Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Multiparametric analysis of etoposide exposed mesenchymal stem cells and Fanconi anemia cells: implications in development of secondary myeloid malignancy.

Secondary acute myeloid leukemia (sAML) may develop following a prior therapy or may evolve from an antecedent hematological disorder such as Fanconi Anemia (FA). Pathophysiology of leukemic evolution is not clear. Etoposide (Eto) is a chemotherapeutic agent implicated in development of sAML. FA is an inherited bone marrow (BM) failure disease characterized by genomic instability and xenobiotic susceptibility. Here, we hypothesized that alterations in the BM niche may play a critical/driver role in development of sAML in both conditions. Expression of selected genes involved in xenobiotic metabolism, DNA double-strand break response, endoplasmic reticulum (ER) stress, heat shock response and cell cycle regulation were determined in BM mesenchymal stem cells (MSCs) of healthy controls and FA patients at steady state and upon exposure to Eto at different concentrations and in recurrent doses. Expression of CYPA1, p53, CCNB1, Dicer1, CXCL12, FLT3L and TGF-Beta genes were significantly downregulated in FA-MSCs compared with healthy controls. Eto exposure induced significant alterations in healthy BM-MSCs with increased expression of CYP1A1, GAD34, ATF4, NUPR1, CXCL12, KLF4, CCNB1 and nuclear localization of Dicer1. Interestingly, FA-MSCs did not show significant alterations in these genes upon Eto exposure. As opposed to healthy MSCs DICER1 gene expression and intracellular localization was not altered on FA BM-MSCs after Eto treatment. These results showed that Eto is a highly potent molecule and has pleiotropic effects on BM-MSCs, FA cells show altered expression profile compared to healthy controls and Eto exposure on FA cells shows differential profile than healthy controls.

Human Mastadenovirus A Infection in a Child During the Course of Hematopoietic Stem Cell Transplant.

Following primary infection, human mastadeno- viruses can persist in various tissues. We report a case of a pediatric patient with Fanconi anemia who had a complicated posttransplant course after allogeneic hematopoietic stem cell transplant that was associated with human mastadenovirus infection. Human mastadenovirus reactivation was detected with metagenomic analysis during a 3-month follow- up period; the predominant rate of occurrence of human mastadenoviruses was 1.1% on day 0, 84% on day +15, 90% on day +30, and 42% on day +82. Virus shedding continued up to 3 months after transplant. At 36 months after hematopoietic stem cell transplant, the patient was in good clinical condition with full donor chimerism. Long-term follow-up studies for human mastadenoviruses are needed to determine latency period.

Hematopoietic stem cell transplantation for adolescents and adults with inborn errors of immunity: an EBMT IEWP study.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the gold standard curative therapy for infants and children with many inborn errors of immunity (IEI), but adolescents and adults with IEI are rarely referred for transplant. Lack of published HSCT outcome data outside small, single-center studies and perceived high risk of transplant-related mortality have delayed the adoption of HSCT for IEI patients presenting or developing significant organ damage later in life. This large retrospective, multicenter HSCT outcome study reports on 329 IEI patients (age range, 15-62.5 years at HSCT). Patients underwent first HSCT between 2000 and 2019. Primary endpoints were overall survival (OS) and event-free survival (EFS). We also evaluated the influence of IEI-subgroup and IEI-specific risk factors at HSCT, including infections, bronchiectasis, colitis, malignancy, inflammatory lung disease, splenectomy, hepatic dysfunction, and systemic immunosuppression. At a median follow-up of 44.3 months, the estimated OS at 1 and 5 years post-HSCT for all patients was 78% and 71%, and EFS was 65% and 62%, respectively, with low rates of severe acute (8%) or extensive chronic (7%) graft-versus-host disease. On univariate analysis, OS and EFS were inferior in patients with primary antibody deficiency, bronchiectasis, prior splenectomy, hepatic comorbidity, and higher hematopoietic cell transplant comorbidity index scores. On multivariable analysis, EFS was inferior in those with a higher number of IEI-associated complications. Neither age nor donor had a significant effect on OS or EFS. We have identified age-independent risk factors for adverse outcome, providing much needed evidence to identify which patients are most likely to benefit from HSCT.

