Kinetics Of Progenitor Cells Research Articles

Altered kinetics of circulating vascular progenitors in patients with cardio-pulmonary bypass associated vasoplegia

Background and Aims: Patients undergoing cardio-pulmonary bypass (CPB) surgery exhibit endothelial dysfunction and vascular damage, often leading to post-operative vasoplegia. Circulating vascular progenitors (CVPs) and endothelial progenitor cells (EPCs) are known to routinely home into the site of damaged endothelium and vessel wall for repair. The aim of this pilot study was to enumerate real time kinetics of progenitor cells during different stages of CPB and to determine the association of the trend with severity of vasoplegia.

Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds

In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.

Keratinocyte Growth Factor (KGF) Modulates Epidermal Progenitor Cell Kinetics through Activation of p63 in Middle Ear Cholesteatoma.

The basal stem/progenitor cell maintains homeostasis of the epidermis. Progressive disturbance of this homeostasis has been implicated as a possible cause in the pathogenesis of epithelial disease, such as middle ear cholesteatoma. In many cases of stem/progenitor cell regulation, the importance of extracellular signals provided by the surrounding cells is well-recognized. Keratinocyte growth factor (KGF) is a mesenchymal-cell-derived paracrine growth factor that specifically participates in skin homeostasis; however, the overexpression of KGF induces middle ear cholesteatoma. In this study, two kinds of thymidine analogs were transferred at different time points and we investigated the effects of overexpressed KGF on the cell kinetics of stem/progenitor cells in vivo. As a result, BrdU(+)EdU(+) cells (stem/progenitor cells) were detected in the thickened epithelium of KGF-transfected specimens. The use of a high-resolution microscope enabled us to analyze the phosphorylated level of p63 in individual nuclei, and the results clearly demonstrated that BrdU(+)EdU(+) cells are regarded as progenitor cells. In the overexpression of KGF, the stimulation of progenitor cell proliferation was inhibited by SU5402, an inhibitor for tyrosine kinase of KGFR. These findings indicate that KGF overexpression may increase stem/progenitor cell proliferation and block terminal differentiation, resulting in epithelial hyperplasia, which is typical in middle ear cholesteatoma.

Protein Segregation Between Dividing Hematopoietic Progenitor Cells in the Determination of the Symmetry/Asymmetry of Cell Division

In the present study, we investigated how the symmetry/asymmetry of cell division in mitotic CD34(+) cells can be evaluated by determining the plane of cell division and the potential distribution of proteins between daughter cells. The orientation of the mitotic spindle is dependent upon the positioning of the centrosomes, which determine the plane of cell division and the sharing of proteins. If the functions of unequally shared proteins are relevant to the kinetics of cell division, they could determine whether the daughter cells undergo self-renewal or differentiation. The kinetic function of the proteins of interest was investigated using a colony-replating assay and carboxyfluorescein succinimidyl ester (CFSE) staining. We used Notch/Numb as a model system, since they have a role in balancing symmetric/asymmetric divisions. Mitotic cells were examined microscopically and centrosomal markers γ-tubulin/pericentrin were used with activated Notch-1 and Numb. We monitored the first crucial divisions by CFSE staining and found an inverse relationship between activated Notch and Numb expression, suggesting a reciprocal regulation. We suggest that the subpopulations expressing activated Notch or Numb have different cell fates. To determine the influence of Notch signaling on progenitor cell self-renewal, we used the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester (DAPT). DAPT influences self-renewal/differentiation outcome by affecting the frequency of symmetric renewal divisions without affecting the rate of divisions. Overall, the purpose of this study was to establish a cellular system for predicting the symmetry/asymmetry of hematopoietic progenitor divisions at the level of centrosomes and protein distribution and to investigate the influence of these proteins on progenitor cell kinetics.

CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

AbstractAbstract 1452The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment.The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations.At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures:Sakamoto:Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

Temporal changes in stem cells in the circulation and myocardium of mice with Coxsackie virus B3-induced myocarditis

Our goal was to investigate temporal changes in stem cell in the circulation and myocardium of mice with Coxsackie virus B3-induced myocarditis. Groups of mice were administered Eagle's minimal essential medium or virus solution. The animals were further divided into six subgroups based on the following time points post-inoculation: 1, 3, 7, 14, 21, and 28 days. Ten animals were studied in each subgroup. Circulating blood mononuclear cells were collected from the heart and analyzed using flow cytometry. Myocardial inflammation, stem cell expression, and cell proliferation were detected by histology and immunofluorescence. H&E staining revealed neutrophil infiltration and bleeding by day 3 post-infection. Myeloperoxidase and reactive oxygen species levels peaked by day 3 and were followed by myocyte loss and collagen deposition. Circulating mesenchymal stem cells also peaked by day 3. In contrast, hematopoietic stem cells remained sustained increase within day 14. Immunohistochemical microscopy also showed a marked increase in cardiac stem cells by day 14. The kinetics of this increase was consistent with a rise in proliferating cells expressing nuclear and cytoplasmic proteins that are typical of cardiomyocyte or vascular endothelial cells. These results demonstrate the rapid kinetics of progenitor cells during viral myocarditis and suggest that the optimal time to administer cell therapy to induce heart repair is within 2 weeks after viral infection.

