Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Long-term bovine hematopoietic engraftment with clone-derived stem cells

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.     

Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells

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长期和短期限制性束缚对小鼠造血干细胞的不同影响

慢性心理应激会导致机体多种功能紊乱,其中包括免疫力下降。慢性心理应激能够负调节免疫系统,但机制尚未完全阐明。免疫细胞,包括髓系来源细胞,由骨髓造血干细胞(hematopoietic stem cell, HSC)分化而来,在机体免疫中发挥重要作用。本实验采用短期高强度束缚和长期温和束缚慢性心理应激小鼠模型,探讨不同限制性束缚模式对小鼠骨髓HSC和髓系来源细胞的影响。长期温和束缚模型中,对小鼠连续束缚4周,每天束缚2次,每次束缚2 h (9:00到17:00间完成);短期高强度束缚模型中,小鼠连续束缚5天,每天束缚16 h (当日17:00到次日9:00)。束缚完成后,取小鼠骨髓和外周血进行白细胞计数,流式细胞术检测小鼠骨髓HSC (Lin^-CD117^+Sca1^+CD150^+CD48^-)和髓系细胞(CD11b^+Ly6C^+)的比例和绝对数,以及外周血髓系细胞的比例和绝对数,BrdU掺入实验检测小鼠HSC增殖能力。实验结果表明,长期温和应激导致小鼠骨髓HSC的比例和绝对数增加,而短期高强度应激导致小鼠骨髓HSC的绝对数下降,伴有HSC的增殖下降。两种束缚模式都使小鼠骨髓和外周血CD11b^+Ly6C^+细胞的总数增加或呈现增加的趋势。综上所述,长期温和应激和短期高强度应激对小鼠HSC的比例和绝对数影响不同;两种应激模型都可以使CD11b^+Ly6C^+细胞总数增加,HSC增多可能并不是CD11b^+Ly6C^+细胞增多的主要机制。     

Stemness,fusion and renewal of hematopoietic and embryonic stem cells

Development of replacement cell therapies awaits the identification of factors that regulate nuclear reprogramming and the mechanisms that control stem cell renewal and differentiation. Once such factors and signals will begin to be elucidated, new technologies will have to be envisaged where uniform differentiation of adult or embryonic stem cells along one differentiation pathway can be induced. Controlled differentiation of stem cells will require the engineering of niches and extracellular signal combinations that would amplify a particular signaling network and allow uniform and selective differentiation. Three recent advances in stem cell research open the possibility to approach engineering studies for cell replacement therapies. Fusion events between stem cells and adult cells or between adult and embryonic stem cells have been shown to result in altered fates and nuclear reprogramming of cell hybrids. Hematopoietic stem cells were shown to require Wnt signaling in order to renew. The purification of Wnt proteins would allow their use as exogenous purified cytokines in attempts to amplify stem cells before bone marrow transplantation. The homeodomain protein Nanog has been shown to be crucial for the embryonic stem cell renewal and pluripotency. However, the cardinal question of how stemness is preserved in the early embryo and adult stem cells remains opened.     

Reduced dimethyl sulfoxide concentrations successfully cryopreserve human hematopoietic stem cells with multi-lineage long-term engraftment ability in mice

Background aimsThe cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured.MethodsCryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33⁺ myeloid, CD19⁺ B-lymphoid, CD235a⁺ erythroid and CD34⁺ progenitors; (ii) engraftment of the same four populations plus CD41⁺CD42b⁺ platelets; (iii) engraftment of CD34⁺⁺CD133⁺ cells; and (iv) engraftment of CD34⁺⁺CD38⁻ cells.ResultsHematopoietic colony-forming, CD34^++/+, CD34⁺⁺CD133⁺ and CD34⁺⁺CD38⁻ cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs.ConclusionCryopreservation of HSCs in DMSO concentrations as low as 5% is effective.     

Factors affecting engraftment of allogeneic hematopoietic stem cells after reduced-intensity conditioning

Several factors influence the engraftment of allogeneic hematopoietic stem cells (HSC). Recently, there has been increased utilization of transplant-conditioning regimens that use reduced doses of chemotherapy and radiation that are considered to be non-myeloablative. These non-myeloablative (or reduced-intensity) allogeneic HSC transplants (RIST) decrease early post-transplant complications, but they are associated with higher incidences of mixed chimerism and graft rejection compared with transplantation after myeloablative condition-ing. RIST provides a unique opportunity to study allogeneic HSC engraftment. In particular, host immune status and stem cell graft composition have emerged as important factors affecting engraftment after RIST Based on these observations, it has been hypothesized that conditioning regimens and allograft composition can be tailored to an individual patients immune and disease status prior to transplant.     

