Bone Marrow-derived Hematopoietic Stem Cells Research Articles

Current Perspectives and Progress in Preoperative Portal Vein Embolization with Stem Cell Augmentation (PVESA).

Portal vein embolization with stem cell augmentation (PVESA) is an emerging approach for enhancing the growth of the liver segment that will remain after surgery (i.e., future liver remnant, FLR) in patients with liver cancer. Conventional portal vein embolization (PVE) aims to induce preoperative FLR growth, but it has a risk of failure in patients with underlying liver dysfunction and comorbid illnesses. PVESA combines PVE with stem cell therapy to potentially improve FLR size and function more effectively and efficiently. Various types of stem cells can help improve liver growth by secreting paracrine signals for hepatocyte growth or by transforming into hepatocytes. Mesenchymal stem cells (MSCs), unrestricted somatic stem cells, and small hepatocyte-like progenitor cells have been used to augment liver growth in preclinical animal models, while clinical studies have demonstrated the benefit of CD133 + bone marrow-derived MSCs and hematopoietic stem cells. These investigations have shown that PVESA is generally safe and enhances liver growth after PVE. However, optimizing the selection, collection, and application of stem cells remains crucial to maximize benefits and minimize risks. Additionally, advanced stem cell technologies, such as priming, genetic modification, and extracellular vesicle-based therapy, that could further enhance efficacy outcomes should be evaluated. Despite its potential, PVESA requires more investigations, particularly mechanistic studies that involve orthotopic animal models of liver cancer with concomitant liver injury as well as larger human trials.

Long-term and real-world safety and efficacy of retroviral gene therapy for adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency leads to severe combined immunodeficiency (SCID). Previous clinical trials showed that autologous CD34+ cell gene therapy (GT) following busulfan reduced-intensity conditioning is a promising therapeutic approach for ADA-SCID, but long-term data are warranted. Here we report an analysis on long-term safety and efficacy data of 43 patients with ADA-SCID who received retroviral ex vivo bone marrow-derived hematopoietic stem cell GT. Twenty-two individuals (median follow-up 15.4 years) were treated in the context of clinical development or named patient program. Nineteen patients were treated post-marketing authorization (median follow-up 3.2 years), and two additional patients received mobilized peripheral blood CD34+ cell GT. At data cutoff, all 43 patients were alive, with a median follow-up of 5.0 years (interquartile range 2.4-15.4) and 2 years intervention-free survival (no need for long-term enzyme replacement therapy or allogeneic hematopoietic stem cell transplantation) of 88% (95% confidence interval 78.7-98.4%). Most adverse events/reactions were related to disease background, busulfan conditioning or immune reconstitution; the safety profile of the real world experience was in line with premarketing cohort. One patient from the named patient program developed a T cell leukemia related to treatment 4.7 years after GT and is currently in remission. Long-term persistence of multilineage gene-corrected cells, metabolic detoxification, immune reconstitution and decreased infection rates were observed. Estimated mixed-effects models showed that higher dose of CD34+ cells infused and younger age at GT affected positively the plateau of CD3+ transduced cells, lymphocytes and CD4+ CD45RA+ naive T cells, whereas the cell dose positively influenced the final plateau of CD15+ transduced cells. These long-term data suggest that the risk-benefit of GT in ADA remains favorable and warrant for continuing long-term safety monitoring. Clinical trial registration: NCT00598481 , NCT03478670 .

