Human CD34-negative hematopoietic stem cells: The current understanding of their biological nature.

Hematopoietic stem cell (HSC) heterogeneity and hierarchy are a current topic of interest, having major implications for clinical HSC transplantation and basic research on human HSCs. It was long believed that the most primitive HSCs in mammals, including mice and humans, were CD34 antigen positive (CD34+). However, 2 decades ago, it was reported that murine long-term multilineage reconstituting HSCs were lineage marker negative (Lin-, i.e., c-kit+Sca-1+CD34low/-), known as CD34low/- KSL cells. In contrast, human CD34- HSCs, a counterpart of murine CD34low/- KSL cells, were hard to identify for a long time mainly because of their rarity. We previously identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method and proposed the new concept that CD34- SRCs (HSCs) reside at the apex of the human HSC hierarchy. Through a series of studies, we identified two positive/enrichment markers: CD133 and GPI-80. The combination of these two markers enabled the development of an ultrahigh-resolution purification method for CD34- as well as CD34+ HSCs and the successful purification of both HSCs at the single-cell level. Cell population purity is a crucial prerequisite for reliable biological and molecular analyses. Clonal analyses of highly purified human CD34- HSCs have revealed their potent megakaryocyte/erythrocyte differentiation potential. Based on these observations, we propose a revised road map for the commitment of human CB-derived CD34- HSCs. This review updates the current understanding of the stem cell nature of human CB-derived primitive CD34- as well as CD34+ HSCs.

[Updated human hematopoietic stem cell findings: purification of human hematopoietic stem cells and elucidation of their hierarchy].

Hematopoietic stem cell (HSC) biology is a current topic of interest having significant implications for clinical HSC transplantation and basic HSC research. It was long believed that the most primitive HSCs in mammals, including those in mice and humans, were CD34 antigen-positive (CD34+) cells. However, Nakauchi et al. reported that murine long-term lymphohematopoietic reconstituting HSCs were lineage marker-negative (Lin-) c-kit+Sca-1+CD34-low/negative (CD34low/-), known as CD34low/-KSL cells. We have previously identified very primitive human cord blood (CB) -derived CD34-negative (CD34-) severe combined immunodeficiency (SCID) -repopulating cells (SRCs) utilizing an intra-bone marrow injection method and have proposed a new concept that CD34-SRCs (HSCs) reside at the apex of human HSC hierarchy. Recently, we developed an ultra-high-resolution purification method using the two positive/enrichment markers CD133 and GPI-80 and succeeded in purifying CD34+/-HSCs on a single-cell level. On the basis of these data, we propose a revised roadmap for the commitment of human CD34-HSCs. This review updates the concept of the stem cell nature of human CB-derived primitive CD34+/-HSCs.

The number of CD34+CD133+ hematopoietic stem cells residing in umbilical cord blood (UCB) units is not correlated with the numbers of total nucleated cells and CD34+ cells: a possible new indicator for quality evaluation of UCB units.

Umbilical cord blood transplantation (UCBT) is often associated with delayed neutrophil and platelet recovery. Engraftment failure is another major obstacle. Several factors influence these serious complications, including the numbers of total nucleated cells (TNCs) and CD34+ cells which have been used as reliable factors for selecting UCB units for transplantation. However, whether both factors are reliable indices of the hematopoietic stem cell (HSC) activity of UCB units remains unknown. To evaluate the quality of UCB units, we quantified the actual number of transplantable CD34+CD133+ HSCs (tHSCs) residing in UCB units. The number of tHSCs was not correlated with the numbers of TNCs or CD34+ cells. These results strongly suggest that neither factor reflects the numbers of tHSCs residing in UCB units. To validate the significance of the number of tHSCs, further analysis is required to determine whether the number of tHSCs residing in UCB units is useful as a new indicator for the quality assessment of UCB units.

A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells.

