Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells.

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.

The putative anti-leukemic effects of anti-thymocyte globulins in patients with CD7-positive acute myeloid leukemia.

Polyclonal anti-thymocyte globulins (ATGs) are widely used in allogeneic stem cell transplantation (allo-SCT) for GvHD prophylaxis. ATGs exerted anti-tumor effects in in vitro experiments, but in vivo studies are lacking. We experienced a case of relapsed AML with cells positive for CD7 who underwent haploidentical SCT and unexpectedly achieved a significant reduction of AML cells in the peripheral blood after receiving ATGs before the administration of other drugs in the conditioning regimen. This patient achieved long-term survival after haploidentical SCT. To assess the impact of ATGs on clinical outcomes in patients with AML, we performed a retrospective analysis of allo-SCT for relapsed/refractory AML and divided 132 patients into four groups according to the expression of CD7 in AML cells and use of ATGs as part of the conditioning regimen, as follows: CD7-positive ATG group (n = 15), CD7-positive no-ATG group (n = 32), CD7-negative ATG group (n = 19), and CD7-negative no-ATG group (n = 66). The overall survival rates in the CD7-positive ATG group were significantly higher than those in the CD7-positive no-ATG group, whereas these rates did not differ statistically between the CD7-negative ATG and CD7-negative no-ATG groups. Our results indicate a possible anti-leukemic effect of ATGs against CD7-positive AML in humans.

Splenic marginal zone lymphoma complicated by cold agglutinin disease.

Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoma accounting for less than 1% of lymphoid neoplasms and is often associated with autoimmune disorders. A 48-year-old woman presented with severe anemia due to steroid-refractory cold agglutinin disease (CAD), and was referred to our hospital for management of progressive systemic illness and high fever. On admission, she showed elevated serum soluble IL-2R and mild splenomegaly. PET/CT revealed FDG accumulation in the spleen and bone. She was pathologically diagnosed as having splenic marginal zone lymphoma by splenectomy and received 8 cycles of rituximab every 2 weeks, resulting in marked improvement of anemia. Lymphoma cells were positive for CD20/CD11c/CD13, phenotypically compatible with SMZL. We report herein the characteristic features of SMZL, and appropriate procedures for diagnosis and treatment.

[Autologous peripheral blood stem cell transplantation for double-refractory myeloma with K-RAS and N-RAS mutations].

The prognosis of multiple myeloma (MM) has been improved due to the introduction of novel agents like proteasome inhibitors and immunomodulatory drugs (IMiDs). However, some cases are refractory to the use of novel agents, and the prognosis of such cases is poor. A 53-year-old male was diagnosed with MM and categorized as follows: Bence-Jones protein lambda type MM, Durie-Salmon IIIA, international staging system (ISS) stage II, and revised ISS stage II. Mutations in K-RAS and IGH/FGFR3 translocation were detected at diagnosis. His tumor was refractory to seven therapeutic regimens including bortezomib, IMiDs (lenalidomide, thalidomide, pomalidomide), conventional chemotherapy, and radiation therapy. N-RAS mutations, CKS1B gains, and C-MYC split signals were detected after treatment. We performed high-dose melphalan/autologous stem cell transplantation (HD-MEL/ASCT) as a salvage therapy and achieved very good partial response. The correlation between K-RAS mutations and poor prognosis or between N-RAS mutations and reduced sensitivity to bortezomib is reported. However, RAS mutations are reported as a favorable factor for HD-MEL/ASCT. In general, mutations of both the K-RAS and N-RAS are known to be mutually exclusive. This rare MM case has mutations in both K-RAS and N-RAS, and the possible relevance of these mutations to both the refractoriness to novel therapies and sensitivity to HD-MEL/ASCT is suggested.

Multicolor staining of globin subtypes reveals impaired globin switching during erythropoiesis in human pluripotent stem cells.

Adult hemoglobin composed of α- and ß-globin reflects a change from expression of embryonic ε- and fetal γ-globin to adult ß-globin in human erythroid cells, so-called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ-globin with little ß-globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of ß-globin and the corresponding silencing of γ-globin at the single-cell level during cord blood CD34(+) cell-derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous ß-globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ-globin. These hPSC-derived erythroid cells also displayed reduced expression of BCL11A-L. However, doxycycline-induced overexpression of BCL11A-L in selected hPSCs promoted γ-globin silencing. These results strongly suggest that impaired γ-globin silencing is associated with downregulated BCL11A-L in hPSC-derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.

Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells.

The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4-5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b(+) platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application.

Immortalization of erythroblasts by c-MYC and BCL-XL enables large-scale erythrocyte production from human pluripotent stem cells.

The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.