The Role of Hematopoietic Stem Cells in Lung Cancer Response to Radiation Therapy

Non-small cell lung carcinoma (NSCLC) comprises approximately 85% of all lung cancer cases in the United States. We demonstrated that after radiation therapy (RT), bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) are recruited to the NSCLC tumor microenvironment (TME). These HSPCs seemed to positively affect tumor regrowth as their numbers directly correlated with regrowth rates. The TME is comprised of tumor tissue, stromal cells, immune cells, bone marrow-derived cells, and extra cellular matrix (ECM) proteins and has emerged as one of the predominant defensive weapons with which cancer can resist various forms of therapeutic intervention. The ability of tumor-associated HSPCs to maintain hematopoietic potency in tumors suggested a prominent role of these cells in tumor physiology, however; what was missing was a mechanistic understanding of: 1) how HSPCs participate in tumor growth post-RT and 2) how HSPCs are maintained within tumors. To answer these questions, we established NSCLC tumors in mice using Lewis Lung Carcinoma (LLC) cells and assessed tumor-associated HSPCs activity and molecular changes in the TME following RT. We observed that RT induces the TME to produce colony stimulating factor (CSF)-1, a factor that promotes the differentiation of HSPCs into tumor supportive M2-macrophages. Adoptive transplantation of dsRed labeled HSPCs into tumor bearing mice followed by RT directly showed the differentiation of HSPCs into M2-macrophages and a concomitant increase in the regrowth of tumors post-RT. Use of a therapeutic inhibitor of the CSF-1 receptor (GW2580) prevented HSPC differentiation in to M2-macrophages and significantly decreasing regrowth rates. We next assessed how tumor-associated HSPCs maintain their hematopoietic potency within the TME. Analysis of tumors demonstrated high expression levels of the laminin 5-1-1 isoform, which is known to bind CD49f (integrin alpha 6), a marker of functional HSPCs. Tumors exposed to RT demonstrated significantly reduced expression of both laminin 5-1-1 and CD49f. When bound, CD49f inhibits phosphorylation of focal adhesion kinase (FAK) resulting in maintenance of pluripotent stem cells. Analysis of tumor-associated HSPCs demonstrated significantly higher levels of phosphorylated FAK following tumor exposure to RT. Since RT induces HSPC differentiation in to M2-macophages, this observation suggests that tumor-associated HSPC functionality is maintained through the interaction between laminin 5-1-1- and CD49f. The identification of single HSPCs that differentiated in to M2-macrophages and which expressed phosphorylated FAK supports this conclusion. Taken together, this data province's evidence of a mechanism wherein RT disrupts laminin 5-1-1/CD49f interactions releasing tumor-associated HSPCs from maintenance signals. Concomitantly, RT induces the production of CSF-1 within the TME, which induces these “released” HSPCs to differentiate into tumor supportive M2-macrophages and promote tumor regrowth post-RT. Overall, these studies define a new understanding of tumor biology in which HSPCs are significant mediators of tumor response to RT. These findings also identify new cellular and molecular pathways to target in the treatment of NSCLC to achieve more optimal clinical outcomes.

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Abstract 18645: Phenylacetylglutamine Causes a Pathologic Hyperinflammatory State and Enhances Atherosclerosis Through the ß2-Adrenergic Receptor in Diabetes

Background: Type 2 diabetes (T2DM) affects billions of patients and has a pathophysiologically important inflammatory component. Macrophage plasticity in diabetes is impaired, and remains in a persistent proinflammatory state; yet currently, the cause of the inflammatory state is unknown. Phenylacetylglutamine (PAGln) is an amino acid metabolism end product that rises in T2DM patients and is associated with incident major adverse cardiovascular events. However, the role of PAGln in the inflammatory component of T2DM remains unknown. Purpose: Here, we aim to identify blood-based systemic factors for pathologic inflammation status and risk of atherosclerosis as they are highly reproducible at low cost and search for effective and safe agents to attenuate diabetic inflammatory complications. Methods: In vivo, phenylacetylglutamine was dissolved in normal saline and injected in mice daily (ip). The levels of PAGln were analyzed by liquid chromatography-mass spectrometry. The effect of PAGln on atherosclerosis progression was analyzed by Ldlr-/- mice, followed by a Western diet feeding for 12 weeks. In vitro, primary mice bone marrow-derived macrophages (BMDMs) and haematopoietic stem cells (HSCs) were treated with phenylacetylglutamine (100 uM) for the indicated time. Results: In vivo, increased immune cells and inflammatory macrophages were detected in PAGln-treated mice blood, spleens, bone marrow, and arteries. In vitro, PAGln leads to macrophage M1 polarization. The result of bone marrow transplantation on Ldlr-/- mice further confirmed the effect of PAGln on the central immune system of mice. Besides, the negative effects of PAGln could be rescued by ß-blockers. Conclusion: Our study unravels a novel mechanism by which PAGln accelerates atherosclerosis progression and causes pathologic inflammation by activating the ß2-adrenergic receptor. The negative effect of PAGln could be rescued by carvedilol (ß blocker). Thus, our recognition of chronic inflammation in diabetes needs to be revised, and understanding these mechanisms can inform alternative treatment strategies and refine atherosclerosis repair therapies for diabetic patients.

