High-quality nuclei isolation from postmortem human heart muscle tissues for single-cell studies.

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

High-Quality Nuclei Isolation from Postmortem Human Heart Muscle Tissues for Single-Cell Studies.

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

The developmental stage of the hematopoietic niche regulates lineage in MLL-rearranged leukemia.

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2 axis.

For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development.

Next-Generation Sequencing Reveals Restriction and Clonotypic Expansion of Treg Cells in Juvenile Idiopathic Arthritis.

OBJECTIVE: Treg cell-mediated suppression of Teff cells is impaired in juvenile idiopathic arthritis (JIA); however, the basis for this dysfunction is incompletely understood. Animal models of autoimmunity and immunodeficiency demonstrate that a diverse Treg cell repertoire is essential to maintain Treg cell function. The present study was undertaken to investigate the Treg and Teff cell repertoires in JIA. METHODS: Treg cells (CD4+CD25+CD127(low) ) and Teff cells (CD4+CD25-) were isolated from peripheral blood and synovial fluid obtained from JIA patients, healthy controls, and children with Lyme arthritis. Treg cell function was measured in suppressive assays. The T cell receptor ß chain (TRB) was amplified by multiplex polymerase chain reaction and next-generation sequencing was performed, with amplicons sequenced using an Illumina HiSeq platform. Data were analyzed using ImmunoSEQ, International ImMunoGeneTics system, and the Immunoglobulin Analysis Tools. RESULTS: Compared to findings in controls, the JIA peripheral blood Treg cell repertoire was restricted, and clonotypic expansions were found in both blood and synovial fluid Treg cells. Skewed usage and pairing of TRB variable and joining genes, including overuse of gene segments that have been associated with other autoimmune conditions, was observed. JIA patients shared a substantial portion of synovial fluid Treg cell clonotypes that were private to JIA and not identified in Lyme arthritis. CONCLUSION: We identified restriction and clonotypic expansions in the JIA Treg cell repertoire with sharing of Treg cell clonotypes across patients. These findings suggest that abnormalities in the Treg cell repertoire, possibly engendered by shared antigenic triggers, may contribute to disease pathogenesis in JIA.

Perivascular deletion of murine Rac reverses the ratio of marrow arterioles and sinusoid vessels and alters hematopoiesis in vivo.

Hematopoietic stem cells (HSCs) are localized within specialized microenvironments throughout the BM. Nestin-expressing (Nestin(+)) mesenchymal stromal cells (MSCs) are important in the perivascular space. Rac is critical for MSC cell shape in vitro, whereas its function in MSCs in vivo remains poorly characterized. We hypothesized that deletion of Rac in the Nestin(+) cells would perturb the perivascular space, altering HSC localization and hematopoiesis. Nestin-Cre-directed excision of Rac1 in Rac3(-/-) mice reduces Nestin(+) cells in the marrow. We observed a 2.7-fold decrease in homing of labeled wild-type hematopoietic cells into Rac1(Δ/Δ)Rac3(-/-) mice compared with control mice. Rac1(Δ/Δ)Rac3(-/-) mice demonstrated a marked decrease in arterioles and an increase in the number and volume of venous sinusoids in the marrow that was associated with a reduction in the numbers of immunophenotypically and functionally-defined long-term HSCs in the marrow, a decrease in colony-forming cells and a reduction in circulating progenitors. Rac-deleted animals demonstrated a significant increase in trabecular bone. These data demonstrate that Rac GTPases play an important role in the integrity of perivascular space. Increased trabecular bone and sinusoidal space and decreased arteriolar volume in this model were associated with decreased HSC, underscoring the complexity of regulation of hematopoiesis in the perivascular space.

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment.

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.