Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures

Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11b(bright), CD8alpha(-)) and "lymphoid-related" (CD11b(dull), CD8alpha(+)), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)-dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c(+) population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro-derived DCs, when treated with interferon-alpha or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8alpha, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti-interleukin-6 (IL-6) antibody, but not anti-GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.     

Development of plasmacytoid dendritic cells in bone marrow stromal cell niches requires CXCL12-CXCR4 chemokine signaling

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producingcells, are thought to play central roles in antiviral immunity and the pathogenesis of some autoimmune diseases. pDCs are produced from hematopoietic stem cells in bone marrow. However, the environmental regulation of the development of pDCs is not fully understood. Here, we show that the numbers of pDCs and their earliest progenitors are severely reduced in the absence of CXCR4, the primary physiologic receptor for CXC chemokine ligand 12 (CXCL12), also known as stromal cell-derived factor-1 (SDF-1) in vivo. In vitro, CXCL12 induces a significant increase in pDC numbers generated from primitive hematopoietic cells, and pDCs and their progenitors migrate to CXCL12. In addition, most pDCs are in contact with CXCL12-abundant reticular (CAR) cells in the intersinal space of bone marrow, although many primitive hematopoietic cells adjoin CAR cells surrounding sinusoidal endothelial cells or residing near the bone surface. Thus we identified CXCL12 as a key regulator of pDC development produced by cellular niches, providing new targets for pDC therapeutic control.     

氧化苦参碱对小鼠树突状细胞成熟和功能的影响

目的:研究氧化苦参碱(OXY)对小鼠树突状细胞(DC)成熟、表型及功能的影响。方法:流式细胞术检测DC表面分子CD40的表达;混合淋巴细胞反应(MLR)检测DC对T淋巴细胞的刺激能力;ELISA法检测MLR上清中细胞因子IFN-γ的分泌。结果:第0天OXY处理组较对照组DC表面分子CD40的表达明显升高(P<0.01),刺激T细胞能力增强,分泌细胞因子IFN-γ明显升高(P<0.05),对LPS诱导的DC成熟,与DC LPS组对照显著升高(P<0.05)。结果:OXY对DC的成熟和功能有一定的促进作用。     

Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells

The migratory properties of dendritic cells (DCs) are important for their functions. Although several chemokines and their receptors have been implicated in DC migration, the downstream signaling molecules are largely unknown. Here we show that DOCK2, a hematopoietic cell-specific CDM family protein, is indispensable for migration of plasmacytoid DCs (pDCs), but not myeloid DCs (mDCs). Although DOCK2-deficiency did not affect development of pDCs, DOCK2-deficient (DOCK2^–/–) mice exhibited a severe reduction of pDCs in the spleen and lymph nodes. Adoptive transfer experiments revealed that DOCK2^–/– pDCs failed to migrate into the periarteriolar lymphoid sheaths of the spleen. In DOCK2^–/– pDCs, chemokine-induced Rac activation was severely impaired, resulting in the reduction of motility and the loss of polarity during chemotaxis. In contrast, DOCK2^–/– mDCs did not show any defects in Rac activation and migration. These results indicate that pDCs and mDCs use distinct molecules to activate Rac during chemotaxis.     

Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation

Following a report of skeletal muscle regeneration from bone marrow cells, we investigated whether hepatocytes could also derive in vivo from bone marrow cells. A cohort of lethally irradiated B6D2F1 female mice received whole bone marrow transplants from age-matched male donors and were sacrificed at days 1, 3, 5, and 7 and months 2, 4, and 6 posttransplantation (n = 3 for each time point). Additionally, 2 archival female mice of the same strain who had previously been recipients of 200 male fluorescence-activated cell sorter (FACS)-sorted CD34(+)lin(-) cells were sacrificed 8 months posttransplantation under the same protocol. Fluorescence in situ hybridization (FISH) for the Y-chromosome was performed on liver tissue. Y-positive hepatocytes, up to 2.2% of total hepatocytes, were identified in 1 animal at 7 days posttransplantation and in all animals sacrificed 2 months or longer posttransplantation. Simultaneous FISH for the Y-chromosome and albumin messenger RNA (mRNA) confirmed male-derived cells were mature hepatocytes. These animals had received lethal doses of irradiation at the time of bone marrow transplantation, but this induced no overt, histologically demonstrable, acute hepatic injury, including inflammation, necrosis, oval cell proliferation, or scarring. We conclude that hepatocytes can derive from bone marrow cells after irradiation in the absence of severe acute injury. Also, the small subpopulation of CD34(+)lin(-) bone marrow cells is capable of such hepatic engraftment.     

