Ferritin-mediated mitochondrial iron homeostasis is essential for the survival of hematopoietic stem cells and leukemic stem cells.

Iron metabolism plays a crucial role in cell viability, but its relationship with adult stem cells and cancer stem cells is not fully understood. The ferritin complex, responsible for intracellular iron storage, is important in this process. We report that conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system reduced the number and repopulation capacity of hematopoietic stem cells (HSCs). These effects were associated with a decrease in cellular iron level, leading to impaired mitochondrial function and the initiation of apoptosis. Iron supplementation, antioxidant, and apoptosis inhibitors reversed the reduced cell viability of Fth1-deleted hematopoietic stem and progenitor cells (HSPCs). Importantly, leukemic stem cells (LSCs) derived from MLL-AF9-induced acute myeloid leukemia (AML) mice exhibited reduced Fth1 expression, rendering them more susceptible to apoptosis induced by the iron chelation compared to normal HSPCs. Modulating FTH1 expression using mono-methyl fumarate increased LSCs resistance to iron chelator-induced apoptosis. Additionally, iron supplementation, antioxidant, and apoptosis inhibitors protected LSCs from iron chelator-induced cell death. Fth1 deletion also extended the survival of AML mice. These findings unveil a novel mechanism by which ferritin-mediated iron homeostasis regulates the survival of both HSCs and LSCs, suggesting potential therapeutic strategies for blood cancer with iron dysregulation.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Impact of macrophage differentiation on hematopoietic function enhancement by Shenzhu ErKang Syrup.

Shenzhu Erkang Syrup (SZEK) is a traditional Chinese medicine that improves spleen and stomach function, tonifying the Qi and activating the blood; however, its therapeutic effects in hematopoietic dysfunction and their underlying mechanism remain unexplored. In this study, mice were given cyclophosphamide (100 mg/kg) by intraperitoneal injections for three days to produce hematopoietic dysfunction model. We investigated the hematopoietic effect and mechanism of SZEK in mice with hematopoietic dysfunction via histopathological examination, flow cytometry, enzyme-linked immunosorbent assay, and Western blotting combined with intestinal flora and serum metabolomics analysis. In mice with hematopoietic dysfunction, SZEK (gavage, 0.3 mL/25 g) alleviated pathological damage to the bone marrow and spleen; increased the number of naïve cells (Lin-), hematopoietic stem cells (Lin-Sca-1+c-Kit+), long-term self-renewing hematopoietic stem cells (Lin-Sca-1+c-Kit+CD48-CD150+), B lymphocytes (CD45+CD19+), and macrophages (CD11b+F4/80+) in the bone marrow; and reduced inflammation. Preliminary intestinal flora and serum metabolome analyses indicated that the pro-hematopoietic mechanism of SZEK was associated with macrophage differentiation. Further validation revealed that SZEK promoted hematopoiesis by decreasing the number of M2 macrophages and inhibiting the secretion of negative hematopoietic regulatory factors in mice with hematopoietic dysfunction.

Effects of blood components of preoperative autologous blood donation on functions of myelopoietic stem cells.

OBJECTIVE: To explore the effects of preoperative autologous whole blood donation and autologous pure red blood cell (RBC) donation on hematopoietic stem cells (HSC), clarify the effects of transfusion by different blood components on HSC, and improve the treatment effect of autotransfusion. METHODS: Forty healthy male New Zealand rabbits were divided into five groups (n = 8) at random: control (Group A), surgery alone (Group B), surgery + blood sampling (Group C), surgery + preoperative autologous whole blood autotransfusion (Group D), and surgery + preoperative autologous pure RBC autotransfusion (Group E). The rabbits' bone marrow was collected before surgery (T1), 6 h after surgery (T2), and 24 h after surgery (T3) to detect the CD34+ cell count, the reactive oxygen species (ROS) concentration, the ratio of senescent cells, and the expression of HSC-related proteins (p53 and p21). RESULTS: Compared with groups A and B, the percentage of CD34+ cells in groups D and E at each time point was significantly increased, while the proportion of senescent cells, ROS, p53 and p21 were significantly decreased (P<0.05). Compared with Group C, the percentage of CD34+ cells at T2 and T3 rose significantly, while the ratio of senescent cells, the ROS concentration, and the content of p53 and p21 declined significantly in Groups D and E (P < 0.05). Compared with Group D, the ratio of senescent cells at T2 declined significantly, the percentage of CD34+ cells at T3 rose significantly, and the ROS concentration at T2 and T3 declined significantly in Group E (P < 0.05). CONCLUSION: From the perspective of HSC, collection and transfusion of pure RBC is more beneficial to postoperative recovery of patients than whole blood transfusion.

