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Gerald A Colvin

Publications and source records attributed to Gerald A Colvin.

At least 19 recordsLinked to original sources
PubMed2007

Murine allogeneic in vivo stem cell homing(,).

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2-mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin-Sca-1+ cells were labeled with CFSE and injected in non-myeloablated BALB/c mice. Fluorescent cell detection was via high-speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony-forming cell (HPP-CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP-CFC. Combining experiments and normalizing the 1-h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 +/- 29%, 72 +/- 21%, 84 +/- 35% of the 1 h level at 3, 6, and 24 h respectively. HPP-CFC assay showed no significant variation as a homing surrogate over 1-6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells.

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PubMed2007

Stem cell continuum: directed differentiation hotspots.

OBJECTIVE: The purpose of this study was to evaluate the technique of stem cell-directed differentiation in the context of cell-cycle position. The hypothesis was that stem cells would have different sensitivities to an identical inductive signal through cell-cycle transit and that this would affect the outcome of its progeny. MATERIALS AND METHODS: Differentiation of murine marrow lineage(negative)rhodamine-123(low-)Hoechst-33342(low) (LRH) stem cells was determined at different points in cell cycle under stimulation by thrombopoietin, flt3 ligand, and steel factor. LRH stem cells were subcultured in granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and steel factor at different points in cell cycle and differentiation determined 14 days later. RESULTS: There was a significant, reproducible, and pronounced reversible increase in differentiation to megakaryocytes in early S-phase and to nonproliferative granulocytes in mid S-phase. Megakaryocyte hotspots also were seen on a clonal basis. Elevations of the transcription factor FOG-1 were seen at the hotspot along with increases in Nfe2 and Fli1. CONCLUSIONS: We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell-cycle transit, suggesting that stem cell regulation is not based on the classic hierarchical model, but instead on a functional continuum. We propose that there is a tight linkage of commitment to a lineage and a particular phase of cell cycle. Thus, windows of vulnerability for commitment can open and close depending on the phase of cell cycle. These data indicate that stem cell differentiation occurs on a cell-cycle-related continuum with fluctuating windows of transcriptional opportunity.

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PubMed2006

Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype.

OBJECTIVE: Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. METHODS: In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin(-/+), c-kit(-/+), Sca-1(-/+)) from GFP(+) (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). RESULTS: The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP(+)/CD45(-)) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP(+)/CD45(-) cells were also cytokeratin(+). Mobilization further increased GFP(+)/CD45(-) cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP(+)/CD45(-) in radiation-injured mice transplanted with Lin(-), c-kit(+), or Sca-1(+) marrow cells. Lin(+), c-kit(-), and Sca-1(-) subpopulations did not significantly engraft the lung. CONCLUSIONS: We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell-producing marrow cell has the phenotype of a hematopoietic stem cell.

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PubMed2005

Critical variables in the conversion of marrow cells to skeletal muscle.

We have studied conversion of marrow cells to skeletal muscle in cardiotoxin-injured anterior tibialis muscle in a green fluorescent protein (GFP) to C57BL/6 transplantation model and ascertained that total body irradiation (TBI) with establishment of chimerism is a critical factor. Local irradiation has little effect in lower doses and was detrimental at higher doses. Whole body (1000 cGy) with shielding of the leg or a combination of 500 cGy TBI and 500 cGy local radiations was found to give the best results. In non-obese diabetic-severe combined immunodeficient (NOD-SCID) recipients, we were able to show that conversion could occur without radiation, albeit at relatively lower levels. Within 3 days of cardiotoxin injury, GFP-positive mononuclear cells were seen in the muscle, and within 2 weeks GFP-positive muscle fibers were identified. Conversion rates were increased by increasing donor-cell dose. Timing of the cardiotoxin injury relative to the transplantation was critical. These studies show that variables in transplantation and injury are critical features of marrow-to-muscle conversions. Irradiation primarily effects conversion by promoting chimerism. These data may explain the differences in the literature for the frequency of marrow-to-skeletal muscle conversion and can set a platform for future models and perhaps clinical protocols.

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PubMed2005

Lineage-negative bone marrow cells travel bidirectionally in the olfactory migratory stream but maintain hematopoietic phenotype.

The mammalian olfactory system is a physiologically plastic region of the brain with the potential to support implanted stem cells. We performed direct injection of lineage-negative (lin-neg), green fluorescent protein-positive (GFP+) bone marrow cells into the olfactory bulb to assess cell survival and motility within the central nervous system (CNS). Before direct injection of 100,000 lin-neg cells, some of the C57/Bl mice received 1,000 cGy brain irradiation with the aim of disabling the endogenous reservoir of periventricular neural progenitor cells. Brain harvest took place up to 2 weeks after cell implantation. Brains were evaluated for presence of GFP positivity via fluorescence microscopy. Many GFP+ cells were identified within the turbinate neuroepithelium, olfactory bulb, and frontal lobe. Most of the cells that had traveled from the implantation site adopted an elongated, arborizing morphology consistent with cellular extensions arrayed in the direction of the rostral migratory stream (RMS). No difference was seen in brain-irradiated versus non-irradiated mice. Antibody staining revealed that these cells did not take on a neural, glial, or endothelial phenotype, while largely retaining their hematopoietic lineage as demonstrated by CD45 positivity.

