Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening.

Mitochondria are key regulators of hematopoietic stem cell (HSC) homeostasis. Our research identifies the transcription factor Nynrin as a crucial regulator of HSC maintenance by modulating mitochondrial function. Nynrin is highly expressed in HSCs under both steady-state and stress conditions. The knockout Nynrin diminishes HSC frequency, dormancy, and self-renewal, with increased mitochondrial dysfunction indicated by abnormal mPTP opening, mitochondrial swelling, and elevated ROS levels. These changes reduce HSC radiation tolerance and promote necrosis-like phenotypes. By contrast, Nynrin overexpression in HSCs diminishes irradiation (IR)-induced lethality. The deletion of Nynrin activates Ppif, leading to overexpression of cyclophilin D (CypD) and further mitochondrial dysfunction. Strategies such as Ppif haploinsufficiency or pharmacological inhibition of CypD significantly mitigate these effects, restoring HSC function in Nynrin-deficient mice. This study identifies Nynrin as a critical regulator of mitochondrial function in HSCs, highlighting potential therapeutic targets for preserving stem cell viability during cancer treatment.

International Society for Cell & Gene Therapy Stem Cell Engineering Committee report on the current state of hematopoietic stem and progenitor cell-based genomic therapies and the challenges faced.

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent ß-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions.

Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Exploring the potential of predicted miRNAs on the genes involved in the expansion of hematopoietic stem cells.

A major challenge in therapeutic approaches applying hematopoietic stem cells (HSCs) is the cell quantity. The primary objective of this study was to predict the miRNAs and anti-miRNAs using bioinformatics tools and investigate their effects on the expression levels of key genes predicted in the improvement of proliferation, and the inhibition of differentiation in HSCs isolated from Human umbilical cord blood (HUCB). A network including genes related to the differentiation and proliferation stages of HSCs was constructed by enriching data of text (PubMed) and StemChecker server with KEGG signaling pathways, and was improved using GEO datasets. Bioinformatics tools predicted a profile from miRNAs containing miR-20a-5p, miR-423-5p, and chimeric anti-miRNA constructed from 5'-miR-340/3'-miR-524 for the high-score genes (RB1, SMAD4, STAT1, CALML4, GNG13, and CDKN1A/CDKN1B genes) in the network. The miRNAs and anti-miRNA were transferred into HSCs using polyethylenimine (PEI). The gene expression levels were estimated using the RT-qPCR technique in the PEI + (miRNA/anti-miRNA)-contained cell groups (n = 6). Furthermore, CD markers (90, 16, and 45) were evaluated using flow cytometry. Strong relationships were found between the high-score genes, miRNAs, and chimeric anti-miRNA. The RB1, SMAD4, and STAT1 gene expression levels were decreased by miR-20a-5p (P < 0.05). Additionally, the anti-miRNA increased the gene expression level of GNG13 (P < 0.05), whereas the miR-423-5p decreased the CDKN1A gene expression level (P < 0.01). The cellular count also increased significantly (P < 0.05) but the CD45 differentiation marker did not change in the cell groups. The study revealed the predicted miRNA/anti-miRNA profile expands HSCs isolated from HUCB. While miR-20a-5p suppressed the RB1, SMAD4, and STAT1 genes involved in cellular differentiation, the anti-miRNA promoted the GNG13 gene related to the proliferation process. Notably, the mixed miRNA/anti-miRNA group exhibited the highest cellular expansion. This approach could hold promise for enhancing the cell quantity in HSC therapy.

Reconstitution of human microglia and resident T cells in the brain of humanized DRAGA mice.

Humanized mouse models are valuable tools for investigating the human immune system in response to infection and injury. We have previously described the human immune system (HIS)-DRAGA mice (HLA-A2.HLA-DR4.Rag1KO.IL-2RgKO.NOD) generated by infusion of Human Leukocyte Antigen (HLA)-matched, human hematopoietic stem cells from umbilical cord blood. By reconstituting human cells, the HIS-DRAGA mouse model has been utilized as a "surrogate in vivo human model" for infectious diseases such as Human Immunodeficiency Virus (HIV), Influenza, Coronavirus Disease 2019 (COVID-19), scrub typhus, and malaria. This humanized mouse model bypasses ethical concerns about the use of fetal tissues for the humanization of laboratory animals. Here in, we demonstrate the presence of human microglia and T cells in the brain of HIS-DRAGA mice. Microglia are brain-resident macrophages that play pivotal roles against pathogens and cerebral damage, whereas the brain-resident T cells provide surveillance and defense against infections. Our findings suggest that the HIS-DRAGA mouse model offers unique advantages for studying the functions of human microglia and T cells in the brain during infections, degenerative disorders, tumors, and trauma, as well as for testing therapeutics in these pathological conditions.

