Loss of sphingosine kinase 2 promotes the expansion of hematopoietic stem cells by improving their metabolic fitness.

Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.

Immune System Influence on Hematopoietic Stem Cells and Leukemia Development.

Hematopoietic stem cells (HSCs) are the source for all blood cells, including immune cells, and they interact dynamically with the immune system. This chapter will explore the nature of stem cells, particularly HSCs, in the context of their immune microenvironment. The dynamic interactions between stem cells and the immune system can have profound implications for current and future therapies, particularly regarding a potential "immune-privileged" HSC microenvironment. Immune/stem cell interactions change during times of stress and injury. Recent advances in cancer immunotherapy have overturned the long-standing belief that, being derived from the self, cancer cells should be immunotolerant. Instead, an immunosurveillance system recognizes and eliminates emergent pre-cancerous cells. Only in the context of a failing immunosurveillance system does cancer fully develop. Combined with the knowledge that stem cells or their unique properties can be critically important for cancer initiation, persistence, and resistance to therapy, understanding the unique immune properties of stem cells will be critical for the development of future cancer therapies. Accordingly, the therapeutic implications for leukemic stem cells (LSCs) inheriting an immune-privileged state from HSCs will be discussed. Through their dynamic interactions with a diverse immune system, stem cells serve as the light and dark root of cancer prevention vs. development.

Human Peripheral Blood Mononucleocyte Derived Myeloid Committed Progenitor Cells Mitigate H-ARS by Exosomal Paracrine Signal.

Radiation-induced loss of the hematopoietic stem cell progenitor population compromises bone marrow regeneration and development of mature blood cells. Failure to rescue bone marrow functions results in fatal consequences from hematopoietic injury, systemic infections, and sepsis. So far, bone marrow transplant is the only effective option, which partially minimizes radiation-induced hematopoietic toxicities. However, a bone marrow transplant will require HLA matching, which will not be feasible in large casualty settings such as a nuclear accident or an act of terrorism. In this study we demonstrated that human peripheral blood mononuclear cell-derived myeloid committed progenitor cells can mitigate radiation-induced bone marrow toxicity and improve survival in mice. These cells can rescue the recipient's hematopoietic stem cells from radiation toxicity even when administered up to 24 h after radiation exposure and can be subjected to allogenic transplant without GVHD development. Transplanted cells deliver sEVs enriched with regenerative and immune-modulatory paracrine signals to mitigate radiation-induced hematopoietic toxicity. This provides a natural polypharmacy solution against a complex injury process. In summary, myeloid committed progenitor cells can be prepared from blood cells as an off-the-shelf alternative to invasive bone marrow harvesting and can be administered in an allogenic setting to mitigate hematopoietic acute radiation syndrome.

Ribosomal DNA copy number loss and sequence variation in cancer.

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Maternal imprinting at the H19-Igf2 locus maintains adult haematopoietic stem cell quiescence.

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and ß-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of ß-catenin alone results in apoptosis of HSCs. However, together, the Wnt/ß-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, ß-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/ß-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.

BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS: We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS: CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

FGF signaling facilitates postinjury recovery of mouse hematopoietic system.

Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.

SHP-1 inhibition targets leukaemia stem cells to restore immunosurveillance and enhance chemosensitivity by metabolic reprogramming.

Leukaemia stem cells (LSCs) in acute myeloid leukaemia present a considerable treatment challenge due to their resistance to chemotherapy and immunosurveillance. The connection between these properties in LSCs remains poorly understood. Here we demonstrate that inhibition of tyrosine phosphatase SHP-1 in LSCs increases their glycolysis and oxidative phosphorylation, enhancing their sensitivity to chemotherapy and vulnerability to immunosurveillance. Mechanistically, SHP-1 inhibition leads to the upregulation of phosphofructokinase platelet (PFKP) through the AKT-ß-catenin pathway. The increase in PFKP elevates energy metabolic activities and, as a consequence, enhances the sensitivity of LSCs to chemotherapeutic agents. Moreover, the upregulation of PFKP promotes MYC degradation and, consequently, reduces the immune evasion abilities of LSCs. Overall, our study demonstrates that targeting SHP-1 disrupts the metabolic balance in LSCs, thereby increasing their vulnerability to chemotherapy and immunosurveillance. This approach offers a promising strategy to overcome LSC resistance in acute myeloid leukaemia.

PD-1 signalling defines and protects leukaemic stem cells from T cell receptor-induced cell death in T cell acute lymphoblastic leukaemia.

