Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia

The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O₂ (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O₂, resulting in a higher fold increase after 12 days under hypoxia (40 ± 10 vs. 30 ± 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O₂ and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O₂ presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O₂ favored CFU‐F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O₂, compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010. © 2009 Wiley‐Liss, Inc.     

Changes in egg and body temperature indicate triggering of egg desertion at a body mass threshold in fasting incubating blue petrels (Halobaena caerulea)

The triggering of transitory egg desertion in fasting and incubating blue petrels (Halobaena caerulea, nocturnal burrowing seabirds living in the subantarctic region) was investigated by continuously monitoring both body temperature (T _sto) and egg temperature (T _egg) with a telemetry system, and by measuring body mass (BM) loss. The birds were kept captive in their burrow and incubated day and night without any interruption; there was no day-night cycle in T _sto and T _egg, which averaged 39.9 °C and 32.0 °C, respectively. There was no evidence of hypothermia as a way to save energy in this fasting situation. Egg desertion occurred at night and was an abrupt and definitive phenomenon reflected by a simultaneous fall in T _egg and a peak in T _sto. After egg desertion, a distinct day-night cycle of body temperature was observed, T _sto being 0.6 °C higher during night-time (P < 0.05), probably reflecting increased nocturnal activity. BM at egg desertion averaged 166.7 ± 3.8 g in telemetered birds and 164.4 ± 1.6 g in␣a group of free-living birds. Throughout fasting, the␣specific daily BM loss remained at 46 ± 1 g · kg⁻¹ · day⁻¹, but increased sharply below a critical BM of 160.0 ± 2.5 g. Thus, fasting incubating blue petrels spontaneously desert their egg when reaching a BM threshold. This BM is very close to a critical value in fasting birds and mammals that corresponds to a critical depletion of fat stores and to a shift from lipid to protein utilization. This strongly suggests that such a metabolic shift triggers behavioural changes leading to egg desertion and refeeding, which is of great relevance to the understanding of the long-term control of food intake and BM. Accepted: 16 July 1998     

Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources

Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O₂, compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.     

Environmental and genetic influences on body mass and resting metabolic rates (RMR) in a natural population of weasel Mustela nivalis

Body mass (BM) and resting metabolic rates (RMR) are two inexorably linked traits strongly related to mammalian life histories. Yet, there have been no studies attempting to estimate heritable variation and covariation of BM and RMR in natural populations. We used a marker‐based approach to construct a pedigree and then the ‘animal model’ to estimate narrow sense heritability (h²) of these traits in a free‐living population of weasels Mustela nivalis—a small carnivore characterised by a wide range of BM and extremely high RMR. The most important factors affecting BM of weasels were sex and habitat type, whereas RMR was significantly affected only by seasonal variation of this trait. All environmental factors had only small effect on estimates of additive genetic variance of both BM and RMR. The amount of additive genetic variance associated with BM and estimates of heritability were high and significant in males (h² = 0.61), but low and not significant in females (h² = 0.32), probably due to small sample size for the latter sex. The results from the two‐trait model revealed significant phenotypic (r_P = 0.62) and genetic correlation (r_A = 0.89) between BM and whole body RMR. The estimate of heritability of whole body RMR (0.54) and BM corrected RMR (0.45) were lower than estimates of heritability for BM. Both phenotypic and genetic correlations between BM corrected RMR and BM had negative signals (r_P = ?0.42 and r_A = ?0.58). Our results indicate that total energy expenditures of individuals can quickly evolve through concerted changes in BM and RMR.     

First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor

Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% (v/v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000.     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Regulation of bone marrow cell survival in short-term cultures: a new macrophage function

The involvement of macrophages (M phi) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied. We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells. 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells. Upon 24 hr of culture in conventional medium, more than 50% of BM cells died. In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal M phi, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival. We found that coculture of BM cells with M phi dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival. The ability to promote BM cell survival, here designated nurse activity, represented a novel function of M phi and was further characterized. The stage of activation of M phi did not influence their nurse activity, since M phi elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guérin, as well as resident M phi unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells. BM-derived M phi (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity. Moreover, nurse activity was also exerted across the histocompatibility barriers. Supernatants from M phi cultures or killed M phi were ineffective. We propose that the nurse effect of M phi on BM is a primitive function that may play an important role in the development of the hemopoietic system.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrids (African violet) cultured in vitro

Leaf discs fromSaintpaulia ionantha xconfusa hybrids (African violet) were transferred between basal medium (BM) containing no hormones and shoot-inducing medium (SIM) containing 2.0 mg 1^–1 indole acetic acid and 0.08 mg l^–1 6-benzylaminopurine to determine whether there is a window of competence for shoot regeneration. Leaf discs precultured on BM prior to transfer to SIM formed buds 3 days earlier than the controls (leaf discs not precultured) regardless of whether the discs were placed upside down or right side up on the medium. This suggests that cultured leaf cells were not competent for shoot induction during the first 3 days of culture. Leaf discs cultured right side up (abaxial surface to the medium) did not form buds on BM alone, unlike discs cultured upside down. Leaf disc survival was affected by a delay in hormonal exposure, but surviving leaf discs produced as many shoots as control leaf discs. This suggests that in the absence of exogenous plant hormones, cellular competence to regenerate shoots is not lost in excised leaf discs of African violet.Abbreviations BM Basal medium - SIM_Sho Shoot-inducing medium     

