Tissue registration and exploration user interfaces in support of a human reference atlas

Seventeen international consortia are collaborating on a human reference atlas (HRA), a comprehensive, high-resolution, three-dimensional atlas of all the cells in the healthy human body. Laboratories around the world are collecting tissue specimens from donors varying in sex, age, ethnicity, and body mass index. However, harmonizing tissue data across 25 organs and more than 15 bulk and spatial single-cell assay types poses challenges. Here, we present software tools and user interfaces developed to spatially and semantically annotate ( register ) and explore the tissue data and the evolving HRA. A key part of these tools is a common coordinate framework, providing standard terminologies and data structures for describing specimen, biological structure, and spatial data linked to existing ontologies. As of April 22, 2022, the registration user interface has been used to harmonize and publish data on 5,909 tissue blocks collected by the Human Biomolecular Atlas Program (HuBMAP), the Stimulating Peripheral Activity to Relieve Conditions program (SPARC), the Human Cell Atlas (HCA), the Kidney Precision Medicine Project (KPMP), and the Genotype Tissue Expression project (GTEx). Further, 5,856 tissue sections were derived from 506 HuBMAP tissue blocks. The second exploration user interface enables consortia to evaluate data quality, explore tissue data spatially within the context of the HRA, and guide data acquisition. A companion website is at https://cns-iu.github.io/HRA-supporting-information/

Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19

This article is made available for unrestricted research re-use and secondary analysis in any form or be any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Diabetes is associated with increased mortality from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2), the key entry factor for SARS-CoV-2 infection. Specifically, we analyzed five public scRNA-seq pancreas datasets and performed fluorescence in situ hybridization, western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) cases. These in silico and ex vivo analyses demonstrated prominent expression of ACE2 in pancreatic ductal epithelium and microvasculature, but we found rare endocrine cell expression at the mRNA level. Pancreata from individuals with COVID-19 demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression that was primarily limited to ducts. These results suggest SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, is an unlikely central pathogenic feature of COVID-19-related diabetes.We thank the families of the organ donors and autopsy subjects for the gift of tissues. We also thank Jill K. Gregory, CMI (Icahn School of Medicine at Mount Sinai, New York, NY) for preparing the graphical abstract. These efforts were supported by NIH P01 AI042288 and UC4 DK108132 (M.A.A.); JDRF (M.A.A.); NIH R01 DK122160 (M.C.-T.); NIH R01 AI134971 and P30 DK020541 (D.H.); JDRF 3-PDF-2018-575-A-N (V.V.D.H.); R01 DK093954 , R21 DK119800-01A1 , UC4 DK104166 , and U01 DK127786 (C.E.-M.); VA Merit Award I01BX001733 (C.E.-M.); Imaging Core of NIH/ NIDDK P30 DK097512 (C.E.-M.); gifts from the Sigma Beta Sorority , the Ball Brothers Foundation , and the George and Frances Ball Foundation (C.E.-M.); the Network for Pancreatic Organ Donors with Diabetes ( nPOD ; RRID: SCR_014641 ) ( 5-SRA-2018-557-Q-R ); and The Leona M. & Harry B. Helmsley Charitable Trust ( 2018PG-T1D053 ). The authors also wish to acknowledge the Islet and Physiology Core of the Indiana Diabetes Research Center ( P30DK097512 ). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Abstract: Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions

Spatial gradients of blood vessels and hematopoietic stem and progenitor cells within the marrow cavities of the human skeleton

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34+ and CD117+ hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34+ or CD117+ HSPC to the nearest trabecular surface was measured and binned in 50-μm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34+ HSPC population, 60% of the CD117+ HSPC population, and 72% of the BVFs were found within 100 μm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche

Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa(–/–) mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 10(12) particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways

Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects

Disrupting Inflammation-Associated CXCL8-CXCR1 Signaling Inhibits Tumorigenicity Initiated by Sporadic- and Colitis-Colon Cancer Stem Cells

Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics is developed, reducing sample loss and processing time, allowing the phosphoproteome profiling of mass-limited samples at the low nanogram level