Seiji Sakano

Bone marrow-derived stem cells initiate pancreatic regeneration.

We show that transplantation of adult bone marrow–derived cells expressing c-kit reduces hyperglycemia in mice with streptozotocin-induced pancreatic damage. Although quantitative analysis of the pancreas revealed a low frequency of donor insulin-positive cells, these cells were not present at the onset of blood glucose reduction. Instead, the majority of transplanted cells were localized to ductal and islet structures, and their presence was accompanied by a proliferation of recipient pancreatic cells that resulted in insulin production. The capacity of transplanted bone marrow–derived stem cells to initiate endogenous pancreatic tissue regeneration represents a previously unrecognized means by which these cells can contribute to the restoration of organ function.

Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes

IgM is the first antibody to be produced in a humoral immune response and plays an important role in the primary stages of immunity. Here we describe a mouse Fc receptor, designated Fcα/μR, and its human homolog, that bind both IgM and IgA with intermediate or high affinity. Fcα/μR is constitutively expressed on the majority of B lymphocytes and macrophages. Cross-linking Fcα/μR expressed on a pro-B cell line Ba/F3 transfectant with soluble IgM or IgM-coated microparticles induced internalization of the receptor. Fcα/μR also mediated primary B lymphocyte endocytosis of IgM-coated Staphylococcus aureus. Thus, Fcα/μR is involved in the primary stages of the immune response to microbes.

Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system

In Drosophila embryogenesis, the slit gene has been shown to play a critical role in CNS midline formation. However, no slit homologues have been reported in vertebrates. Here, we have identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3, and rat Slit-1). Each Slit gene encodes a putative secreted protein, which contains conserved protein-protein interaction domains including leucine-rich repeats (LRR) and epidermal growth factor (EGF)-like motifs, like that of the Drosophila protein. Northern blot analysis revealed that the human Slit-1, -2, and -3 mRNAs are exclusively expressed in the brain, spinal cord, and thyroid, respectively. In situ hybridization studies indicated that the rat Slit-1 mRNA is specifically expressed in the neurons of fetal and adult forebrains. Our data suggest that Slit genes form an evolutionary conserved group in vertebrates and invertebrates, and that the mammalian Slit proteins may participate in the formation and maintenance of the nervous and endocrine systems by protein-protein interactions.

The Association of Notch2 and NF-κB Accelerates RANKL-Induced Osteoclastogenesis

ABSTRACT Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis.

A novel Fc receptor for IgA and IgM is expressed on both hematopoietic and non‐hematopoietic tissues

By contrast to well‐defined Fcγ and Fcϵ receptors, the structural and functional characteristics of Fcμ receptor are unclear. We have recently described a novel mouse Fc receptor, designated Fcα/μ receptor, and its human homologue, which bind both IgM and IgA. Here we show that the Fcα/μ receptor is expressed on mature, but not immature, B lymphocytes and acquires the ability to bind IgM and IgA antibodies after stimulation of B lymphocytes. Moreover, stimulation with phorbol 12‐myristate 13‐acetate increased endocytosis of IgM‐coated microparticles mediated by the Fcα/μ receptor expressed on pro‐B cell line Ba/F3 cells. We also show that the Fcα/μ receptor is expressed in secondary lymphoid organs, such as lymph node and appendix, kidney and intestine, suggesting an important role of the receptor for immunity in these organs.

Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially regulate activation of peripheral T helper cells.

The Notch pathway is involved in cell differentiation processes in various organs and at several developmental stages. The importance of Notch for early T lymphocyte development is well established. Recently, Notch has been implicated in directing naive T helper cell differentiation towards the Th1, Th2 or regulatory T cell lineages. However, the molecular events underlying these processes are poorly understood.We show that the Notch ligands Delta‐like1, Delta‐like4 and Jagged1 differentially affect early T cell activation and proliferation following T cell receptor cross‐linking. Delta‐like1 and Jagged1 induce a dose‐dependent inhibition of early activation markers CD69 and CD25, as well as inhibition of proliferation after anti‐CD3 stimulation of purified CD4+ T cells. Similarly, the rapid activation of transcription factors NF‐AT, AP‐1 and NF‐κB is suppressed. In contrast, triggering of Notch by Delta‐like4 enhances T cell activation and proliferation. The observed effects are dependent on simultaneous cross‐linking of TCR and Notch but independent of γ‐secretase‐mediated cleavage of Notch. These data suggest direct interference between Notch and early TCR signal transduction events, independent of the classical Notch pathway via release of the Notch intracellular domain. A Notch‐mediated alteration of TCR signaling strength may contribute to the recently described modulation of naïve T cell differentiation by Notch ligands.

