An NAD+-dependent metabolic checkpoint regulates hematopoietic stem cell activation and aging.

How hematopoietic stem cells (HSCs) maintain metabolic homeostasis to support tissue repair and regeneration throughout the lifespan is elusive. Here, we show that CD38, an NAD+-dependent metabolic enzyme, promotes HSC proliferation by inducing mitochondrial Ca2+ influx and mitochondrial metabolism in young mice. Conversely, aberrant CD38 upregulation during aging is a driver of HSC deterioration in aged mice due to dysregulated NAD+ metabolism and compromised mitochondrial stress management. The mitochondrial calcium uniporter, a mediator of mitochondrial Ca2+ influx, also supports HSC proliferation in young mice yet drives HSC decline in aged mice. Pharmacological inactivation of CD38 reverses HSC aging and the pathophysiological changes of the aging hematopoietic system in aged mice. Together, our study highlights an NAD+ metabolic checkpoint that balances mitochondrial activation to support HSC proliferation and mitochondrial stress management to enhance HSC self-renewal throughout the lifespan, and links aberrant Ca2+ signaling to HSC aging.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates.

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

Real-World Experience of Cryopreserved Allogeneic Hematopoietic Grafts during the COVID-19 Pandemic: A Single-Center Report.

In response to the widespread COVID-19 pandemic, cryopreservation of allogeneic donor apheresis products was implemented to mitigate the challenges of donor availability and product transport. Although logistically beneficial, the impact of cryopreservation on clinical outcomes and graft composition remains unclear. In this study, we compared outcomes and graft composition with cryopreserved versus fresh allografts in the setting of allogeneic hematopoietic cell transplantation (allo-HCT). We retrospectively analyzed the clinical outcomes of 30 consecutive patients who received cryopreserved allografts between March and August 2020 and 60 consecutive patients who received fresh allografts before the COVID-19 pandemic. Primary endpoints were hematopoietic engraftment and graft failure (GF), and secondary outcomes were overall survival (OS), relapse-free survival (RFS) and nonrelapse mortality (NRM). In addition, extended immunophenotype analysis was performed on cryopreserved and prospectively collected fresh apheresis samples. Compared with recipients of fresh allografts, both neutrophil and platelet recovery were delayed in recipients of cryopreserved reduced-intensity conditioning (RIC) allo-HCT, with a median time to engraftment of 24 days versus 18 days (P = .01) for neutrophils and 27 days versus 18 days (P = .069) for platelets. We observed primary GF in 4 of 30 patients in the cryopreserved cohort (13.3%) versus only 1 of 60 patients (1.7 %) in the fresh cohort (P = .03). Cryopreserved RIC allo-HCT was associated with significantly lower median total, myeloid, and T cell donor chimerism at 1 month. OS and RFS were inferior for cryopreserved graft recipients (hazard ratio [HR], 2.16; 95% confidence interval [CI], 1.00 to 4.67) and HR, 1.90; 95% CI, 0.95 to 3.79, respectively. Using an extended immunophenotype analysis, we compared 14 samples from the cryopreserved cohort to 6 prospectively collected fresh apheresis donor samples. These analyses showed both a decrease in total cell viability and a significantly reduced absolute number of natural killer cells (CD3-CD56+) in the cryopreserved apheresis samples. In this single-institution study, we found delayed engraftment and a trend toward clinical inferiority of cryopreserved allografts compared with fresh allografts. Further evaluation of the use of cryopreserved allografts and their impact on clinical and laboratory outcomes is warranted.

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

Transplantation of donor grafts with defined ratio of conventional and regulatory T cells in HLA-matched recipients.

BACKGROUNDIn preclinical murine and early clinical studies of hematopoietic cell transplantation, engineering of donor grafts with defined ratios of CD4+CD25+FoxP3+ Tregs to conventional T cells (Tcons) results in the prevention of graft-versus-host disease and improved immune reconstitution. The use of highly purified primary graft Tregs for direct cell infusion has potential advantages over impure immunomagnetic selection or culture expansion, but has not been tested clinically. We performed a phase I study of the timed addition of CD34-selected hematopoietic stem cells and Tregs, followed by Tcons for the treatment of patients with high-risk hematological malignancies.METHODSWe present interim evaluation of a single-center open phase I/II study of administration of human leukocyte-matched Tregs and CD34-selected hematopoietic cells, followed by infusion of an equal ratio of Tcons in adult patients undergoing myeloablative hematopoietic stem cell transplantation (HCT) for high-risk or active hematological malignancies. Tregs were purified by immunomagnetic selection and high-speed cell sorting.RESULTSHere we report results for the first 12 patients who received Tregs of between 91% and 96% purity. Greater than grade II GVHD was noted in 2 patients in the first cohort of 5 patients, who received cryopreserved Tregs, but neither acute nor chronic GVHD was noted in the second cohort of 7 patients, who received fresh Tregs and single-agent GVHD prophylaxis. Patients in the second cohort appeared to have normal immune reconstitution compared with patients who underwent transplantation and did not develop GVHD.CONCLUSIONOur study shows that the use of highly purified fresh Tregs is clinically feasible and supports continued investigation of the strategy.TRIAL REGISTRATIONClinicalTrials.gov NCT01660607.FUNDINGNIH NHBLI R01 HL114591 and K08HL119590.

Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming.

Somatic cells can be reprogrammed into pluripotent stem cells using the Yamanaka transcription factors. Reprogramming requires both epigenetic landscape reshaping and global remodeling of cell identity, structure, basic metabolic processes, and organelle form and function. We hypothesize that variable regulation of the proteostasis network and its influence upon the protein-folding environment within cells and their organelles is responsible for the low efficiency and stochasticity of reprogramming. We find that the unfolded protein response of the endoplasmic reticulum (UPRER), the mitochondrial UPR, and the heat shock response, which ensure proteome quality during stress, are activated during reprogramming. The UPRER is particularly crucial, and its ectopic, transient activation, genetically or pharmacologically, enhances reprogramming. Last, stochastic activation of the UPRER predicts reprogramming efficiency in naïve cells. Thus, the low efficiency and stochasticity of cellular reprogramming are due partly to the inability to properly initiate the UPRER to remodel the ER and its proteome.

Flow-cytometric analysis of human monocyte subsets targeted by Mycobacterium bovis BCG before granuloma formation.

Infection with Mycobacterium tuberculosis (Mtb) is characterized by an inflammatory response resulting in the formation of granulomas. These tight aggregates of immune cells play an important role in bacterial containment and in the eventual outcome of infection. Monocytes are a major component of the early immune response to Mtb and contribute to the cellular matrix of the newly forming granuloma. Therefore, defining which monocyte subset is the target of mycobacterial infection is critical. Here, we describe a flow-cytometry-based assay to analyze infectivity in vitro of monocyte subsets by Mycobacterium bovis BCG before granuloma formation. We identified CD14+CD16- monocytes as the main target of infection in peripheral blood mononuclear cells from six healthy donors. CD14+CD16+ monocytes displayed the lowest infection rates and remained uninfected in some donors. We found that a longer infection time resulted in an increase of the percentage of monocytes infected and of the number of granulomas produced. We did not observe changes in monocyte cell death or subset expansion upon infection. Future experiments with our in vitro method could help define Mtb infectivity of monocyte subsets. Our study provides a platform to investigate how early infection of different monocyte subsets may alter granuloma formation and outcomes of Mtb infection.

Sox9-Meis1 Inactivation Is Required for Adipogenesis, Advancing Pref-1+ to PDGFRα+ Cells.

Adipocytes arise from the commitment and differentiation of adipose precursors in white adipose tissue (WAT). In studying adipogenesis, precursor markers, including Pref-1 and PDGFRα, are used to isolate precursors from stromal vascular fractions of WAT, but the relation among the markers is not known. Here, we used the Pref-1 promoter-rtTA system in mice for labeling Pref-1+ cells and for inducible inactivation of the Pref-1 target Sox9. We show the requirement of Sox9 for the maintenance of Pref-1+ proliferative, early precursors. Upon Sox9 inactivation, these Pref-1+ cells become PDGFRα+ cells that express early adipogenic markers. Thus, we show that Pref-1+ cells precede PDGFRα+ cells in the adipogenic pathway and that Sox9 inactivation is required for WAT growth and expansion. Furthermore, we show that in maintaining early adipose precursors, Sox9 activates Meis1, which prevents adipogenic differentiation. Our study also demonstrates the Pref-1 promoter-rtTA system for inducible gene inactivation in early adipose precursor populations.

Mitochondrial Subtype Identification and Characterization.

Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a single cell, and thus interrogation of mitochondrial features at the organelle level is necessary to better understand and accurately measure heterogeneity. Here we describe a new flow cytometric methodology that enables the quantification and classification of mitochondrial subtypes (via their Δψ, size, and substructure) using the small animal model C. elegans. Future application of this methodology should allow research to discern the bioenergetic and mitochondrial component in a number of human disease and aging models, including, C. elegans, cultured cells, small animal models, and human biopsy samples. © 2018 by John Wiley & Sons, Inc.

STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection.

There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen.

Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry.

Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.

Transcriptome Signature and Regulation in Human Somatic Cell Reprogramming.

Reprogramming of somatic cells produces induced pluripotent stem cells (iPSCs) that are invaluable resources for biomedical research. Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming.

Development of the fetal bone marrow niche and regulation of HSC quiescence and homing ability by emerging osteolineage cells.

Hematopoietic stem cells (HSCs) reside within a specialized niche where interactions with vasculature, osteoblasts, and stromal components regulate their self-renewal and differentiation. Little is known about bone marrow niche formation or the role of its cellular components in HSC development; therefore, we established the timing of murine fetal long bone vascularization and ossification relative to the onset of HSC activity. Adult-repopulating HSCs emerged at embryonic day 16.5 (E16.5), coincident with marrow vascularization, and were contained within the c-Kit(+)Sca-1(+)Lin(-) (KSL) population. We used Osterix-null (Osx(-/-)) mice that form vascularized marrow but lack osteolineage cells to dissect the role(s) of these cellular components in HSC development. Osx(-/-) fetal bone marrow cells formed multilineage colonies in vitro but were hyperproliferative and failed to home to and/or engraft transplant recipients. Thus, in developing bone marrow, the vasculature can sustain multilineage progenitors, but interactions with osteolineage cells are needed to regulate long-term HSC proliferation and potential.

Nonstochastic reprogramming from a privileged somatic cell state.

Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:

An extensive network of TET2-targeting MicroRNAs regulates malignant hematopoiesis.

The Ten-Eleven-Translocation 2 (TET2) gene, which oxidates 5-methylcytosine in DNA to 5-hydroxylmethylcytosine (5hmC), is a key tumor suppressor frequently mutated in hematopoietic malignancies. However, the molecular regulation of TET2 expression is poorly understood. We show that TET2 is under extensive microRNA (miRNA) regulation, and such TET2 targeting is an important pathogenic mechanism in hematopoietic malignancies. Using a high-throughput 3' UTR activity screen, we identify >30 miRNAs that inhibit TET2 expression and cellular 5hmC. Forced expression of TET2-targeting miRNAs in vivo disrupts normal hematopoiesis, leading to hematopoietic expansion and/or myeloid differentiation bias, whereas coexpression of TET2 corrects these phenotypes. Importantly, several TET2-targeting miRNAs, including miR-125b, miR-29b, miR-29c, miR-101, and miR-7, are preferentially overexpressed in TET2-wild-type acute myeloid leukemia. Our results demonstrate the extensive roles of miRNAs in functionally regulating TET2 and cellular 5hmC and reveal miRNAs with previously unrecognized oncogenic potential. Our work suggests that TET2-targeting miRNAs might be exploited in cancer diagnosis.

Very small embryonic-like stem cells from the murine bone marrow differentiate into epithelial cells of the lung.

The view that adult stem cells are lineage restricted has been challenged by numerous reports of bone marrow (BM)-derived cells giving rise to epithelial cells. Previously, we demonstrated that nonhematopoietic BM cells are the primary source of BM-derived lung epithelial cells. Here, we tested the hypothesis that very small embryonic like cells (VSELs) are responsible for this engraftment. We directly compared the level of BM-derived epithelial cells after transplantation of VSELs, hematopoietic stem/progenitor cells, or other nonhematopoietic cells. VSELs clearly had the highest rate of forming epithelial cells in the lung. By transplanting VSELs from donor mice expressing H2B-GFP under a type 2 pneumocyte-specific promoter, we demonstrate that this engraftment occurs by differentiation and not fusion. This is the first report of VSELs differentiating into an endodermal lineage in vivo, thereby potentially crossing germ layer lineages. Our data suggest that Oct4+ VSELs in the adult BM exhibit broad differentiation potential.

Dynamic migration and cell-cell interactions of early reprogramming revealed by high-resolution time-lapse imaging.

Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate that E-cadherin is required for proper cellular interactions from an early stage of reprogramming, including the two-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.

An in vivo functional screen uncovers miR-150-mediated regulation of hematopoietic injury response.

Hematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control hematopoietic injury response is largely unknown. We report an in vivo gain-of-function screen and the identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150 null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150-forced expression. Our data highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of in vivo functional screens for studying microRNAs in normal tissue physiology.

TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors.

A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versus-host disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)-matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and event-free survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graft-versus-tumor reactions. Active trial registration currently at clinicaltrials.gov under IDs of NCT00185640 and NCT00186615.