Yoshiaki Sonoda

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

OBJECTIVEnTissue 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.nnnMETHODSnDental 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThese 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.

CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.

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

OBJECTIVEnWe 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.nnnMATERIALS AND METHODSnThe 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.nnnRESULTSnA 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.nnnCONCLUSIONSnWe 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.

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, TGFβ3, 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. Stem Cells 2015;33:1554–1565

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 G0 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 G0. 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. STEM CELLS 2011;29:1783–1791

In vivo dynamics of human cord blood-derived CD34 − SCID-repopulating cells using intra-bone marrow injection

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. This study investigated the long-term repopulating capacity and redistribution kinetics of human cord blood-derived CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) and compared them with those of CD34+CD38+ and CD34+CD38− SRCs using the intra-bone marrow injection (IBMI) to clarify the characteristics of CD34− SRCs. On the basis of the limiting dilution analyses data, estimated numbers of CD34+CD38+, CD34+CD38−, and CD34− SRCs were transplanted to NOD/SCID mice by IBMI. The human cell repopulation at the site of injection and the other bones were serially investigated. Interestingly, CD34+CD38+, CD34+CD38−, and CD34− SRCs began to migrate to other bones 2 and 5 weeks after the transplantation, respectively. Accordingly, the initiation of migration seemed to differ between the CD34+ and CD34− SRCs. In addition, CD34+CD38+ SRCs only sustained a short-term repopulation. However, both CD34+CD38− and CD34− SRCs had longer-term repopulation capacity. Taken together, these findings showed that CD34− SRCs show different in vivo kinetics, thus suggesting that the identified CD34− SRCs are a distinct class of primitive HSCs in comparison to the CD34+CD38+ and CD34+CD38− SRCs.

Human cord blood-derived primitive CD34-negative hematopoietic stem cells (HSCs) are myeloid-biased long-term repopulating HSCs

Human cord blood-derived primitive CD34-negative hematopoietic stem cells (HSCs) are myeloid-biased long-term repopulating HSCs

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.

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°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.

Human CD34-negative Hematopoietic Stem Cells

Based on the recent development of fluorescence-activated cell sorting (FACS) technology, murine hematopoietic stem cells (HSCs) can be purified at the single cell level. The immunophenotype of murine HSCs is CD34low/−c-kit+Sca-1+Lin− (CD34− KSL) cells. However, the characterization of primitive human HSCs has not been fully elucidated. The biology of human HSCs is a current topic of interest that has important implications for clinical HSC transplantation as well as basic research on HSCs. Recently, human cord blood (CB)-derived CD34− HSCs, a counterpart of murine CD34low/− KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review aims to update the concept of the immunophenotype and functional characteristics of human primitive CD34− HSCs. In addition, the significance of the application of the IBMI technique in clinical CB stem cell transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs, thereby clarifying the human HSC hierarchy, in the near future.