Implications of hemopoietic progenitor cell kinetics and experimental leukemogenesis: Relevance to Gompertzean mortality as possible hematotoxicological endpoint

The aim of this study is to investigate a possible implication in cell kinetics of the hematopoietic progenitors to the experimental leukemogenesis to elucidate the relevance of various leukemic mode of action to Gompertzean survival curves, a new parameter based on the lifespan. Mice, C3H/He, and C57BL/6 strain, male and female, with or without genetic modifications, e.g., p53-deficiency or thioredoxin overexpression were used in the present hemopoietic stem/progenitor research, radiation- or benzene-induced leukemogenesis followed by histopathological examination. A lethal dose of radiation for bone marrow transplantation, and a graded increased dose up to 5 Gy of x-rays for induction of hematopoietic malignancies were given. For caloric restriction studies, 77 kcal/week was maintained in accordance to different restriction-timing. For assays of hematopoietic colonization, colony-forming unit spleen and colony-forming unit granulocyte macrophage were evaluated. Hematopoietic progenitor cell-specific kinetics were studied by continuous labeling of bromodeoxyuridine for cycling cells, followed by ultraviolet (UV) exposure and hemopoietic colonization (bromodeoxyuridine UV [BUUV] method). Various experimental survival curves were applied to a mathematical analysis by Gompertz-Makeham law of mortality. Referring current authors' studies on leukemogenesis induced by ionizing radiation and benzene exposure, implications of hematopoietic progenitor cell kinetics to the experimental leukemogenesis were evaluated by means of a novel experimental tool, the BUUV method. Comparative studies to elucidate relevancies of these data, including two prevention studies, one on caloric restriction and the other on antioxidative thioredoxin overexpression, to those Gompertzean survival curves of experimental animals were analyzed. The Gompertzean expression may elucidate an appropriate toxicological endpoint for evaluating the effect of radiation and/or benzene-exposure on the lifespan and its modification by various experimental preventive measures.

A role for the Fas/Fas ligand apoptotic pathway in regulating myeloid progenitor cell kinetics.

Bone marrow from wild-type mice and mice with mutated Fas (lpr) or mutated Fas ligand (gld) was used to investigate the role of the Fas/FasL system in the regulation of myeloid progenitor cell kinetics.Granulocyte-macrophage colony-forming cells (CFU-GM) were measured by a standard colony assay and the proliferative activity of CFU-GM was measured by replating primary colonies and observing secondary colony formation. Fas expression was restored to lpr mouse bone marrow cells by retrovirus-mediated gene transfer and gld mouse marrow cells were treated with soluble FasL. Wild-type marrow cells were treated with YVAD (a caspase inhibitor) or anti-Fas monoclonal antibodies. There were greater frequencies of myeloid progenitor cells (CFU-GM) in lpr and gld mouse marrow compared to wild-type (WT) marrow (p = 0.0008). The proliferative capacity of CFU-GM was also significantly greater for lpr and gld CFU-GM compared to WT CFU-GM (p = 0.0003 and 0.0001, respectively). Retrovirus-mediated restoration of Fas into lpr marrow, and provision of soluble FasL (sFasL) to gld CFU-GM reduced CFU-GM proliferation to WT levels. Treatment of WT CFU-GM with YVAD or anti-FasL monoclonal antibody increased CFU-GM proliferation to the levels found in lpr and gld CFU-GM. YVAD significantly increased and anti-Fas significantly reduced the proliferative capacity of human CFU-GM (p = 0.015 and 0.04, respectively).Fas, FasL, and caspase activation may play an important role in regulating myeloid progenitor cell kinetics.