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.

The release of osteoclast-stimulating factors on supraphysiological loading by osteoprogenitors coincides with expression of genes associated with inflammation and cytoskeletal arrangement

Supraphysiological loading induced by unstable orthopedic implants initiates osteoclast formation, which results in bone degradation. We aimed to investigate which mechanosensitive cells in the peri-implant environment produce osteoclast-stimulating factors and how the production of these factors is stimulated by supraphysiological loading. The release of osteoclast-stimulating factors by different types of isolated bone marrow-derived hematopoietic and mesenchymal stem cells from six osteoarthritic patients was analyzed after one hour of supraphysiological loading (3.0 ± 0.2 Pa, 1 Hz) by adding their conditioned medium to osteoclast precursors. Monocytes produced factors that enhanced osteoclastogenesis by 1.6 ± 0.07-fold and mesenchymal stem cells by 1.4 ± 0.07-fold. Medium from osteoprogenitors and pre-osteoblasts enhanced osteoclastogenesis by 1.3 ± 0.09-fold and 1.4 ± 0.03-fold, respectively, where medium from four patients elicited a response and two did not. Next generation sequencing analysis of osteoprogenitors revealed that genes encoding for inflammation-related pathways and cytoskeletal rearrangements were regulated differently between responders and non-responders. Our data suggest that released osteoclast-stimulating soluble factors by progenitor cells in the bone marrow after supraphysiological loading may be related to cytoskeletal arrangement in an inflammatory environment. This connection could be relevant to better understand the aseptic loosening process of orthopedic implants.

Genetic correction of concurrent \u03b1- and \u03b2-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology

BackgroundThalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above.MethodsIn the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays.ResultsThese mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential.ConclusionsWe designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia.

Long-term Persistence of Allosensitization After Islet Allograft Failure.

Allosensitization has been reported after discontinuation of immunosuppression following graft failure in islet transplantation (ITx) recipients, though duration of its persistence is unknown. We evaluated 35 patients with type 1 diabetes who received ITx, including 17 who developed graft failure (ITx alone, n = 13; ITx plus bone marrow-derived hematopoietic stem cells, n = 4) and 18 with persistent graft function. Panel-reactive antibody (PRA) was measured yearly for the duration of graft function within 1 y after graft failure at enrollment and yearly thereafter. In ITx alone graft failure patients, 61% (8/13) were PRA-positive at 6 y postgraft failure, and 46% (6/13) developed donor-specific anti-HLA antibodies (DSA to 2 ± 1 donors) during follow-up. The degree of sensitization was variable (cPRA ranging between 22% and 100% after graft failure). Allosensitization persisted for 7-15 y. Three subjects (3/13) were not allosensitized. In ITx plus bone marrow-derived hematopoietic stem cell recipients, cPRA-positivity (88%-98%) and DSA positivity persisted for 15 y in 75% (3/4) of subjects. Allosensitization was minimal while subjects remained on immunosuppression, but after discontinuation of immunosuppressive therapy, the majority of subjects (77%) became allosensitized with persistence of PRA positivity for up to 15 y. Persistence of allosensitization in this patient population is of clinical importance as it may result in longer transplant waiting list times for identification of a suitable donor in the case of requiring a subsequent transplant.