Intestinal mycobiota composition and changes in children with thalassemia who underwent allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) alters the diversity of the intestinal bacterial microbiota. This study aimed to evaluate human mycobiota composition pre-HSCT and post-HSCT in children with thalassemia. METHOD: Ten children with thalassemia undergoing allogeneic HSCT were enrolled. The stool samples were collected before the transplantation regimen, before the transplant day, and +15, +30 days, and three months after transplantation. Stool samples were also collected from the donor and the patient's caregivers. Gut mycobiota composition was evaluated with metagenomic analysis. RESULTS: Pretransplant mycobiota of children with thalassemia (the predominant genus was Saccharomyces, 64.1%) has been shown to approximate the diverse mycobiota compositions of healthy adult donors but becomes altered (lower diversity) following transplant procedures. Three months after HSCT, phyla Ascomycota and Basidiomycota were 83.4% and 15.6%, respectively. The predominant species were Saccaharomyces_uc and Saccharomyces cerevisiae (phylum Ascomycota); we also observed Malassezia restricta and Malassezia globosa (phylum Basidiomycota) (∼13%). On day 90 after HSCT, we observed 65.3% M. restricta and 18.4% M. globosa predominance at the species level in a four-year-old boy with acute graft-versus-host disease (GVHD) (skin and gut involvement) 19 days after transplantation included. CONCLUSION: The mycobiota composition of children with thalassemia altered after HSCT. We observed Malassezia predominance in a child with GVHD. Further studies in children with GVHD will identify this situation.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Long Term Follow-Up of the Patients with Severe Combined Immunodeficiency After Hematopoietic Stem Cell Transplantation: A Single-Center Study.

BACKGROUND: We aimed to evaluate hematopoietic stem cell transplantation (HSCT) related outcomes of patients with severe combined immunodeficiency (SCID). METHODS: We retrospectively collected data from SCID patients who were diagnosed, followed up and survived at least 2 years after HSCT. RESULTS: Forty four SCID patients were included in the study. Median age of HSCT and follow-up period after HSCT were 7.1 months and 8.7 years, respectively. Human leukocyte antigen (HLA) identical donors were used in 77.3% (n = 34) of the patients (23 siblings, six fathers, two mothers, three extended family donors), HLA 1-2 mismatched family donors in 11.3% (n = 5), and haploidentical family donors in 11.3% (n = 5). CD3 and CD19 counts were normal in more than 90% and in 45.4% at last follow-up, respectively. Intravenous immunoglobulin (IVIG) could be stopped in 72.7% (n = 32) after HSCT. B+ SCID patients had better CD19 counts than B- (p < .001). T cell numbers, lymphocyte proliferation, IVIG need, immunoglobulin levels, antibody responses did not differ among B- and B+ immunophenotypes. Acute graft-versus-host disease (GVHD) was less in bone marrow transplanted patients (19.4%) than peripheral stem cell (58.3%) transplanted ones (p = .024). There was no correlation between age at transplantation and immune reconstitution. At the last follow-up, 70.2% and 78.3% of the patients had body weight and height above 3rd percentile, respectively. CONCLUSION: The immune reconstitution and the growth were normal in the majority of SCID patients after HSCT. It may be rational to use bone marrow instead of peripheral stem cell, as acute GVHD was less in bone marrow transplanted patients.

Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71-CD34+ CD38mo/lo], megakaryocyte-erythroid progenitor cells (MEP) [CD71-CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.

Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells.

BACKGROUND: Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2. METHODS: Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media. RESULTS: All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage. CONCLUSIONS: Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.

Stem Cell Applications in Lysosomal Storage Disorders: Progress and Ongoing Challenges.