How aging influences the gut-bone marrow axis and alters hematopoietic stem cell regulation.

The gut microbiome has come to prominence across research disciplines, due to its influence on major biological systems within humans. Recently, a relationship between the gut microbiome and hematopoietic system has been identified and coined the gut-bone marrow axis. It is well established that the hematopoietic system and gut microbiome separately alter with age; however, the relationship between these changes and how these systems influence each other demands investigation. Since the hematopoietic system produces immune cells that help govern commensal bacteria, it is important to identify how the microbiome interacts with hematopoietic stem cells (HSCs). The gut microbiota has been shown to influence the development and outcomes of hematologic disorders, suggesting dysbiosis may influence the maintenance of HSCs with age. Short chain fatty acids (SCFAs), lactate, iron availability, tryptophan metabolites, bacterial extracellular vesicles, microbe associated molecular patterns (MAMPs), and toll-like receptor (TLR) signalling have been proposed as key mediators of communication across the gut-bone marrow axis and will be reviewed in this article within the context of aging.

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.

The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

ZMAT3 participated in benzene-caused disruption in self-renewal and differentiation of hematopoietic stem cells via TNF-α/NF-κB pathway.

Benzene is a common environmental and occupational pollutant, benzene exposure causes damage to hematopoietic system. ZMAT3 is a zinc finger protein which has important biological functions. In this study, benzene-exposed mouse model and ZMAT3 overexpression and low expression hematopoietic stem cells (HSCs) models were constructed to explore the mechanism of ZMAT3 in benzene-induced hematopoietic toxicity. The results showed that benzene increased the expression of ZMAT3 in mouse bone marrow (BM) cells, HSCs and peripheral blood (PB) leukocyte, and the changes in HSCs were more sensitive than BM and PB cells. In addition, overexpression of ZMAT3 decreased the self-renewal ability of HSCs and reduced the HSCs differentiation into myeloid hematopoietic cells, while low expression has the opposite effect. Besides, over and low expression of ZMAT3 both increased the HSCs differentiation into lymphoid progenitor cells. Moreover, bioinformatics analysis suggested that ZMAT3 was associated with TNF-α signaling pathway, and the correlation was confirmed in mouse model. Meanwhile, the results indicated that ZMAT3 promoted TNF-α mRNA processing by binding to the ARE structural domain on TNF-α and interacting with hnRNP A2/B1 and hnRNP A1 proteins, ultimately activating the NF-κB signaling pathway. This study provides a new mechanism for the study of benzene toxicity.

Advancements in Hematopoietic Stem Cell Gene Therapy: A Journey of Progress for Viral Transduction.

Hematopoietic stem cell (HSC) transduction has undergone remarkable advancements in recent years, revolutionizing the landscape of gene therapy specifically for inherited hematologic disorders. The evolution of viral vector-based transduction technologies, including retroviral and lentiviral vectors, has significantly enhanced the efficiency and specificity of gene delivery to HSCs. Additionally, the emergence of small molecules acting as transduction enhancers has addressed critical barriers in HSC transduction, unlocking new possibilities for therapeutic intervention. Furthermore, the advent of gene editing technologies, notably CRISPR-Cas9, has empowered precise genome modification in HSCs, paving the way for targeted gene correction. These striking progresses have led to the clinical approval of medicinal products based on engineered HSCs with impressive therapeutic benefits for patients. This review provides a comprehensive overview of the collective progress in HSC transduction via viral vectors for gene therapy with a specific focus on transduction enhancers, highlighting the latest key developments, challenges, and future directions towards personalized and curative treatments.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Imaging Flow Cytometry and Convolutional Neural Network-Based Classification Enable Discrimination of Hematopoietic and Leukemic Stem Cells in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring.

Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging.

Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.

Facilitating the ethical sourcing of donor hematopoietic stem cells for cell and gene therapy research and development.

Aim: Unrelated stem cell donor registries (DRs) are increasingly engaging in the field of cell and gene therapy (CGT). This study aims to explore the values, concerns, needs and expectations of donors and members of the public on donating hematopoietic stem cells (HSCs) for CGT. Methods: Seven focus groups were conducted in 2019 with members of the public, prospective donors and donors on the Anthony Nolan DR in the UK. Results: Participants expressed concerns over increased frequency of donation and incidental findings and required more information on the type of research including the purpose and possible outcomes. Conclusion: Addressing donors' concerns, needs and expectations on donating cellular materials for CGT research and development is essential to maintaining the highest standards for donor care and safety within this rapidly emerging field.

