Terumasa Umemoto

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2.

Many types of organ‐specific stem cells have been recently shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. Because stem cells from corneal epithelium reside in the basal layer of the limbal epithelium, the purpose of this study was to examine whether the limbal epithelium contains SP cells. The ATP‐binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in cells from several diverse sources. Here we show data from fluorescence‐activated cell sorting and real‐time quantitative RT‐PCR analysis showing that harvested limbal epithelial cells contain SP cells expressing ABCG2. Immunofluorescence revealed that a portion of limbal epithelial basal cells expressed ABCG2. Data indicate that ABCG2 positive limbal epithelial cells are putative corneal epithelial stem cells.

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state.

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side‐population (SP) cells with the ability to efflux the DNA‐binding dye Hoechst 33342 have stem cell–like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell–like properties. SP cells, separated by fluorescence‐activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi‐1, and nestin but no expression of markers for differentiated CE cells compared with non‐SP cells. Cell‐cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.

Expression of Integrin β3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin β3 (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin β3 is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage− (Lin−)/integrin β3high (β3high) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit+/Sca-1+/Lin− SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin β3 subunit that is present in SP cells having the properties of HSCs, is associated with integrin αv (CD51). Therefore, our results demonstrate that high expression of integrin β3 is correlated to the properties of quiescent HSCs and suggest that the integrin β3 subunit is available as a common surface marker of tissue-specific stem cells.

Unipotent Megakaryopoietic Pathway Bridging Hematopoietic Stem Cells and Mature Megakaryocytes

Recent identification of platelet/megakaryocyte‐biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41+ cells in the lineage Sca1+cKit+ (LSK) fraction (subCD41+LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41+LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3+CD41−LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41+LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO‐mediated phosphorylation. On increased demand of platelet production after 5‐FU treatment, a part of CD41+LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41+CD42b+LSK are straightforward progenies of megakaryocytes/platelet‐biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196–2207

Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin αvβ3

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the ‘niche’. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3(+) cells were significantly enriched among CD61(+)rLE-SP cells as compared to CD61(-)rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.

Computational promoter modeling identifies the modes of transcriptional regulation in hematopoietic stem cells.

Extrinsic and intrinsic regulators are responsible for the tight control of hematopoietic stem cells (HSCs), which differentiate into all blood cell lineages. To understand the fundamental basis of HSC biology, we focused on differentially expressed genes (DEGs) in long-term and short-term HSCs, which are closely related in terms of cell development but substantially differ in their stem cell capacity. To analyze the transcriptional regulation of the DEGs identified in the novel transcriptome profiles obtained by our RNA-seq analysis, we developed a computational method to model the linear relationship between gene expression and the features of putative regulatory elements. The transcriptional regulation modes characterized here suggest the importance of transcription factors (TFs) that are expressed at steady state or at low levels. Remarkably, we found that 24 differentially expressed TFs targeting 21 putative TF-binding sites contributed significantly to transcriptional regulation. These TFs tended to be modulated by other nondifferentially expressed TFs, suggesting that HSCs can achieve flexible and rapid responses via the control of nondifferentially expressed TFs through a highly complex regulatory network. Our novel transcriptome profiles and new method are powerful tools for studying the mechanistic basis of cell fate decisions.

Stripe-Patterned Thermo-responsive Cell Culture Dish for Cell Separation without Cell Labeling

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 μm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry.

Promotion of mouse ameloblast proliferation by Lgr5 mediated integrin signaling

Rodent incisors grow throughout the animals lives, and the tooth‐forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre‐ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61−/−), also known as integrin β3, were significantly shorter than those of the wild‐type mice at 8‐week‐old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre‐ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2‐week‐old CD61−/− mice. The proliferation of pre‐ameloblasts was reduced in CD61−/− ameloblasts. The siRNA‐mediated suppression of CD61 (siCD61) reduced the proliferation of pre‐ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre‐ameloblastic cells through Lgr5. J. Cell. Biochem. 114: 2138–2147, 2013.