Melissa van Pel

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Differential role of CD97 in interleukin-8-induced and granulocyte-colony stimulating factor-induced hematopoietic stem and progenitor cell mobilization

CD97 is a transmembrane receptor involved in neutrophil migration. This study shows that CD97 plays a role in interleukin-8-induced hematopoietic stem cell and progenitor mobilization. CD97 is broadly expressed on hematopoietic cells and is involved in neutrophil migration. Since neutrophils are key regulators in HSC/HPC mobilization, we studied a possible role for CD97 in interleukin-8 and granulocyte-colony stimulating factor-induced HSC/HPC mobilization. Mobilization was absent in mice receiving CD97 mAb followed by interleukin-8, while granulocyte-colony stimulating factor-induced mobilization remained unaltered following anti-CD97 administration. Furthermore, combined administration of CD97 mAb and IL-8 induced a significant reduction in the neutrophilic compartment. We hypothesize that the absence of interleukin-8-induced HSC/HPC mobilization after CD97 mAb administration is due to its effect on neutrophil function.

Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration

Background Flt3-ligand is a cytokine that induces relatively slow mobilization of hematopoietic cells in animals and humans in vivo. This provides a time-frame to study hematopoietic stem and progenitor cell migration kinetics in detail. Design and Methods Mice were injected with Flt3-ligand (10 μg/day, intraperitoneally) for 3, 5, 7 and 10 days. Mobilization of hematopoietic stem and progenitor cells was studied using colony-forming-unit granulocyte/monocyte and cobblestone-area-forming-cell assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells was studied by transplantation of 1.5×106 Flt3-ligand-mobilized peripheral blood mononuclear cells into lethally irradiated (9.5 Gy) recipients. Results Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of Flt3-ligand produced a steady increase in mobilized progenitor cells. Compared to Flt3-ligand administration for 5 days, the administration of Flt3-ligand for 10 days led to a 5.5-fold increase in cobblestone-area-forming cells at week 4 and a 5.0-fold increase at week 5. Furthermore, transplantation of peripheral blood mononuclear cells mobilized by 5 days of Flt3-ligand administration did not radioprotect lethally irradiated recipients, whereas peripheral blood mononuclear cells mobilized by 10 days of Flt3-Ligand administration did provide 100% radioprotection of the recipients with significant multilineage donor chimerism. Compared to the administration of Flt3-ligand or interleukin-8 alone, co-administration of interleukin-8 and Flt3-ligand led to synergistic enhancement of hematopoietic stem and progenitor cell mobilization on days 3 and 5. Conclusions These results indicate that hematopoietic stem and progenitor cells show different mobilization kinetics in response to Flt3-ligand, resulting in preferential mobilization of hematopoietic progenitor cells at day 5, followed by hematopoietic stem cell mobilization at day 10.

CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells.

CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) receptor family. This study shows that murine hematopoietic cells expressing high levels of c-Kit and intermediate levels of CD97 have repopulating and radioprotective capacity. This phenotype allows simple and rapid purification of murine hematopoietic stem cells. Background CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of adhesion receptors and is broadly expressed on hematopoietic cells. The aim of this study was to investigate the expression of CD97 on hematopoietic stem- and progenitor cells (HSC/HPC). Design and Methods CD97 expression on hematopoietic stem- and progenitor cells was studied in BALB/c, C57BL/6 and DBA/1 mice using flow cytometry. Functional hematopoietic stem- and progenitor cell characteristics were investigated in vitro and in vivo by progenitor cell assays, cobblestone area forming cell assays and bone marrow cell transplantation. Results Analysis of CD97 expression on murine bone marrow cells showed three major populations i.e. CD97HI, CD97INT and CD97NEG cells. Functional studies revealed that radioprotective capacity and cobblestone area forming cell day 28–35 activity resides in the CD97INT bone marrow cell fraction while CFU-GM colony-forming capacity mainly resides in the CD97NEG population in all strains. In C57BL/6 and DBA/1 mice CD97NEG and CD97HI bone marrow cells show hematopoietic stem cell characteristics as well. Further functional analysis of BALB/c CD97INT bone marrow cells revealed that c-KitHICD97INT bone marrow cells exhibit HSC activity and are 1.5-fold enriched for cobblestone area forming cell-day 35 activity compared to c-KitHI bone marrow cells. Moreover, phenotypical analysis showed that BALB/c and C57BL/6 HSC are CD97INT, while DBA/1 HSC are CD97HI. Conclusions CD97 is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells. Committed progenitor cell activity is largely comprised in the CD97NEG fraction, while the CD97INT population contains hematopoietic stem cell activity. In BALB/c mice, CD97 expression can be applied to almost completely separate colony-forming cells and cells exhibiting radioprotective capacity. In addition we propose that the CD97INTc-KitHI phenotype allows simple and rapid purification of murine hematopoietic stem cells.

Mesenchymal stem cells in autoimmune diseases: hype or hope?

