The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.     

Effects of zinc supplementation on diabetes mellitus: a systematic review and meta-analysis

ABSTRACT: The number of people with diabetes and pre-diabetes are exponentially increasing. Studies on humans have shown the beneficial effects of Zinc supplementation in patients with diabetes. The present study aims to systematically evaluate the literature and meta-analyze the effects of Zinc supplementation on diabetes. A systematic review of published studies reporting the effects of Zinc supplementations on diabetes mellitus was undertaken. The literature search was conducted in the following databases; PubMed, Web of Science and SciVerse Scopus. A meta-analysis of studies examining the effects of Zinc supplementation on clinical and biochemical parameters in patients with diabetes was performed. The total number of articles included in the present review is 25, which included 3 studies on type-1 diabetes and 22 studies on type-2 diabetes. There were 12 studies comparing the effects of Zinc supplementation on fasting blood glucose in patients with type-2 diabetes. The pooled mean difference in fasting blood glucose between Zinc supplemented and placebo groups was 18.13mg/dl (95%CI:33.85,2.41; p<0.05). 2-h post-prandial blood sugar also shows a similar distinct reduction in (34.87mg/dl [95%CI:75.44; 5.69]) the Zinc treated group. The reduction in HbA1c was 0.54% (95%CI:0.86;0.21) in the Zinc treated group. There were 8 studies comparing the effects of Zinc supplementation on lipid parameters in patients with type-2 diabetes. The pooled mean difference for total cholesterol between Zinc supplemented and placebo groups was 32.37mg/dl (95%CI:57.39,7.35; p<0.05). Low-density lipoprotein cholesterol also showed a similar distinct reduction in the Zinc treated group, the pooled mean difference from random effects analysis was 11.19mg/dl (95%CI:21.14,1.25; p<0.05). Studies have also shown a significant reduction in systolic and diastolic blood pressures after Zinc supplementation. This first comprehensive systematic review and meta-analysis on the effects of Zinc supplementation in patients with diabetes demonstrates that Zinc supplementation has beneficial effects on glycaemic control and promotes healthy lipid parameters. Further studies are required to identify the exact biological mechanisms responsible for these results.     

Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells

The effects of stem cell factor (SCF) have been tested on a murine bone marrow subpopulation (RH123lo, Lin-, Ly6A/E+) that is highly enriched for long-term hematopoietic repopulating cells. SCF maintained cells from this population with long-term repopulating ability for up to 10 days in vitro. However, compared with freshly isolated cells, the level of engraftment in vivo by the cultured cells declined during the in vitro culture period, suggesting that SCF alone was unable to stimulate the self-renewal of long-term repopulating cells. By direct visualization of cultures, only small numbers of cells survived and rarely underwent cell division. However, SCF did directly stimulate proliferation of a population (Rh123med/hi,Lin-,Ly6A/E+) enriched for short-term repopulating cells. These data suggest that stem cell differentiation is associated with the development of mitogenic activity by SCF at least in some progenitor cell populations.     

The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.     

Heterogeneity in human neutrophil, macrophage and eosinophil progenitor cells demonstrated by velocity sedimentation separation

Progenitor cells of neutrophils, monocyte-macrophages, and eosinophils in human marrow were enumerated in agar cultures stimulated by placental conditioned medium or white cell underlayers. Fractionation of marrow populations by velocity sedimentation showed that the profiles of neutrophil and macrophage colony-forming cells shifted from a peak of 8-9 mm/hr in 7-day cultures to a peak of 6-7 mm/hr in 14-day cultures. This shift was due to degeneration of some early colonies formed by rapidly sedimenting cells and the delayed formation of colonies by slowly sedimenting cells. Eosinophil colony formation was delayed until the second week of incubation. Further evidence of heterogeneity was the observation that rapidly sedimenting colony forming cells were more responsive to stimulation than more slowly sedimenting cells. In the macrophage and eosinophil populations, cluster-forming cells were partially segregatable form colony-forming cells. The observed heterogeneity was similar to the described previously in the mouse and suggests that separate subpopulations of progenitor cells may exist within each hemopoietic family that could possibly give rise to functionally different progeny.     

Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization

Murine bone marrow cells were fractionated by fluorescence-activated cell sorting into Rh123lo Lin- c-kit+ Ly6A+, Rh123hi Lin-c-kit+ Ly6A+, and Lin- c-kit+ Ly6A- populations within which most, if not all, of the hematopoietic activities of the marrow resided. The Rh123lo Lin- c- kit+Ly6A+ cells, which consist exclusively of small- or medium-sized lymphocyte-like cells, are highly enriched for long-term hematopoietic in vivo repopulating cells. The enrichment factor for these cells from the marrow was estimated as 2,000-fold. The Rh123hi Lin- c-kit+ Ly6A+ cells, although also highly enriched for day-12 spleen colony-forming units, were relatively depleted of long-term in vivo repopulation capacity. Most, if not all Lin- c-kit+ Ly6A- cells were Rb123hi. In contrast to both Rh123lo and Rh123hi Lin- c-kit+ Ly6A+ stem cell populations, the Lin- c-kit+ Ly6A- cells can be stimulated to proliferate in vitro in the presence of single cytokines, which is a characteristic of committed progenitor cells. No marked synergistic interactions between individual cytokines were observed with this cell population. Both Rh123hi Lin- c-kit+ Ly6A+ mature stem cell and Lin- c- kit+ Ly6A- progenitor cell populations displayed in vivo repopulation kinetics resembling those of the putative short-term hematopoietic repopulating cells.     

Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors

A microculture assay for murine granulocyte-macrophage colony- stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five- fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM- CSF for several months. Microassay results for several sources of GM- CSF at different stages of purification are compared to the results obtained from the soft agar assay.     

High titer anti-HIV antibody reactivity associated with a paraprotein spike in a homosexual male with AIDS related complex

We observed a human immunodeficiency virus (HIV)-infected homosexual male with AIDS related complex (ARC) who had a serum globulin level of 80 g/L. Serum protein electrophoresis revealed a gamma globulin fraction of 40 g/L, of which 50% (20 g/L) was contained within a paraprotein spike, comprised predominantly of IgG kappa. This patient also had high titer anti-HIV antibodies in his serum, which were Western blot reactive at a final dilution of 1:500,000, and recognized gp120env, p66pol, p55gag, p53pol, p41gag, and p24gag. Because paraproteins in the past have been shown to be directed against specific antigens, we purified this patient's paraprotein using a modified high performance liquid chromatography (HPLC)-hydroxylapatite procedure and tested the purified paraprotein for anti-HIV antibody activity. The purified paraprotein retained anti-HIV antibody activity to a final dilution of 1:100,000, and recognized p66pol, p55gag, p53pol, p41gag, and p24gag. The recognition of both "gag" and "pol" gene products suggested that the purified paraprotein might not be monoclonal in origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified paraprotein contained at least two immunoglobulin light chain species (Mol wt 30 to 33 Kd). Affinity chromatography of the purified paraprotein using a p24- Sepharose 4B matrix separated the "gag" and "pol" antibody activities. Immunoglobulin gene rearrangement analysis of a bone marrow aspirate (which contained 15% plasma cells) failed to reveal a clonal population of immunoglobulin producing cells. We conclude that this patient's paraprotein accounted for most of the anti-HIV activity present in whole serum, and that this paraprotein was not monoclonal in origin.