Fraction Of Bone Marrow Cells Research Articles

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N= 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished invitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid invitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

Knockout of HMGA1 Promotes ATRA-Induced Differentiation in MLL-Rearranged Leukemia

Introduction: HMGA1 is a small non-histone chromatin protein characterized by 3 AT-hooks, which modulates transcription by altering the chromatin architecture. It tends to be highly expressed in poorly differentiated cells including hematopoietic stem cells. In various kinds of solid tumors, HMGA1 has been reported to play as an oncogene, and its upregulation was reported to be associated with poorer clinical outcomes. However, although recent study demonstrated that HMGA1 was associated with progression of JAK2V617F positive myeloproliferative neoplasms to myelofibrosis or leukemia, the roles of HMGA1 in myeloid malignancies were still unclear. Therefore, we analyzed its function in myeloid malignancies using HMGA1 knockout (KO) mice. We could not detect clear differences in peripheral blood counts or bone marrow cells, and generated various kinds of models of acute myeloid leukemia (AML). Knockout of HMGA1 could not delay the onset of these leukemia, but we could demonstrate that leukemic cells with MLL-ENL fusion gene which lacked in HMGA1 gene were more susceptible to all-trans retinoic acid (ATRA)-induced expression of CD11b, which suggested that HMGA1 could be associated with the differentiation process induced by ATRA. Method: We generated Vav-iCre HMGA1flox/flox mice, and we first analyzed the fraction of peripheral blood cells and bone marrow cells of these mice using flow cytometry. Next, to evaluate roles of HMGA1 in AML, we made various murine models of AML. We collected c-kit positive fractions of bone marrow cells from the KO mice or Vav-iCre(-) HMGA1flox/flox mice and retrovirally introduced MLL-ENL, MOZ-TIF2, BCL2-MYC fusions. Primary transplantation was performed to sublethally irradiated (5Gy) mice with 4.0×105 infected cells for each fusion gene. Secondary transplantation was also performed for MLL-ENL and BCL2-MYC with 1.0×104 cells for each. Blood samples were collected weekly, and disease-free survivals were compared by Kaplan-Meier method with Vav-iCre(-) HMGA1flox/flox mice. Also, leukemic cells infected with MLL-ENL were collected, and these cells were cultured for four days with different concentrations (1μM, 5μM or 10μM) of ATRA. Flow cytometry analysis was performed to evaluate the differentiation induced by ATRA with antibodies to murine CD11b, c-kit and Gr-1. Result: There was no significant difference in birth rate between Vav-iCre(+) and Vav-iCre(-) mice, which suggested that HMGA1 was not essential for fetal hematopoiesis. Peripheral blood cell counts were measured at week 8 and week 20, and fractions of peripheral blood and bone marrow cells were assessed at week 8 using flow cytometry, but no significant differences were observed. As for AML models, there were no significant differences in survival periods after primary or secondary transplantation. Leukemic cells retrovirally induced with MLL-ENL were collected from primary recipients, and these cells were treated with ATRA. Although growth rates were not show significantly different, expression levels of CD11b after exposure to ATRA were significantly higher in Vav-Cre (+) mice (5μM: 70% vs 44%, respectively; p=0.035. 10μM: 80% vs 46%, respectively; p=0.0006) as shown in the following figure. Discussion: We established HMGA1 KO mice and assessed the function of HMGA1 both in normal and leukemic hemopoiesis. Although HMGA1 might have little effect on normal hematogenesis, it seemed that HMGA1 had inhibitory effect on ATRA-induced differentiation process of MLL-ENL leukemic cells. Though more conditions including AML types and differentiation methods should be tested and the molecular mechanisms should be scrutinized, there is a possibility that inhibition of HMGA1 is effective in combination with differentiation treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.

Nestin-GFP transgene labels immunoprivileged bone marrow mesenchymal stem cells in the model of ectopic foci formation.

