Hematopoietic stem cells preferentially traffic misfolded proteins to aggresomes and depend on aggrephagy to maintain protein homeostasis.

Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). However, how HSCs purge misfolded proteins is unknown. Here, we show that in contrast to most cells that primarily utilize the proteasome to degrade misfolded proteins, HSCs preferentially traffic misfolded proteins to aggresomes in a Bag3-dependent manner and depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. When autophagy is disabled, HSCs compensate by increasing proteasome activity, but proteostasis is ultimately disrupted as protein aggregates accumulate and HSC function is impaired. Bag3-deficiency blunts aggresome formation in HSCs, resulting in protein aggregate accumulation, myeloid-biased differentiation, and diminished self-renewal activity. Furthermore, HSC aging is associated with a severe loss of aggresomes and reduced autophagic flux. Protein degradation pathways are thus specifically configured in young adult HSCs to preserve proteostasis and fitness but become dysregulated during aging.

Simplified murine multipotent progenitor isolation scheme: Establishing a consensus approach for multipotent progenitor identification.

The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.

Hsf1 promotes hematopoietic stem cell fitness and proteostasis in response to ex vivo culture stress and aging.

Maintaining proteostasis is key to resisting stress and promoting healthy aging. Proteostasis is necessary to preserve stem cell function, but little is known about the mechanisms that regulate proteostasis during stress in stem cells, and whether disruptions of proteostasis contribute to stem cell aging is largely unexplored. We determined that ex-vivo-cultured mouse and human hematopoietic stem cells (HSCs) rapidly increase protein synthesis. This challenge to HSC proteostasis was associated with nuclear accumulation of Hsf1, and deletion of Hsf1 impaired HSC maintenance ex vivo. Strikingly, supplementing cultures with small molecules that enhance Hsf1 activation partially suppressed protein synthesis, rebalanced proteostasis, and supported retention of HSC serial reconstituting activity. Although Hsf1 was dispensable for young adult HSCs in vivo, Hsf1 deficiency increased protein synthesis and impaired the reconstituting activity of middle-aged HSCs. Hsf1 thus promotes proteostasis and the regenerative activity of HSCs in response to culture stress and aging.

Developmental Stage-Specific Changes in Protein Synthesis Differentially Sensitize Hematopoietic Stem Cells and Erythroid Progenitors to Impaired Ribosome Biogenesis.

Adult hematopoietic stem cell (HSC) self-renewal requires precise control of protein synthesis, but fetal and adult HSCs have distinct self-renewal mechanisms and lineage outputs. This raises the question of whether protein synthesis rates change with age. Here, we show that protein synthesis rates decline during HSC ontogeny, yet erythroid protein synthesis rates increase. A ribosomal mutation that impairs ribosome biogenesis (Rpl24Bst/+) disrupts both fetal and adult HSC self-renewal. However, the Rpl24Bst/+ mutation selectively impairs fetal erythropoiesis at differentiation stages that exhibit fetal-specific attenuation of protein synthesis. Developmental changes in protein synthesis thus differentially sensitize hematopoietic stem and progenitor cells to impaired ribosome biogenesis.

Hematopoietic stem cell regulation by the proteostasis network.

PURPOSE OF REVIEW: Protein homeostasis (proteostasis) is maintained by an integrated network of physiological mechanisms and stress response pathways that regulate the content and quality of the proteome. Maintenance of cellular proteostasis is key to ensuring normal development, resistance to environmental stress, coping with infection, and promoting healthy aging and lifespan. Recent studies have revealed that several proteostasis mechanisms can function in a cell-type-specific manner within hematopoietic stem cells (HSCs). Here, we review recent studies demonstrating that the proteostasis network functions uniquely in HSCs to promote their maintenance and regenerative function. RECENT FINDINGS: The proteostasis network is regulated differently in HSCs as compared with restricted hematopoietic progenitors. Disruptions in proteostasis are particularly detrimental to HSC maintenance and function. These findings suggest that multiple aspects of cellular physiology are uniquely regulated in HSCs to maintain proteostasis, and that precise control of proteostasis is particularly important to support life-long HSC maintenance and regenerative function. SUMMARY: The proteostasis network is uniquely configured within HSCs to promote their longevity and hematopoietic function. Future work uncovering cell-type-specific differences in proteostasis network configuration, integration, and function will be essential for understanding how HSCs function during homeostasis, in response to stress, and in disease.

