Keisuke Sumide

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.

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

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.

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

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

Summary 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 β-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 β2 (TGF-β2) 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-β antibodies. These results suggest that, in part, old DPMSCs accelerate cellular senescence through TGF-β signaling.

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-2Rγcnull (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.

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.Single CD34 negative haematopoietic stem cells (HSCs) can fully reconstitute lympho-myeloid hematopoiesis in mice. Here, using single cell transplantation and gene expression analyses, the authors show that CD34 negative HSCs lie at the apex of haematopoiesis in human cord blood and that they can give rise to erythroid megakaryocytic progenitors.