Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation.

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

The characterization of distinct populations of murine skeletal cells that have different roles in B lymphopoiesis.

Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin-CD31-Sca-1-CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⍺ and ß (PDGFR⍺ and PDGFRß). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.

[Development of low-cost ex vivo hematopoietic stem cell expansion].

Hematopoietic stem cells (HSC) have self-renewal as well as multilineage differentiation capacity and maintain hematopoiesis throughout life. HSC transplantation (HSCT) is performed as a curative therapy for hematopoietic malignancies and nonmalignant hematopoietic disorders. Furthermore, bone marrow, mobilized peripheral blood, and cord blood are available sources for HSCT. HLA compatibility is the most critical factor for a successful HSCT. The HSC number in a graft is also invaluable for engraftment. Moreover, it is challenging to obtain an abundant number of HSC for patients with obesity, particularly, in cord blood. HSC ex vivo expansion is an appropriate solution for this problem. Extrinsic factors to expand and maintain HSCs, such as cytokines are identified from analysis of HSCs and their niche. Thus, HSC ex vivo expansion is improved by adding them in culture medium; however, it is still difficult for therapeutic applications. Recently, several small molecular compounds have been reported to facilitate ex vivo expansion of HSC. Clinical trials that transplant ex vivo expanded cord blood have been already expanded, and some trials demonstrate reduction of time to hematopoietic recovery. Thus, we anticipate that ex vivo expanded cord blood transplantation will be applied widely in the future.

In Vivo Rescue of the Hematopoietic Niche By Pluripotent Stem Cell Complementation of Defective Osteoblast Compartments.

Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSC), are capable of differentiating into osteoblasts. To determine the capacity of stem cells needed to rescue aberrant skeletal development and bone marrow hematopoiesis in vivo, we used a skeletal complementation model. Mice deficient in Runx2, a master transcription factor for osteoblastogenesis, fail to form a mineralized skeleton and bone marrow. Wild-type (WT) green fluorescent protein (GFP)+ ESCs and yellow fluorescent protein (YFP)+ iPSCs were introduced into Runx2-null blastocyst-stage embryos. We assessed GFP/YFP+ cell contribution by whole-mount fluorescence and histological analysis and found that the proportion of PSCs in the resulting chimeric embryos is directly correlated with the degree of mineralization in the skull. Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis. We validated this finding in a separate model with diphtheria toxin A-mediated ablation of hypertrophic chondrocytes and osteoblasts. Remarkably, chimeric embryos harboring as little as 37.5% WT PSCs revealed grossly normal skeletal morphology, suggesting a near-complete rescue of skeletogenesis. In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo. Stem Cells 2017;35:2150-2159.

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-Gsα(OsxKO) mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 µg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-Gsα(OsxKO) mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-Gsα(OsxKO) mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.

Purging myeloma cell contaminants and simultaneous expansion of peripheral blood-mobilized stem cells.

Human hematopoietic stem cells (HSCs) are widely used as a cellular source for hematopoietic stem cell transplantation (HSCT) in the clinical treatment of hematological malignancies. After transplantation therapy, delays in hematopoietic recovery due to insufficient donor-derived HSCs can lead to increased risks of life-threatening infections and bleeding. Our previous studies developed an efficient ex vivo expansion culture medium (3a medium) for umbilical cord blood-derived HSCs (CBSCs), offering a potential solution to this problem. Nevertheless, the broader applicability of our culture method to alternative cell sources and, of greater significance, its efficacy in eliminating potentially disease-associated contaminated tumor cells, especially in autologous transplantation, raise critical clinical questions. In this study, we modified the 3a medium by incorporating UM729 to replace UM171, adding FMS-like tyrosine kinase 3 (Flt3) ligand, and adjusting the concentrations of butyzamide, 740Y-P, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG, Soluplus) to create the modified-3a medium. This sophistication allowed the efficient expansion of not only CBSCs but also peripheral blood-mobilized HSCs (PBSCs). Additionally, we successfully removed contaminated myeloma cells by adding bortezomib and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at appropriate concentrations, although we maintained HSCs through the addition of lenalidomide. Our research findings present the potential for widespread clinical application of the modified-3a medium and suggest a safe ex vivo culture technique for expanding human HSCs within peripheral blood-derived donor grafts used for autologous HSCT.

Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling.

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation in vivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the in vivo effects of MSC transplantation.