Lysosomal storage disorders (LSDs) are rare inborn errors of metabolism caused by defects in lysosomal function. These diseases are characterized by accumulation of completely or partially degraded substrates in the lysosomes leading to cellular dysfunction of the affected cells. Currently, enzyme replacement therapies (ERTs), treatments directed at substrate reduction (SRT), and hematopoietic stem cell (HSC) transplantation are the only treatment options for LSDs, and the effects of these treatments depend strongly on the type of LSD and the time of initiation of treatment. However, some of the LSDs still lack a durable and curative treatment. Therefore, a variety of novel treatments for LSD patients has been developed in the past few years. However, despite significant progress, the efficacy of some of these treatments remains limited because these therapies are often initiated after irreversible organ damage has occurred.Here, we provide an overview of the known effects of LSDs on stem cell function, as well as a synopsis of available stem cell-based cell and gene therapies that have been/are being developed for the treatment of LSDs. We discuss the advantages and disadvantages of use of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and induced pluripotent stem cell (iPSC)-related (gene) therapies. An overview of current research data indicates that when stem cell and/or gene therapy applications are used in combination with existing therapies such as ERT, SRT, and chaperone therapies, promising results can be achieved, showing that these treatments may result in alleviation of existing symptoms and/or prevention of progression of the disease. All together, these studies offer some insight in LSD stem cell biology and provide a hopeful perspective for the use of stem cells. Further development and improvement of these stem cell (gene) combination therapies may greatly improve the current treatment options and outcomes of patients with a LSD.

Genomic Spectrum and Phenotypic Heterogeneity of Human IL-21 Receptor Deficiency.

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.

Characterization of human bone marrow niches with metabolome and transcriptome profiling.

Bone marrow (BM) niches are special microenvironments that work in harmony with each other for the regulation and maintenance of hematopoiesis. Niche investigations have thus far been limited to various model organisms and animal studies; therefore, little is known about different niches in healthy humans. In this study, a special harvesting method for the collection of BM from two different anatomical regions in the iliac crest of humans was used to investigate the presence of different niches in BM. Additionally, metabolomic and transcriptomic profiles were compiled using comparative 'omics' technologies, and the main cellular pathways and corresponding transcripts and metabolites were identified. As a result, we found that the energy metabolism between the regions was different. This study provides basic broad data for regenerative medicine in terms of the design of the appropriate microenvironment for in vitro hematopoietic niche modeling, and identifies the normal reference values that can be compared in hematological disease.

CXCR4 expression by mesenchymal stromal cells is lost after use of enzymatic dissociation agents, but preserved by use of non-enzymatic methods.

In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.

G-CSF treatment of healthy pediatric donors affects their hematopoietic microenvironment through changes in bone marrow plasma cytokines and stromal cells.

Although G-CSF mobilized peripheral blood stem cell (PBSC) transplantation is commonly used in adults, bone marrow (BM) is still the preferred stem cell source in pediatric stem cell transplantation. Despite the fact that G-CSF is increasingly being used to enhance the hematopoietic stem/progenitor cell (HSPC) yield in BM transplantation (G-BM), the direct effects of G-CSF on the pediatric BM microenvironment have never been investigated. The BM hematopoietic niche provides the physical space where the HSPCs reside. This BM niche regulates HSPC quiescence and proliferation through direct interactions with other niche cells, including Mesenchymal Stromal Cells (MSCs). These cells have been shown to secrete a wide range of hematopoietic cytokines (CKs) and growth factors (GFs) involved in differentiation, retention and homing of hematopoietic cells. Here, we assessed changes in the BM microenvironment by measuring levels of 48 different CKs and GFs in G-BM and control BM (C-BM) plasma from pediatric donors. In addition, the effect of G-CSF on cell numbers and characteristics of HSPCs and MSCs was assessed. IL-16, SCGF-b, MIP-1b (all >1000 pg/mL) and RANTES (>10.000 pg/mL) were highly expressed in healthy donor pediatric BM plasma. Levels of IL-3, IL-18, GROa, MCP-3 (p<0.05) were increased in G-BM, whereas levels of RANTES (p<0.001) decreased after G-CSF treatment. We found a negative correlation with increasing age for IL2-Ra and LIF (p<0.05). In addition, a concomitant increase in the number of both hematopoietic and fibroblast colony forming units was observed, indicating that G-CSF affects both HSPC and MSC numbers. In conclusion, G-CSF treatment of healthy pediatric donors affects the hematopoietic BM microenvironment by expansion of HSPC and MSC numbers and modifying local CK and GF levels.

