The mouse homolog of the mutant WASp responsible for human X-linked neutropenia renders granulopoiesis ineffective.

Severe congenital neutropenia (SCN) is characterized by severe neutropenia and recurrent critical infections. X-linked neutropenia (XLN) is caused by a gain-of-function mutation in the Wiskott-Aldrich syndrome gene (WAS), the product of which (WASp) is expressed only in blood cells, especially during neutrophil maturation. To investigate the mechanism of neutropenia, we established a novel knock-in mouse line expressing WASp-I292T. WASp-I292T neutrophils exhibited activated (dysregulated) actin polymerization. Although WASp-I292T mice did not recapitulate neutropenia, neutrophil levels were increased in the bone marrow, and extramedullary hematopoiesis was observed. Bone marrow neutrophils from WASp-I292T mice exhibited attenuated transmigration. These abnormalities were associated with downregulation of NFκB and TP53 and faulty activation of their downstream pathways.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Activated HoxB4-induced hematopoietic stem cells from murine pluripotent stem cells via long-term programming.

Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or indirectly arising from defects within the HSC compartment. Pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) can give rise to all embryonic cell types; however, efficient in vitro differentiation of HSCs from PSCs remains challenging. HoxB4 is a key regulator orchestrating the differentiation of PSCs into all cells types across the mesodermal lineage, including HSCs. Moreover, the ectopic expression of HoxB4 enhances the in vitro generation and expansion of HSCs. However, several aspects of HoxB4 biology including its regulatory functions are not fully understood. Here, we describe the role of HoxB4 in indirectly inhibiting the emergence of mature CD45+ HSCs from iPSCs in vitro. Forced activation of HoxB4 permitted long-term maintenance of functional hematopoietic stem and progenitor cells (HSPCs), which efficiently reconstituted hematopoiesis upon transplantation. Our method enables an easy and scalable in vitro platform for the generation of HSCs from iPSCs, which will ultimately lead to a better understanding of HSC biology and facilitate preparation of the roadma for producing an unrestricted supply of HSCs for several curative therapies.

In vivo leukemogenic potential of an interleukin 7 receptor α chain mutant in hematopoietic stem and progenitor cells.

Somatic gain-of-function mutations in interleukin 7 receptor α chain (IL7Rα) have been described in pediatric T and B acute lymphoblastic leukemias (T/B-ALLs). Most of these mutations are in-frame insertions in the extracellular juxtamembrane-transmembrane region. By using a similar mutant, a heterozygous in-frame transmembrane insertional mutation (INS), we validated leukemogenic potential in murine hematopoietic stem/progenitor cells, using a syngeneic transplantation model. We found that ectopic expression of INS alone in hematopoietic stem/progenitor cells caused myeloproliferative disorders, whereas expression of INS in combination with a Notch1 mutant led to the development of much more aggressive T-ALL than with wild-type IL7Rα. Furthermore, forced expression of INS in common lymphoid progenitors led to the development of mature B-cell ALL/lymphoma. These results demonstrated that INS has significant in vivo leukemogenic activity and that the lineage of the resulting leukemia depends on the developmental stage in which INS occurs, and/or concurrent mutations.

A versatile drug delivery system using streptavidin-tagged pegylated liposomes and biotinylated biomaterials.

Here we have developed a versatile liposome-mediated drug delivery system (DDS) allowing a strong bridge between the streptavidin-tagged liposome (SAL) and biotin (Bi)-tagged biomaterials which has strong affinity to surface proteins expressed in restricted cell lineages. This DDS was effective and specific for many leukemia cells in vitro and in vivo. When examining 6 human leukemia cell lines using calcein-encapsulated SALs in combination with Bi-granulocyte colony-stimulating factor (G-CSF), Bi-anti-CD33 monoclonal antibody (MAb) or Bi-anti-CD7 MAb, the fluorescent positive rate of each cell line was in almost proportion to degree of G-CSF receptor, CD33 or CD7 expression, respectively. More importantly, the binding ability was shown to be well maintained in a mouse xenograft model. Furthermore the cytosine arabinoside (AraC)-encapsulated SALs could kill the corresponding cells much more effectively in combination with Bi-biomaterials than free AraC, as expected. These findings strongly indicate that our SAL/Bi-biomaterial system could allow various types of medical agents to be delivered reliably and stably to the cells targeted.