Rejuvenated iPSC-derived GD2-directed CART Cells Harbor Robust Cytotoxicity Against Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is exceptionally aggressive, with limited treatment options. Disialoganglioside (GD2) is highly expressed on SCLC and is considered a good target for chimeric antigen receptor (CAR) T cells (CART). Although GD2-directed CARTs (GD2-CART) exhibit cytotoxicity against various GD2-expressing tumors, they lack significant cytotoxicity against SCLC. To enhance cytotoxicity of GD2-CARTs against SCLC, we introduced GD2-CAR into induced pluripotent stem cells (iPSC)-derived rejuvenated cytotoxic T lymphocytes (GD2-CARrejT). GD2-CARrejTs acted much more strongly against SCLC cells than did GD2-CARTs both in vitro and in vivo. Single-cell RNA sequencing elucidated that levels of expression of TIGIT were significantly lower and levels of expression of genes associated with cytotoxicity were significantly higher in GD2-CARrejTs than those in GD2-CARTs. Dual blockade of TIGIT and programmed death-1 (PD-1) increased the cytotoxicity of GD2-CARTs to some extent, suggesting that low TIGIT and PD-1 expression by GD2-CARrejTs is a major factor required for robust cytotoxicity against SCLC. Not only for robust cytotoxicity but also for availability as "off-the-shelf" T-cell therapy, iPSC-derived GD2-CARrejTs are a promising novel treatment for SCLC. SIGNIFICANCE: This research introduces iPSC-derived rejuvenated GD2-CARTs (GD2-CARrejT) as a novel approach to combat SCLC. Compared with conventional GD2-CARTs, GD2-CARrejTs with reduced TIGIT and PD-1 expression demonstrate robust cytotoxicity against SCLC and would be a promising therapy for SCLC.

Parathyroid hormone 1 receptor signaling mediates breast cancer metastasis to bone in mice.

Bone metastases are a common complication of breast cancer. We have demonstrated that intermittent administration of parathyroid hormone (PTH[1-34]) reduces the incidence of bone metastases in murine models of breast cancer by acting on osteoblasts to alter the bone microenvironment. Here, we examined the role of signaling mediated by PTH 1 receptor (PTH1R) in both osteoblasts and breast cancer cells in influencing bone metastases. In mice with impaired PTH1R signaling in osteoblasts, intermittent PTH did not reduce bone metastasis. Intermittent PTH also did not reduce bone metastasis when expression of PTH1R was knocked down in 4T1 murine breast cancer cells by shRNA. In 4T1 breast cancer cells, PTH decreased expression of PTH-related protein (PTHrP), implicated in the vicious cycle of bone metastases. Knockdown of PTHrP in 4T1 cells significantly reduced migration toward MC3T3-E1 osteoblasts, and migration was further inhibited by treatment with intermittent PTH. Conversely, overexpression of PTHrP in 4T1 cells increased migration toward MC3T3-E1 osteoblasts, and this was not inhibited by PTH. In conclusion, PTH1R expression is crucial in both osteoblasts and breast cancer cells for PTH to reduce bone metastases, and in breast cancer cells, this may be mediated in part by suppression of PTHrP.

Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Loss of Parathyroid Hormone Receptor Signaling in Osteoprogenitors Is Associated With Accumulation of Multiple Hematopoietic Lineages in the Bone Marrow.

Osteoblasts and their progenitors play an important role in the support of hematopoiesis within the bone marrow (BM) microenvironment. We have previously reported that parathyroid hormone receptor (PTH1R) signaling in osteoprogenitors is required for normal B cell precursor differentiation, and for trafficking of maturing B cells out of the BM. Cells of the osteoblast lineage have been implicated in the regulation of several other hematopoietic cell populations, but the effects of PTH1R signaling in osteoprogenitors on other maturing hematopoietic populations have not been investigated. Here we report that numbers of maturing myeloid, T cell, and erythroid populations were increased in the BM of mice lacking PTH1R in Osx-expressing osteoprogenitors (PTH1R-OsxKO mice; knockout [KO]). This increase in maturing hematopoietic populations was not associated with an increase in progenitor populations or proliferation. The spleens of PTH1R-OsxKO mice were small with decreased numbers of all hematopoietic populations, suggesting that trafficking of mature hematopoietic populations between BM and spleen is impaired in the absence of PTH1R in osteoprogenitors. RNA sequencing (RNAseq) of osteoprogenitors and their descendants in bone and BM revealed increased expression of vascular cell adhesion protein 1 (VCAM-1) and C-X-C motif chemokine ligand 12 (CXCL12), factors that are involved in trafficking of several hematopoietic populations. © 2022 American Society for Bone and Mineral Research (ASBMR).

[Exacerbation of autoimmune neutropenia to agranulocytosis in association with severe autoimmune thrombocytopenia and hemolytic anemia in a patient with Sjögren's syndrome].