Molecular pathways in bone marrow homing: dominant role of α4β1 over β2-integrins and selectins

AbstractThe specific retention of intravenously administered hemopoietic cells within bone marrow is a complex multistep process. Despite recent insights, the molecular mechanics governing this process remain largely undefined. This study explored the influence of β2-integrins on the homing to bone marrow and repopulation kinetics of progenitor cells. Both antifunctional antibodies and genetically deficient cells were used. In addition, triple selectin-deficient mice were used as recipients of either deficient (selectin or β2) or normal cells in homing experiments. The homing patterns of either β2 null or selectin null cells into normal or selectin-deficient recipients were similar to those of normal cells given to normal recipients. Furthermore, spleen colony-forming units and the early bone marrow repopulating activity for the first 2 weeks after transplantation were not significantly different from those of control cells. These data are in contrast to the importance of β2-integrin and selectins in the adhesion/migration cascade of mature leukocytes. The special bone marrow flow hemodynamics may account for these differences. Although early deaths after transplantation can be seen in recipients deficient in CD18 and selectin, these are attributed to septic complications rather than homing defects. However, when β2- or selectin-null donor cells are treated with anti-α4 antibodies before their transplantation to normal or selectin-deficient recipients, a dramatic inhibition of homing (>90%) was found. The data suggest that the α4β1/vascular cell adhesion molecule-1 pathway alone is capable of providing effective capture of cells within the bone marrow, but if its function is compromised, the synergistic contribution of other pathways, that is, β2-integrins or selectins, is uncovered.

Portofino International Conference on Chronic Myeloid Leukemia.

The 4 th International Conference on Chronic Myeloid Leukemia, held September 22–25, 2000 in Rapallo-Portofino, was designed to address and reach consensus on the most important issues concerning this disease. Some of the world’s leading research physicians and scientists gathered in this idyllic setting. Their task was to assess the “state of the art” on recent data concerning the structure and function of the BCR-ABL gene and mechanisms of cellular transformation. At the cell biology level, new contributions covered the kinetics of progenitor cells, abnormalities in t/he adhesion/integrin system, leukemogenesis in murine models, telomere length, and characterization of quiescient stem cell populations. Other topics discussed in detail focused on conventional therapy with recombinant -interferon, allogeneic transplantation (including mini-allografting), and autologous transplantation with unselected or in vivo mobilized diploid cells. The situation has become even more complex with the discovery and the initial results of the tyrosine kinase inhibitors. A summary of the presentations and discussion of these results follows.

Timing of recombinant human granulocyte colony-stimulating factor administration on neutropenia induced by cyclophosphamide in normal mice.

The effects of altering the timing of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration on neutropenia induced by cyclophosphamide (CPA) were studied experimentally in a mouse model. Experimental mice were divided into three groups: (a) treatment with rhG-CSF after CPA administration (post-treatment group); (b) treatment with rhG-CSF both before and after CPA administration (pre- and post-treatment group); and (c) treatment with saline after CPA administration (control group). The results were as follows. Mice receiving rhG-CSF on the 2 days preceding CPA treatment, in which progenitor cell counts outside the S-phase when CPA was administered were the lowest of all the groups, showed accelerated neutrophil recovery but decreased neutrophil nadirs compared with the control group despite rhG-CSF treatment. The pre- and post-treatment group, consisting of mice who received rhG-CSF treatment on days -4 and -3 before CPA treatment, and in which progenitor cell counts when CPA was administered were increased to greater levels than in the other groups, showed remarkably accelerated neutrophil recovery and the greatest increase in the neutrophil nadirs of all the groups. These results suggested that the kinetics of progenitor cell populations when chemotherapeutic agents were administered seemed to play an important role in neutropenia after chemotherapy, and that not only peripheral neutrophil cell and total progenitor cell counts but also progenitor cell kinetics should be taken into consideration when administering rhG-CSF treatment against the effects of chemotherapy.

Circulating Myeloid Progenitor Cell Kinetics during Hematologic Recovery from Chemotherapy and Subsequent Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Administration

Hematopoietic recovery from chemotherapy may be associated with an increase in circulating myeloid progenitor cell concentration (CFU-GM); these cells may be harvested by apheresis and used for autologous transplantation after high-dose cytoreductive therapy. Not all patients will demonstrate this increase, possibly due to damage to the stem cell compartment from prior chemoradiotherapy. Elevated circulating CFU-GM has also been reported in patients after short-term administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF); whether elevation would persist during longer duration is unknown. We measured circulating CFU-GM (by both limiting dilution in liquid culture and colony formation in semisolid media) in patients with sarcoma who began infusion of rhGM-CSF during recovery from chemotherapy. Patients with elevated circulating CFU-GM did not sustain these levels during subsequent rhGM-CSF infusion. By contrast, patients without rebound elevation of circulating CFU-GM following chemotherapy recovery did increase CFU-GM levels with rhGM-CSF administration. The proportion of marrow CFU-GM in cell cycle during chemotherapy recovery was elevated in both patient groups and remained elevated with rhGM-CSF administration. Both marrow and peripheral blood limiting dilution assays demonstrated linear growth kinetics, indicating a direct effect of the in vitro growth factor (also rhGM-CSF) on progenitor cells without excessive influence or dependence on accessory cells in culture. The use of rhGM-CSF to restore circulating CFU-GM for apheresis during recovery in patients lacking such elevation merits further study.

