Resveratrol increases the bone marrow hematopoietic stem and progenitor cell capacity

Resveratrol is a plant‐derived polyphenol that has shown protective effects against many disorders including, several types of cancers and other age‐associated diseases as well as blood disorders in cultured cells and/or animal models. However, whether resveratrol has any impact specifically on normal blood stem cells remains unknown. Here, we show that a 3‐week treatment of resveratrol increases the frequency and total numbers of normal bone marrow hematopoietic stem cells (HSC) without any impact on their competitive repopulation capacity. In addition, we show that resveratrol enhances the bone marrow multipotent progenitor capacity in vivo. These results have therapeutic value for disorders of hematopoietic stem and progenitor cells (HSPC) as well as for bone marrow transplantation settings. Am. J. Hematol. 89:E235–E238, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of aged bone marrow microenvironment on mesenchymal stem cell migration

Mesenchymal stem cells (MSCs) are known to have many notable features, especially their multiple differentiation ability and immunoregulatory capacity. MSCs are important stem cells in the bone marrow (BM), and their characteristics are affected by the BM microenvironment. However, effects of the BM microenvironment on the properties of MSCs are not well understood. In this study, we found that BM from aged mice decreased MSC colony formation. Flow cytometry data showed that the proportion of B220⁺ cells in BM from aged mice was significantly lower than that in BM from young mice, while the proportion of CD11b⁺, CD3⁺, Gr-1⁺, or F4/80⁺ cells are on the contrary. CD11b⁺, B220⁺, and Ter119⁺ cells from aged mice were not the subsets that decreased MSC colony formation. We further demonstrated that both BM from aged mice and young mice exhibited similar effects on the proliferation of murine MSC cell line C3H10T1/2. However, when cocultured with BM from aged mice, C3H10T1/2 showed slower migration ability. In addition, we found that phosphorylation of JNK (c-Jun N-terminal kinases) in C3H10T1/2 cocultured with BM from aged mice was lower than that in C3H10T1/2 cocultured with BM from young mice. Collectively, our data revealed that BM from aged mice could decrease the migration of MSCs from their niche through regulating the JNK pathway.     

Loss of hematopoietic stem cell self-renewal after bone marrow transplantation

The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.     

Multipotential hematopoietic stem cells in the bone marrow

It has been generally held that human hematopoietic stem cells are lineage-negative CD34⁺ CD38^?. However, murine hematopoietic stem cells were reported to be CD34^?. We have characterized the surface phenotypes of murine hematopoietic stem cells by using a murine transplantation model. Our studies revealed that the majority of the stem cells in normal adult mice are CD34^? while a minority (15%–20%) being CD34⁺. Our studies also revealed that stem cells that are activated by injection of 5-fluorouracil in vivo, exposure to cytokines in vitro, or mobilization by G-CSF are CD34⁺ and that CD34 expression is reversible. It has been reported that fetal murine hematopoietic stem cells are CD34⁺. Our studies revealed that stem cells of juvenile mice are CD34+ and that the developmental change from CD34⁺ to CD34^? state takes place between 7 and 10 weeks of age. In adult mice, expression of CD38 by steady-state and activated stem cells was completely reciprocal of CD34 expression. Activated stem cells and the minority population of the stem cells in the normal mice are CD34⁺ CD38^?. In contrast, the majority of stem cells in normal adult mice are CD34- CD38+. Recently, we studied CD38 expression by stem cells of neonatal and juvenile mice. Stem cells of newborn mice are CD38^?. About half of the stem cells of 5-week-old mice are CD38⁺. Finally, our studies indicated that some of the CD34⁺ stem cells in the bone marrow of normal adult mice express lineage markers such as Mac-1 and CD4. These studies in a murine model clearly documented that expression of both CD34 and CD38 by stem cells is under developmental control and may be subject to changes induced by activation of the stem cells. In order to test whether or not these principles apply to human stem cells we tested surface phenotypes of human stem cells using two xenotransplantation techniques. Studies based on human/sheep xenograft model indicated that a significant portion of adult human long-term engrafting cells are CD34^?. Similar to mouse stem cells expression of CD34 by human stem cells was reversible. Studies based on our newborn NOD/SCID/β-microglobulin^null mice indicated that human cord blood stem cells are CD34⁺ CD38^?. These results appear to support the validity of studies of murine stem cells to provide insight into human stem cells.     