A Bloody Feast-Nutritional Regulation of Hematopoiesis.

Hematopoietic stem cells provide us with a lifelong supply of blood cells. Hence, their proper function is absolutely essential for life, and their dysfunction can lead to infectious and malignant diseases. These cells have specific metabolic requirements to enable their lifelong function and blood-producing capacity. With the words of the Roman poet Juvenal "a healthy mind in a healthy body" in mind, it is intriguing to understand the connection between our daily diet and the quality of our blood, with the hope that through specific dietary adjustments we can improve our hematopoietic stem cell function and prevent disease. Nowadays, dietary supplements are an expanding market filled with potential and promises for better health. However, the link between many of those supplements and human physiology is obscure. Several groups have begun to shed light on this by investigating the metabolic regulation of hematopoiesis by specific nutrients. Beyond the link to dietary supplementation, these studies have also significantly improved our understanding of basic hematopoietic stem cell biology. Herein we summarize recent knowledge on the effect of specific vitamins and amino acids, which might be considered as dietary supplements, on normal hematopoiesis and hematopoietic stem cell function. We propose that improving our understanding of the link between nutrition in general and blood physiology can ultimately lead to the optimization of health-care policies, protocols, and standards of care.

The Therapeutic Potential of a Strategy to Prevent Acute Myeloid Leukemia Stem Cell Reprogramming in Older Patients.

Acute myeloid leukemia (AML) is the most common and incurable leukemia subtype. Despite extensive research into the disease's intricate molecular mechanisms, effective treatments or expanded diagnostic or prognostic markers for AML have not yet been identified. The morphological, immunophenotypic, cytogenetic, biomolecular, and clinical characteristics of AML patients are extensive and complex. Leukemia stem cells (LSCs) consist of hematopoietic stem cells (HSCs) and cancer cells transformed by a complex, finely-tuned interaction that causes the complexity of AML. Microenvironmental regulation of LSCs dormancy and the diagnostic and therapeutic implications for identifying and targeting LSCs due to their significance in the pathogenesis of AML are discussed in this review. It is essential to perceive the relationship between the niche for LSCs and HSCs, which together cause the progression of AML. Notably, methylation is a well-known epigenetic change that is significant in AML, and our data also reveal that microRNAs are a unique factor for LSCs. Multiple-targeted approaches to reduce the risk of epigenetic factors, such as the administration of natural compounds for the elimination of local LSCs, may prevent potentially fatal relapses. Furthermore, the survival analysis of overlapping genes revealed that specific targets had significant effects on the survival and prognosis of patients. We predict that the multiple-targeted effects of herbal products on epigenetic modification are governed by different mechanisms in AML and could prevent potentially fatal relapses. Thus, these strategies can facilitate the incorporation of herbal medicine and natural compounds into the advanced drug discovery and development processes achievable with Network Pharmacology research.

Effects of Dopamine on stem cells and its potential roles in the treatment of inflammatory disorders: a narrative review.

Inflammation is the host's protective response against harmful external stimulation that helps tissue repair and remodeling. However, excessive inflammation seriously threatens the patient's life. Due to anti-inflammatory effects, corticosteroids, immunosuppressants, and monoclonal antibodies are used to treat various inflammatory diseases, but drug resistance, non-responsiveness, and severe side effect limit their development and application. Therefore, developing other alternative therapies has become essential in anti-inflammatory therapy. In recent years, the in-depth study of stem cells has made them a promising alternative drug for the treatment of inflammatory diseases, and the function of stem cells is regulated by a variety of signals, of which dopamine signaling is one of the main influencing factors. In this review, we review the effects of dopamine on various adult stem cells (neural stem cells, mesenchymal stromal cells, hematopoietic stem cells, and cancer stem cells) and their signaling pathways, as well as the application of some critical dopamine receptor agonists/antagonists. Besides, we also review the role of various adult stem cells in inflammatory diseases and discuss the potential anti-inflammation function of dopamine receptors, which provides a new therapeutic target for regenerative medicine in inflammatory diseases.