A 73-year-old woman with Sjögren's syndrome and autoimmune neutropenia (AIN) associated with large granular lymphocytosis of the polyclonal T cell type, demonstrated autoimmune thrombocytopenia (AIT) at diagnosis of sigmoid colon cancer. Ten months later, both AIN and AIT had exacerbated to agranulocytosis and severe thrombocytopenia below 10×10(9)/L, respectively. There were no dysplastic features of bone marrow hematopoietic cells. Furthermore, an in vitro assay of hematopoietic progenitors showed normal granuloid and erythroid colony formation. Although we serially treated her with prednisolone (oral), filgrastim, intravenous high-dose immunoglobulin infusion, cyclophosphamide (oral), danazol, cyclosporine A (oral), and rituximab, number of neutrophils and platelets elevated only temporarily. During the course of agranulocytosis and severe thrombocytopenia, the patient also developed autoimmune hemolytic anemia (AIHA). She died of pneumonia 5 months after the onset of agranulocytosis. This case is very unique and novel in terms of autoimmune phenomena simultaneously directed to granulocytes, platelets, and red blood cells under the background of Sjögren's syndrome.

Successful living donor liver transplantation for severe hepatic GVHD histologically resembling autoimmune hepatitis after bone marrow transplantation from the same sibling donor.

A 30-year-old woman developed severe liver dysfunction 1 year after bone marrow transplantation (BMT) from an HLA-identical sibling donor for B lymphoblastic leukemia (B-ALL) during the tapering of cyclosporin A. The histologic picture resembled autoimmune hepatitis (AIH), although neither autoantibody nor hypergammaglobulinemia was detected. She entered hepatic coma, and underwent living donor liver transplantation from the same donor on day 421 after BMT. She is well 18 months after the procedure, showing normal liver function and hematopoiesis. AIH-like hepatic graft-versus-host disease (GVHD) has not been documented. This patient is the second case of living donor liver transplantation for hepatic GVHD from the same donor.

Hepatosplenic alphabeta T cell lymphoma.

A 32-year-old male with chronic hepatitis B was admitted to a hospital with cellulitis in the right leg in September 2006. Pancytopenia, hepatosplenomegaly, and systemic superficial lymph node swelling were noted, and he was referred to our hospital. He developed fever and liver dysfunction in June 2007 and underwent a splenectomy. His pancytopenia subsequently improved. A pathologic diagnosis of hepatosplenic alphabeta T cell lymphoma was made by examining spleen tissue and biopsy specimens of the liver and mesenteric lymph node. He had stage IVB disease because neoplastic T cells were noted in the bone marrow. The response of the lymphoma to conventional chemotherapy including the CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone) and DeVIC (dexamethasone, etoposide, ifoshamide, carboplatin) regimens was poor and transient. A partial remission was obtained with an ESHAP (etoposide, cisplatin, cytarabine, methylprednisolone) regimen. Therefore, we planned a bone marrow transplantation (BMT) from an HLA-haploidentical sibling donor. He was moved to the Department of Hematology, Hyogo Medical College, to receive this BMT as part of a clinical trial. During the conditioning procedure for the transplantation, however, he died of septicemia. Since hepatosplenic alphabeta T cell lymphoma is very rare with only 23 reported cases to date, herein we report this case and discuss the therapeutic strategy.

[Graft-versus-leukemia effect by lymphocytes from the first donor after second cord blood transplantation in a patient with T-lymphoblastic lymphoma].

A 19-year-old girl with T-lymphoblastic lymphoma (T-LBL) was referred to our hospital because of refractory disease. After complete remission was achieved by the JALSG ALL-97 protocol, she received a bone marrow transplantation (BMT) from an unrelated, HLA-matched donor with myeloablative conditioning. Four months after BMT, T-LBL relapsed and donor lymphocyte infusion was ineffective. After partial remission was achieved with l-asparaginase therapy, she received 2 antigen-mismatched cord blood transplantation with non-myeloablative conditioning; however, sustained engraftment of cord blood stem cells has failed. This was associated with the reappearance of the blood cells from the first donor and the disappearance of leukemic cells from both the peripheral blood and bone marrow. Computed tomography showed no enlarged lymph nodes. The patient and the cord blood donor shared two minor histocompatibility antigens (mHAgs), while these mHAgs were not detected in the blood cells of the first donor. TCR analysis disclosed expanded oligoclonal Vbeta2T cells in the peripheral blood at relapse, and these cells secreted IFN-gamma in response to stimulation by the patient's leukemic cells. Moreover, these cells exhibited cytotoxicity against both leukemic cells and cord blood mononuclear cells. These results strongly suggest that Vbeta2T cells, derived from the first donor, may have been cytotoxic lymphocytes against both leukemic cells and cord blood stem cells.

[Therapeutic efficacy of cyclosporin A for refractory angioimmunoblastic T cell lymphoma].