We previously identified CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells as primitive hematopoietic stem cells (HSCs) in human cord blood. In this study, we develop a prospective ultra-high-resolution purification method by applying two positive markers, CD133 and GPI-80. Using this method, we succeed in purifying single long-term repopulating CD34- HSCs with self-renewing capability residing at the apex of the human HSC hierarchy from cord blood, as evidenced by a single-cell-initiated serial transplantation analysis. The gene expression profiles of individual CD34+ and CD34- HSCs and a global gene expression analysis demonstrate the unique molecular signature of CD34- HSCs. We find that the purified CD34- HSCs show a potent megakaryocyte/erythrocyte differentiation potential in vitro and in vivo. Megakaryocyte/erythrocyte progenitors may thus be generated directly via a bypass route from the CD34- HSCs. Based on these data, we propose a revised road map for the commitment of human CD34- HSCs in cord blood.

TGF-ß Signaling Accelerates Senescence of Human Bone-Derived CD271 and SSEA-4 Double-Positive Mesenchymal Stromal Cells.

It is generally thought that the proliferative capacity and differentiation potential of somatic stem cells, including mesenchymal stromal/stem cells (MSCs) and hematopoietic stem cells, decline with age. We investigated the effects of aging on human bone-derived MSCs expressing CD271 and SSEA-4 (double-positive MSCs [DPMSCs]). The percentages of DPMSCs in bone tissue decreased significantly with age. The DPMSCs from elderly patients (old DPMSCs) showed cellular senescence, which was evidenced by low growth potential, high senescence-associated ß-galactosidase activity, and elevated p16 and p21 CDK inhibitor levels. Moreover, old DPMSCs showed weak osteogenic differentiation potential and less hematopoiesis-supporting activity in comparison with young DPMSCs. Interestingly, the addition of transforming growth factor ß2 (TGF-ß2) induced cellular senescence in young DPMSCs. With the exception of the adipogenic differentiation potential, all of the aging phenomena observed in old DPMSCs were reversed by the addition of anti-TGF-ß antibodies. These results suggest that, in part, old DPMSCs accelerate cellular senescence through TGF-ß signaling.

CD34-negative hematopoietic stem cells show distinct expression profiles of homing molecules that limit engraftment in mice and sheep.

We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34-) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin- or 18Lin-) CD34+/- cells from human CB in mice and sheep. Both 13Lin- and 18Lin- CD34+ cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin- nor 18Lin- CD34- cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin- and 18Lin- CD34- cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34- HSCs is significantly more limited than that of CD34+ HSCs. As for 18Lin-, CD34- HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34- HSCs could thus explain in part their reduced ability to home to the BM niche.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFß3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo.

Mouse dental pulp stem cells support human umbilical cord blood-derived hematopoietic stem/progenitor cells in vitro.

It is well documented that specialized mesenchymal stem/stromal cells (MSCs) constitute the hematopoietic stem cell (HSC) niche in the bone marrow (BM), and these MSCs support/maintain the HSCs in an undifferentiated state. A number of studies have demonstrated that BM-derived MSCs (BM-MSCs) can support HSCs in vitro. However, it remains unclear whether nonhematopoietic tissue-derived MSC-like cells, such as dental pulp stem cells (DPSCs), have the ability to support HSCs. In this study, we prospectively isolated DPSCs from mouse mandibular incisors by fluorescence-activated cell sorting (FACS) using BM-MSC markers, such as PDGFRα and Sca-1. The PDGFRα and Sca-1 double-positive DPSCs and BM-MSCs showed similar morphologies and expression patterns of MSC markers. The ability of the DPSCs to support hematopoietic stem/progenitor cells (HSPCs) was then analyzed by an in vitro coculture system. Moreover, their HSC-supporting activity was evaluated by in vivo xenotransplantation assays using NOD/Shi-scid/IL-2Rγc(null) (NOG) mice. Interestingly, the DPSCs supported human cord blood (CB)-derived CD34-positive (CD34(+)), as well as CD34-negative (CD34(-)), HSCs. The supporting activities of DPSCs for human CB-derived CD34(+) and CD34(-) HSCs were comparable to those of BM-MSCs. The results of the present study demonstrated, for the first time, that prospectively isolated murine PDGFRα and Sca-1 double-positive DPSCs could support primitive human CD34(+) and CD34(-) HSCs in vitro.