Immune privileges are demonstrated for different types of quiescent stem cells of adult mammalian organisms. Mesenchymal stem cells (MSCs) are believed to have immune privileges; however, an accurate experimental confirmation hasn’t been presented. Here, we provide direct experimental evidence that MSCs of C57Black/6J murine bone marrow (BM) are immune privileged in vivo and retain their functionality after prolonged exposure to the uncompromised immune system. The BM of Nes-Gfp transgenic mice was implanted as a tissue fragment under the kidney capsule in isogenic C57Black/6J immunocompetent recipients. Nestin-Gfp strain provides a fluorescent immunogenic marker for a small fraction of BM cells, including GFP+CD45– MSCs. Despite the exposure of xenogenically marked MSCs to the fully-functional immune system, primary ectopic foci of hematopoiesis formed. Six weeks after implantation, multicolor fluorescence cytometry revealed both GFP+CD45– and GFP+CD45+ cells within the foci. GFP+CD45– cells proportion was 2.0 × 10–5 ×÷9 and it didn’t differ significantly from syngenic Nes-GFP transplantation control. According to current knowledge, the immune system of the recipients should eliminate GFP+ cells, including GFP+ MSCs. These results show that MSCs evade immunity. Primary foci were retransplanted into secondary Nes-GFP recipients. The secondary foci formed, in which CD45–GFP+ cells proportion was 6.7 × 10–5 ×÷2.2, and it didn’t differ from intact Nes-GFP BM. The results demonstrate that MSCs preserve self-renewal and retain their functionality after prolonged immune exposure. The success of this study relied on the implantation of BM fragments without prior dissociation of cells and the fact that the vast majority of implanted cells were immunologically equivalent to the recipients.

Current State and Issues of Regenerative Medicine for Rheumatic Diseases.

The prognosis of rheumatic diseases is generally better than that of malignant diseases. However, some cases with poor prognoses resist conventional therapies and cause irreversible functional and organ damage. In recent years, there has been much research on regenerative medicine, which uses stem cells to restore the function of missing or dysfunctional tissues and organs. The development of regenerative medicine is also being attempted in rheumatic diseases. In diseases such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and rheumatoid arthritis, hematopoietic stem cell transplantation has been attempted to correct and reconstruct abnormalities in the immune system. Mesenchymal stem cells (MSCs) have also been tried for the treatment of refractory skin ulcers in SSc using the ability of MSCs to differentiate into vascular endothelial cells and for the treatment of systemic lupus erythematosus SLE using the immunosuppressive effect of MSCs. CD34-positive endothelial progenitor cells (EPCs), which are found in the mononuclear cell fraction of bone marrow and peripheral blood, can differentiate into vascular endothelial cells at the site of ischemia. Therefore, EPCs have been used in research on vascular regeneration therapy for patients with severe lower limb ischemia caused by rheumatic diseases such as SSc. Since the first report of induced pluripotent stem cells (iPSCs) in 2007, research on regenerative medicine using iPSCs has been actively conducted, and their application to rheumatic diseases is expected. However, there are many safety issues and bioethical issues involved in regenerative medicine research, and it is essential to resolve these issues for practical application and spread of regenerative medicine in the future. The environment surrounding regenerative medicine research is changing drastically, and the required expertise is becoming higher. This paper outlines the current status and challenges of regenerative medicine in rheumatic diseases.

The Disparity Observed Between Murine Short-term and Long-term Hematopoietic Stem Cells in Their Ability to Migrate and Engraft When Introduced IV is Due to Distinct Adhesion Molecule Expression