Nicotinamide riboside intervention alleviates hematopoietic system injury of ionizing radiation-induced premature aging mice.

Radiotherapy destroys cancer cells and inevitably harms normal human tissues, causing delayed effects of acute radiation exposure (DEARE) and accelerating the aging process in most survivors. However, effective methods for preventing premature aging induced by ionizing radiation are lacking. In this study, the premature aging mice of DEARE model was established after 6 Gy total body irradiation (TBI). Then the therapeutic effects and mechanism of nicotinamide riboside on the premature aging mice were evaluated. The results showed that 6 Gy TBI induced premature aging of the hematopoietic system in mice. Nicotinamide riboside treatment reversed aging spleen phenotypes by inhibiting cellular senescence and ameliorated serum metabolism profiles. Further results demonstrated that nicotinamide riboside supplementation alleviated the myeloid bias of hematopoietic stem cells and temporarily restored the regenerative capacity of hematopoietic stem cells probably by mitigating the reactive oxygen species activated GCN2/eIF2α/ATF4 signaling pathway. The results of this study firstly indicate that nicotinamide riboside shows potential as a DEARE therapeutic agent for radiation-exposed populations and patients who received radiotherapy.

Gold Nanoparticles Coated with SH-PEG-NH2 and Loaded with Ziyuglycoside I for Promoting Autophagy in Hematopoietic Stem Cells.

Introduction: Radiotherapy and chemotherapy are the fundamental causes of myelosuppression in cancer patients, which usually induce a serious hematopoietic system toxicity, causing the hemocytes and immunity decline of patients. Ziyuglycoside I (ZgI), an active ingredient isolated from traditional Chinese medicine Sanguisorba officinalis L, has been demonstrated to increase the leucocytes and protect hematopoietic stem cells, which is related to its promotion of autophagy in hematopoietic stem cells. Methods: In the present study, we formulated the SH-PEG-NH2-coated gold nanoparticles loading ZgI (ZgI-AuNPs) with a enhanced autophagy promotion in hematopoietic stem cells. ZgI-AuNPs were prepared by HAuCl4-sodium citrate reduction method, and the synthesis of ZgI-AuNPs was validated by XRD, FT-IR, DSC, and TEM findings. Furthermore, the drug loading rate and the release of ZgI were evaluated, and the ZgI-AuNPs' effects on autophagy and immunofluorescence staining for LC3B were tested. Finally, the effect of ZgI-AuNPs on the autophagy and hematopoietic ability of HSCs in vivo was also carried out. Results: The prepared ZgI-AuNPs have an irregular cubic crystal structure by TEM observation, and the average particle size was 340 ± 16.5 nm determined by DLS. The XRD, FT-IR and DSC detection showed that the ZgI had been well loaded in AuNPs, and the AuNPs can load the ZgI at a content of 160.63 ± 1.35 µg·mg-1. Meanwhile, the AuNPs can reduce the drug release rate of ZgI. Importantly, the ZgI-AuNPs enhanced autophagy of HSCs both in vitro and in vivo. At the same time, the gold nanoparticles enhance the hematopoietic effect of ZgI on mice HSCs. Conclusion: Our research suggests that SH-PEG-NH2-coated gold nanoparticles loading ZgI has potential application in myelosuppression therapy.

Human growth hormone supplement promotes human lymphohematopoietic cell reconstitution in immunodeficient mice.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

Humanized mice generated by human hematopoietic stem cell transplantation play crucial roles in biomedical investigations. One of the factors hindering the efficacy of their construction is the lack of or insufficient interaction of human cells to mouse cytokines and growth hormones (GHs) that are crucial for hematopoiesis and immune cell differentiation. In this study, we show that injection of human GH significantly improved human hematopoietic stem cell engraftment and function, as well as immune cell reconstitution in humanized mice. Our findings indicate that human cells may not efficiently respond to mouse GH, and generation of immunodeficient mice producing human GH may improve the efficacy of humanized mouse construction.

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Meteorin links the bone marrow hypoxic state to hematopoietic stem/progenitor cell mobilization.