Articular Cartilage Regeneration Utilizing Decellularized Human Placental Scaffold, Mesenchymal Stem Cells and Platelet Rich Plasma.

BACKGROUND: Articular cartilage repair has been a challenge in orthopedic practice due to the limited self-regenerative capability. Optimal treatment method for cartilage defects has not been defined. We investigated the effect of decellularized human placental (DHP) scaffold, mesenchymal stem cells (MSC) and platelet-rich plasma (PRP) on hyaline cartilage regeneration in a rat model. METHODS: An osteochondral defect was created in trochlea region of the femur in all groups, bilaterally. No additional procedure was performed in control group (n = 14). Only the DHP scaffold was applied to the P group (n = 14). The DHP scaffold and 1 × 106 MSCs were applied to the PS group (n = 14). The DHP scaffold and PRP were applied to the PP group (n = 14). The DHP scaffold, 1 × 106 MSCs and PRP were applied to the PSP group (n = 14). Outcome measures at 12 weeks included Pineda histology score and qualitative histology. RESULTS: The mean Pineda scores of P, PS, PP, and PSP groups were significantly better than the control group (p = 0.031, p = 0.002, p < 0.001, p < 0001, respectively). There was no statistically difference in mean Pineda scores of P, PS, PP, and PSP groups (p > 0.05). CONCLUSION: In conclusion, the DHP scaffold appears to be a promising scaffold on hyaline cartilage regeneration. The augmentation of DHP scaffold with MSCs and PRP combinations did not enhance its efficacy on articular cartilage regeneration.

Outcome of patients with Fanconi anemia developing myelodysplasia and acute leukemia who received allogeneic hematopoietic stem cell transplantation: A retrospective analysis on behalf of EBMT group.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is curative for bone marrow failure in patients with Fanconi anemia (FA), but the presence of a malignant transformation is associated with a poor prognosis and the management of these patients is still challenging. We analyzed outcome of 74 FA patients with a diagnosis of myelodysplastic syndrome (n = 35), acute leukemia (n = 35) or with cytogenetic abnormalities (n = 4), who underwent allo-HSCT from 1999 to 2016 in EBMT network. Type of diagnosis, pre-HSCT cytoreductive therapies and related toxicities, disease status pre-HSCT, donor type, and conditioning regimen were considered as main variables potentially influencing outcome. The 5-year OS and EFS were 42% (30-53%) and 39% (27-51%), respectively. Patients transplanted in CR showed better OS compared with those transplanted in presence of an active malignant disease (OS:71%[48-95] vs 37% [24-50],P = .04), while none of the other variables considered had an impact. Twenty-two patients received pre-HSCT cytoreduction and 9/22 showed a grade 3-4 toxicity, without any lethal event or negative influence on survival after HSCT(OS:toxicity pre-HSCT 48% [20-75%] vs no-toxicity 51% [25-78%],P = .98). The cumulative incidence of day-100 grade II-IV a-GvHD and of 5-year c-GvHD were 38% (26-50%) and 40% (28-52%). Non-relapse-related mortality and incidence of relapse at 5-years were 40% (29-52%) and 21% (11-30%) respectively, without any significant impact of the tested variables. Causes of death were transplant-related events in most patients (34 out of the 42 deaths, 81%). This analysis confirms the poor outcome of transformed FA patients and identifies the importance of achieving CR pre-HSCT, suggesting that, in a newly diagnosed transformed FA patient, a cytoreductive approach pre-HSCT should be considered if a donor have been secured.

Neuropeptide Y is involved in the regulation of quiescence of hematopoietic stem cells.

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G0 phase and an increase in the gene expression levels of FOXO3, DICER1, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence.