MAP kinase: a switch in fate determination of stem cells.
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Animals↗
Biomedical subjects
V P Kale
Publications and source records attributed to V P Kale.
Differential activation of MAPK signaling pathways by TGF-beta1 forms the molecular mechanism behind its dose-dependent bidirectional effects on hematopoiesis.
We have earlier reported that transforming growth factor-beta1 (TGF-beta1), a well-known inhibitor of hematopoiesis, stimulated colony formation from adult human bone marrow mononuclear cells (BM MNC) when used at low concentrations. We examined the possible molecular mechanism behind this bidirectional effect using CD34+ cells isolated from human BM for clonal assays and the KG1a cell line as a model system for analysis of proteins for signaling pathways by immunoblotting. We found that TGF-beta1 at low doses (picogram levels) stimulated the colony formation from CD34+ cells, indicating that these progenitors form the direct target of stimulatory action of TGF-beta1. CD34+ cells were found to be more sensitive to the TGF-beta1 concentration than the total MNC. We used the KG1a cell line as a model system for identification of mitogen-activated protein kinase (MAPK) and AKT signaling pathways involved in the process. Low doses strongly induced p44/42 MAPK phosphorylation, whereas high doses induced p38 activation. Use of specific p44/42 MAPK inhibitor PD 98059 in the colony assay abrogated the stimulatory effect of low TGF-beta1. On the other hand, use of p38 MAPK inhibitor SB 203580 along with low TGF-beta1 concentrations had a synergistic effect on stimulation of colony formation. Treatment of BM MNC with Anisomycin, which activates stress kinases, resulted in a dose-dependent inhibition of colony formation. This inhibition could not be rescued by stimulatory doses of TGF-beta1. Phosphorylation of AKT was found to occur in a dose-dependent way but declined slightly at the highest concentration used (10 ng/ml). Inhibition of the AKT pathway by LY 294002 strongly suppressed colony formation. These data indicate clearly that sustained activation of p44/42 MAPK perhaps forms the stimulatory signal induced by low TGF-beta1, whereas activation of p38 forms the inhibitory pathway.
Anisomycin↗
Molecular mechanisms behind the dose-dependent differential activation of MAPK pathways induced by transforming growth factor-beta1 in hematopoietic cells.
Transforming growth factor-beta (TGF-beta) controls a wide range of cellular responses, including cell proliferation, lineage determination, differentiation, and apoptosis, and figures prominently in animal development. It is considered as a pleiotropic factor because it can exert a positive or negative effect on various cellular processes depending on developmental stage of the target cell, its microenvironment, and also its biochemical make up. It has been shown to have a strong inhibitory effect on hematopoietic stem cell proliferation and differentiation. We have earlier shown that TGF-beta1 exerts a bidirectional effect on hematopoietic cell proliferation as a function of its concentration. Although it acted as an inhibitor at high concentrations, at low concentrations it stimulated the stem/progenitor cells. We also provided evidence that the differential activation of mitogen-activated protein kinase pathways was responsible for the observed bidirectional effect. In the present study, we examined the molecular mechanism behind this phenomenon. We observed that the high inhibitory concentrations of TGF-beta1 induced a strong phosphorylation of SMAD 3 and also activated stress kinase-related transcription factors, namely c-Jun and ATF-2. On the other hand, low stimulatory concentrations acted in a SMAD 3-independent pathway and activated STAT proteins. Our results clearly show that differential activation of signal transduction pathways by TGF-beta1 as a function of its concentration underlies its bidirectional effect on hematopoietic cells.
Adaptor Proteins, Signal Transducing↗
Colony-stimulating activity of fetal liver cells: synergistic role of stem cell factor in bone marrow recovery from aplastic anemia.
Previously, we and others have shown that fetal liver infusion (FLI) leads to autologous hematopoietic improvement in 40-54% of patients with aplastic anemia. However, whether this recovery was spontaneous or the effect of the infused liver cells was not clear. To dissect the role of FLI in autologous hematopoietic recovery, the colony-supporting potential of fetal liver-conditioned medium (FLCM) was evaluated in bone marrow (BM) cells of normal adult and aplastic anemia patients. In both cases, each sample of FLCM supported the growth of colony-forming cells in semi solid culture medium. The FLCM was assayed for the presence of four principal colony-stimulating cytokines, namely stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). While GM-CSF, IL-3, and Epo were present in insignificant amounts or were altogether absent, 50-635 pg/ml of SCF was found in 8 of the 13 FLCM samples tested. Preliminary results of bioneutralization assay indicated the possible role of SCF, secreted by the FL cells, in colony-supporting activity of aplastic anemia and normal BM cells. Overall, our in vitro study implicates the paracrine role of infused FL cells in regenerating autologous hematopoiesis in aplastic anemia patients.
Anemia, Aplastic↗
Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium.
Cord blood (CB) and fetal liver (FL) cells are two alternative sources of human hematopoietic stem cells. Optimization of cryopreservation protocols is an important aspect in the banking of these tissues. Out of the multiple factors responsible for cryodamage of cells, membrane leakage and oxygen free-radical generation have been shown to contribute substantially toward the process. We have studied the effect of certain additives, like membrane stabilizers and bioantioxidants, to the conventional freezing medium on viability, nucleated cell recovery, and clonogenic potential of frozen cells. Our results show that trehalose, a membrane stabilizer, when used in combination with 10% dimethyl sulfoxide (DMSO) affords better cryoprotection as evidenced by significantly increased colony formation as compared to 10% DMSO alone. The cryoprotection afforded by trehalose persists at least for 1.5 years and there is no bias toward protection of a particular lineage. We also found that this increased cryoprotective effect of trehalose is seen both at -196 degrees C and -80 degrees C storage temperatures. Addition of taurine, an amino acid, another membrane stabilizer, and a natural cryoprotectant to the traditional freezing medium also results in beneficial effect. Of the three bioantioxidants tested, i.e., ascorbic acid, alpha-tocopherol acetate, and catalase, catalase shows maximum cryoprotective effect both at -196 degrees C and at -80 degrees C. Because the mode of cryoprotective action of catalase and trehalose are totally different, we tried a combination of these two compounds along with 10% DMSO. At -196 degrees C the protection afforded by the combination was significantly better than that afforded by individual components. At -80 degrees C, however, the combination did not give any added protection as compared to the individual single additives, although it was significantly better than 10% DMSO alone. These results indicate that the addition of membrane stabilizers and antioxidants to the conventional freezing medium may help to improve post thaw recovery of hematopoietic cells.
Antioxidants↗
Stimulation of adult human bone marrow by factors secreted by fetal liver hematopoietic cells: in vitro evaluation using semisolid clonal assay system.
Fetal liver infusion (FLI) therapy has been used in various disorders, such as aplastic anemia, leukemia, metabolic disorders, etc., and has been shown to result in stimulation of autologous hematopoiesis in many cases. The aim of the present study was to elucidate the mechanism of stimulation of adult hematopoiesis by fetal liver hematopoietic cells (FLHC) and to identify the factors involved in the process using a clonal assay system in vitro. The effect of FLHC on the clonal growth of bone marrow cells was studied using a co-culture system consisting of mitomycin C-treated FLHC with 2 x 10(5) bone marrow (BM) mononuclear cells. It was observed that FLHC induced a two- to four-fold increase in the BM colony formation. A further increase in the number of FLHC did not, however, result in an equivalent fold increase in the colony formation, indicating that the number of cells in the BM population responsive to FLHC was perhaps the limiting factor. When the effect of fetal liver cell conditioned medium (FLCM) was examined in a similar fashion, it was observed that the FLCM showed a 1.5- to 4-fold increase in the colony formation when used at 1%-5% along with limiting amounts of growth factors. Higher concentrations of conditioned medium resulted in inhibitory responses. One of the principal factors responsible for the stimulatory activity of FLCM was shown to be transforming growth factor-beta1 (TGF-beta1), by a variety of experiments such as its quantitation in FLCM by enzyme-linked immunosorbent assay, antibody neutralization, and reconstruction experiments using purified TGF-beta1 and normal medium. In these reconstitution experiments, TGF-beta1 stimulated the colony formation when it was applied at 1-50 pg/ml, but at higher concentration it induced an inhibitory effect, mimicking the behavior earlier seen with FLCM. Our data strongly suggest that one of the mechanisms in stimulation of a recipient's hematopoiesis could be mediated by the action of TGF-beta1 secreted by infused FLHC and could provide a rational framework on which FLI therapy can be further evaluated.
Adult↗
Comparison of biochemical characterisation of ICRC bacilli with M. leprae: effect of substrate alteration in the medium.
ICRC-bacilli strain C-44 when grown in Dubos medium of its equivalent, express M. avium taxonomic biochemical characters. Assuming that difference in characters of M. leprae and ICRC bacilli, could be due to 'in vivo' and 'in vitro' milieu, we altered the substrates in the medium. The bacilli grow well in the new medium containing selenium, ferric nitrate, magnesium chloride and deleting Tween 80. The ICRC strain C-44 grown in new medium expressed characters: 9/10 similarity with M. leprae. The 10 day tween hydrolysis reaction in weak but positive. It is probable that 'M. leprae culture isolate', may have acquired 'in vitro' growth potential by recombination with M. avium, an ubiquitous mycobacterium. The M. leprae culture isolate thus may express some characters of both M. leprae and M. avium.
Culture Media↗
Antigeneic cross-reactivity between ICRC-bacilli and M. leprae--"in vitro" evaluation.
Leucocyte migration inhibition, in presence of specific particulate antigen, is a good correlate of CMI. It can detect small differences in related antigens. In the present study, LMI was used to study the cross-reactivity between ICRC bacilli and M. leprae and also to examine the antigenic relationship amongst different ICRC isolates. The study showed a close antigenic cross-reactivity in lepromin and ICRC-in. LMI has brought out strain differences in two ICRC culture isolates, C-44 and C-75. The data appear to elucidate the possible strain specific antigenic relationship of M. leprae with its culture isolate e.g. ICRC strains. This may have important bearing on the development effective antileprosy vaccine for human use.
Animals↗
Dopa-oxidase activity on ICRC bacilli.
Presence of O-phenoloxidase is regarded as M. leprae specific character. This enzyme activity was found to be present in ICRC bacilli, Strain C-44. Though this strain is cultivable 'in vitro', the expression of DOPA-Oxidase activity strongly suggests that it carries M. leprae genome. The ICRC bacilli, therefore, may thus from a group of M. leprae culture isolates, distinct from other known cultivable mycobacteria which do not possess this enzyme activity.
Catechol Oxidase↗