Human umbilical cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications.

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

Gene products promoting remyelination are up-regulated in a cell therapy product manufactured from banked human cord blood.

BACKGROUND AIMS: DUOC-01, a cell product being developed to treat demyelinating conditions, is composed of macrophages that arise from CD14+ monocytes in the mononuclear cell (MNC) population of banked cord blood (CB). This article demonstrates that expression of multiple gene products that promote remyelination is rapidly up-regulated during manufacturing of DUOC-01 from either MNC or purified CB CD14+ monocytes. METHODS: Cell cultures were initiated with MNC or with immunoselected CD14+ monocytes isolated from the same CB unit. Cell products present in these cultures after 2 and 3 weeks were compared by three methods. First, quantitative polymerase chain reaction was used to compare expression of 77 transcripts previously shown to be differentially expressed by freshly isolated, uncultured CB CD14+ monocytes and DUOC-01. Second, accumulation of 16 soluble proteins in the culture medium was measured by Bioplex methods. Third, whole transcriptomes of the cell products were compared by microarray analysis. RESULTS: Key transcripts in multiple pathways that promote remyelination were up-regulated in DUOC-01, and substantial secretion of proteins corresponding to many of these transcripts was detected. Cell products manufactured from MNC or from CD14+ monocytes were similar with regard to all metrics. Upregulation of gene products characteristic of DUOC-01 was largely completed within 14 days of culture. CONCLUSION: We demonstrate that expression of multiple gene products that promote remyelination is up-regulated during the first 2 weeks of manufacturing of DUOC-01. Measuring these mechanistically important transcripts and proteins will be useful in monitoring manufacturing, evaluating manufacturing changes, and developing mechanism-based product potency assays.

A cord blood monocyte-derived cell therapy product accelerates brain remyelination.

Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells.

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.

A CALM-derived nuclear export signal is essential for CALM-AF10-mediated leukemogenesis.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

The PICALM protein plays a key role in iron homeostasis and cell proliferation.

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

An ankyrin-based mechanism for functional organization of dystrophin and dystroglycan.

beta-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and beta-DG. Dystrophin binds ankyrin-B and ankyrin-G, while beta-DG binds ankyrin-G. Dystrophin and beta-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and beta-DG remain intracellular in ankyrin-B-depleted muscle, where beta-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and beta-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.