas are incurable brain neoplasms with dismal prognoses and near-universal fatality, with minimal therapeutic progress despite billions of dollars invested in research and clinical trials over the last 2 decades. Many glioma studies have utilized disparate histologic and genomic platforms to characterize the stunning genomic, transcriptomic, and immunologic heterogeneity found in gliomas. Single-cell and spatial omics technologies enable unprecedented characterization of heterogeneity in solid malignancies and provide a granular annotation of transcriptional, epigenetic, and microenvironmental states with limited resected tissue. Heterogeneity in gliomas may be defined, at the broadest levels, by tumors ostensibly driven by epigenetic alterations (IDH- and histone-mutant) versus non-epigenetic tumors (IDH-wild type). Epigenetically driven tumors are defined by remarkable transcriptional programs, immunologically distinct microenvironments, and incompletely understood topography (unique cellular neighborhoods and cell-cell interactions). Thus, these tumors are the ideal substrate for single-cell multiomic technologies to disentangle the complex intra-tumoral features, including differentiation trajectories, tumor-immune cell interactions, and chromatin dysregulation. The current review summarizes the applications of single-cell multiomics to existing datasets of epigenetically driven glioma. More importantly, we discuss future capabilities and applications of novel multiomic strategies to answer outstanding questions, enable the development of potent therapeutic strategies, and improve personalized diagnostics and treatment via digital pathology.

Challenges and advice for MD/PhD applicants who are underrepresented in medicine.

The importance of diversity is self-evident in medicine and medical research. Not only does diversity result in more impactful scientific work, but diverse teams of researchers and clinicians are necessary to address health disparities and improve the health of underserved communities. MD/PhD programs serve an important role in training physician-scientists, so it is critical to ensure that MD/PhD students represent diverse backgrounds and experiences. Groups who are underrepresented in medicine and the biomedical sciences include individuals from certain racial and ethnic backgrounds, individuals with disabilities, individuals from disadvantaged backgrounds, and women. However, underrepresented students are routinely discouraged from applying to MD/PhD programs due to a range of factors. These factors include the significant cost of applying, which can be prohibitive for many students, the paucity of diverse mentors who share common experiences, as well as applicants' perceptions that there is inadequate support and inclusion from within MD/PhD programs. By providing advice to students who are underrepresented in medicine and describing steps programs can take to recruit and support minority applicants, we hope to encourage more students to consider the MD/PhD career path that will yield a more productive and equitable scientific and medical community.