CD34 has traditionally been used as a marker to enrich human bone marrow stem cells for clinical bone marrow transplants for over five decades. However, several studies support that the most primitive hematopoietic stem cells (HSCs) responsible for long-term hematopoiesis-long-term HSCs (LT-HSCs) -are found within the CD34 negative (CD34 neg) fraction of bone marrow cells. LT-HSCs are challenging to study due to their scarcity and lack of specific markers to identify them. Once these stem cells enter the circulation, they must find their way, through multistep process controlled by a variety of cell surface adhesion molecules, into the bone marrow. In contrast to the CD34 pos stem cells, studies have suggested that LT-HSCs have trouble migrating and engrafting when introduced intravenously. In order to better understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by various murine hematopoietic stem cell populations to migrate. To this end, we assayed the homing effectors that are either necessary in the first step, i.e., the cell surface adhesion molecule Sialyl Lewis X (sLe x) and its interaction with selectins-mainly E-selectin-on the bone marrow endothelium, or the second step, i.e. the chemokine receptor CXCR4 and its binding to the bone marrow stroma resident chemokine SDF-1α, of cell migration.Specifically focusing on murine populations of short term HSCs (ST-HSCs; Flk2 negCD34 pos) and LT-HSCs (Flk2 negCD34 neg), flow cytometric and gene expression analysis clearly illustrated distinctive expression patterns of homing effectors with the highest contrast observed in sLe x where Flk2 negCD34 neg HSCs exhibited low expression (< 10%) and Flk2 negCD34 pos HSCs exhibited much higher expression (> 60%) which correlated to higher binding to E-selectin. Of the common E-selectin ligands identified, both Flk2 negCD34 pos and Flk2 negCD34 neg HSCs expressed CD44 and PSGL-1 to equivalent levels while CD43 was expressed at significantly lower levels on Flk2 negCD34 neg HSCs. Moreover, our data indicates that the variation observed in sLe x expression/E-selectin binding correlates with differences in the expression of key glycosyltransferase genes involved in the creation of the sLe x epitope on these E-selectin ligands. mRNA expression analysis of glycosyltransferases revealed that, in general, sialyltransferase (ST3Gals) genes were expressed at higher levels in Flk2 negCD34 neg HSCs while only one fructosyltransferase (FT4) gene was expressed at higher levels in this HSC population. Thus, these data suggest that α2,3 sialylation is intact in Flk2 negCD34 neg HSCs but α1,3 fucosylation is lacking. The expression levels of homing molecules involved in the stages of cell migration after E-selectin binding were also assessed. No differences in the expression of the integrins CD49d, CD49e and CD29 were observed between Flk2 negCD34 neg and Flk2 negCD34 pos HSCs. Interestingly, higher levels of the chemokine receptor CXCR4 were measured on the surface of Flk2 negCD34 pos HSCs compared to Flk2 negCD34 neg HSCs. Likewise, this was reflected in a much lower ability of the Flk2 negCD34 neg HSCs to migrate towards the CXCR4 ligand, SDF-1, in a transwell assay.Based on these findings that show that both E-selectin binding and CXCR4 mediated migration are compromised in Flk2 negCD34 neg HSCs, we sought to enhance this LT-HSC populations ability to migrate using well-known treatments including recombinant human fucosyltransferase 6 (rhFVI) to create the glycans necessary for selectin binding, as well as the CD26 inhibitor (Diprotin A; Dip A) to enhance the binding of chemokines to their receptors.Interestingly, although both CD26 inhibition and cell surface fucosylation remarkably enhanced the expression of homing molecules in both HSC populations in vitro, only pretreatment of Flk2 negCD34 neg HSCs with Dip A significantly enhanced engraftment in vivo after transplantation into C57BL/6N mice.This study represents the first direct analysis of E-selectin ligand expression on murine LT-HSC populations and strongly support that by temporarily enhancing the expression of migration-associated molecules on the surface of these cells, prolonged and efficient HSC engraftment can be achieved. We hope our finding will potentially shed light on methods to optimally utilize these very valuable long-term HSCs in clinical bone marrow and cord blood transplants worldwide. DisclosuresNo relevant conflicts of interest to declare.

Cell Therapy of Pathological Gastrointestinal Lesions in Modeled NSAID-Induced Enterocolitis

Wistar rats with NSAID-induced (dexketoprofen) chronic enterocolitis of the gastrointestinal tract were treated with a cultured allogeneic cell suspension consisting of mononuclear cells (40 millions) and multipotent mesenchymal stromal cells (10 millions). The suspension was transplanted intraperitoneally, 2 times with an interval of 30 days. Cultured allogeneic fractions of bone marrow cells accelerate the regeneration of long-term non-healing gastrointestinal lesions by reducing the duration and severity of the inflammatory phase and activating the regenerative phase of the ulcerative process. It was found that the simultaneous administration of the studied cultured stem cells can be used for the treatment of chronic, long-term non-healing, poorly-scarring ulcerative necrotic gastrointestinal pathologies.

Subretinal versus intravitreal administration of human CD34+ bone marrow-derived stem cells in a rat model of inherited retinal degeneration.