Vaginal epithelial dendritic cells renew from bone marrow precursors

Dendritic cells (DCs) represent key professional antigen-presenting cells capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by nonmonocyte bone-marrow-derived precursors, with a half-life of 13 days under steady-state conditions. Upon infection with HSV-2, the Gr-1(hi) monocytes were found to give rise to VEDCs. Furthermore, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b(+)F4/80(hi), CD11b(+)F4/80(int), and CD11b(-)F4/80(-). Importantly, these VEDC populations expressed CD207 at low levels and had a constitutively more activated phenotype compared with the skin LCs. Collectively, our results revealed mucosa-specific features of the VEDCs with respect to their phenotype, activation status, and homeostatic renewal potential.     

Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II- restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T- cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM- derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.     

TET2 mutations,myelodysplastic features,and a distinct immunoprofile characterize blastic plasmacytoid dendritic cell neoplasm in the bone marrow

Distinguishing blastic plasmacytoid dendritic cell neoplasm (BPDCN) from acute myeloid leukemia (AML) is gaining increased importance because of emerging differences in therapeutic approaches, and this distinction can be problematic in bone marrow specimens. We identified retrospectively 16 patients with bone marrow involvement by BPDCN: 11 men and 5 women with a median age of 62.5 years (range, 19–86 years). Myelodysplastic changes were observed in five patients. Immunophenotypic analysis showed that the neoplastic cells were positive for CD4, CD123, TCL‐1, and HLA‐DR and were negative for CD3, CD8, CD13, CD19, CD34, and myeloperoxidase. Other antigens expressed by subsets of BPDCN cases included the following: CD56 (13/15; 81%), CD33 (7/10; 70%), CD7 (11/14; 69%), TdT (5/15; 33%), CD2 (5/11; 31%), CD117 (2/9; 22%), and CD5 (2/13; 15%). Conventional cytogenetic analysis showed chromosomal abnormalities in 6 of 13 (46%) cases analyzed, of which 3 cases had ?13/13q?. Targeted next‐generation sequencing performed on five BPDCN cases identified TET2 (ten eleven translocation 2) mutations and no other AML‐associated mutations. In conclusion, BPDCN in the bone marrow has a characteristic immunoprofile (CD4+, CD56+, CD123+, and TCL‐1+) and appears to be commonly associated with myelodysplastic features and a high frequency of TET2 mutations in the absence of other mutations commonly observed in AML. Am. J. Hematol. 88:1055–1061, 2013. © 2013 Wiley Periodicals, Inc.     

Multiparameter analysis of murine bone marrow side population cells

We describe the multiparameter flow cytometric analysis of the relationship between side population (SP) formation and well-characterized, antigen-defined stem cell subsets. We also compared the competitive repopulation ability of subsets defined by the SP profile to those identified by antigenic markers. The vast majority of SP cells possessed a primitive cell phenotype (c-kit+, SCA-1+, Thy-1+, CD31+, CD135neg, lineage neg), but only a minority of antigen-defined subsets were SP cells. Hence, although SP cells are identified independently of antigenic markers, they are not distinct from established stem cell phenotypes, but are a small subset of them. Approximately half of SP cells expressed CD34 at readily detectable levels, and one-third of SP cells possessed the primitive c-kit+, SCA-1+, lineage neg, CD34neg cell phenotype. Since most SP cells are a subset of c-kit+, Thy-1+, lineage neg, SCA-1+ cells (KTLS), we determined whether the SP cell subset represents a further enrichment in long-term repopulating cell content. The SP+ subset of KTLS+ cells was more enriched for competitive repopulation units than the SP- fraction of KTLS+ cells. Hence, the SP cell fraction highlights a subset of the long-term repopulating cells found within the already highly purified KTLS fraction.     

Mechanisms of TRAIL-induced apoptosis in leukemic plasmacytoid dendritic cells

OBJECTIVE: Dendritic cells play a central role in regulating the innate and adaptive immune responses. Plasmacytoid dendritic cells (PDC) represent a newly identified kind of DC with specialized functions aimed at fighting against viral infections. Recently, we have shown that CD4+CD56+ malignancies were leukemia arising from PDC, with a particularly aggressive clinical course. Hence, we asked whether these malignant PDC could be killed via TRAIL, a death-inducing ligand that belongs to a new class of anticancer drugs currently under development. MATERIALS AND METHODS: In this study we used a PDC line (GEN2.2) we recently developed from leukemic PDC as a model. RESULTS: We show that GEN2.2 PDC are sensitive to TRAIL-induced apoptosis and can be killed in vitro by TRAIL-expressing NK cells. Our results suggest that TRAIL binds to Death Receptor 5 (DR5) expressed by GEN2.2 and induces apoptosis mainly via caspases 10, 8, and 3. Interestingly, during infection with influenza, DR5 decreases on GEN2.2 cell surface, which consequently become resistant to TRAIL-induced apoptosis. Moreover, we confirmed the expression of DR5 or DR4 on half of LPDC tested, suggesting the possibility to kill these cells via TRAIL. Hopefully, normal PDC expressed neither DR4 nor DR5. CONCLUSION: These results suggest that TRAIL agonists represent a therapeutic alternative for the treatment of LPDC.     

