Stefan O. Peters

Lymphohematopoietic stem cell engraftment

Abstract: Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation.

Influence of Short-term use of Dexamethasone on the Pharmacokinetics of Ifosfamide in Patients

Dexamethasone induces the hepatic cytochrome P450 3A and, therefore, is predicted to change the pharmacokinetics, activities, and side effects of drugs metabolized by cytochrome P450 3A. The aim of this study was to determine whether the pharmacokinetics of the cytochrome P450 3A-dependent oxazaphosphorine cytostatic drug ifosfamide is influenced by short-term antiemetic use of dexamethasone in patients. The peak concentration and area under the curve (AUC) were determined for the parent compound and the metabolites 4-hydroxyifosfamide and chloracetaldehyde in eight patients who received two cycles of ICE chemotherapy (ifosfamide 5 g/m2 day 1, carboplatin 300 mg/m2 day 1, etoposide 100 mg/m2 days 1-3). One cycle included concomitant administration of dexamethasone (40 mg over 30 min, 16 h and 1 h before chemotherapy), whereas the other did not. The half-lives of ifosfamide, 4-hydroxyifosfamide, and chloracetaldehyde were shorter with concomitant administration of dexamethasone, but the differences were not statistically significant. In addition, there were no significant differences in the ifosfamide and active 4-hydroxyifosfamide peak concentrations and AUCs when dexamethasone was included. After dexamethasone administration, the chloracetaldehyde peak concentration was slightly increased by 1.5-fold and the AUC by 1.3-fold; however, these increases were not statistically significant. In conclusion, dexamethasone comedication in ICE chemotherapy did not change the ifosfamide pharmacokinetics. Thus, dexamethasone can be used safely as an antiemetic drug in ifosfamide chemotherapy.

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.

Cytokine Modulation of Murine Stem Cell Engraftment: The Role of Adherence to Plastic Surfaces

Murine marrow stem cells acquire an engraftment defect when cultured for 48 hours in cytokines, whereas the number of progenitor cells expands. Stem or progenitor cells have been noted to adhere to various surfaces, including plastic. Despite vigorous harvesting by cell scraping, the possibility existed that cytokines might induce selective adhesion of the rare engraftable stem cells to plastic surfaces.We have evaluated whether loss of engraftability by cytokine-treated marrow cells could be due to adhesion to plastic culture vessels. BALB/c marrow cells were cultured in the presence of interleukin 3 (IL-3), IL-6, IL-11, and steel factor for 48 hours in plastic tissue culture flasks from which cells were harvested by standard scraping and washing or after 5 or 10 minutes of additional exposure to trypsin (0.25%), or they were cultured with the same cytokines in nonadherent polytetrafluoroethylene (Teflon) culture bottles. Harvested cultured or fresh-starting male cells were then engrafted into nonirradiated female hosts or were placed in competition with fresh female BALB/c marrow in lethally irradiated female hosts. Defective engraftment was seen in nonmyeloablated or irradiated female hosts 7 to 24 weeks after marrow infusion in all cultured cell groups.These data indicate that cytokine-treated engraftable stem cells do not show significant adherence to plastic surfaces.

Hematopoietic Progenitor Cells Residing in Muscle Engraft into Bone Marrow following Transplantation

Hematopoietic and mesenchymal stem cells can potentially be the same cell type or adhere simultaneously in both bone marrow (BM) and muscle. In this study, we asked whether murine BM-derived cells could be tracked in muscle tissue after BM transplantation and whether muscle-derived cells have hematopoietic potential. To answer the first question, we transplanted BM from male BALB/c mice into irradiated female recipients and analyzed for engraftment. We used quantitative polymerase chain reaction (PCR) and fluorescent in situ hybridization techniques for Y chromosome—specific gene probes. A high number of BM-derived cells were located in both the intravascular and extravascular spaces in muscle tissue after BM transplantation. To answer the second question, we analyzed colony-forming potential in vitro with soft-agar assays and the competitive engraftment potential in vivo of muscle-derived cells. Engraftment levels of male cell populations were tested by quantitative PCR. The long-term engraftment potential of muscle-derived cells was low compared with that of BM. We conclude that there is intensive cellular trafficking between BM and muscle tissue. The engraftment potential of muscle-derived stem cells into BM is low and corresponds to the low amounts of hematopoietic colony-forming cells found in muscle tissue.