Michiko Ichii

Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development

A strict balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is required in order to maintain homeostasis, as well as to efficiently respond to injury and infections. Numbers and fate decisions made by progenitors derived from HSC must also be carefully regulated to sustain large-scale production of blood cells. The complex Wnt family of molecules generally is thought to be important to these processes, delivering critical signals to HSC and progenitors as they reside in specialized niches. Wnt proteins have also been extensively studied in connection with malignancies and are causatively involved in the development of several types of leukemias. However, studies with experimental animal models have produced contradictory findings regarding the importance of Wnt signals for normal hematopoiesis and lymphopoiesis. Here, we will argue that dose dependency of signaling via particular Wnt pathways accounts for much, if not all of this controversy. We conclude that there seems little doubt that Wnt proteins are required to sustain normal hematopoiesis, but are likely to be presented in carefully controlled gradients in a tissue-specific manner.

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells

Considerable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

Functional diversity of stem and progenitor cells with B-lymphopoietic potential.

Summary:  Technical advances have made it possible to separate hematopoietic tissues such as the bone marrow into ever smaller populations, complicating our understanding of immune system replenishment. Patterns of surface marker expression and transcription profiles as well as results obtained with reporter mice suggest that lymphopoietic cells are not closely synchronized, and there is considerable cell to cell variation. Loss of differentiation options is gradual, and ultimate fate can be established at different stages of lineage progression. For example, individual hematopoietic stem cells can be biased such that some are very poor sources of lymphocytes as contrasted to ones with balanced outputs. Still other hematopoietic stem cells are effective at generating B and T cells but are defective with respect to expansion and difficult to distinguish from early lymphoid progenitors. That diversity carries forward to later events, and similar appearing cells in the immune system can arise from alternate differentiation pathways. In fact, new categories of lymphoid progenitors are still being discovered. Heterogeneity provides adaptability as hematopoiesis can be dramatically altered during infections, influencing numbers and types of cells that are produced.

Soluble Frizzled-Related Protein 1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally simulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker− c-kithigh ScaI+, hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen-inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-α was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized β-catenin and blocked early lymphoid progression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differentiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels.

Successful treatment of refractory subcutaneous panniculitis-like T-cell lymphoma with allogeneic peripheral blood stem cell transplantation from HLA-mismatched sibling donor.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is an uncommon type of peripheral T-cell lymphoma (PTCL) usually presenting with erythematous, subcutaneous nodules and fever [1]. It is considered as a cytotoxic T-cell neoplasm with cyotoxic granules and can be cytochemically identified using granzyme B, perforin or TIA-1 [2]. Some cases of SPTCL might be indolent for months or years, but SPTL frequently has an aggressive course, and is often complicated by fatal hemophagocytic syndrome (HPS) [3]. Among the cases of SPTCL, resistance to anthracyclin-based therapy generally means a poor prognosis [4]. The use of high-dose therapy (HDT) with autologous stem cell transplantation (SCT) in patients with SPTCL who are refractory to anthracyclins has yielded good results [4]. However, a cure can only be expected if the disease is at least sensitive to HDT [5]. Allogeneic SCT, however, might yield a cure even in chemo-refractory lymphoma patients via a graft-vs.-lymphoma (GVL) effect [6]. We report a case of successful use of an allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an HLA-mismatched sibling donor in a patient with SPTCL who was refractory to conventional chemotherapy. In August 2000, a 17-year-old woman presented to our hospital because of an intermittent fever and a painful swelling of her hip and right thigh. A biopsy specimen of cutaneous tissue from her right hip was diagnosed as cytophagic histiocytic panniculitis. She was treated with oral prednisolone, which resolved the skin lesions. Three months later, she once again presented with persistent high fever and multiple subcutaneous indurations. Oral prednisolone and etoposide were administered, but only a temporary effect on the skin lesions and fever was seen. A second incisional biopsy of the subcutaneous tissue of her right thigh was performed on 19 March 2001. The biopsy specimen showed massive neoplastic lymphoid infiltration of the subcutaneous fat compatible with a diagnosis of SPTCL (Figure 1A). Immunohistochemically, the neoplastic cells were positive for CD3 and CD45RO (UCHL-1), positive for granzyme B, partially positive for CD4 and CD56 and negative for CD20. A test for Epstein – Barr virus using EBER-1 in situ hybridization was negative. A full blood count revealed mild pancytopenia (white blood cell of 2.4610/l, hemoglobin of 111 g/l and platelets of 74610/l). Her serum lactate dehydrogenase level was 1304 IU/l (normal range 150 – 460 IU/l). Bone marrow aspiration revealed marked hemophagocytosis by activated macrophages without involvement of lymphoma cells (Figure 1B). Two cycles of CHOP-E (cyclophosphamide, adriamycin, vincristine, prednisolone and etoposide) resolved the skin lesions, fever, and HPS. However, 2 weeks after the second course of CHOP-E chemotherapy, the skin lesion and fever recurred. Several treatments, such as high-dose etoposide (500 mg/m on days 1 – 3), L-asparaginase (6000 U/m on days 1 – 7), L-asparaginase (6000 U/m on day 2) plus cytarabine (Ara-C; 3 g/m every 12 h on days 1 – 2), and high-dose Ara-C (1.5 g/m every 12 h on days

Identification of functional domains and novel binding partners of STIM proteins

With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca2+ depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled‐coil domains was massively expressed on the cell surface while mutants with the coiled‐coil domains localized in ER. In addition, STIM1 mutants with the coiled‐coil domains showed a longer half‐life of proteins than those without them. These results are likely to indicate that the coiled‐coil domains of STIM1 are essential for its ER‐retention and its stability. Furthermore, we tried to comprehensively identify STIM1‐associated molecules with mass spectrometry analysis of co‐immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein‐transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca2+ control as well as a useful strategy to develop therapeutic drugs for intracellular Ca2+‐related diseases including inflammation and allergy. J. Cell. Biochem. 112: 147–156, 2011.