BackgroundTo evaluate whether subretinal or intravitreal injection of human CD34+ bone marrow-derived stem cells (BMSC) can have protective effects on retinal degeneration that may be enhanced by coadministration of exosomes harvested from human bone marrow mesenchymal stem cells (MSCs).MethodsHuman CD34+ cells were harvested from the mononuclear cell fraction of bone marrow using magnetic beads and labeled with EGFP. Exosomes were harvested from cultured human MSCs under hypoxic conditions. Royal College of Surgeons (RCS) 3-weeks-old rats, immunosuppressed with cyclosporine A, received subretinal or intravitreal injection of CD34+ cells (50,000 cells), CD34+ cells with exosomes (50,000 cells+10 µg), exosomes alone (10 µg), or PBS. Retinal function was examined using electroretinography (ERG), and the eyes were harvested for histologic and immunohistochemical analysis.ResultsThe b-wave amplitude of ERG at 2 weeks after injection was significantly higher in eyes with subretinal or intravitreal CD34+ BMSC alone or in combination with exosomes when compared to PBS injected eyes or untreated contralateral eyes. At 4 weeks after injection, the ERG signal decreased in all groups but eyes with subretinal CD34+ BMSCs alone or combined with exosomes showed partially preserved ERG signal and preservation of the outer nuclear layer of the retina near the injection site on histology when compared to eyes with PBS injection. Immunohistochemical analysis identified the human cells in the outer retina. Subretinal or intravitreal exosome injection had no effect on retinal degeneration when administered alone or in combination with CD34+ cells.ConclusionsBoth subretinal and intravitreal injection of human CD34+ BMSCs can provide functional rescue of degenerating retina, although the effects were attenuated over time in this rat model. Regional preservation of the outer retina can occur near the subretinal injection site of CD34+ cells. These results suggest that CD34+ cells may have therapeutic potential in retinal degeneration.

Effects of intravitreal injection of human CD34+ bone marrow stem cells in a murine model of diabetic retinopathy

Human CD34 + stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 + stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 + BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 + cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 + BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 + BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 + BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 + BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 + BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 + BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

The overexpression of IGF-1 is a poor prognostic factor in multiple myeloma

Introduction . Insulin-like growth factors (IGF) are one of the widely studied factors in oncology. For tumors with a high expression level of IGF typical postoperative relapse, they are invasive and give distant metastases. There are also data on the participation of IGF in the emergence of resistance to anticancer drugs. The mechanisms that determine the influence of insulin-like growth factors on the progression of a number of malignant neoplasms remain undisclosed and carrying out fundamental research in this direction is relevant. Objective: to study the role of IGF type 1 (IGF-1) in multiple myeloma (MM).Materials and methods . 26 samples of bone marrow aspirates received from 26 patients – 14 men and 12 women – were studied in the work. All patients were diagnosed with stage III ММ. The age of patients ranged from 52 to 72 years. From the obtained bone marrow aspirates, using centrifugation in the Ficoll gradient, a mononuclear fraction of bone marrow cells containing plasma cells was obtained. Then we carried out the procedure of extracting RNA and using polymerase chain reaction with reverse transcription, we studied the expression of mRNA of the genes of IGF-1 and MDR1/ABCB1.Results . The paper analyzes the overall survival (OS) of patients with MM depending on the expression of the gene IGF-1. It is shown that for patients with MM who have a high level of IGF-1 expression, a decrease in OS is characteristic and, conversely, with a weak expression of IGF-1 or in the absence of its expression, an increase in OS is observed. Studies of expression of IGF-1 gene and MDR1/ABCB1 gene responsible for the occurrence of multiple drug resistance showed that these genes are co-expressed in patients with MM. Conclusion . The obtained results indicate that the high level of IGF-1 gene expression may be a poor prognostic factor in ММ. IGF-1 may participate in regulation of the mechanisms of emergence of multiple drug resistance in patients with MM.