The prognosis of angioimmunoblastic T cell lymphoma (AITL) is poor because of chemotherapy-resistance and the short duration of remission. In recent years, cyclosporin A (CyA) has been employed as a alternative treatment for AITL in some patients with the rationale that CyA inhibits the activity and proliferation of neoplastic T cells. We herein report 4 chemotherapy-resistant AITL patients who were treated with oral CyA. The dosage of CyA was individually determined in each patient in order to achieve a blood CyA concentration of around 200 ng/ml at the trough level. A patient in whom AITL had relapsed 3 months after high dose chemotherapy with autologous hematopoietic stem cell transplantation (HSCT) achieved a sustained complete remission (CR) with CyA and underwent allogeneic HSCT. In 2 patients who had failed to respond to conventional chemotherapies, the circulating lymphoma cells rapidly disappeared after the initiation of CyA, and one of these patients demonstrated a durable CR. The other patient showed a good response to CyA, but the agent was discontinued because of infection. The remaining one patient with advanced AITL did not respond to CyA and died of disease progression. To our knowledge, the efficacy of CyA for chemotherapy-resistant AITL, even in a leukemic state, has not previously been reported.

Successful treatment with autologous peripheral blood stem cell transplantation for acquired immunodeficiency syndrome (AIDS)-related malignant lymphoma.

A 62-year-old man was diagnosed with human immunodeficiency virus (HIV) infection while suffering from recurrent herpes zoster infection. Laboratory examination revealed CD4(+) lymphocyte count 16 cells/mul and HIV loading 150,000 copies/ml at presentation. In addition, he had multiple lymph node swelling. Histologic diagnosis of a biopsied lymph node was diffuse, large, B cell-type malignant lymphoma. The karyotype of the lymphoma cells was t(8;14)(q24;q32), which was confirmed by G-banding and fluorescent in situ hybridization. Positron emission tomography (PET)-combined CT scanning revealed systemic extranodal tumors involving the gastrointestinal tract, pancreas, and bone marrow. The clinical stage of the lymphoma was IVB and the international prognosis index was categorized as high. Complete remission (CR) of the lymphoma was obtained after 2 courses of CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone) chemotherapy and 4 subsequent courses of rituximab-combined CHOP (R-CHOP). Highly active antiretroviral therapy (HAART) was started at the initiation of CHOP. Because of the poor prognosis of AIDS-related lymphoma, he received autologous peripheral blood stem cell transplantation with the MEAM protocol (ranimustine, etoposide, cytarabine, melphalan) as a conditioning procedure without a severe infectious episode. He remains in CR 24 months after the transplantation.

[Angioimmunoblastic T-cell lymphoma complicated by recurrent drug-induced agranulocytosis].

A 73-year-old man was hospitalized with fever, erythema, generalized superficial lymphadenopathy and marked neutropenia in July 2007. Hematologic examination demonstrated a white blood cell count of 1,400/microl with 0% neutrophils, and 18% abnormal lymphocytes. A bone marrow aspirate showed marked myeloid hypoplasia. A diagnosis of drug-induced agranulocytosis was made. Although neutrophil counts immediately returned to normal levels in response to filgrastim, fever, skin rash and systemic lymphadenopathy were all persistent. He also developed autoimmune hemolytic anemia and a second episode of agranulocytosis. The causative agent of the both episodes of agranulocytosis appeared to be acetaminophen. The histologic picture of a biopsied lymph node showed diffuse infiltration of polymorphous lymphoid cells with clear cytoplasm and proliferation of arborizing capillary vessels. Based on the histologic findings, PCR, and immunohistologic analyses, he was diagnosed with angioimmunoblastic T cell lymphoma (AILT) in leukemic state. The response of the lymphoma to conventional chemotherapy (CHOP and ESHAP) was poor. We next performed an immunomodulatory therapy using cyclosporine A to suppress cytokine production by neoplastic T cells. The treatment resulted in a partial remission of AILT including disappearance of circulating lymphoma cells. To our knowledge, this is the first published report of AILT complicated by drug-induced agranulocytosis.

Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration.

Appropriate and abundant sources of bone-forming osteoblasts are essential for bone tissue engineering. Pluripotent stem cells can self-renew and thereby offer a potentially unlimited supply of osteoblasts, a significant advantage over other cell sources. We generated mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) from transgenic mice expressing rat 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP), a reporter of the osteoblast lineage. We demonstrated that Col2.3GFP ESCs and iPSCs can be successfully differentiated to osteoblast lineage cells that express Col2.3GFP in vitro. We harvested GFP+ osteoblasts differentiated from ESCs. Genome wide gene expression profiles validated that ESC- and iPSC-derived osteoblasts resemble calvarial osteoblasts, and that Col2.3GFP expression serves as a marker for mature osteoblasts. Our results confirm the cell identity of ESC- and iPSC-derived osteoblasts and highlight the potential of pluripotent stem cells as a source of osteoblasts for regenerative medicine.