This study aims to explore the values, concerns, needs and expectations of people who donate, or consider donating, their stem cells (cells that can develop into many different types of cells) for research that could lead to new medical treatments. We focused on the thoughts of these donors about providing their cells for use in cell and gene therapy (CGT) research, a field that is rapidly advancing but still forming its rules and ethical guidelines. In 2019, we conducted seven focus groups (FGs) with a total of 73 people in the UK. This included individuals who are registered as potential stem cell donors on the Anthony Nolan unrelated stem cell donor register (DR), those who have already donated stem cells and members of the general public. We explored their thoughts about their donated cells being used for research to develop new therapies rather than for direct treatment of patients. Questions during the FGs touched on topics such as the roles of various organizations in managing donated cells, the commercial use of these cells and where responsibilities lie in ensuring ethical practices. Participants expressed a strong desire for openness and clear communication regarding how their donated cells are used in research. They wanted to ensure that any use of their cells aligns with their personal values and the ethical standards of the organizations handling the donations. Participants expected DRs like Anthony Nolan to safeguard their interests and the ethical use of their cells. This study highlights that while donors are generally willing to contribute to advancements in CGT research, they need clear, understandable information about how their donations are used. This is crucial for maintaining their trust and willingness to donate. Overall, this study underscores the importance of ethical practices and donor engagement in the growing field of CGT, ensuring that donor contributions are respected and used responsibly.

Synergistic effect and molecular mechanism of nicotinamide and UM171 in ex vivo expansion of long-term hematopoietic stem cells.

Introduction: Several approaches to expand human hematopoietic stem cells (HSCs) have been reported, but the ability of these methods to expand long-term hematopoietic stem cells (LT-HSCs) remains to be improved, which limits the application of HSCs-based therapies. Methods: CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and then cultured for 12 d in a serum-free medium. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Colony-forming cell (CFC) assays can evaluate multi-lineage differentiation potential of HSCs. Real-time polymerase chain reaction was employed to detect the expression of genes related to self-renewal programs and antioxidant activity. DCFH-DA probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. Results: Here, we show a combination, Nicotinamide (NAM) combined with pyrimidoindole derivative UM171, can massively expand LT-HSCs ex vivo, and the expanded cells maintained the capability of self-renewal and multilineage differentiation. Additionally, our data indicated that UM171 promoted self-renewal of HSCs by inducing HSCs entry into the cell cycle and activating Notch and Wnt pathways, but the infinite occurrence of this process may lead to mitochondrial metabolism disorder and differentiation of HSCs. NAM kept HSCs in their primitive and dormant states by reducing intracellular ROS levels and upregulating the expression of stemness related genes, so we believed that NAM can act as a brake to control the above process. Conclusions: The discovery of the synergistic effect of NAM and UM171 for expanding LT-HSCs provides a new strategy in solving the clinical issue of limited numbers of HSCs.

Endogenous stem cell mobilization and localized immunosuppression synergistically ameliorate DSS-induced Colitis in mice.

BACKGROUND: Stem cell therapy is a promising alternative for inflammatory diseases and tissue injury treatment. Exogenous delivery of mesenchymal stem cells is associated with instant blood-mediated inflammatory reactions, mechanical stress during administration, and replicative senescence or change in phenotype during long-term culture in vitro. In this study, we aimed to mobilize endogenous hematopoietic stem cells (HSCs) using AMD-3100 and provide local immune suppression using FK506, an immunosuppressive drug, for the treatment of inflammatory bowel diseases. METHODS: Reactive oxygen species (ROS)-responsive FK506-loaded thioketal microspheres were prepared by emulsification solvent-evaporation method. Thioketal vehicle based FK506 microspheres and AMD3100 were co-administered into male C57BL6/J mice with dextran sulfate sodium (DSS) induced colitis. The effect of FK506-loaded thioketal microspheres in colitis mice were evaluated using disease severity index, myeloperoxidase activity, histology, flow cytometry, and gene expression by qRT-PCR. RESULTS: The delivery of AMD-3100 enhanced mobilization of HSCs from the bone marrow into the inflamed colon of mice. Furthermore, targeted oral delivery of FK506 in an inflamed colon inhibited the immune activation in the colon. In the DSS-induced colitis mouse model, the combination of AMD-3100 and FK506-loaded thioketal microspheres ameliorated the disease, decreased immune cell infiltration and activation, and improved body weight, colon length, and epithelial healing process. CONCLUSION: This study shows that the significant increase in the percentage of mobilized hematopoietic stem cells in the combination therapy of AMD and oral FK506 microspheres may contribute to a synergistic therapeutic effect. Thus, low-dose local delivery of FK506 combined with AMD3100 could be a promising alternative treatment for inflammatory bowel diseases.