T cell acute lymphoblastic leukaemia (T-ALL) is an aggressive malignancy with poor prognosis, but a decisive marker and effective treatment for leukaemia stem cells (LSCs) remain unclear. Here, using lineage tracing, limiting dilution assays and in vivo live imaging approaches, we identify rare inhibitory receptor programmed cell death 1 (PD-1)-expressing cells that reside at the apex of leukaemia hierarchy for initiation and relapse in T-ALL. Ablation of PD-1-expressing cells, deletion of PD-1 in T-ALL cells or blockade of PD-1 or PD-1 ligand 1 significantly eradicated LSCs and suppressed disease progression. Combination therapy using PD-1 blockade and chemotherapy substantially extended the survival of mice engrafted with mouse or human T-ALL cells. Mechanistically, PD-1+ LSCs had high NOTCH1-MYC activity for disease initiation. Furthermore, PD-1 signalling maintained quiescence and protected LSCs against T cell receptor-signal-induced apoptosis. Overall, our data highlight the hierarchy of leukaemia by identifying PD-1+ LSCs and provide a therapeutic approach for the elimination of LSCs through PD-1 blockade in T-ALL.

Propagating concentration polarization and ionic current rectification in a nanochannel-nanofunnel device.

We study ionic current rectification observed in a nanofluidic device with a nanofunnel positioned between two straight nanochannels. Ion transport is simulated by resolving the coupled three-dimensional Nernst-Planck, Poisson, and Navier-Stokes equations. In the modeled system, the electric double layer extends into the channel, and consequently, the funnel tip exhibits charge-selective properties, which results in the formation of enriched and depleted concentration polarization (CP) zones within the nanofunnel in the high- and low-conductance states, respectively. This scenario is similar to the one observed for ion transport through a charged conical nanopore connecting two macroscopic reservoirs. However, the presence of the adjacent straight nanochannels allows the CP zones to propagate out of the funnel into the adjoining channels. The condition for propagation of the CP zones is determined by several parameters, including the electroosmotic flow velocity. We demonstrate that in the high-conductance regime the modeled system is characterized by increased ionic concentrations in the entire cathodic nanochannel, whereas in the low-conductance state the depleted CP zone does not propagate out of the funnel and remains localized. The required three-dimensional modeling scheme is implemented on a parallel computational platform, is general as well as numerically efficient, and will be useful in the study of more advanced nanofluidic device designs for tailoring ionic current rectification.

3D nanofluidic channels shaped by electron-beam-induced etching.

In-plane nanofluidic channels with 3D topography are fabricated. Nanochannel masters are written by electron beam lithography in SU-8 resist and shaped by electron-beam-induced etching (EBIE) with water as the precursor gas. Nanofunnel replicas cast from unmodified and EBIE-modified masters show that the funnel tip dimensions decrease from a 150-nm depth and 80-nm width to a 70-nm depth and 40-nm width.

An optimized chromatin immunoprecipitation protocol using Staph-seq for analyzing genome-wide protein-DNA interactions.

Genome-wide mapping of transcription factors (TFs) is critical to understand their functions. In chromatin immunoprecipitation (ChIP)-seq assay, it's challenging to study recruitment of low-abundant TFs transiently boud to the genome. Here, we present an optimized protocol using ChIP Next-Gen Seq Sepharose (Staph-seq) to efficiently pull down chromatin complexes. The double size selection promotes sensitive capture of genome-wide protein-DNA associations while eliminating potential Staph A contamination, which is a common problem in protocols using Staph A cells. For complete details on the use and execution of this protocol, please refer to Tao et al. (2020).1.

Child physical abuse and COVID-19: Trends from nine pediatric trauma centers.

BACKGROUND: Economic, social, and psychologic stressors are associated with an increased risk for abusive injuries in children. Prolonged physical proximity between adults and children under conditions of severe external stress, such as witnessed during the COVID-19 pandemic with "shelter-in-place orders", may be associated with additional increased risk for child physical abuse. We hypothesized that child physical abuse rates and associated severity of injury would increase during the early months of the pandemic as compared to the prior benchmark period. METHODS: We conducted a nine-center retrospective review of suspected child physical abuse admissions across the Western Pediatric Surgery Research Consortium. Cases were identified for the period of April 1-June 30, 2020 (COVID-19) and compared to the identical period in 2019. We collected patient demographics, injury characteristics, and outcome data. RESULTS: There were no significant differences in child physical abuse cases between the time periods in the consortium as a whole or at individual hospitals. There were no differences between the study periods with regard to patient characteristics, injury types or severity, resource utilization, disposition, or mortality. CONCLUSIONS: Apparent rates of new injuries related to child physical abuse did not increase early in the COVID-19 pandemic. While this may suggest that pediatric physical abuse was not impacted by pandemic restrictions and stresses, it is possible that under-reporting, under-detection, or delays in presentation of abusive injuries increased during the pandemic. Long-term follow-up of subsequent rates and severity of child abuse is needed to assess for unrecognized injuries that may have occurred.

Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.

The production of mature cells necessitates that lineage-committed progenitor cells be constantly generated from multipotential progenitors. In addition, the ability to respond rapidly to physiologic stresses requires that the signals that regulate the maintenance of progenitor populations be coordinated with the signals that promote differentiation of progenitors. Here we examine the signals that are necessary for the maintenance of the BMP4-dependent stress erythropoiesis pathway. Our previous work demonstrated that BMP4, stem cell factor, and hypoxia act in concert to promote the expansion of a specialized population of stress erythroid progenitors in the spleen during the recovery from acute anemia. Our analysis shows that acute anemia leads to an almost complete mobilization of BMP4-responsive stress erythroid burst-forming units; therefore, new stress progenitors must be recruited to the spleen to replenish this system. We show that bone marrow cells can home to the spleen and, in response to a signal in the spleen microenvironment, Hedgehog, they develop into BMP4-responsive stress progenitors. Hedgehog induces the expression of BMP4, and together these 2 signals are required for the development of BMP4-responsive stress progenitors. These data demonstrate that the interplay between these 2 signals is crucial for maintenance of this stress response pathway.

Nanoparticles by decomposition of long chain iron carboxylates: from spheres to stars and cubes.

In this paper, we report the influence of reaction conditions and the chain length on the nanoparticle (NP) size and morphology for thermal decomposition of long-chain iron carboxylates such as Fe(III) oleate, palmitate, and myristate. In the majority of cases, spherical NPs are obtained; however, nonspherical morphologies were observed in some "extreme" conditions. For example, iron oxide nanostars are formed in eicosane at the Fe oleate/oleic acid ratio of 0.49 g/mL: the highest oleic acid content when NPs still form. The cubic NPs with flat facets are obtained by decomposition of iron palmitate at the lowest palmitic acid fractions, but the most monodisperse cubes are formed at the Fe palmitate/palmitic acid ratio of 1.19 g/mL. Elliptical NPs are formed from Fe myristate with the most well-defined structure. Easy transformation of these NPs from wüstite to maghemite without aggregation and loss of solubility makes them excellent candidates for biomedical applications after proper functionalization described in our preceding papers.

Evaluation of the association of health care system access with kidney cancer surgical outcomes for hispanic and non-hispanic white patients.

OBJECTIVE: To determine the impact of health care system access on outcomes for Hispanic and Non-Hispanic White patients with renal cell carcinoma (RCC). METHODS: We retrospectively analyzed Hispanic and non-Hispanic White patients diagnosed with localized RCC between 2007 and 2020. We used Health Resources and Services Administration criteria to identify patients living in Medically Underserved Areas (MUA). Primary outcome all-cause mortality and cancer-specific survival using Log Rank test on Kaplan Meier Analysis. Secondary outcome was all-cause mortality and cancer specific survival on Cox Regression when adjusting for risk factors. RESULTS: We analyzed 774 patients, 246 (31.8%) Hispanic patients and 528 (68.2%) Non-Hispanic White patients. Hispanic ethnicity was associated with lower risk of ACM (HR 0.53, P = 0.019) and there was no difference for cancer specific survival (HR 0.57, P = 0.059). Living in a MUA was associated with worse all-cause mortality (P = 0.010) but not cancer specific survival (CSS) (P = 0.169). Comparing Hispanic and Non-Hispanic Whites, KMA revealed no difference in 5-year all-cause mortality (83.1% vs. 78.8%, P = 0.254) and 5-year CSS (85.7% vs. 85.4%, P = 0.403). CONCLUSIONS: Hispanics had lower all-cause mortality risk and no significant differences in 5-year overall survival and CSS compared to non-Hispanic Whites. Our findings indicate that tertiary referral centers may help mitigate inequalities in access to care.

Overcoming resistance to immunotherapy by teaching old drugs new tricks.

Cancer stem cells (CSCs) underlie resistance to therapy. Cancer develops only in the context of failing immunosurveillance, and stem cells occupy immune privileged microenvironments. Recent evidence demonstrates that CSCs borrow immune privilege from their normal counterparts. However, low doses of doxorubicin can target CSCs by restoring anticancer immunity.