Evaluation and Significance of CD25 Expression in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells in Chronic Myelogenous Leukemia Patients

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs maintain the depth of response; however, the life-long use of TKI has also been associated with late complications such as cardiovascular events and huge financial burden impairing their quality of life. To overcome these issues, investigators have been attempting to discontinue TKIs after durable molecular remission. However, the optimal timing to stop TKIs remains to be elucidated. We previously demonstrated that CD25 was highly expressed in murine and human CML-leukemia initiating cells (LICs) (Kobayashi CI et al., Blood, 2014). In this study we tried to clarify whether the proportion of CD25-positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to select patients who are likely to obtain durable treatment-free remission after stopping TKIs.Methods: Bone marrow samples were obtained from the patients with CML in chronic phase who were treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL1 was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25-positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction (%CD25+) was evaluated by flow cytometry. The response to TKIs at the time of analysis was determined according to as follows: complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL1 by quantitative and qualitative PCR.Results: Bone marrow samples obtained from 109 patients were evaluated (median age, 52 years; male/female, 76/33). Analysis was performed prior to TKI exposure in 26 patients and under TKI therapy in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 19 patients were treatment free because they were enrolled into a clinical trial of TKI discontinuation. At diagnosis (n=26), %CD25+ were significantly correlated with hemoglobin level and platelet count (Table). The %CD25+ was significantly lower in patients with post TKI exposure than those at diagnosis without TKIs (p<0.00001). In nine patients with available samples before and after TKI therapy, the %CD25+ at diagnosis was significantly higher than after TKI therapy (Mean 34.7%, SD 24.8% vs. Mean 4.96%, SD 4.17%, p<0.01, Fig.a). In addition, %CD25+ was significantly correlated with copy number of BCR/ABL1 (P<0.001, Fig.b).Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the disease status, and may be useful as a LIC minimal residual disease marker. [Display omitted] DisclosuresKasahara:Chugai: Research Funding. Sakurai:Bristol-Myers Squibb K.K.: Speakers Bureau. Kikuchi:Celgene: Speakers Bureau; Takeda: Speakers Bureau; Ono: Speakers Bureau. Shimizu:Bristol-Myers Squibb K.K: Honoraria. Mori:Astella Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Novartis Pharma: Research Funding; MSD: Research Funding; MSD: Honoraria; Janssen: Honoraria; SHIONOGI: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Celgene: Honoraria; Ono: Honoraria; Eisai: Honoraria; Novartis Pharma: Honoraria; Shire Japan: Honoraria; CHUGAI: Honoraria; Asahi Kasei: Research Funding; Japan Blood Products Organization: Honoraria; Pfizer: Honoraria. Okamoto:Pfizer Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Mismatch repair deficient hematopoietic stem cells are preleukemic stem cells.

Whereas transformation events in hematopoietic malignancies may occur at different developmental stages, the initial mutation originates in hematopoietic stem cells (HSCs), creating a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels transform the PLSC into lymphoma/leukemia initiating cells (LIC). Thymic lymphomas have been thought to develop from developing thymocytes. T cell progenitors are generated from HSCs in the bone marrow (BM), but maturation and proliferation of T cells as well as T-lymphomagenesis depends on both regulatory mechanisms and microenvironment within the thymus. We studied PLSC linked to thymic lymphomas. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Following BM transplantation, we found that MSH2-/- BM cells from young mice are able to fully reconstitute multiple hematopoietic lineages of lethally irradiated wild-type recipients. However, all recipients developed thymic lymphomas within three and four months post transplantation. Transplantation of different fractions of BM cells or thymocytes from young health MSH2-/- mice showed that an HSC enriched fraction always reconstituted hematopoiesis followed by lymphoma development. In addition, lymphomas did not occur in thymectomized recipients of MSH2-/- BM. These results suggest that HSCs with DNA repair defects such as MSH2-/- are PLSCs because they retain hematopoietic function, but also carry an obligate lymphomagenic potential within their T-cell progeny that is dependent on the thymic microenvironment.

Time course of VCAM-1 expression in reperfused myocardial infarction in swine and its relation to retention of intracoronary administered bone marrow-derived mononuclear cells.

