Stem Cell Suspension Research Articles

Comparison of tissue culture and whole plant responses to salinity in potato

Salt sensitivities of six potato cultivars using six levels of sodium chloride (0.0 to 0.25M) were studied in a greenhouse. Responses of these cultivars were also determined in tissue culture by studying rooting of stem segments, increase in length of cultured roots and inhibition of growth of cell suspension cultures using similar salt concentrations. Responses of cultured stem segments and cell suspensions differed from those expressed by whole plants. A close similarity was observed between the salt stress response of whole plants and of cultured roots. The latter technique may provide a preliminary screening method for assessing salt tolerance in potato genotypes.

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergism between recombinant growth factors, GM-CSF and G-CSF, acting on the blast cells of acute myeloblastic leukemia [published erratum appears in Blood 1987 Jul;70(1):339

The genes for the hemopoietic growth factors, GM colony-stimulating factor (CSF) and G-CSF have been cloned, and recombinant material is available for both. We tested these recombinant factors for their effects on the blast cells of acute myeloblastic leukemia (AML). Culture methods are available that support both colony formation by AML blasts and the growth of blast stem cells in suspension. Recombinant GM- CSF is active in both culture systems, although to a varying degree. We found that recombinant G-CSF was also effective; however, the two recombinant factors showed striking synergism for the stimulation of blast growth of cells from five of eight AML patients. In these cases, the combination was equivalent to the stimulating activity of supernatants from the continuous cell line 5637. This conditioned medium (HTB9-CM) is considered the standard for blast growth. Blasts from one of the patients grew without added factor. In another instance, recombinant GM-CSF alone was almost as effective as HTB9-CM. In the third case, both recombinant factors were active, but synergism was not observed and their combined effect was not equivalent to that of HTB9-CM. Both GM-CSF and G-CSF were active on normal bone marrow granulopoietic progenitors, but synergism was not observed. We conclude that the marked heterogeneity observed when AML blasts are examined by other criteria is also observed when their response to growth factors is evaluated.

Synergism Between Recombinant Growth Factors, GM-CSF and G-CSF, Acting on the Blast Cells of Acute Myeloblastic Leukemia

The genes for the hemopoietic growth factors, GM colony-stimulating factor (CSF) and G-CSF have been cloned, and recombinant material is available for both. We tested these recombinant factors for their effects on the blast cells of acute myeloblastic leukemia (AML). Culture methods are available that support both colony formation by AML blasts and the growth of blast stem cells in suspension. Recombinant GM-CSF is active in both culture systems, although to a varying degree. We found that recombinant G-CSF was also effective; however, the two recombinant factors showed striking synergism for the stimulation of blast growth of cells from five of eight AML patients. In these cases, the combination was equivalent to the stimulating activity of supernatants from the continuous cell line 5637. This conditioned medium (HTB9-CM) is considered the standard for blast growth. Blasts from one of the patients grew without added factor. In another instance, recombinant GM-CSF alone was almost as effective as HTB9-CM. In the third case, both recombinant factors were active, but synergism was not observed and their combined effect was not equivalent to that of HTB9-CM. Both GM-CSF and G-CSF were active on normal bone marrow granulopoietic progenitors, but synergism was not observed. We conclude that the marked heterogeneity observed when AML blasts are examined by other criteria is also observed when their response to growth factors is evaluated.

Survey of experimental data on fetal liver transplantation.

Fetal liver transplantation has been shown to induce hematological and immunological reconstitution in irradiated rodents, dogs, horses, and sheep. Engraftment and reconstitution without GvHD has been readily obtained using histocompatible donors. When mismatched fetal donors were used, a comparatively larger number of donor cells was required, in addition to pre-treatment of host with higher doses of irradiation or irradiation plus chemotherapy. Stem cell suspensions devoid of any T lymphocyte can be transplanted across major histocompatibility barrier without inducing overt GvHD. The transplanted animals become tolerant to both donor and host grafts.