Post-Transcriptional Regulation of Homeostatic, Stressed, and Malignant Stem Cells.

Cellular identity is not driven by differences in genomic content but rather by epigenomic, transcriptomic, and proteomic heterogeneity. Although regulation of the epigenome plays a key role in shaping stem cell hierarchies, differential expression of transcripts only partially explains protein abundance. The epitranscriptome, translational control, and protein degradation have emerged as fundamental regulators of proteome complexity that regulate stem cell identity and function. Here, we discuss how post-transcriptional mechanisms enable stem cell homeostasis and responsiveness to developmental cues and environmental stressors by rapidly shaping the content of their proteome and how these processes are disrupted in pre-malignant and malignant states.

Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Cell-type-specific quantification of protein synthesis in vivo.

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8-10 h.

Living Up to the Hype: Protein Synthesis Promotes Hypertranscription in Embryonic Stem Cells.

The rapid proliferation and unlimited self-renewal of embryonic stem cells depends upon a permissive chromatin landscape that enables hypertranscription. In this issue of Cell Stem Cell, Bulut-Karslioglu et al. report that euchromatin and transcriptional output are enhanced by protein synthesis in embryonic stem cells (Bulut-Karslioglu et al., 2018).

CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing.

Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1F22-24/F22-24) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1BRCA1/BRCA1) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1F22-24/5382insC) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction.

The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.

Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis.

Pten Cell Autonomously Modulates the Hematopoietic Stem Cell Response to Inflammatory Cytokines.

Pten negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway and is required to maintain quiescent adult hematopoietic stem cells (HSCs). Pten has been proposed to regulate HSCs cell autonomously and non-cell autonomously, but the relative importance of each mechanism has not been directly tested. Furthermore, the cytokines that activate the PI3K pathway upstream of Pten are not well defined. We sought to clarify whether Pten cell autonomously or non-cell autonomously regulates HSC mobilization. We also tested whether Pten deficiency affects the HSC response to granulocyte colony-stimulating factor (G-CSF) and interferon-α (IFNα) since these cytokines induce HSC mobilization or proliferation, respectively. We show that Pten regulates HSC mobilization and expansion in the spleen primarily via cell-autonomous mechanisms. Pten-deficient HSCs do not require G-CSF to mobilize, although they are hyper-sensitized to even low doses of exogenous G-CSF. Pten-deficient HSCs are similarly sensitized to IFNα. Pten therefore modulates the HSC response to inflammatory cytokines.

Suppression of the SWI/SNF Component Arid1a Promotes Mammalian Regeneration.

Mammals have partially lost the extensive regenerative capabilities of some vertebrates, possibly as a result of chromatin-remodeling mechanisms that enforce terminal differentiation. Here, we show that deleting the SWI/SNF component Arid1a substantially improves mammalian regeneration. Arid1a expression is suppressed in regenerating tissues, and genetic deletion of Arid1a increases tissue repair following an array of injuries. Arid1a deficiency in the liver increases proliferation, reduces tissue damage and fibrosis, and improves organ function following surgical resection and chemical injuries. Hepatocyte-specific deletion is also sufficient to increase proliferation and regeneration without excessive overgrowth, and global Arid1a disruption potentiates soft tissue healing in the ear. We show that Arid1a loss reprograms chromatin to restrict promoter access by transcription factors such as C/ebpα, which enforces differentiation, and E2F4, which suppresses cell-cycle re-entry. Thus, epigenetic reprogramming mediated by deletion of a single gene improves mammalian regeneration and suggests strategies to promote tissue repair after injury.

Lens regeneration using endogenous stem cells with gain of visual function.

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

Cellular differences in protein synthesis regulate tissue homeostasis.

Although sometimes considered a "house-keeping" function, multiple aspects of protein synthesis are regulated differently among somatic cells, including stem cells, and can be modulated in a cell-type-specific manner. These differences are required to establish and maintain differences in cell identity, cell function, tissue homeostasis, and tumor suppression.