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1‐Fc Chimeric Protein

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long‐term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133‐sorted cells using a soluble form of Delta1. After a 3‐week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt‐3 ligand, interleukin (IL)‐3, and IL‐6/soluble IL‐6 receptor chimeric protein (FP6) in a serum‐ and stromal cell‐free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self‐renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/γcnull mice, which showed myeloid, B, T, and natural killer cells as well as CD133+CD34+ cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133‐sorted cells contained approximately 4.5 times more SRCs than the CD34‐sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

Expression and function of the Delta-1/Notch-2/Hes-1 pathway during experimental acute kidney injury

The Notch signaling pathway consists of several receptors and their ligands Delta and Jagged and is important for embryogenesis, cellular differentiation and proliferation. Activation of Notch receptors causes their cleavage yielding cytoplastic domains that translocate into the nucleus to induce target proteins such as the basic-loop-helix proteins Hes and Hey. Here we sought to clarify the significance of the Notch signaling pathway in acute kidney injury using a rat ischemia-reperfusion injury model and cultured NRK-52E cells. Analysis of the whole kidney after injury showed increased expression of Delta-1 and Hes-1 mRNA and protein along with processed Notch-2. Confocal microscopy, using specific antibodies, showed that Delta-1, cleaved Notch-2 and Hes-1 colocalized in the same segments of the injured renal proximal tubules. Recombinant Delta-1 significantly stimulated NRK-52E cell proliferation. Our study suggests that the Delta-1/Notch-2/Hes-1 signaling pathway may regulate the regeneration and proliferation of renal tubules during acute kidney injury.

A role of EphB4 receptor and its ligand, ephrin-B2, in erythropoiesis.

Erythropoiesis is regulated not only by erythropoietin but also by microenvironments which are composed of transmembrane molecules. We have previously shown that a receptor tyrosine kinase EphB4 is predominantly expressed on human erythroid progenitors in bone marrow. EphB4 is expressed in approximately 45% of hematopoietic progenitor cells, which are CD34-positive and c-Kit-positive in human umbilical cord blood (hUCB). The transmembrane ligand for EphB4 or ephrin-B2 is expressed on bone marrow stromal cells and arterial endothelial cells. When such EphB4-positive hematopoietic progenitor cells were co-cultured with stromal cells which express ephrin-B2, they were immediately detached from stromal cells and differentiated to mature erythroid cells. At that time, expression of EphB4 immediately down-regulated. In contrast, on ephrin-B2 non-expressing stromal cells, they remained EphB4-positive cells and the generated number of mature erythroid cells was less than that on ephrin-B2 expressing stromal cells. Additionally, ephrin-B2 expression on endothelial cells up-regulated under hypoxic condition. Taken together, we propose that one of the molecular cues that regulate erythropoiesis is ephrin-B2 on stromal cells.

Differential response of primitive human CD34- and CD34+ hematopoietic cells to the Notch ligand Jagged-1

Recent reports indicate that activation of the Notch signaling pathway delays the differentiation of hematopoietic progenitors, suggesting that Notch may be used to develop novel ex vivo culture conditions for the expansion of primitive cells to be used in clinical transplantation. Here, we compare Notch expression and the effects of Jagged-1 treatment on highly purified subfractions of primitive CD34+ and CD34− human hematopoietic cells. Unlike response of cultured CD34+ cells, Jagged-1 treatment did not enhance the proliferation of CD34− cells, or promote differentiation of CD34− cells into CD34+ cells. While CD34+ and AC133−CD34− cells were shown to express all known forms of Notch receptors, Notch-3 and Notch-4 were not detected in AC133+CD34− cells. Similarly, CD34+ progeny of differentiated CD34− cells did not upregulate Notch-3 or Notch-4 upon differentiation, although transcripts for these genes were expressed in CD34+ arising from CD34+ CD38− parents, suggesting that the Notch receptor expression is tightly and differentially controlled. Fringe, known to inhibit Notch signaling in response to specific Notch ligands, was expressed in parent CD34− and CD34+ cells as well as their CD34+ progeny. We suggest that the inability of primitive CD34− cells to positively respond to Jagged-1 may be due in part to the absence of Notch-3 and Notch-4. Taken together, our study illustrates functional distinctiveness of the primitive CD34− subsets to CD34+ counterparts in relation to Jagged-1 response, and represents the first demonstration of a molecular difference among de novo isolated CD34+ compared to in vitro generated CD34+ cells arising from primitive CD34− or CD34+ parents.