Cellular specificity of the cure for osteopetrosis: Isolation of and treatment with pluripotent hemopoietic stem cells

Osteopetrosis in the ia rat is the result of reduced bone resorption due to abnormal osteoclasts. Studies in the ia mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. Recent studies demonstrated that the Ficoll-Hypaque isolates of spleen, bone marrow, and newborn liver were effective in curing the skeletal disease. These results suggest that the cellular source of the cure is a pluripotent hemopoietic stem cell (PHSC). Goldschneider et al. (1980) demonstrated that stem cells from rat bone marrow relatively large mononuclear cells that are strongly positive for Thy 1.1 antigen. Splenic and bone marrow samples from normal rats were treated with a monoclonal antibody developed against the rat Thy 1.1 antigen and guinea pig complement. This procedure was cytotoxic for 47% of the mononuclear spleen cells and 74% of the mononuclear bone marrow cells. When the Thy 1.1 depleted samples were transplanted into ia littermates, they were ineffective in reversing the osteoclast and bone resorption defects. Using the parameters described by Goldschneider, and fluorescence-activated cell sorting (FACS), the PHSC were isolated from normal rat bone marrow. The isolated cells were assayed for colony forming units — spleen (CFU-S) and were found to contain approximately 65–70% PHSC. Two, 3, and 5-week-old ia rats were given an intraperitoneal injection of normal bone marrow stem cells (1.4 × 10 4 to 7.0 × 10 4 cells). The effects of treatment were evaluated by radiography, by measuring the size of the tibial marrow cavity, and by identifying active (phenotypically normal) osteoclasts at 3 weeks after transplantation. At all of the ages transplanted and at all of the cell numbers tested, the stem cell suspensions were effective in curing the osteopetrotic condition. These positive findings and the fact that the Thy 1.1 depleted cells were ineffective suggests that the PHSC is a cellular source of the cure in the ia mutation.

Plant regeneration of Solanum lycopersicoides Dun. from stem explants, callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l− NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l−1 zeatin + 0.1 mg l−1 gibberellic acid.

Peroxidase Activity and Isoperoxidase Composition in Cultured Stem Tissue, Callus and Cell Suspensions of Actinidia chinensis

An investigation of peroxidase activity and isoperoxidase composition in cultured stem tissue, subcultures and cell suspensions of a dioecious plant, Actinidia chinensis , is reported. Soluble total peroxidase activity is higher in callus tissue, subcultures and cell suspensions obtained from female plants than in male plants. Moreover, callus tissue, subcultures and cell suspensions obtained from female plants contain characteristic isoperoxidase fractions. A slight peroxidase activity and isoperoxidases are detected in the residual nutritient medium collected from cell suspensions.

Effects of preezing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of pvp, sucrose, or glycerol

Based on results with microorganisms, one theory of freezing damage in cells states that the survival of a frozen-thawed cell is critically dependent on the rate at which it is cooled to subzero temperatures. The hypothesis suggests that the ultimate survival of a frozen and thawed cell is a function of the interaction of two factors that are oppositely dependent on cooling rate: 1) exposure to solution effects such as increased electrolyte concentration, pH changes, and dehydration and 2) the formation of intracellular ice during cooling and the extent of its recrystallization during warming. Using the colony-forming ability of the stem cells of mouse marrow as an assay of viability, we have sought to test the applicability of this theory to freezing damage in a nucleated mammalian cell. Stem cells suspended in each of three concentrations of glycerol, two of polyvinylpyrrolidone (mol wt, 40,000), or one of sucrose were frozen at rates varying from 0.3 to 500°C per min to −196°C, held for about 1 hr, and then thawed rapidly at about 900°C per min. In a second type of experiment, cells cooled at various rates were warmed either rapidly or slowly. Under all conditions, the survival of the colony-forming ability of the stem cells varied as a function of cooling rate, showing a distinct maximum at one rate. The optimum rate, however, varied with the specific additive and its concentration. For example, maximum survival was obtained for cells suspended in 1.25 m, 0.8 m, and 0.4 m glycerol and cooled at 1.5°C per min, 15°C per min, and 100°C per min, respectively. If cells in 1.25 m glycerol were cooled at 1.5°C per min, survival was about 65%, regardless of whether the suspensions were thawed at 2°C per min or 900°C per min, but if these cells were cooled at 200°C per min survival was 25% when the cells were thawed at 900°C per min and only 5% when they were thawed at 2°C per min. Good survival was also obtained in two additives that we believe to be nonpermeating, namely PVP and sucrose. Here, too, survivals showed distinct maxima as a function of cooling rate. Moreover, since the maximum survival was about 55% for cells suspended in 0.35 m sucrose but only 20% for cells in 0.4 m glycerol, it appears as if a nonpermeating additive is more effective on a molar basis in preventing freezing damage than is glycerol which has been assumed to permeate nucleated cells. All of these results are consistent with the “two-factor” theory of freezing damage developed from findings with microorganisms.