Bioimaging analysis of nuclear factor-κB activity in Philadelphia chromosome-positive acute lymphoblastic leukemia cells reveals its synergistic upregulation by tumor necrosis factor-α-stimulated changes to the microenvironment.

To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.

Gaussia luciferase for bioluminescence tumor monitoring in comparison with firefly luciferase.

Gaussia luciferase (Gluc) is a secreted reporter, and its expression in living animals can be assessed by in vivo bioluminescence imaging (BLI) or blood assays. We characterized Gluc as an in vivo reporter in comparison with firefly luciferase (Fluc). Mice were inoculated subcutaneously with tumor cells expressing both Fluc and Gluc and underwent Fluc BLI, Gluc BLI, blood assays of Gluc activity, and caliper measurement. In Gluc BLI, the signal from the tumor peaked immediately and then decreased rapidly. In the longitudinal monitoring, all measures indicated an increase in tumor burden early after cell inoculation. However, the increase reached plateaus in Gluc BLI and Fluc BLI despite a continuous increase in the caliper measurement and Gluc blood assay. Significant correlations were found between the measures, and the correlation between the blood signal and caliper volume was especially high. Gluc allows tumor monitoring in mice and should be applicable to dual-reporter assessment in combination with Fluc. The Gluc blood assay appears to provide a reliable indicator of viable tumor burden, and the combination of a blood assay and in vivo BLI using Gluc should be promising for quantifying and localizing the tumors.

Comparison of subcutaneous and intraperitoneal injection of D-luciferin for in vivo bioluminescence imaging.

PURPOSE: We compared subcutaneous (SC) injection and intraperitoneal (IP) injection of D-luciferin for in vivo bioluminescence imaging (BLI) to determine the utility of SC injection. METHODS: Mice bearing SC tumours stably expressing firefly luciferase underwent in vivo BLI using SC and IP injection of D: -luciferin. BLI studies were repeated at an interval of 3 h using a given injection route to assess repeatability and using different injection routes to assess correlation. In mice bearing both SC and IP tumours, BLI was performed successively using intravenous (IV), SC, and IP injection of D: -luciferin. Haematological malignancy model mice underwent BLI using SC and IP injection. RESULTS: In SC tumours, the peak time was slightly shorter and the peak signal was greater using SC injection than using IP injection. The repeatability of determining peak signals was comparable between the two injection routes, and a good correlation was observed between them. In mice bearing both SC and IP tumours, signals from IP tumours relative to those from SC tumours were much greater using IP injection than using IV or SC injection. In the haematological malignancy model, signals from the spleen relative to those from the bone marrow were greater using IP injection than using SC injection. CONCLUSION: In addition to rare injection failure, the IP injection of D: -luciferin led to the overestimation of signals from IP tissues. For BLI, SC injection was shown to be a convenient alternative to IP injection.

Evaluation of gadoxetate disodium as a contrast agent for mouse liver imaging: comparison with gadobenate dimeglumine.

We investigated the characteristics of gadoxetate disodium (Gd-EOB-DTPA) as a contrast agent for magnetic resonance imaging of the mouse liver. Mice were imaged sequentially under isoflurane anesthesia using a T1-weighted, three-dimensional fast low-angle shot (3D FLASH) sequence after an intravenous injection of Gd-EOB-DTPA or gadobenate dimeglumine (Gd-BOPTA), and the time course of the contrast effect was examined. The time course of the contrast effect of Gd-EOB-DTPA was also assessed after intravenous injection under pentobarbital anesthesia and after subcutaneous injection while awake or under isoflurane or pentobarbital anesthesia. Moreover, different doses of Gd-EOB-DTPA or Gd-BOPTA were injected subcutaneously into conscious mice, and the clarity of the liver border was evaluated visually. Intravenous injection under isoflurane anesthesia caused rapid contrast enhancement in the liver with both Gd-EOB-DTPA and Gd-BOPTA, and the contrast effect was 41% stronger with Gd-EOB-DTPA. Subcutaneous injection of Gd-EOB-DTPA caused delayed but favorable contrast enhancement in the liver. Washout of Gd-EOB-DTPA was faster in mice injected while awake than in those injected under anesthesia. After intravenous injection, washout was faster under pentobarbital anesthesia than under isoflurane anesthesia. The peak liver contrast was 11% and 18% stronger under pentobarbital anesthesia than under isoflurane anesthesia, after intravenous and subcutaneous injections, respectively. Subcutaneous injection of Gd-EOB-DTPA or Gd-BOPTA caused dose-dependent contrast effects in the liver. At a given dose, the contrast effect tended to be stronger and liver demarcation tended to be clearer with Gd-EOB-DTPA than with Gd-BOPTA. In conclusion, intravenous or subcutaneous injection of Gd-EOB-DTPA produces a favorable contrast effects in the mouse liver, indicating its potential in investigating mouse models of liver diseases. The contrast effects vary between conscious mice and anesthetized mice and among anesthetic agents used.

