Aya Fujishiro

Accelerated apoptosis of peripheral blood monocytes in Cebpb-deficient mice.

The CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb(-/-) mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb(-/-) bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb(-/-) mice. These results indicate that C/EBPβ is required for the survival of monocytes in peripheral blood.

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34(+) hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation.

Graft‐Versus‐Host Disease Amelioration by Human Bone Marrow Mesenchymal Stromal/Stem Cell‐Derived Extracellular Vesicles Is Associated with Peripheral Preservation of Naive T Cell Populations

A substantial proportion of patients with acute graft‐versus‐host disease (aGVHD) respond to cell therapy with culture‐expanded human bone marrow mesenchymal stromal/stem cells (BM‐MSCs). However, the mechanisms by which these cells can ameliorate aGVHD‐associated complications remain to be clarified. We show here that BM‐MSC‐derived extracellular vesicles (EVs) recapitulated the therapeutic effects of BM‐MSCs against aGVHD. Systemic infusion of human BM‐MSC‐derived EVs prolonged the survival of mice with aGVHD and reduced the pathologic damage in multiple GVHD‐targeted organs. In EV‐treated GVHD mice, CD4+ and CD8+ T cells were suppressed. Importantly, the ratio of CD62L‐CD44+ to CD62L + CD44‐ T cells was decreased, suggesting that BM‐MSC‐derived EVs suppressed the functional differentiation of T cells from a naive to an effector phenotype. BM‐MSC‐derived EVs also preserved CD4 + CD25 + Foxp3+ regulatory T cell populations. In a culture of CD3/CD28‐stimulated human peripheral blood mononuclear cells with BM‐MSC‐derived EVs, CD3+ T cell activation was suppressed. However, these cells were not suppressed in cultures with EVs derived from normal human dermal fibroblasts (NHDFs). NHDF‐derived EVs did not ameliorate the clinical or pathological characteristics of aGVHD in mice, suggesting an immunoregulatory function unique to BM‐MSC‐derived EVs. Microarray analysis of microRNAs in BM‐MSC‐derived EVs versus NHDF‐derived EVs showed upregulation of miR‐125a‐3p and downregulation of cell proliferative processes, as identified by Gene Ontology enrichment analysis. Collectively, our findings provide the first evidence that amelioration of aGVHD by therapeutic infusion of BM‐MSC‐derived EVs is associated with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of BM‐MSC‐derived EVs. Stem Cells 2018;36:434–445

Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells

The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×108) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.