Natural killer cells generated from bone marrow culture

By using anti-Nk-1 antiserum, we detected a significant proportion of Nk-1+ cells in bone marrow (BM) with low lytic activity that can be slightly enhanced with interferon (IF). These BM Nk-1+ cells also bind to YAC-1 cells. Because Nk-1 antigen has been found to mark NK cells, BM appears to harbor immature precursors to natural killer (NK) cells. We therefore used concanavalin A (Con A)-conditioned medium to culture BM cells to induce differentiation of the putative NK precursors. After 3 to 4 days in culture, cytotoxic activity to YAC, that peaked at 6 to 7 days, was consistently generated, and the activity still could be detected after 10 to 14 days in culture. In contrast, experiments using spleen cultures, performed in a similar manner, showed peak activity after 4 to 5 days and the activity declined thereafter. The cytotoxic activity of cultured BM cells was also higher than that of cultured spleen cells. Cultures of BM cells from old mice have good cytotoxic activity. The cytotoxic cells generated were Nk-1+ and Qa-5+. Furthermore, these culture conditions did not maintain the proliferation of CFU-C cells.     

Spectroscopic,structural and antibacterial properties of copper(II) complexes with bio-relevant 5-methyl-3-formylpyrazole N(4)-benzyl-N(4)-methylthiosemicarbazone

The coordination behaviour of the title ligand, 5-methyl-3-formylpyrazole N(4)-benzyl-N(4)-methylthiosemicarbazone(HMPz4BM), is reported with solid state isolation of copper(II) complexes, [Cu(HMPz4BM)X₂] (X = Cl, Br, NO₃, ClO₄ and BF₄) which have been spectroscopically and structurally characterised. I.r. data for the free ligand and its Cu(II) complexes indicate that HMPz4BM exhibits a neutral NNS tridentate function via the pyrazolyl nitrogen(tertiary), azomethine nitrogen and thione sulphur. Electronic spectral data are suggestive of a square pyramidal environment for the seemingly pentacoordinated Cu(II) species. E.s.r parameters (RT and LNT) of the reported copper(II) complexes are indicative of a dx_x2–y2 ground state for the reported species. Cyclic voltammograms of Cu(II) complexes show a quasireversible Cu^II/Cu^III couple and also an irreversible Cu^II/Cu^I couple. X-ray crystallography of a representative species, [Cu(HMPz4BM)(NO₃)₂] (C2/c, monoclinic ), has unambiguously documented the conjectural findings from i.r. data that coordinating sites of the title ligand are pyrazolyl (tertiary)nitrogen, azomethine nitrogen and the thione sulphur (NNS); and the oxygen of one of the nitrate ions has occupied the basal plane; the fifth coordination position has been occupied by the oxygen of another nitrate ion in a square pyramidal geometry. The antibacterial properties of the ligand and its copper(II) complexes studied on microorganism, Staphylococcus aureus have pointed out that most of the complexes have higher activities than that of the free ligand.     

Cell Signaling-Based Classifier Predicts Response to Induction Therapy in Elderly Patients with Acute Myeloid Leukemia

Single-cell network profiling (SCNP) data generated from multi-parametric flow cytometry analysis of bone marrow (BM) and peripheral blood (PB) samples collected from patients >55 years old with non-M3 AML were used to train and validate a diagnostic classifier (DX_SCNP) for predicting response to standard induction chemotherapy (complete response [CR] or CR with incomplete hematologic recovery [CRi] versus resistant disease [RD]). SCNP-evaluable patients from four SWOG AML trials were randomized between Training (N = 74 patients with CR, CRi or RD; BM set = 43; PB set = 57) and Validation Analysis Sets (N = 71; BM set = 42, PB set = 53). Cell survival, differentiation, and apoptosis pathway signaling were used as potential inputs for DX_SCNP. Five DX_SCNP classifiers were developed on the SWOG Training set and tested for prediction accuracy in an independent BM verification sample set (N = 24) from ECOG AML trials to select the final classifier, which was a significant predictor of CR/CRi (area under the receiver operating characteristic curve AUROC = 0.76, p = 0.01). The selected classifier was then validated in the SWOG BM Validation Set (AUROC = 0.72, p = 0.02). Importantly, a classifier developed using only clinical and molecular inputs from the same sample set (DX_CLINICAL2) lacked prediction accuracy: AUROC = 0.61 (p = 0.18) in the BM Verification Set and 0.53 (p = 0.38) in the BM Validation Set. Notably, the DX_SCNP classifier was still significant in predicting response in the BM Validation Analysis Set after controlling for DX_CLINICAL2 (p = 0.03), showing that DX_SCNP provides information that is independent from that provided by currently used prognostic markers. Taken together, these data show that the proteomic classifier may provide prognostic information relevant to treatment planning beyond genetic mutations and traditional prognostic factors in elderly AML.     

Structural Insights into Mitochondrial Antiviral Signaling Protein (MAVS)-Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Signaling

In response to viral infection, cytosolic retinoic acid-inducible gene I-like receptors sense viral RNA and promote oligomerization of mitochondrial antiviral signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins, including TRAF6, to activate an antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood, and atomic details are lacking. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2; amino acids 455–460). Further, we solved the crystal structure of MAVS T6BM2 in complex with the TRAF6 TRAF_C domain at 2.95 Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS^−/− mouse embryonic fibroblast cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.     

Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic “shield” from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K_m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.     

A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes

Summary A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7–10 days of culture. The antibody BM8 belonged to the IgG_2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.