Characterization of fibroblastic stromal cells from murine bone marrow

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.     

Increased generation of pre-plasmacytoid dendritic cells in bone marrow of rheumatoid arthritis

Abstract

Objective. Plasmacytoid dendritic cells (pDCs) have been found to be accumulated in synovial tissues in rheumatoid arthritis (RA). Since pDCs originate from bone marrow (BM), we explored the differentiation of pDC in BM in RA and osteoarthritis (OA).

Methods. BM mononuclear cells (BMMNCs) of the posterior ileac crest from 25 RA patients and 22 OA patients were examined for the expression of BDCA2 and CD34 by flow cytometry. The degree of synovial proliferation was assessed on light microscopy in 10 of 25 RA patients.

Results. There were no significant differences in percentages of CD34 + cells or BDCA2 + cells within BMMNC between RA and OA. However, RA BMMNC contained higher percentages of BDCA2 + CD34 + cells (pre-pDCs) than OA BMMNCs. Accordingly, percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly higher in RA than in OA. Finally, the percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly correlated with the degree of synovial proliferation in RA.

Conclusion. These results indicate that the generation of pre-pDC from BM CD34 + cells is increased in RA compared with OA. Moreover, the data suggest that the increased output of pDC from BM might be involved in the synovial proliferation in RA.     

Expression of alpha-smooth muscle actin in murine bone marrow stromal cells

Human fibrotic bone marrow (BM) stroma has been shown to contain alpha-smooth muscle actin (alpha-SMA)-positive cells. These closely resemble myofibroblasts that were described in other fibrotic tissues. We studied the expression of alpha-SMA in a series of murine BM-derived stromal cell lines to investigate the cellular origin and functional significance of myofibroblast-like cells in hematopoietic tissues. Although these cell lines differed in their biologic properties, most of them expressed alpha-SMA under certain conditions. Cells expressing alpha-SMA constituted a minor population in post-confluent, growth-arrested cultures. However, the incidence of cells expressing alpha-SMA increased significantly when cultures were transferred to nonconfluent conditions. A similar increase in alpha-SMA-positive cells occurred after a strip of cells was scraped away from the confluent cell layer; the cells of the affected area acquired alpha-SMA-positive contractile phenotype. The relationship between alpha-SMA expression and hematopoietic activity was studied using a cloned cell line of BM origin (14F1.1). The ability of these endothelial-adipocyte cells to support hematopoiesis in vitro was maximal under confluent conditions, whereas their expression of alpha-SMA under such conditions was residual. Moreover, in long-term BM cultures supported by confluent 14F1.1 cells, stromal areas associated with proliferating hematopoietic precursors, known as "cobblestone areas," were devoid of alpha-SMA-positive cells. These observations suggest that the expression of alpha-SMA is reversible and inversely related to hematopoietic activity.     

Persistence of murine myeloid leukaemic cells in long-term bone marrow cultures

Patterns of growth in long-term bone marrow culture were compared for a number of murine myeloid leukaemias. A leukaemic pattern of haematopoiesis was maintained in culture for a period of at least 15 weeks for one of the lines. However in the other five murine myeloid leukaemic lines studies, the leukaemic cells appeared not to survive in culture and normal haematopoiesis was established. Transplantation of cells from these established cultures at weeks 7 or 11 into syngeneic recipients revealed that leukaemic cells were present. Thus leukaemic cells seem to persist in long-term bone marrow cultures even in morphologically normal cultures.     

Stimulated plasmacytoid dendritic cells impair human T-cell development

Thymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into the thymus, allowing for interactions between thymic pDCs and precursor cells. We demonstrate that in vitro-generated pDCs stimulated with CpG or virus impaired the development of human autologous CD34(+)CD1a(-) thymic progenitor cells into the T-cell lineage. Rescue by addition of neutralizing type I interferon (IFN) antibodies strongly implies that endogenously produced IFN-alpha/beta is responsible for this inhibitory effect. Consistent with this notion, we show that exogenously added IFN-alpha had a similar impact on IL-7- and Notch ligand-induced development of thymic CD34(+)CD1a(-) progenitor cells into T cells, because induction of CD1a, CD4, CD8, and TCR/CD3 surface expression and rearrangements of TCRbeta V-DJ gene segments were severely impaired. In addition, IL-7-induced proliferation but not survival of the developing thymic progenitor cells was strongly inhibited by IFN-alpha. It is evident from our data that IFN-alpha inhibits the IL-7R signal transduction pathway, although this could not be attributed to interference with either IL-7R proximal (STAT5, Akt/PKB, Erk1/2) or distal (p27(kip1), pRb) events.     