Hematopoietic stem/progenitor cells (HSPCs) are supported and regulated by niche cells in the bone marrow with an important characterization of physiological hypoxia. However, how hypoxia regulates HSPCs is still unclear. Here, we find that meteorin (Metrn) from hypoxic macrophages restrains HSPC mobilization. Hypoxia-induced factor 1α and Yin Yang 1 induce the high expression of Metrn in macrophages, and macrophage-specific Metrn knockout increases HSPC mobilization through modulating HSPC proliferation and migration. Mechanistically, Metrn interacts with its receptor 5-hydroxytryptamine receptor 2b (Htr2b) to regulate the reactive oxygen species levels in HSPCs through targeting phospholipase C signaling. The reactive oxygen species levels are reduced in HSPCs of macrophage-specific Metrn knockout mice with activated phospholipase C signaling. Targeting the Metrn/Htr2b axis could therefore be a potential strategy to improve HSPC mobilization for stem cell-based therapy.

Effects of Soy-Whey Protein Nutritional Supplementation on Hematopoiesis and Immune Reconstitution in an Allogeneic Transplanted Mice.

Profound malnutrition and immunodeficiency are serious negative effects of radiotherapy and bone marrow transplantation for hematologic malignancy patients. This study aimed to evaluate the effects of nutritional supplementation with a soy-whey protein mixture on hematopoietic and immune reconstitution in an allogeneic transplant mouse model. Male BALB/c (H-2Kd) mice, 6-8 weeks-old, were divided randomly into five groups and then provided with different protein nutrition support. After 28 days, blood samples, bone marrow, spleen, and thymus were harvested to measure the effects. The results showed that soy-whey blended protein supplements promoted hematopoietic stem cell engraftment, body weight recovery, and the recovery of white blood cells, lymphocytes, and neutrophils; triggered the expansion of hematopoietic stem cells and progenitor cell pools by increasing the numbers of the c-kit+ progenitor, Lin-Sca1+c-kit+, short-term hematopoietic stem cells, and multipotent progenitors; enhanced thymus re-establishment and splenic subset recovery in both organ index and absolute number; improved overall nutritional status by increasing total serum protein, albumin, and globulin; protected the liver from radiation-induced injury, and increased antioxidant capacity as indicated by lower concentrations of alanine aminotransferase, aspartate aminotransferase, malondialdehyde, and 4-hydroxynonenal. This study indicated that soy-whey blended protein as important nutrients, from both plant and animal sources, had a greater positive effect on patients with hematological malignancies to accelerate hematopoiesis and immune reconstitution after bone marrow transplantation.

The Impact of Human Microbiotas in Hematopoietic Stem Cell and Organ Transplantation.

The human microbiota heavily influences most vital aspects of human physiology including organ transplantation outcomes and transplant rejection risk. A variety of organ transplantation scenarios such as lung and heart transplantation as well as hematopoietic stem cell transplantation is heavily influenced by the human microbiotas. The human microbiota refers to a rich, diverse, and complex ecosystem of bacteria, fungi, archaea, helminths, protozoans, parasites, and viruses. Research accumulating over the past decade has established the existence of complex cross-species, cross-kingdom interactions between the residents of the various human microbiotas and the human body. Since the gut microbiota is the densest, most popular, and most studied human microbiota, the impact of other human microbiotas such as the oral, lung, urinary, and genital microbiotas is often overshadowed. However, these microbiotas also provide critical and unique insights pertaining to transplantation success, rejection risk, and overall host health, across multiple different transplantation scenarios. Organ transplantation as well as the pre-, peri-, and post-transplant pharmacological regimens patients undergo is known to adversely impact the microbiotas, thereby increasing the risk of adverse patient outcomes. Over the past decade, holistic approaches to post-transplant patient care such as the administration of clinical and dietary interventions aiming at restoring deranged microbiota community structures have been gaining momentum. Examples of these include prebiotic and probiotic administration, fecal microbial transplantation, and bacteriophage-mediated multidrug-resistant bacterial decolonization. This review will discuss these perspectives and explore the role of different human microbiotas in the context of various transplantation scenarios.

New Approach for Targeted Treatment of Mild COVID-19 by Honeysuckle through Network Pharmacology Analysis.