BackgroundIntracoronary infusion of autologous bone marrow-derived mononuclear cells (BMMNC), after acute myocardial infarction (AMI), has been shown to improve myocardial function. However, therapeutic efficacy is limited, possibly because cell retention rates are low, suggesting that optimization of cell retention might increase therapeutic efficacy. Since retention of injected BMMNC is observed only within infarcted, but not remote, myocardium, we hypothesized that adhesion molecules on activated endothelium following reperfusion are essential. Consequently, we investigated the role of vascular cell adhesion molecule 1 (VCAM-1) in BMMNC retention in swine undergoing reperfused AMI produced by 120 min of percutaneous left circumflex coronary occlusion.Methods and resultsVCAM-1 expression in the infarct and remote region was quantified at 1, 3, 7, 14, and 35 days, post-reperfusion (n≥6 swine per group). Since expression levels were significantly higher at 3 days (2.41±0.62%) than at 7 days (0.98±0.28%; p<0.05), we compared the degree of cell retention at those time points in a follow-up study, in which an average of 43·106 autologous BMMNCs were infused intracoronary at 3, or 7 days, post-reperfusion (n = 6 swine per group) and retention was histologically quantified one hour after intracoronary infusion of autologous BMMNCs. Although VCAM-1 expression correlated with retention of BMMNC within each time point, overall BMMNC retention was similar at day 3 and day 7 (2.3±1.3% vs. 3.1±1.4%, p = 0.72). This was not due to the composition of infused bone marrow cell fractions (analyzed with flow cytometry; n = 5 per group), as cell composition of the infused BMMNC fractions was similar.ConclusionThese findings suggest that VCAM-1 expression influences to a small degree, but is not the principal determinant of, BMMNC retention.

Correlation of Expression of CD25 in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells with Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia Patients

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy.Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR.Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P<0.01). In addition, the proportion of CD25 positive cells in HSPC fraction significantly correlated the level of quantitative PCR (Figure, P<0.0001), and MMR was also dividable from CMR by the proportion of CD25 positive cells (MMR; n=29, Mean 4.6%, SD 4.8% vs CMR; n=49, Mean 2.6%, SD 2.5%, P<0.05). The expression of CD25 was still detected in the majority of patients who achieved CMR, including those who sustained CMR after the discontinuation of TKIs.Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. [Display omitted] DisclosuresKarigane:Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Leukemic Stem Cell Quantification in Newly Diagnosed Patients With Chronic Myeloid Leukemia Predicts Response to Nilotinib Therapy.

Leukemic stem cells (LSCs) may harbor important resistance to tyrosine kinase inhibitors in chronic myelogenous leukemia (CML). We identified Philadelphia chromosome (Ph)-positive CD34(+)CD38(-) bone marrow cells (here denoted LSCs) and addressed their response-predictive value in patients with CML (n = 48) subjected to nilotinib in the ENEST1st trial (NCT01061177). Two flow cytometry-based cell sorting methods were used with multiparameter-directed CD45- (MPFC) and BCR-ABL1 probe-linked (FISH) identification of Ph-positive cells, respectively. We observed a positive correlation between the proportion of LSCs at diagnosis and established prognostic markers (blast count, spleen size, Sokal score, and hemoglobin). Conversely, a high LSC burden predicted for an inferior molecular response at 3 (MPFC and FISH), 6 (MPFC), 9 (FISH), and 15 months (FISH). During nilotinib therapy, the proportion of LSCs decreased rapidly. At 3 months, a median of only 0.3% LSCs remained among CD34(+)CD38(-) cells, and in 33% of the patients the LSC clone was not detectable anymore (FISH). The response kinetics was similar in LSC fractions as it was in the progenitor and unseparated bone marrow cell fractions. The proportion of LSCs at diagnosis, as analyzed by two independent methodologies, reflects the biology of the disease and appeared as a prognostic and response-predictive marker in patients with CML subjected to first-line nilotinib therapy. Clin Cancer Res; 22(16); 4030-8. ©2016 AACR.

The Role of WT1 in Embryonic Development and Normal Organ Homeostasis.

The Wilms' tumor suppressor gene 1 (Wt1) is critically involved in a number of developmental processes in vertebrates, including cell differentiation, control of the epithelial/mesenchymal phenotype, proliferation, and apoptosis. Wt1 proteins act as transcriptional and post-transcriptional regulators, in mRNA splicing and in protein-protein interactions. Furthermore, Wt1 is involved in adult tissue homeostasis, kidney function, and cancer. For these reasons, Wt1 function has been extensively studied in a number of animal models to establish its spatiotemporal expression pattern and the developmental fate of the cells expressing this gene. In this chapter, we review the developmental anatomy of Wt1, collecting information about its dynamic expression in mesothelium, kidney, gonads, cardiovascular system, spleen, nervous system, lung, and liver. We also describe the adult expression of Wt1 in kidney podocytes, gonads, mesothelia, visceral adipose tissue, and a small fraction of bone marrow cells. We have reviewed the available animal models for Wt1-expressing cell lineage analysis, including direct Wt1 expression reporters and systems for permanent Wt1 lineage tracing, based on constitutive or inducible Cre recombinase expression under control of a Wt1 promoter. Finally we provide a number of laboratory protocols to be used with these animal models in order to assess reporter expression.