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PubMed2005

The stem cell continuum.

Hematopoietic stem cells have been felt to exist in a hierarchical structure with a relatively fixed phenotype at each stage of differentiation. Recent studies on the phenotype of the marrow hematopoietic stem cell indicate that it is not a fixed entity, but rather that it fluctuates and shows marked heterogeneity. Past studies have shown that stem cell engraftment characteristics, adhesion protein, and gene expression varies with the phase of the cell cycle. More recently, we demonstrated that progenitor numbers and differentiation potential also vary reversibly during one cytokine-induced cell cycle transit. We have also shown high levels of conversion of marrow cells to skeletal muscle and lung cells, indicating a different level of plasticity. Recently, we demonstrated that homing to lung and conversion to lung cells in a mouse transplant model also fluctuates reversibly with cell cycle transit. This could be considered plasticity squared. These data indicate that marrow stem cells are regulated on a continuum related to the cell cycle both as to hematopoietic and to nonhematopoietic differentiation.

Cell Cycle↗
PubMed2004

Intrinsic hematopoietic stem cell/progenitor plasticity: Inversions.

Traditional concepts indicate that stem cells give rise to progenitor cells in a hierarchical system. We studied murine engraftable stem cells (ESCs) and progenitors in in vitro and found that ESC and progenitors exist in a reversible continuum, rather then a hierarchy. B6.SJL and BALB/c marrow cells were serially cultured with thrombopoietin (TPO), FLT-3 ligand (FLT-3L), and steel factor through cell cycle. Progenitors (high-proliferative potential colony-forming cells (HPP-CFC) and colony-forming unit culture (CFU-c)) and ESC capacity was determined. The cell cycle status of purified lineage(negative)rhodamine(low)Hoechst(low) stem cells was determined under the same conditions using tritiated thymidine incorporation and cell counts. We found an inverse relationship between progenitors and ESC, which occurred during the first cell cycle transit and was reversible. We have termed these progenitor/stem cell inversions and found that these inversions were consistently seen at 28-32 h of culture, representing early S-phase. We observed 13 major reversible increases in progenitor numbers from one time-point to another during the first cell cycle transit; this was coupled with 11 major ESC decreases and in 2 instances ESC were at baseline. These studies indicate that primitive marrow cells reversibly shift from ESC to progenitors without differentiation occurring. They exist as a fluctuating continuum.

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PubMed2004

Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: a multifactorial process.

OBJECTIVE: Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the "robustness" of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow-to-muscle conversion. METHODS: We transplanted green fluorescence protein (GFP)-transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopoietic markers. RESULTS: Irradiation was essential to conversion, although whether by injury or induction of chimerism is not clear. Cardiotoxin- and, to a lesser extent, PBS-injected muscles showed significant number of GFP(+) muscle fibers, while uninjected muscles showed only rare GFP(+) cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent by G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57BL/6 and GFP to ROSA26 mice showed fusion of donor cells to recipient muscle. High numbers of donor-derived muscle colonies and up to 12% GFP(+) muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels that could be clinically significant in developing strategies for the treatment of muscular dystrophies. CONCLUSION: In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

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PubMed2003

Akt signaling regulates side population cell phenotype via Bcrp1 translocation.

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3' end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from "stem cell genes" being highly expressed at 0 h and turned off at 48 h, while "cell division" genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

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PubMed2003

The latest treatment advances for acute myelogenous leukemia.

The research into pathogenesis and mechanisms behind AML is advancing rapidly, but in general, translation into global application for the majority of patients is wanting. As more becomes known about the cytogenetic and molecular characteristics of leukemia cells and the pathways of leukemogenesis are further elucidated, it is hoped that future therapies will be directed more specifically toward the least toxic method to eradicate clonal malignant cells. HLA-haploidentical and alloBMT using KIR mismatch may dramatically improve survival for many more patients.

Antineoplastic Agents↗
PubMed2002

The chiaroscuro stem cell: a unified stem cell theory.

Hematopoiesis has been considered hierarchical in nature, but recent data suggest that the system is not hierarchical and is, in fact, quite functionally plastic. Existing data indicate that engraftment and progenitor phenotypes vary inversely with cell cycle transit and that gene expression also varies widely. These observations suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. This may, in turn, be dependent on shifting chromatin and gene expression with cell cycle transit. If the phenotype of these primitive marrow cells changes from engraftable stem cell to progenitor and back to engraftable stem cell with cycle transit, then this suggests that the identity of the engraftable stem cell may be partially masked in nonsynchronized marrow cell populations. A general model indicates a marrow cell that can continually change its surface receptor expression and thus responds to external stimuli differently at different points in the cell cycle.

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PubMed2002

H2-mismatched transplantation with repetitive cell infusions and CD40 ligand antibody infusions without myeloablation.