OBJECTIVE: To investigate the potential pharmacological value of extracts from honeysuckle on patients with mild coronavirus disease 2019 (COVID-19) infection. METHODS: The active components and targets of honeysuckle were screened by Traditional Chinese Medicine Database and Analysis Platform (TCMSP). SwissADME and pkCSM databases predict pharmacokinetics of ingredients. The Gene Expression Omnibus (GEO) database collected transcriptome data for mild COVID-19. Data quality control, differentially expressed gene (DEG) identification, enrichment analysis, and correlation analysis were implemented by R toolkit. CIBERSORT evaluated the infiltration of 22 immune cells. RESULTS: The seven active ingredients of honeysuckle had good oral absorption and medicinal properties. Both the active ingredient targets of honeysuckle and differentially expressed genes of mild COVID-19 were significantly enriched in immune signaling pathways. There were five overlapping immunosignature genes, among which RELA and MAP3K7 expressions were statistically significant (P < 0.05). Finally, immune cell infiltration and correlation analysis showed that RELA, MAP3K7, and natural killer (NK) cell are with highly positive correlation and highly negatively correlated with hematopoietic stem cells. CONCLUSION: Our analysis suggested that honeysuckle extract had a safe and effective protective effect against mild COVID-19 by regulating a complex molecular network. The main mechanism was related to the proportion of infiltration between NK cells and hematopoietic stem cells.

Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness.

Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal.

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

An experimental study on the effect of preoperative autologous blood donation on bone marrow hematopoiesis and hematopoietic stem cells.

To evaluate the effect of autologous blood transfusion (ABT) on hematopoietic stem cells through the observation of the changes in the number and activity of bone marrow CD34+ cells after preoperative autologous blood donation (PABD). Rabbit bone marrow specimens were collected preoperatively (T2), 6 h postoperatively (T3), and 24 h postoperatively (T4). Next, the percentages of CD34+ cells, the cell cycle, and the relative expression of telomeric DNA were measured in each group of rabbits. Peripheral blood specimens were collected before PABD (T1) and at T4 to measure reticulocytes. At T3 and T4, the percentages of CD34+ cells and the expressions of telomeric DNA were significantly higher, and the percentages of cells in the G1 phase were significantly lower in each experimental group compared with those in the blank control group (group A) (P<0.05). Compared with the surgical blood collection group (group C), the CD34+ cells and the expressions of telomeric DNA were significantly higher, and the percentages of cells in the G1 phase were significantly lower in the preoperative autologous whole blood group (group D) and the preoperative autologous blood component group (group E) (P<0.05). Compared with group D, the CD34+ cells and the expressions of telomeric DNA were significantly lower, and the percentages of cells in the G1 phase were significantly higher in group E (P<0.05). At T4, the reticulocyte percentages in the surgery group (group B) and group C were significantly higher than in group E and group D, and the reticulocyte percentages in group E were higher than in group D (P<0.05). This study's findings indicated that ABT suppressed bone marrow hematopoiesis, while autologous blood component transfusion had less of an effect than that of whole blood transfusion. Therefore, PABD blood component transfusion would be superior to autologous whole blood transfusion.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

Ascorbic acid supports ex vivo generation of plasmacytoid dendritic cells from circulating hematopoietic stem cells.

Plasmacytoid dendritic cells (pDCs) constitute a rare type of immune cell with multifaceted functions, but their potential use as a cell-based immunotherapy is challenged by the scarce cell numbers that can be extracted from blood. Here, we systematically investigate culture parameters for generating pDCs from hematopoietic stem and progenitor cells (HSPCs). Using optimized conditions combined with implementation of HSPC pre-expansion, we generate an average of 465 million HSPC-derived pDCs (HSPC-pDCs) starting from 100,000 cord blood-derived HSPCs. Furthermore, we demonstrate that such protocol allows HSPC-pDC generation from whole-blood HSPCs, and these cells display a pDC phenotype and function. Using GMP-compliant medium, we observe a remarkable loss of TLR7/9 responses, which is rescued by ascorbic acid supplementation. Ascorbic acid induces transcriptional signatures associated with pDC-specific innate immune pathways, suggesting an undescribed role of ascorbic acid for pDC functionality. This constitutes the first protocol for generating pDCs from whole blood and lays the foundation for investigating HSPC-pDCs for cell-based immunotherapy.