The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

TREM2 promotes natural killer cell development in CD3\u2212CD122+NK1.1+ pNK cells

BackgroundTriggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells.ResultsHere, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice.ConclusionsCollectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

PROGRESSION OF PULMONARY HYPERTENSION IS ATTENUATED BY COMBINED TREATMENT WITH GRANULOCYTE COLONY-STIMULATING FACTOR AND BONE MARROW-DERIVED MESENCHYMAL STEM CELLS

<h3>Background</h3> Pulmonary arterial hypertension (PAH) is a cardiovascular disease characterized by increased resting pulmonary pressure levels. The prognosis remains poor, with a 5-years mortality rate estimated at 40%. Mesenchymal stem cells (MSCs) transplantation has demonstrated promising therapeutic effects in the treatment of pulmonary hypertensive rodents, as well as the administration of CXCR4/SDF inhibitor granulocyte-colony-stimulating factor (G-CSF). However, it remains unclear whether the association of MSCs and G-CSF therapies provides additional salutary effects in the treatment of PAH compared to the isolated treatments. <h3>Methods</h3> PAH was induced in adult male Wistar rats (Protocol number 087/15) by intraperitoneal injection of monocrotaline (MCT, 60mg/kg IP), while the Control group (CTL) received vehicle. Two weeks after the MCT injection, a subset of MCT-treated rats was treated with vehicle, MSCs (5 × 106 cells), G-CSF (50 µg/kg/d for 14 days), or the combination of both, totaling five groups (CTL n=8, MCT n=7, MSCs n=10, G-CSF n=13, and MSCs+G-CSF n=13). Electrocardiographic, echocardiographic, and hemodynamic recordings were performed. Blood samples were collected for the characterization of circulating bone marrow-derived hematopoietic stem cells (HSC). Data were analyzed with One-way and Two-way ANOVA (Prism®, GraphPad) and were expressed as mean ± SEM. <h3>Results</h3> Compared to the CTL group, the MCT group exhibited a decreased pulmonary acceleration time/pulmonary ejection time (CTL 0.46±0.01 vs MCT 0.22±0.02, <i>p</i><0.05), while it was not affected by neither therapeutic approach (<i>p</i>>0.05). Whereas the MCT group exhibited a decreased velocity-time index versus the CTL group, it was improved by the MSCs+G-CSF therapy (CTL 4±0.2 vs MCT 2.1±0.4 vs MSCs 2.5±0.5 vs G-CSF 3.1±0.4 vs MSCs+G-CSF 4.9±0.5 cm, p<0.05). The G-CSF group exhibited decreased levels of RV systolic pressure versus the MCT group (MCT 39.4±3.15 vs G-CSF 28.7±1.1, <i>p</i><0.05). The QTc interval was prolonged in groups versus CTL group (CTL 164.2±7.2 vs MCT 198.6±5.5 vs MSCs 190.7±6.4 vs G-CSG 193.4±7.1 vs MSCs+G-CSF 183.9±9.6 ms, <i>p</i><0.05). Furthermore, the relative cardiac weight was significantly decreased by the MSCs therapy versus the MCT group (CTL 3.6±0.3 vs MCT 4.7±0.3 vs MSCs 3.1±0.2 G-CSF 4.2±0.2 vs MSCs+G-CSF 3.9±0.3, <i>p</i><0.05). <h3>Conclusion</h3> Our study indicates that G-CSF and MSCs have therapeutic potential in the treatment of PAH either when administered isolated or combined.

Cigarette smoke extract affects methylation status and attenuates Sca-1 expression of mouse endothelial progenitor cell in vitro.