CD61 enriches long-term repopulating hematopoietic stem cells

Among the subsets that define hematopoietic stem cells (HSCs), CD34⁻ c-kit⁺ Sca-1⁺ lineage marker⁻ (CD34⁻KSL) cells are regarded as one of the populations that have the highest enrichment of HSCs in adult mouse bone marrow. Here, we demonstrate that long-term repopulating hematopoietic stem cells (LTR-HSCs) have high expression of CD61 (integrin β₃) within the CD34⁻KSL population. Approximately 60% of CD34⁻KSL cells showed high expression of CD61. CD61^HighCD34⁻KSL populations also exhibited significantly greater properties of HSC, such as expression of HSC markers, the side population (SP) phenotype, and ability for long-term repopulation. In both SP cells and non-SP (NSP) cells, CD61^HighCD34⁻KSL cells also contained significantly more LTR-HSCs than CD61^Low/−CD34⁻KSL cells. Our results indicate that CD61 is exploitable for HSC enrichment as a supportive positive cell surface marker.     

Parvovirus infection suppresses long-term repopulating hematopoietic stem cells

The functional disturbance of self-renewing and multipotent hematopoietic stem cells (HSCs) in viral diseases is poorly understood. In this report, we have assessed the susceptibility of mouse HSCs to strain i of the autonomous parvovirus minute virus of mice (MVMi) in vitro and during persistent infection of an immunodeficient host. Purified 5FU(r) Lin(-) Sca-1(+) primitive hematopoietic precursors were permissive for MVMi genome replication and the expression of viral gene products. The lymphoid and myeloid repopulating capacity of bone marrow (BM) cells was significantly impaired after in vitro infection, although the degree of functional effect proportionally decreased with the posttransplantation time. This indicated that MVMi targets the heterogeneous compartment of repopulating cells with differential affinity and suggests that the virus may persist in some primitive HSCs in the quiescent stage, killing those eventually recruited for proliferative activity. Immunodeficient SCID mice oronasally infected with MVMi were cured of the characteristic virus-induced lethal leukopenia by transplantation of immunocompetent BM grafts. However, two double-stranded viral DNA species, probably uncommon replicative intermediates, remained in the marrow of every transplanted mouse months after infectious virus clearance. Genetic analysis of the rescued mice showed that the infection ensured a stable engraftment of donor hematopoiesis by markedly depleting the pool of endogenous HSCs. The MVMi-induced suppression of HSC functions illustrates the accessibility of this compartment to infection during a natural viral hematological disease. These results may provide clues to understanding delayed hematopoietic syndromes associated with persistent viral infections and to prospective gene delivery to HSCs in vivo.     

Wild-type measles virus interferes with short-term engraftment of human CD34+ hematopoietic progenitor cells

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.     

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca²⁺ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K⁺ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.     

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues. A. Gerasimou, R. Ramella and A. Brero contributed equally to this paper.     

Number of megakaryocytic progenitors and adhesion molecule expression of stem cells predict platelet engraftment after allogeneic hematopoietic stem cell transplantation

BACKGROUND: The mechanism of platelet recovery after hematopoietic stem cell transplantation and the factors that influence its time-course are not fully understood. Rapid hematopoietic recovery results in a reduction of transplantation-related complications. In the present study, we questioned and analyzed whether there were important factors predicting the speed of platelet engraftment. METHODS: Thirty-seven patients with various hematologic diseases transplanted with allogeneic BM between January 2002 and December 2005 were included. We investigated the differences in mononuclear cell counts (MNC), numbers of infused CD34(+), CD34(+) CD41(+) and CD34(+) CD61(+) cells and phenotypic analysis of homing-associated cell adhesion molecules (CXCR4, CD49d and CD49e). The number of megakaryocytes formed in vitro (colony-forming unit-megakaryocytes; CFU-Mk) was also measured. RESULTS: Median days of ANC >/=0.5x10(9)/L and platelet count >/=20x10(9)/L were 14.8 and 17.3, respectively. The number of infused CD34(+) CD41(+) and CD34(+) CD61(+) cells correlated much better with the time to platelet engraftment than that of infused CD34(+)cells (P<0.05 each). Rapid platelet recovery also occurred in patients receiving both higher homing-associated cell adhesion molecule doses and CFU-Mk (P<0.05 each). DISCUSSION: Rapid platelet recovery has several advantages, including reducing the cost of supportive therapy and reducing the risk of fatal bleeding as a result of severe thrombocytopenia. Our findings suggest that phenotypic and clonogenic assessment of infused progenitor cells can identify patients in whom platelet engraftment is likely to be significantly delayed, and new strategies to overcome related problems might be employed in the very near future.     

Effect of long-term cytostatic therapy on the hematopoietic stem cells]

CFU-C and diffusion chamber studies were performed in patients with teratocarcinoma, who underwent long term chemotherapy. No significant decline of bone marrow CFU-C or diffusion chamber cell recovery was found during twelve months of cytotoxic treatment. In contrast to the results in these patients the CFU-C-content of the remission marrow in leukemic patients showed a significant decrease in relation to the duration of remission and chemotherapy.     

The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A₁ and A₃ receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N ⁶-cyclopentyladenosine (CPA), an adenosine A₁ receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N ⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A₃ receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.     

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.