Graft rejection and graft-versus-host disease are major problems in mismatched marrow transplants along with toxicity from standard myeloablative host treatments. We have established a tolerization model, using 1 Gy irradiation, which reduces stem cell capacity to < 10% of control while causing minimal myelosuppression, donor antigen pre-exposure (spleen cells), CD40-ligand antibody blockade and high levels of marrow (40 x 106 cells), which allows for stable long-term multilineage engraftment in H2-mismatched murine marrow transplants. We now show that the establishment of 'microchimaerism' (0.5-3.8%) sets the stage for macrochimaerism, with subsequent marrow infusions in H2-mismatched mice with CD40-ligand blockade only. Neither irradiation nor spleen cell exposure were necessary. When 40 x 106 bone marrow cells were infused on weeks 0, 12, 14 and 16, blood engraftment was about seven times the single 40 x 106 control. When marrow cells were given on weeks 0, 3, 4, 5 and 6, engraftment at 24 weeks post transplant was 17.9 +/- 1.2%, compared with 2.7 +/- 0.8% for the single 40 x 106 control (P = 0.009). We have shown stable, long-term multilineage chimaerism and established that the schedule of marrow administration, not the total cell dose, is critical for tolerization. This approach indicates that microchimaerism can tolerize for subsequent marrow infusions and produce macrochimaerism. This strategy could be applied in clinical human transplants.

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PubMed2002

Engraftment of post 5-fluorouracil murine marrow into minimally myeloablated (100 cGy) murine hosts.

Minimal myeloablative approaches are now being widely applied in the treatment of different hematological malignancies. One hundred cGy whole-body irradiation is a stem-cell-toxic, relatively non-myelotoxic treatment that allows for relatively high levels of donor chimerism. 5-Fluorouracil (5-FU) treatment leads to a relative concentration of high proliferative potential-colony-forming cell (HPP-CFC) and is an approach that has been used to induce in vivo progenitor/stem cell cycling to facilitate retroviral integration in gene therapy approaches. We have now evaluated the capacity of marrow harvested 1, 2, 6, or 12 days after 5-FU treatment (150 mg/kg) to engraft in 100 cGy-treated female BALB/c mice. Engraftment was assessed at 3, 10, and 24 weeks. A rapid induction of an engraftment defect occurred 1 day post 5-FU and persisted through day 6 with a recovery by day 12. To evaluate cell cycle status of normal and 5-FU-treated marrow cells, male donors received hydroxyurea (900 mg/kg i.v.) or phosphate-buffered saline (PBS), 2 h prior to marrow harvest and transplantation into submyeloablated female recipients. Engraftment levels were similar for hydroxyurea-treated mice and controls. Thus, these studies show transiently defective engraftment of 5-FU-treated marrow into submyeloablated hosts, which may be related to the cell cycle status of the stem cells.

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PubMed2002

Rhythmicity of engraftment and altered cell cycle kinetics of cytokine-cultured murine marrow in simulated microgravity compared with static cultures.

Space flight with associated microgravity is complicated by "astronaut's anemia" and other hematologic abnormalities. Altered erythroid differentiation, red cell survival, plasma volume, and progenitor numbers have been reported. We studied the impact of microgravity on engraftable stem cells, culturing marrow cells in rotary wall vessel (RWV) culture chambers mimicking microgravity and in normal gravity nonadherent Teflon bottles. A quantitative competitive engraftment technique was assessed under both conditions in lethally irradiated hosts. We assessed 8-wk engraftable stem cells over a period spanning at least one cell cycle for cytokine (FLT-3 ligand, thrombopoietin [TPO], steel factor)-activated marrow stem cells. Engraftable stem cells were supported out to 56 h under microgravity conditions, and this support was superior to that seen in normal-gravity Teflon bottle cultures out to 40 h, with Teflon bottle culture support superior to RWV from 40 to 56 h. A nadir of stem cell number was seen at 40 h in Teflon and 48 h in RWV, suggesting altered marrow stem cell cycle kinetics under microgravity. This is the first study of engraftable stem cells under microgravity conditions, and the differences between microgravity and normal gravity cultures may present opportunities for unique future stem cell expansion strategies.

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PubMed2002

The new stem cell biology.

Recent studies have indicated that bone marrow stem cells are capable of generating muscle, cardiac, hepatic, renal, and bone cells. Purified hematopoietic stem cells have generated cardiac and hepatic cells and reversed disease manifestations in these tissues. Hematopoietic stem cells also alter phenotype with cell cycle transit or circadian phase. During a cytokine stimulated cell cycle transit, reversible alterations of differentiation and engraftment occur. Primitive hematopoietic stem cells express a wide variety of adhesion and cytokine receptors and respond quickly with migration and podia extensions on exposure to cytokines. These data suggest an "Open Chromatin" model of stem cell regulation in which there is a fluctuating continuum in the stem cell/progenitor cell compartments, rather than a hierarchical relationship. These observations, along with progress in using low dose treatments and tolerization approaches, suggest many new therapeutic strategies involving stem cells and the creation of a new medical specialty; stemology.

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