Bioluminescent evaluation of the therapeutic effects of total body irradiation in a murine hematological malignancy model.

OBJECTIVE: We investigated the utility of in vivo bioluminescence imaging (BLI) in assessing the therapeutic effects of total body irradiation (TBI) in a murine hematological malignancy model. MATERIALS AND METHODS: The suspension of Ba/F3 cells transduced with firefly luciferase and p190 BCR-ABL genes was exposed to ionizing radiation, and viable cell numbers and bioluminescent signals were measured serially. Mice intravenously inoculated with the cells underwent TBI at various doses. In vivo BLI was performed repeatedly until spontaneous death, and whole-body bioluminescence signals were determined as an indicator of whole-body tumor burden. RESULTS: In the cell culture study, bioluminescence signals generally reflected viable cell numbers, despite some overestimation immediately after irradiation. Sublethal TBI in mice transiently depressed the increase in whole-body signals and prolonged survival. Spontaneous death occurred at similar signal levels regardless of radiation dose. A significant negative correlation was found between survival and whole-body signal early after TBI. Significant dose dependence was demonstrated for both survival and signal increase early after TBI and was more evident for signal increase. Lethally irradiated mice without bone marrow transplantation died while showing weak signals. In mice receiving lethal TBI and syngeneic bone marrow transplantation, signal reduction and prolongation of survival were prominent, and whole-body signals at death were similar to those in nonirradiated or sublethally irradiated mice. CONCLUSION: In vivo BLI allows longitudinal, quantitative evaluation of the response to TBI in mice of a hematological malignancy model. Antitumor effects can be assessed early and reliably using in vivo BLI.

Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice.

We investigated the effect of diet on abdominal autofluorescence detected by in vivo fluorescence imaging (FLI) of living mice. Groups of mice were fed a regular, alfalfa-free, or purified diet, and whole-body FLI was performed without the administration of fluorescent probes. In addition, quantum dots were injected intravenously into mice fed one of the three diets, and FLI was performed 3 and 24 hours later. Intense autofluorescence originating from the animals' intestinal contents was observed in mice fed the regular diet. Intestinal autofluorescence decreased substantially after feeding with the alfalfa-free diet and further after feeding with the purified diet. The decline was rapid and took only 1 to 2 days; however, it may have been affected by an intake of feces. The reticuloendothelial system was clearly delineated using a low dose of quantum dots in mice fed the purified diet. On the other hand, intestinal autofluorescence was visible 24 hours postinjection in mice given the alfalfa-free diet and definitely impaired the image quality in mice fed the regular diet. The use of a low-fluorescence diet, especially a purified diet, rapidly reduces intestinal autofluorescence and is expected to enhance the potential of in vivo FLI.

In vivo fluorescence imaging of the reticuloendothelial system using quantum dots in combination with bioluminescent tumour monitoring.