Peripheral blood vs bone marrow as a source for allogeneic hematopoietic stem cell transplantation.

In this randomized prospective study, we included 30 patients with different hematological diseases (acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome or severe aplastic anemia) to compare peripheral blood stem cells (PBSC) (15 patients; mean age 23) and bone marrow (BM) (15 patients; mean age 21.8) as a source for allogeneic transplantation regarding the tempo of hematopoietic recovery and the incidence of acute graft-versus-host disease (GVHD). In the BM group, the median nucleated cell count harvested was 1.3 x 10(10), while in the PBSC group, the aphereses contained a median of 4.4 x 10(6) CD34+/kg recipient weight. PBSC transplantation (PBSCT) was associated with faster hematopoietic reconstitution measured as absolute neutrophil count (ANC) >0.5 x 10(9)/l (log-rank P value <0.0018) and platelet count >25 x 10(9)/l (log-rank P value <0.0098). Seven patients (46.7%) in the BM group vs only one patient (6.7%) in the PBSC group developed acute GVHD (P = 0.013). Therefore, we conclude that PBSCT is associated with faster hematopoietic recovery and the incidence of acute GVHD does not exceed that seen with BMT.     

Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment

Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Between 4 to 10 weeks after transplantation, eGFP-MSCs that engrafted in murine BM integrated into the hematopoietic microenvironment (HME) of the host mouse. They differentiated into pericytes, myofibroblasts, BM stromal cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells, which constituted the functional components of the BM HME. The presence of human MSCs in murine BM resulted in an increase in functionally and phenotypically primitive human hematopoietic cells. Human MSC-derived cells that reconstituted the HME appeared to contribute to the maintenance of human hematopoiesis by actively interacting with primitive human hematopoietic cells.     

Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line

An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor-dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL-11. This immortalized primate bone marrow stromal cell line may be useful in maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.     

小鼠骨髓造血干细胞转化为肝细胞的实验研究

为进一步明确向肝细胞分化的细胞为骨髓中的那一亚群细胞,我们将纯化后的雄性小鼠造血干细胞移植到雌性受体小鼠的体内,然后在受体小鼠的肝脏内进行检测,以确定有无供体来源的肝细胞存在。一、材料与方法1.实验动物、主要器材和试剂:健康清洁级雌雄性纯系BALB/C鼠,8～12周龄,均由第一军医大学动物实验中心提供。主要试剂及材料:磁性活化细胞分选(magnetic activated cell sorting,MACS)分离用MiniMACS缓冲液,     

Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation

The engraftment of donor bone marrow (BM) cells in nonablated mice is inefficient. Niche availability has been thought to be the reason, and cytoablation with irradiation or cytotoxic agents is routinely used with the belief that this frees the preoccupied niches in recipients. In this study, donor cell redistribution and proliferation in ablated and nonablated mice were compared by implanting donor cells directly into the femur cavity of sedated mice. The redistribution of Lin(-) donor cells into BM was similar between ablated and nonablated mice. Poor engraftment in nonablated mice was shown to be the result of inefficient donor cell proliferation rather than because of a lack of space. Competitive repopulation assays demonstrated that the donor hematopoietic stem cells (HSCs) were present in nonirradiated recipients for at least 6 months after transplantation, but that they did not expand as did their counterparts in lethally irradiated mice. This study suggests that efficient bone marrow transplantation in nonablated recipients may be possible as a result of better understanding of HSC proliferative regulation and appropriate in vitro manipulation.     

Host origin of the human hematopoietic microenvironment following allogeneic bone marrow transplantation

The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA- identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.     

TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation

Hematopoietic stem cells (HSCs) reside in a bone marrow niche in a nondividing state from which they occasionally are aroused to undergo cell division. Yet, the mechanism underlying this unique feature remains largely unknown. We have recently shown that freshly isolated CD34-KSL hematopoietic stem cells (HSCs) in a hibernation state exhibit inhibited lipid raft clustering. Lipid raft clustering induced by cytokines is essential for HSCs to augment cytokine signals to the level enough to re-enter the cell cycle. Here we screened candidate niche signals that inhibit lipid raft clustering, and identified that transforming growth factor-beta (TGF-beta) efficiently inhibits cytokine-mediated lipid raft clustering and induces HSC hibernation ex vivo. Smad2 and Smad3, the signaling molecules directly downstream from and activated by TGF-beta receptors were specifically activated in CD34-KSL HSCs in a hibernation state, but not in cycling CD34+KSL progenitors. These data uncover a critical role for TGF-beta as a candidate niche signal in the control of HSC hibernation and provide TGF-beta as a novel tool for ex vivo modeling of the HSC niche.     

Shwachman-Diamond syndrome: An inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty marrow microenvironment.

Shwachman-Diamond syndrome (SD), an inherited disorder with varying cytopenias and a marked tendency for malignant myeloid transformation, is an important model for understanding genetic determinants in hematopoiesis. To define the basis for the faulty hematopoietic function, 13 patients with SD (2 of whom had myelodysplasia with a clonal cytogenetic abnormality) and 11 healthy marrow donors were studied. Patients with SD had significantly lower numbers of CD34(+) cells on bone marrow aspirates. SD CD34(+) cells plated directly in standard clonogenic assays showed markedly impaired colony production potential, underscoring an intrinsically aberrant progenitor population. To assess marrow stromal function, long-term marrow stromal cell cultures (LTCs) were established. Normal marrow CD34(+) cells were plated over either SD stroma (N/SD) or normal stroma (N/N); SD CD34(+) cells were plated over either SD stroma (SD/SD) or normal stroma (SD/N). Nonadherent cells harvested weekly from N/SD LTCs were strikingly reduced compared with N/N LTCs; numbers of granulocyte-monocyte colony-forming units (CFU-GM) derived from N/SD nonadherent cells were also lower. SD/N showed improved production of nonadherent cells and CFU-GM colonies compared with SD/SD, but much less than N/N. Stem-cell and stromal properties from the 2 patients with SD and myelodysplasia did not differ discernibly from SD patients without myelodysplasia. We conclude that in addition to a stem-cell defect, patients with SD have also a serious, generalized marrow dysfunction with an abnormal bone marrow stroma in terms of its ability to support and maintain hematopoiesis. This dual defect exists in SD with and without myelodysplasia.     

Impaired bone marrow hematopoietic progenitor cell function in rheumatoid arthritis patients candidated to autologous hematopoietic stem cell transplantation

We have evaluated bone marrow morphology, percentage of bone marrow CD34(+) cells, proliferative activity of bone marrow precursors, clonogenic assay (BFU-E and CFU-GM) in short-term bone marrow cultures, and bone marrow cell apoptosis, together with serum TNF-alpha and IL-6, in 16 chronic, refractory RA patients, as well as in five healthy controls. Of 16 RA patients (68.7%), 11 showed a reduced bone marrow cellularity, while it was normal in all the controls. In RA patients, the median percentage of CD34(+) bone marrow cells, the median percentage of proliferating bone marrow myeloid precursors, and the median number of both BFU-E and CFU-GM colonies were significantly lower than observed in the controls. As far as TNF-alpha and IL-6 titers is concerned, the latter did not significantly differ from controls' values, while TNF-alpha titers were significantly lower in healthy controls. Finally, the median apoptotic index of early bone marrow myeloid cells of RA patients was significantly higher compared with controls. These observations may identify the biological risk factors for impaired mobilization and/or engraftment when RA patients are candidates for autologous hematopoietic stem cell grafting.