Intervention with mesenchymal stem cells (MSCs) represents a promising therapeutic tool in treatment-refractory autoimmune diseases. A new report by Schurgers and colleagues in a previous issue of Arthritis Research & Therapy sheds novel mechanistic insight into the pathways employed by MSCs to suppress T-cell proliferation in vitro, but, at the same time, indicates that MSCs do not influence T-cell reactivity and the disease course in an in vivo arthritis model. Such discrepancies between the in vitro and in vivo effects of potent cellular immune modulators should spark further research and should be interpreted as a sign of caution for the in vitro design of MSC-derived interventions in the setting of human autoimmune diseases.

HLA Reduces Killer Cell Ig-like Receptor Expression Level and Frequency in a Humanized Mouse Model

NK cells use NK cell receptors to be able to recognize and eliminate infected, transformed, and allogeneic cells. Human NK cells are prevented from killing autologous healthy cells by virtue of inhibitory NKRs, primarily killer cell Ig-like receptors (KIR) that bind “self” HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. The extreme polymorphism of the KIR and HLA loci, with wide-ranging affinities for individual KIR and HLA allele combinations, has made this issue particularly hard to tackle. In this study, we used a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function in the absence of genetic variation inside and outside the KIR locus. These H-2Kb−/− and H-2Db−/− mice lacked ligands for inhibitory Ly49 receptors and were transgenic for HLA-Cw3 and a KIR B haplotype. In this reductionist system, the presence of HLA-Cw3 reduced the frequency of KIR2DL2+ cells, as well as the surface expression levels of KIR2DL2. In addition, in the presence of HLA-Cw3, the frequency of NKG2A+ cells and the surface expression levels of NKG2A were reduced. In line with these findings, both transgene-encoded KIR and endogenous NKG2A contributed to the rejection of cells lacking HLA-Cw3. These findings support the idea that HLA influences the human KIR repertoire.

Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection

Insight into molecular and cellular mechanisms of innate immunity is critical to understand viral pathogenesis and immunopathology and might be exploited for therapy. Whereas the molecular mechanisms of the IFN defense are well established, cellular mechanisms of antiviral immunity are only emerging, and their pharmacological triggering remains unknown. COAM is a polysaccharide derivative with antiviral activity but without comprehension about its mechanism of action. The COAM mixture was fractionated, and prophylactic treatment of mice with COAM polymers of high MW resulted in a conversion from 100% lethal mengovirus infection to an overall survival rate of 93% without obvious clinical sequelae. Differential and quantitative analysis of peritoneal leukocytes demonstrated that COAM induced a profound influx of neutrophils. Selective cell depletion experiments pointed toward neutrophils and macrophages as key effector cells in the rescue of mice from lethal mengovirus. COAM was able to induce mRNA and protein expression of the mouse neutrophil chemokine GCP‐2. Binding of GCP‐2 to COAM was demonstrated in solution and confirmed by SPR technology. Although COAM was not chemotactic for neutrophils, COAM‐anchored muGCP‐2 retained chemotactic activity for human and mouse neutrophils. In conclusion, this study established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, as proposed through the induction, binding, and concentration of endogenous chemokines. These findings reinforce the role of neutrophils and macrophages as critical cells that can be manipulated toward antiviral defense.

Chimerism levels after stem cell transplantation are primarily determined by the ratio of donor to host stem cells

To the editor: Ever since the concept of the hematopoietic stem cell (HSC) niche was first proposed in 1978, there has been debate whether toxic ablative conditioning is required before transplantation for creating HSC niche space, or whether engraftment is merely determined by stem cell

Serpina1 (α1-AT) is synthesized in the osteoblastic stem cell niche

OBJECTIVE Previously, we identified Serpina1 as a potent inhibitor of hematopoietic stem and progenitor cell (HSC/HPC) mobilization. Serpina1 protein is found in the bone marrow (BM) extracellular fluid and concentrations are decreased during granulocyte colony-stimulating factor-induced HSC/HPC mobilization in mice. In addition, administration of exogenous Serpina1 protein inhibits HSC/HPC mobilization. BM cells responsible for production and secretion of Serpina1 remain unknown. Here, we examined the expression of Serpina1 in order to identify cell populations of the BM that synthesize Serpina1. MATERIALS AND METHODS Osteoblast (OB) and hematopoietic BM cell fractions were isolated from femurs, tibias, and humeri obtained from untreated mice. Subsequently, each BM fraction was examined for the production of Serpina1 messenger RNA and protein by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. RESULTS Quantitative real-time polymerase chain reaction analysis showed that Serpina1 messenger RNA is produced at high levels by OB compared to hematopoietic BM cells. Furthermore, Western blot analysis indicated that Serpina1 protein was secreted by OB. In contrast, no Serpina1 protein could be detected in the supernatant obtained from overnight cultured hematopoietic BM cells. Finally, in BM sections obtained from the femurs of untreated mice, Serpina1 protein was detected in OB cells lining the bone. CONCLUSION Serpina1 protein in the BM extracellular fluid is predominantly produced by OB. This indicates that Serpina1 may play a regulatory role in the maintenance of HSC in the OB stem cell niche.