INTRODUCTIONEndothelial dysfunction appears in many smoking-related diseases, it is also an important pathophysiological feature. Endothelial progenitor cells (EPCs) are precursors of endothelial cells and have a crucial effect on the repair and maintenance of endothelial integrity. Sca-1 is not only common in bone marrow-derived hematopoietic stem cells (HSCs), but it is also expressed in nonhematopoietic organs by tissue-resident stem and progenitor cells. The aim of this study is to investigate the impact of cigarette smoke extract (CSE) on the function of bone marrow-derived EPCs and the expression level of Sca-1 in EPCs, and also whether the methylation of Sca-1 is involved in EPC dysfunction.METHODSWe measured EPC capacities including adhesion, secretion and proliferation, the concentration of endothelial nitric oxide synthase (eNOS) and apoptosis-inducing factor (AIF) in cell culture supernatant, and also Sca-1 expression and promoter methylation in EPCs induced by CSE. Decitabine (Dec) was applied to test whether it could alter the impact caused by CSE.RESULTSThe adhesion, proliferation and secretion ability of EPCs can be induced to be decreased by CSE in vitro, accompanied by decreased concentrations of AIF and eNOS in cell culture supernatant and decreased Sca-1 expression in EPCs. In addition, Dec could partly attenuate the impact described above. There were no significant differences in the quantitative analysis of Sca-1 promoter methylation among different groups.CONCLUSIONSThe decreased Sca-1 expression was related to EPC dysfunction induced by CSE. EPC dysfunction resulting from CSE may be related to methylation mechanism, but not the methylation of Sca-1 promoter.

Antiviral effect of honey extract Camelyn against SARS-CoV-2

This study aimed to evaluate the potential antiviral effects of honey extract “Camelyn” against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The baby hamster kidney cell line 21 (BHK‐21), bone marrow-derived hematopoietic stem cells (HSCs), and splenic cells were used for Camelyn cytotoxicity assay. After the isolation procedures, cell viability was assessed by trypan blue dye exclusion under a microscope using a hemocytometer. The in vitro cell growth rate was carried out using the cell counting Kit 8 (CCK-8) assay. The cells were seeded in growth media with various Camelyn concentrations (35 μg, 50 μg, 70 μg, 100 μg, 150 μg, and 200 μg). The absorbance at 450 nm was determined by the multiplate reader. The antiviral effect was assessed by plaque reduction assay for the determination of drug susceptibility against SARS-CoV-2. Serial dilution of the selected compounds was pre-incubated with 40 to 100 plaque-forming units (PFUs) of SARS-CoV-2. The pre-incubated mix of Camelyn and SARS-CoV-2 was then added to the confluent Vero E6 cells after incubation cells were fixed and stained and the number of PFUs was counted under an inverted microscope and plotted against the logarithm of antiviral concentrations. Our study showed that Camelyn is not cytotoxic, has a stimulatory effect on cell proliferation, and has an inhibitory effect against SARS-CoV-2 with EC50 (half-maximal effective concentration) from 85.7 μg/mL to 192.4 μg/mL depending on product concentration and viral plaque per cell. © 2021 Global Research Online. All rights reserved.

ARID3a expression in human hematopoietic stem cells is associated with distinct gene patterns in aged individuals

BackgroundImmunologic aging leads to immune dysfunction, significantly reducing the quality of life of the elderly. Aged-related defects in early hematopoiesis result in reduced lymphoid cell development, functionally defective mature immune cells, and poor protective responses to vaccines and pathogens. Despite considerable progress understanding the underlying causes of decreased immunity in the elderly, the mechanisms by which these occur are still poorly understood. The DNA-binding protein ARID3a is expressed in a subset of human hematopoietic progenitors. Inhibition of ARID3a in bulk human cord blood CD34+ hematopoietic progenitors led to developmental skewing toward myeloid lineage at the expense of lymphoid lineage cells in vitro. Effects of ARID3a expression in adult-derived hematopoietic stem cells (HSCs) have not been analyzed, nor has ARID3a expression been assessed in relationship to age. We hypothesized that decreases in ARID3a could explain some of the defects observed in aging.ResultsOur data reveal decreased frequencies of ARID3a-expressing peripheral blood HSCs from aged healthy individuals compared with young donor HSCs. Inhibition of ARID3a in young donor-derived HSCs limits B lineage potential, suggesting a role for ARID3a in B lymphopoiesis in bone marrow-derived HSCs. Increasing ARID3a levels of HSCs from aged donors in vitro alters B lineage development and maturation. Finally, single cell analyses of ARID3a-expressing HSCs from young versus aged donors identify a number of differentially expressed genes in aged ARID3A-expressing cells versus young ARID3A-expressing HSCs, as well as between ARID3A-expressing and non-expressing cells in both young and aged donor HSCs.ConclusionsThese data suggest that ARID3a-expressing HSCs from aged individuals differ at both molecular and functional levels compared to ARID3a-expressing HSCs from young individuals.

Effect of N-Acetylcysteine Supplementation on Oxidative Stress-Mediated Cryoinjury of Bone Marrow Derived-Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) transplantation was introduced as curative treatment for various diseases. Cryopreservation of HSCs is crucial for long term storage and maintenance of cellular quality; however, it has been reported that cryopreservation itself causes oxidative stress-driven apoptosis and cell loss. This study investigated impact of supplementing N-acetylcysteine (NAC) as antioxidant during cryopreservation on viability and oxidative stress in HSCs. HSCs were isolated from murine bone marrow, cultured in HSCs growth media and cryopreserved (1×106 cells per vial) together with 10% DMSO and NAC (0, 0.25, 0.5 or 2.0 μM) for 48 h, 2 weeks or 8 weeks at -196°C using controlled-rate-freezing technique. Cell viability and oxidative stress in cryopreserved cells were analysed at each time-point. Cell viability was significantly reduced (p<0.05) following cryopreservation as compared to pre-cryopreservation. NAC supplementation significantly increased cell viability (p<0.05) after 48 h cryopreservation at 0.5 μM and 2.0 μM and after 2 weeks cryopreservation at 0.25 μM compared to the controls. Cryopreservation significantly enhanced GSH level (p<0.05) and reduced MDA level (p<0.05) without affecting SOD activity and PC level in HSCs compared to pre-cryopreservation. NAC supplementation significantly increased GSH level at 0.25 μM in cryopreserved HSCs compared to control. In conclusion, NAC supplementation during cryopreservation showed potential in minimizing cryoinjury by promoting cell viability, increasing antioxidant capacity and reducing oxidative damage in HSCs, however these effects are influenced by both durations of cryopreservation and NAC concentration.

Orphan designation: Autologous human bone marrow-derived haematopoietic and mesenchymal stem cells depleted of erythrocytes, monocytes and lymphocytes, Treatment of spinal cord injury

Orphan designation: Autologous human bone marrow-derived haematopoietic and mesenchymal stem cells depleted of erythrocytes, monocytes and lymphocytes, Treatment of spinal cord injury

A Single-Cell Immunofluorescence Method for the Division Patterns Research of Mouse Bone Marrow-Derived Hematopoietic Stem Cells

How hematopoietic stem cells (HSCs) maintain the balance of self-renewal and differentiation could be partially ascribed to asymmetric and symmetric division patterns. However, a simple and effective method to detect stem cell division patterns is lacking. In this study, we introduce a strategy to describe stem cells division patterns with high spatial resolution at the single-cell level. We show that the fate determinant, Numb, exhibits low expression levels in HSCs that increase upon the initiation of differentiation. Using this single-cell immunofluorescence technique, we found that HSCs mainly undergo symmetric self-renewal in the presence of only stem cell factor, but with the addition of trombopoietin this division pattern is transformed into a symmetric commitment dominant mode in vitro. In addition, our study indicated that the division pattern cannot be defined by cell size or the nuclear/cytoplasm ratio. These findings collectively demonstrate that this single-cell immunofluorescence technique provides a new biological strategy in stem cell division research, and can be more widely applied given its flexibility, easy operability, and inexpensiveness.

Evaluation of a vial adaptor to ensure safe and efficient needle-free transfer of cells post-cryopreservation in preparation for drug delivery

Background & Aim The commercialization of cells for therapeutic use drives a critical need for the refinement of cell storage and handling procedures, ensuring the integrity of cell-based products is maintained throughout the product life cycle. One area for refinement is the transfer of cells from a sealed container system after they have been cryopreserved, stored, shipped and thawed. There are many parameters that may affect either the total volume or the viability of the cells recovered. These include the type of transfer equipment used, the properties of the cryo-formulations that may have different viscosities or rheology, and the proficiency of the manual operator performing the task. This study investigates the comparability of a vial adaptor when compared to a traditional needle and syringe for recovering cells from a sealed cyclic olefin polymer (COP) vials after cryopreservation and storage for one week at -180°C. Methods, Results & Conclusion One experiment was designed to evaluate the effect of operator variability (n = 4) when transferring 1 ml fill volumes containing 1 × 106 cells/ml stored in Cryostor CS10 using a vial adaptor or needle and syringe. Bone marrow-derived human mesenchymal stem cells (hMSCs) and cord blood hematopoietic stem cells (HSCs) were used as exemplar adherent and suspension cell types. Total volume recovery from each system was measured, as was morphology, cell yield and viability after a 4-day outgrowth to determine cell and volume losses from the transfer process. A second experiment looked at the effect different cell densities and cryo-formulations may have during transfer. 1ml vials of hMSCs at different densities (5 e5/ml and 4 e6/ml) were prepared and stored in 4 different formulations with differing colloidal components. The cell yield and viability were measured immediately post-transfer as was total recovered volume. The results showed that recovery of both intact cells and product volume were >80% for recovered volume with a suitable cryo-formulation regardless of transfer method (Figure 1). The vial adaptor was comparable in performance to a needle and syringe, thus providing a safe needle-free alternative for operators during manual transfer. Furthermore, the data highlighted the how cryo-formulation choice and operator variability can impact upon overall product recovery in a cell therapy context.

Non-thermal atmospheric pressure plasma is an excellent tool to activate proliferation in various mesoderm-derived human adult stem cells

Adult stem cells are capable of self-renewal and differentiation into specific cell types in tissues and have high potential for stem cell therapy. Mesenchymal and hematopoietic stem cells are easily attainable from the human body and have become applicable tools for adult stem cell therapy. However, there are still technical barriers for the application of mesenchymal and hematopoietic stem cells for therapy, such as the small number of cell populations, high risk of contamination, and loss of their stemness properties in vitro. In our previous study, we showed that non-thermal atmospheric pressure plasma (NTAPP) promoted the proliferation of adipose tissue-derived stem cells (ASCs) by 1.6-fold on average, while maintaining their stemness. Here, we examined the feasibility of NTAPP as a tool to activate the proliferation of mesenchymal and hematopoietic stem cells in vitro without affecting their stem cell characteristics. NTAPP increased the proliferation of bone marrow-derived stem cells (BM-MSCs) and hematopoietic stem cells (HSCs) by 1.8- and 2-fold, respectively, when compared to that of untreated cells. As observed in ASCs, NTAPP exposure also activated the expression of stem cell-specific surface markers, CD44 and CD105, by 5-fold in BM-MSCs, when compared to that in unexposed control cells in a low glucose medium with a low concentration of basic fibroblast growth factor (b-FGF). In addition, NTAPP exposure highly augmented the mRNA expression of well-known pluripotent genes for stemness, such as Oct4, Sox2, and Nanog in ASCs and BM-MSCs when compared to that in unexposed control cells. When cell cycle progression was examined, the G1-S shift was accelerated, and expression of PCNA was increased in NTAPP-exposed ASCs when compared to that in untreated control cells, suggesting that NTAPP activated G1-S transition. Taken together, these results demonstrated that NTAPP activated the proliferation of various mesodermal-derived human adult stem cells by accelerating the G1-S transition while maintaining their pluripotency and stemness, strongly suggesting that NTAPP can be an efficient tool for expanding the population of various adult stem cells in vitro for medical applications.