PURPOSE: We characterised in vivo fluorescence imaging (FLI) of the reticuloendothelial system using quantum dots (QD) and investigated its use in combination with in vivo bioluminescence imaging (BLI). MATERIALS AND METHODS: In vivo FLI was performed in five mice repeatedly after the intravenous administration of QD without conjugation to targeting ligands. Ex vivo FLI of the excised organs was performed 24 h after QD injection in three mice. Seven days after intravenous inoculation of luciferase-expressing model cells of a haematological malignancy, mice were injected with the QD or saline (n = 5 each), and combined BLI/FLI was performed repeatedly. Additional five mice inoculated with the tumour cells were examined by in vivo BLI/FLI, and the structures harbouring bioluminescent foci were determined by ex vivo BLI. The utility of combining FLI with bioluminescent tumour monitoring was evaluated. RESULTS: In vivo FLI after QD injection allowed long-term, repeated observation of the reticuloendothelial system in individual mice, although fluorescence intensity and image contrast gradually decreased over time. Ex vivo FLI verified selective accumulation in reticuloendothelial structures. The administration of QD did not affect whole-body bioluminescent signal intensities during longitudinal tumour monitoring. In vivo BLI/FLI, accompanied by fusion of both images, improved the accuracy and confidence level of the localisation of the bioluminescent foci. CONCLUSIONS: In vivo FLI using QD provides an overview of the reticuloendothelial system in living mice. In combination with bioluminescent tumour monitoring, fluorescent reticuloendothelial imaging is expected to provide valuable information for lesion localisation.

Gadobenate dimeglumine as a contrast agent for MRI of the mouse liver.

We investigated the characteristics and utility of gadobenate dimeglumine (Gd-BOPTA) for MRI of the mouse liver. Mice were imaged under isoflurane anesthesia using a T(1)-weighted, three-dimensional fast low-angle shot (3D FLASH) sequence before and after intravenous or subcutaneous injection of Gd-BOPTA, and the time course of the contrast effect was examined. The appropriate dose for subcutaneous injection was determined visually, and the inter- and intra-observer reproducibilities in liver volumetry were evaluated with and without contrast injection. When mice were imaged sequentially before and after Gd-BOPTA injection and isoflurane anesthesia was maintained throughout the experiment, a long-lasting contrast effect was noted in the liver. Subcutaneous injection caused delayed, but favorable, enhancement. Washout from the liver was definitely accelerated in conscious mice in comparison with anesthetized mice. Visual evaluation indicated that a dose of 0.1 mmol/kg was appropriate for clear delineation of the entire liver margin, and the application of Gd-BOPTA significantly improved the inter- and intra-observer reproducibilities of liver volumetry. In conclusion, the intravenous or subcutaneous injection of Gd-BOPTA has a favorable contrast effect for the mouse liver, resulting in clear visualization of the liver border and improved reproducibility of liver volumetry. The possible influence of anesthesia on the pharmacokinetics of a contrast agent should be considered in determining the optimal scan timing.

Monitoring of disease progression by bioluminescence imaging and magnetic resonance imaging in an animal model of hematologic malignancy.

OBJECTIVE: We evaluated disease progression in a mouse model of a hematologic malignancy using a multimodality approach that included bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). We aimed to examine the feasibility and capability of BLI and MRI and to establish techniques for quantitative assessment of disease severity. METHODS: Mice were inoculated intravenously with Ba/F3 cells transduced with firefly luciferase and p190 BCR-ABL genes. Disease progression in a given mouse was observed longitudinally by in vivo BLI and MRI (n = 5). Imaging studies, including in vivo BLI and MRI of living mice and ex vivo BLI of excised organs, were also performed at various time points (n = 4, 3, 4, and 4 at 1, 2, 3, and 4 weeks after cell inoculation). RESULTS: Longitudinal studies allowed the assessment of disease progression for each mouse, and an approximately 4-log increase in whole-body BLI signal was shown after initial detection. MRI demonstrated progressive hepatosplenomegaly and growth of hepatic nodules. Ex vivo BLI demonstrated proliferation of the implanted cells in various organs including bone marrow, and the signal for each organ increased with time (Spearman's rank correlation coefficient, R = 0.831-0.914) and as the whole-body signal, observed by in vivo BLI, increased (R = 0.921-0.982). MRI measurements of liver and spleen volumes were shown to have excellent accuracy and volume increases significantly correlated with the BLI organ signal (liver, R = 0.875; spleen, R = 0.971). CONCLUSION: BLI and MRI allow repeated assessment of disease progression in a mouse model of a hematologic malignancy and provide quantitative markers of disease severity. BLI and MRI measurements reveal different details of disease progression and may play complementary roles in comprehensive assessment.