Is There an Optimal Hemoglobin Level?

THE RED CELL mass is an organ suspended in plasma and designed almost exclusively for the purpose of carrying oxygen from the lungs to the tissues. It is composed of erythron units, each originating from a proerythroblast. This precursor cell matures, synthesizes hemoglobin, and over a period of 4 to 5 days, undergoes four mitotic divisions resultingin the formation of 16 long-lived red blood cells (Fig 1). The marrow precursor cells are the end product of erythroid marrow progenitor cells. Our current concept of the kinetics of progenitor cells is that they are derived from a pool of multipotential stem cells capable of lifelong self-renewal (Fig 1). These stem cells can differentiate into a number of unipotential progenitor cells, including the erythroid progenitors. The early erythroid progenitors, the burst forming cells-erythroid (BFU-E), have only a limited capacity for self-renewal, and depend to a great extent on influx of cells from the stem-cell pool. In the presence of various nonspecific growth factors such as granulocyte monocyte-eolony stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin, they will mature and multiply until they reach an end-stage, the colony forming unit-erythroid (CFU-E). In the presence ofthe erythropoietic hormone, erythropoietin, some of these cells will undergo blast transformation to a proerythroblast and start the formation of a new erythron. The total size of the red cell mass can be measured accurately by labeling a small number of red cells with various isotopes, primarily chromium 51, and determining the dilution of label 10 to 15 minutes after their infusion. The major problem with this technique is in the expression of the measured volume in a meaningful manner. The measurement is most often expressed in milliliters per

Erythroid progenitor cell kinetics in chronic haemodialysis patients responding to treatment with recombinant human erythropoietin

The response of bone marrow and peripheral blood erythroid progenitors to human recombinant erythropoietin (rHuEPO) was studied in nine haemodialysed renal failure patients receiving this hormone for the correction of their anaemia. The haematocrit rose in all patients in response to thrice weekly injections of escalating rHuEPO doses (12-192 IU/kg). Both the numbers of CUF-e and BFU-e and their proliferative state in the bone marrow as well as BFU-e numbers in the peripheral blood were estimated before treatment and again after correction of the anaemia, at 16 h following an intravenous dose of rHuEPO. Following treatment bone marrow BFU-e numbers fell to a mean of 24.5% (P less than 0.01) of the pre-treatment values although there was no significant change in CFU-e or circulating BFU-e numbers. The mitotic rate (percentage S-phase cells) estimated by tritiated thymidine suicide rose from 45.2% to 68.4% (P less than 0.05) in the case of CFU-e and from 16.4% to 45.1% (P less than 0.05) for BFU-e following treatment with rHuEPO thus indicating in-vivo sensitivity of both the primitive as well as the mature erythroid progenitors to the hormone. The fall in BFU-e numbers in the bone marrow after several months of treatment may be due to a loss of cells from this progenitor pool by maturation that is uncompensated by replacement from the pluripotential stem cell compartment.

Progenitor cell kinetics during guided tissue regeneration in experimental periodontal wounds.

The present investigation compared the premitotic labelling of cells in the bone and periodontal ligament (PDL) compartments apical to a surgically created fenestration wound containing a “periodontal space.” Buccal fenestration wounds (3x3 mm) were made at midroot level in 4 Macaca fascicularis monkeys. The exposed root surfaces were planed and a piece of millipore filter was placed over each fenestration prior to flap replacement. Wounding in the different dentition segments and sacrifice of monkeys were scheduled to provide observation periods of I h I, 2, 3, 7 and 21 d in each monkey. One hour prior to sacrifice, tritiated thymidine (1 μCi/gm body weight) was administered intravenously. Autoradiographs were used to determine the labeling index (L.I.) of fibroblast‐like cells in each of the three following histologic compartments: PDL, alveolar bone adjacent to the apical border of the fenestration, and the wound proper. No significant differences in the L.I. between the bone and PDL tissue compartments were noted for nearly all of the observation periods. L.I. in the PDL peaked at 2 d while the alveolar bone demonstrated two peaks of activity, one at 2 d and the other at 7 d. Labeled cells first appeared at the edge of the wound proper at 3 d. At 7 d, labeled cells were noted throughout the wound compartment indicating continued activity of migrated cells. The 21‐d specimens demonstrated a few labeled cells in the wound compartment and a reduction of mitotic activity in the adjacent PDL and bone compartments to almost normal levels. The results of the present investigation suggest that both the bone and PDL tissue compartments supply cells to a periodontal wound containing a surgically created periodontal space. Moreover, some of the cells which migrate into the potential space formed by the physical barrier continue to prepare for mitosis as they migrate.