Imaging of plasmacytoid dendritic cell interactions with T cells

Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4(+) T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC-T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.     

Reduced numbers of plasmacytoid dendritic cells in aged blood donors

Dendritic cells (DC) play essential functions in both innate and adaptive immune responses. Peripheral blood DCs are divided into two major subsets, named conventional DC (cDC) and plasmacytoid DC (pDC), which play specific functions in the immune response. The absolute numbers of DCs (and their subsets) in peripheral blood may suffer variations due to physiological or pathological reasons, and therefore the enumeration of DC subsets in blood samples may be of clinical interest. However, to date this enumeration has produced controversial rather than consistent results. Here, using a two-tube platform approach, peripheral blood DCs have been enumerated in samples from healthy blood donors aged 18-65 years old. The results obtained showed a significant age-related decrease in pDC numbers, whilst cDC numbers remained unaltered. The different protocols used to define and enumerate DC subsets from blood samples may account for the controversial results reported before, thus emphasizing the importance of a general consensus to enumerate DCs. Reduced pDC numbers may be related to the higher susceptibility to infection and deficient response to vaccination often observed in aged individuals.     

Characterization of plasmacytoid dendritic cells in inflammatory arthritis synovial fluid

OBJECTIVE: To examine the phenotype of dendritic cell subsets in synovial fluid and peripheral blood from patients with rheumatoid arthritis (RA) or spondyloarthropathy (SpA). METHODS: Multiparameter flow cytometry was used to identify and characterize dendritic cells in mononuclear cell populations isolated from synovial fluid and peripheral blood. RESULTS: Synovial fluid contained two subsets of dendritic cells (DC), myeloid and plasmacytoid. These subsets could also be identified in peripheral blood, but there were lower numbers of DC in peripheral blood compared with synovial fluid. Plasmacytoid DC were distinguished from the myeloid subset by high expression of CD123 and lack of expression of CD11c. In comparison with myeloid dendritic cells, the plasmacytoid subset were less mature, similar to those in peripheral blood. They failed to express CD83 and DC-LAMP, and had relatively low levels of CD40 and CD86. Comparison of dendritic cells in synovial fluid from RA and SpA patients showed increased numbers of the plasmacytoid subset in SpA. CONCLUSIONS: This is the first demonstration of the plasmacytoid subset of dendritic cells in synovial fluid. Since these cells are major producers of type I interferons, their increased numbers in SpA might be relevant to pathogenesis, but the immature phenotype in SpA synovial fluid may also indicate that conditions for maturation of this subset do not pertain in SpA synovium.     

CpG ODN2216刺激慢性乙型肝炎患者浆样树突状细胞表型和功能的变化

目的观察慢性乙型肝炎患者外周血浆样树突状细胞(pDCs)的特点,并探讨pDCs在乙型肝炎病毒(HBV)感染发病机制中的作用。方法收集20例慢性乙型肝炎患者和15名健康人外周抗凝血,应用流式细胞仪分析pDCs的频率；同时检测CpG ODN2216刺激后pDCs表型及分泌干扰素(IFN)α功能的变化；并分析HBV慢性感染患者外周血pDCs频率、数量和功能的变化及其与临床病情的关系。结果应用免疫磁珠分选技术可得到纯度大于95%的pDCs。经CpG ODN2216刺激后纯化的pDCs能高表达共刺激分子CD80、CD86、CD40和CD83,并分泌大量的IFNα。与健康人比较,HBV感染患者外周血中pDCs频率明显下降,分别为0．287%±0．142%和0．192%±0．110%；经CpG ODN2216刺激后,其表面共刺激分子CD80和CD40表达明显下降,产生IFNα的能力也显著降低,分别为(3142．9±1292．3) pg/ml和(972．6±705．5)pg/ml。相关性分析表明,患者pDCs产生的IFNα水平与丙氨酸氨基转移酶水平呈明显负相关,但与血清病毒载量无明显相关性。结论CpG ODN2216可以刺激pDCs成熟并分泌大量IFNα。慢性乙型肝炎患者外周血pDCs频率、表型和功能明显下降,这可能是HBV持续感染的重要致病机制。     

CD4dull+ hematopoietic progenitor cells in murine bone marrow

CD4+ cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1- cells (CD4hiGr-1-) and CD4+ Gr- 1+ cells (CD4loGr-1lo). CD4hiGr-1- cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1lo cells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1lo cells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1lo cells were present in the recipients' BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr- 1lo cells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.