Light emission requires exposure to the atmosphere in ex vivo bioluminescence imaging.

The identification of organs bearing luciferase activity by in vivo bioluminescence imaging (BLI) is often difficult, and ex vivo imaging of excised organs plays a complementary role. This study investigated the importance of exposure to the atmosphere in ex vivo BLI. Mice were inoculated with murine pro-B cell line Ba/F3 transduced with firefly luciferase and p190 BCR-ABL. They were killed following in vivo BLI, and whole-body imaging was done after death and then after intraperitoneal air injection. In addition, the right knee was exposed and imaged before and after the adjacent bones were cut. Extensive light signals were seen on in vivo imaging. The luminescence disappeared after the animal was killed, and air injection restored the light emission from the abdomen only, suggesting a critical role of atmospheric oxygen in luminescence after death. Although no substantial light signal at the right knee was seen before bone cutting, light emission was evident after cutting. In conclusion, in ex vivo BLI, light emission requires exposure to the atmosphere. Bone destruction is required to demonstrate luciferase activity in the bone marrow after death.

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals.

PURPOSE: The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. METHODS: Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. RESULTS: The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. CONCLUSION: Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity.

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus).

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.

Anti-NK cell treatment induces stable mixed chimerism in MHC-mismatched, T cell-depleted, nonmyeloablative bone marrow transplantation.

OBJECTIVE: To clarify natural killer (NK) cell-mediated resistance under cytoreductive conditioning and T cell-depleted bone marrow transplantation, we investigated the effects of host NK cell depletion on engraftment and induction of stable mixed chimerism. METHODS: BALB/c mice (H-2kd) were injected intraperitoneally with anti-asialoGM1 antibody (anti-NK Ab) on day -1. On day 0, they received total body irradiation (TBI) at a dose of 500 cGy, followed by intravenous infusion of 2 x 10(7) T cell-depleted (TCD) bone marrow cells from C57BL/6 mice (H-2kb). Early engraftment and chimerism were determined by the relative ratio of peripheral blood (PB) lymphocytes expressing either H-2kd or H-2kb on day +21. Long-term engraftment and chimerism were evaluated on PB and spleen by multicolor flow cytometry. RESULTS: Although no recipients treated with TBI alone showed engraftment, all the recipients conditioned with anti-NK Ab and TBI showed successful engraftment as well as a donor-dominant pattern of mixed chimerism in both PB and spleen. Spleen cells from recipients with mixed chimerism showed specific tolerance to both host and donor strains, but not to a third party (C3H/He). None of the reconstituted mice showed signs of graft vs host disease, and all survived up to day +330. CONCLUSION: These observations indicate that host NK cell depletion may be used to reduce the intensity of conditioning regimens for engraftment of TCD grafts, and can contribute to establishment of stable mixed chimerism in major histocompatibility complex-mismatched nonmyeloablative transplantation.

Hematopoietic activity of common marmoset CD34 cells isolated by a novel monoclonal antibody MA24.

OBJECTIVE: We focused on a small New World monkey, the common marmoset (Callithrix jacchus), to establish a nonhuman primate model of the treatment of hematological disorders. In this study, we developed the first monoclonal antibodies (MAbs) against marmoset CD34 and tested the in vitro and in vivo hemopoietic activity of cell populations isolated using one of these MAbs. METHODS AND RESULTS: Marmoset cDNA encoding a human CD34 homologue was cloned from bone marrow (BM)-derived RNA using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The amino acid sequence of the marmoset CD34 had 81% homology with the human sequence. Five mouse MAbs were raised against marmoset CD34 transfectant. One representative MAb, MA24 (IgM), reacted with approximately 0.5 to 1% of BM mononuclear cells (MNCs), where the colony-forming unit granulocyte/macrophage (CFU-GM) was enriched approximately 11- to 75-fold as compared with the whole BM MNCs. Multilineage differentiation of marmoset CD34+ cells in NOD/SCID mice was confirmed by flow cytometry 1 month after xenotransplantation. CONCLUSION: These results demonstrated that MA24 is useful for the analysis and enrichment of hematopoietic progenitor cells in the marmoset model for preclinical experiments.