Identification of Recurrence-Related microRNAs from Bone Marrow in Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is a poor-prognosis cancer due to its high rate of recurrence. microRNAs (miRNAs) are a class of small non-coding RNA molecules that affect crucial processes in cancer development. The objective of this study is to identify the role of miRNAs in patient bone marrow (BM) and explore the function of these molecules during HCC progression. We purified miRNAs from bone marrow cells of seven HCC patients, and divided them into three fractions by cell surface markers as follows: CD14+ (macrophage), CD14−/CD45+ (lymphocyte), and CD14−/CD45−/EpCAM+ (epithelial cell). We employed microarray-based profiling to analyze miRNA expression in the bone marrow of patients with HCC. Differentially expressed miRNAs were significantly different between fractions from whole bone marrow, macrophages, and lymphocytes, and depended on stages in tumor progression. Differences in expression of miRNAs associated with cell proliferation also varied significantly between HCC patients with recurrence, multiple tumors, and advanced clinical stages. These results suggest that miRNA profiles in separated fractions of BM cells are associated with HCC progression.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

Background aimsThe diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow–derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. MethodsHuman bone marrow–derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2′-deoxyuridine incorporation assay, respectively. ResultsTotal mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2–dependant. ConclusionsFibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow–derived mononuclear cells and may support the development of improved bone marrow cell–based therapies.

Abstract 1910: Evolution of preleukemia stem cells to lymphoma initiating cells requires thymus in msh2-/- mice

Abstract Thymic lymphomas have been thought to develop from disregulated differentiation and proliferation of developing thymocytes. T cell progenitors are generated from hematopoietic stem cells (HSCs) in the bone marrow (BM). Recent evidence strongly suggests that whereas transformation events may occur at different developmental stages, the initial mutation originates in the HSCs, and creates a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels would transform the PLSC into lymphoma/leukemia initiating cells (LIC). Maturation and proliferation of T cells depend on regulatory mechanisms in the thymus where the T-progenitors must interact with the microenvironment. Here we investigate the requirement of thymus environment for lymphoma development. Defects in the mismatch repair system (MMR) underlie hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. Individuals with no MMR function present with childhood onset of hematological and brain malignancies. Mice carrying a null allele for the MMR gene, Msh2, are preferentially prone to develop thymic lymphomas and to a lesser extent, gastrointestinal tumors. Consistent with these findings in mice, MMR defects have also been observed in sporadic and hereditary hematological malignancies. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Using bone marrow transplantation, we found that limiting dilutions of MSH2-/- HSCs from young mice are able to reconstitute lethally irradiated wild-type recipients, and contribute to development of multiple hematopoietic lineages. However, all the recipients develop thymic lymphomas after a latency of 3-4 months post transplantation. Transplantation of different fractions of bone marrow cells or thymocytes from young MSH2-/- mice showed that only the HSC enriched fraction leads to lymphoma development. The lymphomas are transplantable, limiting dilution experiments showed that even 40 lymphoma cells could initiate T cell leukemia in sublethally irradiated secondary recipients within a month. In contrast, transplantation of the HSC enriched LSK fraction from the BM of lymphoma bearing mice into secondary recipients resulted in thymic lymphomas after a latency of 5 months. However, lymphoma development following transplant of HSC required an intact thymus and was not observed for more than 9 months if recipients were thymectomized, whereas this stromal requirement was not observed when transplanting T cell leukemicthymic lymphoma cells. These results suggested that MSH2-/- HSCs are PLSCs. While they retain full hematopoietic potential, their T-cell progeny gain lymphomagenic potential in the thymic microenvironment and become LICs. In this model, the evolution from PLSC to LIC is stromal-dependent. Citation Format: Yulan Qing, Stanton L. Gerson. Evolution of preleukemia stem cells to lymphoma initiating cells requires thymus in msh2-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2014-1910