Regulation of human B lymphopoiesis by the transforming growth factor-β superfamily in a newly established coculture system using human mesenchymal stem cells as a supportive microenvironment

OBJECTIVES To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology. MATERIALS AND METHODS We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development. RESULTS Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells. CONCLUSIONS hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.

The density of CD10 corresponds to commitment and progression in the human B lymphoid lineage.

Background Requirements for human B lymphopoiesis are still poorly understood, and that has hampered investigation of differentiation events. For example, there are few cell surface antigens that can be used as milestones of lineage progression. The CD10 ectoenzyme is one such marker and has been used to define CLP, but we found substantial tissue specific variations in CD10 levels, and there was no information about how that corresponded to differentiation options. Methodology/Principal Findings The aim of the present study was to use recently developed culture methods to assess the nature and differentiation potential of progenitors sorted according to CD10 density from umbilical cord blood (CB), adult bone marrow (BM) or G-CSF mobilized peripheral blood (PB). Many CD34+ cells in BM express high levels of CD10, while low or low/negative CD10 densities were found on CD34+ cells in CB or G-CSF mobilized PB, respectively. The relative abundance of CD10Lo versus CD10Hi cells only accounts for some CB versus BM differences. Almost all of the CD34+ CD10Hi cells expressed CD19 and lymphocyte transcription factors and corresponded to loss of myeloid potential. A high degree of immunoglobulin DH-JH gene rearrangements was characteristic only of the CD10Hi subset. In contrast, the CD34+ CD10Lo progenitors efficiently produced plasmacytoid and conventional dendritic cells as well as myeloid cells. These findings suggest a positive correlation between CD10 density and degree of differentiation. Although freshly isolated CD34+ CD10Hi cells were in cycle, those from CB or BM expanded poorly in culture, suggesting regulators of populations remain to be discovered. Conclusions/Significance Steps in human B lymphopoiesis have not been sufficiently studied, and we now show that increased CD10 expression corresponds to differentiation potential and stage. CD34+ CD10Hi progenitors are obviously in the B lineage but may have progressed beyond the point where they can be expanded in culture.

Identification of amino-terminal region of adiponectin as a physiologically functional domain

Adiponectin is an abundant adipose‐specific protein, which acts as an anti‐diabetic, anti‐atherogenic, and anti‐inflammatory adipokine. Although recent advances in the field of adiponectin have been made by the identification of adiponectin receptors and by the understanding about relationship between its multimerization and functions, detailed molecular background remains unclear. Our established anti‐human adiponectin antibodies, ANOC 9103 and ANOC 9104, blocked some adiponectin functions such as the growth inhibition of B‐lymphocytes on stromal cells and the inhibition of acetylated LDL uptake in macrophages, suggesting that they may recognize important functional regions of adiponectin. As a result of epitope mapping based on the ability to bind to the deleted adiponectin mutants, we identified that these antibodies recognize amino‐terminal region of adiponectin before the beginning of the collagen‐like domain. Notably, a peptide fragment (DQETTTQGPGVLLPLPKGACTGWMA) corresponding to amino acid residues 17–41 of human adiponectin could bind to restricted types of cells and block adiponectin‐induced cyclooxygenase‐2 gene expression and prostaglandin E2 production in MS‐5 stromal cells. Moreover, the deletion of its amino‐terminal region reduced the abilities to inhibit not only collagen‐induced platelet aggregation but also diet‐induced hepatic steatosis. These data indicate that amino‐terminal region of adiponectin is a physiologically functional domain and that a novel receptor, which recognizes amino‐terminal region of adiponectin, may exist on some types of cells. Further investigations will contribute to the understanding of molecular mechanisms about adiponectin functions as well as to the designing of novel strategies for the treatment of patients with insulin‐resistance, vascular dysfunction, and chronic inflammation. J. Cell. Biochem. 98: 194–207, 2006.

Stromal cell-free conditions favorable for human B lymphopoiesis in culture.

Progress has been slow in defining molecular requirements for human B lymphopoiesis in part because of differences from experimental animals and also because of the lack of culture conditions that efficiently support the process. We recently found that human CD10+ lymphocytes were produced when CD34+ hematopoietic stem and progenitor cells were cultured in contact with human mesenchymal stem cells (hMSC). Further investigation revealed that it occurred even when progenitors were separated from hMSC by membrane filters. Experiments with neutralizing antibodies suggested that important heat labile factors produced by hMSC are unlikely to be IL-7, TSLP, CXCL12 or hemokinin-1. Further manipulation of culture conditions revealed that optimal lymphopoiesis required careful selection of fetal calf serum lots, maintenance of high cell densities, as well as recombinant cytokines (SCF, FL and G-CSF). G-CSF was particularly important when adult bone marrow rather than umbilical cord blood derived CD34+ cells were used to initiate the cultures. These improved methods should facilitate identification of molecules that can be used to speed regeneration of the humoral immune system following chemotherapy and might suggest ways to inhibit growth of B lineage malignancies.