Low level of c-kit expression marks deeply quiescent murine hematopoietic stem cells.

Although c-kit is expressed highly on murine hematopoietic stem cells (HSCs) and essential for bone marrow (BM) hematopoiesis, the significance of the high level of expression of c-kit on HSCs was not well determined. We show here that CD150(+) CD48(-) Lineage(-) Sca-1(+) c-kit(+) HSCs in adult BM are distributed within the range of roughly a 20-fold difference in the expression level of c-kit, and that c-kit density correlates with the cycling status of the HSC population. This predisposition is more evident in the BM of mice older than 30 weeks. The HSCs in G(0) phase express a lower level of c-kit both on the cell surface and inside the cells, which cannot be explained by ligand receptor binding and internalization. It is more likely that the low level of c-kit expression is a unique property of HSCs in G(0). Despite functional differences in the c-kit gradient, the HSCs are uniformly hypoxic and accessible to blood perfusion. Therefore, our data indicate the possibility that the hypoxic state of the HSCs is actively regulated, rather than them being passively hypoxic through a simple anatomical isolation from the circulation.

CXCL8 enhances the angiogenic activity of umbilical cord blood-derived outgrowth endothelial cells in vitro.

OECs (outgrowth endothelial cells), also known as late-EPCs (late-endothelial progenitor cells), have a high proliferation potential in addition to in vitro tube formation capability. In ischaemic animal models, injected OECs were integrated into regenerating blood vessels and improved neovascularization. Previous reports have demonstrated the expression of CXCL8 to be up-regulated in ischaemic tissues. It has also been documented that CXCL8 stimulates the angiogenic activity of mature ECs (endothelial cells). Therefore, it has been suggested that CXCL8 plays an important role in neovascularization in ischaemic tissues. However, it is still uncertain whether CXCL8 also stimulates the angiogenic activity of OECs. This study evaluated the effects of CXCL8 on the angiogenic activity of OECs in vitro. OECs were isolated from human UCB (umbilical cord blood)-derived mononuclear cells. Phenotypes of the OECs were assessed by flow cytometry, immunostaining, and real-time RT (reverse transcription)-PCR. The effects of CXCL8 on OECs were investigated by transwell migration assay and capillary tube formation assay on Matrigel. The OEC clones isolated from UCB expressed OEC phenotypes. In addition, CXCL8 receptors (CXCR1 and CXCR2) were expressed on these OEC clones. CXCL8 significantly stimulated the transwell migration and capillary tube formation of OECs. Neutralizing antibody against CXCR2, but not CXCR1, abolished a transwell migration of OECs induced by CXCL8, suggesting the involvement of CXCL8/CXCR2 axis in transwell migration. These results demonstrate that CXCL8 stimulates the angiogenic activity of UCB-derived OECs in vitro.

Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells.

OBJECTIVE: We have successfully identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid repopulating ability using the intrabone marrow injection method. In our previous study, a limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in CB-derived 13lineage-negative (Lin(-)) CD34(-) cells to be approximately 1/25,000. In this study, we intended to develop a high-resolution purification method to obtain highly purified CD34(-) SRCs. MATERIALS AND METHODS: The pooled CB-derived Lin(-) cells were stained with 13 reported Lin monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA, and CD127. Then 18Lin(-)CD34(high), 18Lin(-)CD34(-), and 13Lin(-)CD34(high)CD38(-) cells were sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractions of cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of this new purification method. RESULTS: A limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in these 18Lin(-)CD34(-) cells to be approximately 1/1,000, which is associated with a seeding efficiency 25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/IL-2Rγc(null) mice that received transplants of only two CD34(-) SRCs were highly engrafted with human lymphomyeloid cells at 24 weeks after primary transplantation and showed secondary multilineage repopulating abilities. CONCLUSIONS: We succeeded to highly purify the CD34(-) SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification method is indispensable to precisely characterize a distinct class of primitive human CB-derived CD34(-) hematopoietic stem cells.

5-Aza-2'-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells.

OBJECTIVE: Tissue stem cells in dental pulp are assumed to possess differentiation potentials similar to mesenchymal stem cells (MSCs). The aim of this in vitro study is to examine the differentiation potentials of mouse dental pulp stem cells (DPSCs) and develop the appropriate differentiation assay systems for skeletal myogenic differentiation of these cells. METHODS: Dental pulps were extracted from mandible sections of C57/BL6 mice, and adherent dental pulp cells were isolated in culture. These cells were cultured in osteogenic or adipogenic induction medium to induce osteogenic and adipogenic differentiation. On the other hand, the skeletal myogenic differentiation potential of these cells was investigated using different conditions, such as serum-free medium, Myod1 overexpression, or 5-Aza-2'-deoxycytidine (5-Aza) treatment for DNA demethylation. Muscle-specific transcriptional factor expression was evaluated by RT-PCR, and myotube formation and myosin heavy chain expression were evaluated by phase-contrast microscopy and immunofluorescence staining, respectively. RESULTS: The adherent dental pulp cells exhibited a proliferative capacity and they showed osteogenic and adipogenic differentiation as seen in previous studies. Although the expression of Myod1 mRNA and myotube formation was not detected in serum-free conditions, the forced expression of Myod1 up-regulated the expression of Myogenin and Pax7 mRNA. However, myotube formation was not confirmed. Interestingly, myosin heavy chain expression and myotube formation were observed following 5-Aza treatment of these cells. CONCLUSIONS: These results demonstrated that mouse DPSCs possess MSC-like differentiation potential. DNA demethylation induced by 5-Aza treatment resulted in the skeletal muscle differentiation in mouse DPSCs, suggesting that DNA demethylation might trigger this differential induction of mouse DPSCs.

Marginal expression of CXCR4 on c-kit(+)Sca-1 (+)Lineage (-) hematopoietic stem/progenitor cells.

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 are the key regulatory molecules of hematopoietic stem cell (HSC) migration and engraftment to the bone marrow (BM) microenvironment. However, the significance of the ligand-receptor complex on HSC in steady-state BM is not clear. There is currently a lack of information as to how CXCR4 is expressed on HSCs. We herein demonstrate that c-kit(+)Sca-1(+)Lineage(-) (KSL) cells freshly isolated from BM expressed very low to undetectable levels of CXCR4. Two hours of incubation at 37 degrees C quickly up-modulated the receptor expression on KSL cells. Protein synthesis was not required for this early stage up-regulation, thus suggesting the emergence of intracellularly pooled receptors to the cell surface. However, protein synthesis was involved at the later stage of up-regulation. The up-regulated CXCR4 was functional, as evidenced by the fact that the incubated KSL cells more efficiently migrated to the SDF-1 gradient in vitro. Therefore, although KSL cells are able to express functional CXCR4, the receptors are only marginally expressed in the steady-state BM microenvironment. These observations therefore indicate the limited role of the SDF-1-CXCR4 axis on HSC functionality in a static BM environment.

Smoking prevalence among dentists in Hyogo, Japan 2003.

We examined smoking prevalence among dentists in Hyogo, Japan, as smoking would influence their smoking cessation interventions to encourage their patients to stop smoking. In 2003, a self-administered questionnaire was mailed to all members of the Hyogo Dental Association (HDA) in Japan. Of the 1,133 members of the HDA, 327 were current smokers (28.9%). Smoking prevalence among HDA members was significantly higher than that among Japan Medical Association (JMA) members in 2004, as previously reported (16.2%) (p<0.01). Although smoking prevalence among HDA members decreased overall in 2003 in comparison with 2000, smoking prevalence among dentists aged 20-39 yr increased. These findings indicate that smoking was more prevalent among dentists in Hyogo Prefecture than among Japanese medical doctors. It is important to promote smoking cessation among Japanese dentists so that dentists will be more likely to encourage their patients to quit smoking.