Julie P. Goff

Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts

In vivo and in vitro studies indicate that a subpopulation of human marrow‐derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17–90 years). We tested the effect of age on senescence‐associated β‐galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence‐associated β‐galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7‐fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age‐related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age‐related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age‐related decrease in generation of osteoblasts both in the STRO‐1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age‐dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence‐associated β‐galactosidase‐positive cells and apoptosis in hMSCs. Up‐regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcεRI or FcγRI

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Modeling Stem Cell Population Growth: Incorporating Terms for Proliferative Heterogeneity

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle‐derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Two Strategies for the Development of Mitochondrion-Targeted Small Molecule Radiation Damage Mitigators

PURPOSE To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules. METHODS AND MATERIALS We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53(+/+) and p53(-/-) murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI). RESULTS Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53(+/+) (p = 0.0396 and p = 0.0071, respectively) and p53(-/-) cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53(-/-) cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively). CONCLUSION Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

Automated measurement of cell motility and proliferation

BackgroundTime-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data.ResultsWe have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth.ConclusionThese results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype.

Lack of DNA Polymerase θ (POLQ) Radiosensitizes Bone Marrow Stromal Cells In Vitro and Increases Reticulocyte Micronuclei after Total-Body Irradiation

Abstract Mammalian POLQ (pol θ) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq+/+ and Polq−/− mice. Aging of bone marrow cultures was not altered, but Polq−/− cells were more sensitive to γ radiation than were Polq+/+ cells. The D0 was 1.38 ± 0.06 Gy for Polq+/+ cells compared to 1.27 ± 0.16 and 0.98 ± 0.10 Gy (P  =  0.032) for two Polq−/− clones. Polq−/− cells were moderately more sensitive to bleomycin than Polq+/+ cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in γ-irradiated Polq−/− cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq+/+ cells slightly but did not affect Polq−/− cells. Polq−/− mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq+/+ and +/− mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks.

Synergistic Effects of Hepatocyte Growth Factor on Human Cord Blood CD34+ Progenitor Cells are the Result of c‐met Receptor Expression

Hepatocyte growth factor (HGF) is a pleiotropic growth factor which, in addition to its mitogenic potency for primary hepatocytes, also has a role in the regulation of cell motility, cell growth and morphogenesis. In the present study, we show that c‐met, the high‐affinity receptor for HGF, is expressed on human cord blood (CB) CD34+ progenitor cells and CD34+Thy‐1+ Lin− (lin−) cells. We have investigated the capacity of HGF to synergize with other growth factors to induce colony formation by CB CD34+ progenitor cells. CD34+ cells were cultured in semisolid medium containing serum with increasing concentrations of GM‐CSF, G‐CSF, macrophage colony‐stimulating factor (M‐CSF), stem cell factor (SCF), interleukin 3 (IL‐3) and IL‐11 alone or in combination with HGF. HGF acted as a potent synergist and enhanced, up to fourfold, colony formation induced by GM‐CSF, G‐CSF or M‐CSF. HGF in combination with SCF, IL‐3 or IL‐11 did not induce proliferation of colony forming units‐granulocyte macrophage (CFU‐GM) above control levels. In serum‐deprived cultures, HGF was only detectably synergistic with IL‐11, and all other culture combinations showed no proliferation. To determine whether the stimulatory effect of IL‐11 and the synergistic effect of HGF in the absence of serum could be attributed to the effect of these two cytokines on stem cells, IL‐11‐stimulated and unstimulated lin− cells were analyzed for expression of c‐met. CD34+Thy‐1+Lin− cells were positive for c‐met, both in the presence and absence of IL‐11 stimulation, and Northern analysis indicated that c‐met RNA expression was upregulated in response to IL‐11 compared to unstimulated controls. These results provide strong evidence for upregulation of the HGF receptor on primitive hematopoietic cells by IL‐11, and for the synergistic role of HGF in colony formation by hematopoietic stem cells.

Effects of recombinant cytokines on colony formation by irradiated human cord blood CD34+ hematopoietic progenitor cells

The role of recombinant hematopoietic growth factors in radiation repair has become a subject of increasing interest in both clinical and basic radiobiology. Combinations of cytokines such as hepatocyte growth factor, interleukin (IL)-3, IL-11, kit ligand, GM-CSF and erythropoietin were used to study the in vitro radiation dose response of human cord blood CD34+ hematopoietic progenitor cells using clonogenic survival assays. CD34+ cells were isolated by immunomagnetic selection and irradiated at 8 cGy/min. Irradiated cells were plated in methylcellulose with or without added cytokines, and hematopoietic colonies including CFU-GM, BFU-E and CFU-GEMM were scored on day 14. The radiation response characteristics of BFU-E and CFU-GEMM were similar for all culture conditions tested. The D0 values for BFU-E ranged between 1.29 and 2.40 Gy and n between 1.0 and 1.4. The D0 values for CFU-GEMM ranged from 86 cGy to 2.02 Gy and n between 1.0 and 1.5. The D0 for CFU-GM grown without added factors was 1.03 Gy. With single cytokine stimulation (IL-3, IL-11 or varying concentrations of HGF), D0 values ranged from 1.11 to 1.44 Gy. With the combination of IL-3, GM-CSF, kit ligand and HGF, D0 values were not significantly altered and ranged between 1.61 and 2.60 Gy. In contrast, the combination of IL-11 and HGF produced an increase in the shoulder of the radiation survival curve (n = 3.35). No increase in the shoulder was detected for any of the other conditions tested (n = 1.0-1.7). Thus the combination of HGF and IL-11 increased the radiation survival of hematopoietic progenitor cells forming CFU-GM. Understanding the mechanism by which combinations of early-acting growth factors support postirradiation recovery of primitive clonogenic hematopoietic cells may be relevant to the design of clinical protocols for improving hematopoietic recovery after total-body irradiation.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Intraesophageal manganese superoxide dismutase-plasmid liposomes ameliorates novel total-body and thoracic radiation sensitivity of NOS1-/- mice.

Abstract The effect of deletion of the nitric oxide synthase 1 gene (NOS1−/−) on radiosensitivity was determined. In vitro, long-term cultures of bone marrow stromal cells derived from NOS1−/− were more radioresistant than cells from C57BL/6NHsd (wild-type), NOS2−/− or NOS3−/− mice. Mice from each strain received 20 Gy thoracic irradiation or 9.5 Gy total-body irradiation (TBI), and NOS1−/− mice were more sensitive to both. To determine the etiology of radiosensitivity, studies of histopathology, lower esophageal contractility, gastrointestinal transit, blood counts, electrolytes and inflammatory markers were performed; no significant differences between irradiated NOS1−/− and control mice were found. Video camera surveillance revealed the cause of death in NOS1−/− mice to be grand mal seizures; control mice died with fatigue and listlessness associated with low blood counts after TBI. NOS1−/− mice were not sensitive to brain-only irradiation. MnSOD-PL therapy delivered to the esophagus of wild-type and NOS1−/− mice resulted in equivalent biochemical levels in both; however, in NOS1−/− mice, MnSOD-PL significantly increased survival after both thoracic and total-body irradiation. The mechanism of radiosensitivity of NOS1−/− mice and its reversal by MnSOD-PL may be related to the developmental esophageal enteric neuronal innervation abnormalities described in these mice.