Bone marrow origin of esophageal stem cells

Purpose: Single fraction or fractionated irradiation of C3H/HeNsd mouse esophagus produces apoptosis of basal lining cells, microulceration, sloughing of epithelial tissues, and a syndrome of dehydration and weight loss. We have previously demonstrated that manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intraesophageal injection prior to single fraction 37 Gy reduces the magnitude of cytokine elevation, as well as the histopathological and cell biological changes associated with esophagitis, and improves survival [Epperly et al, Radiat. Res. 155:2-14, 2001]. To determine if specific cells in the esophagus represented targets for MnSOD-PL transgene protection, we isolated a population of self-renewing ESCs and determined if intraesophageal injection of an epitope-tagged hemagglutinin (HA)-MnSOD-PL (HA-MnSOD-PL) preparation targeted these cells, and was correlated with irradiation reduction in cell numbers. Materials and Methods: Adult C3H/HeNsd female mice were sacrificed and esophagus removed. Single cell suspensions of whole esophagus were sorted by the Hoechst/PI method of Goddell, or the serial preplate method of Huard, and a population of nonadherent cells with the phenotype of Sca1-lin-Thy1+ isolated. These cells, termed esophageal stem cells (ESCs), were also isolated from male C57BL/6J, GFP+ mice. ESCs were isolated from male C57BL/6J mice that had been intraesophageally injected 24 hours earlier with HA-MnSOD-PL, stained with an anti-HA antibody, and counted. Bone marrow from male C57BL/6J, GFP+ mice was prepared as a single cell suspension and injected (in comparison to ESCs) into female C57BL/6J, GFP- recipient mice that received 10 Gy TBI, and an additional 19 Gy boost to the esophagus. Serial transfer of ESCs isolated from mice receiving BMT after 19 Gy esophageal irradiation involved transplant into a second generation of 10 Gy TBI (with an additional 19 Gy esophageal boost) recipients. In vitro quantitation of ESC differentiation was carried out on 1% gelatin-coated plates with growth factors stimulatory for squamous cells, and also in semisolid agar containing medium in assays for CFU-GEMM, and for AFT024 fetal liver stromal cell coculture to assay CFU cobblestones. Results: The population of ESCs from C57BL/6J, GFP+ mice demonstrated a direct cell dose-dependent engraftment capacity into the esophagus of female C57BL/6J, GFP- recipient mice. Female C57BL/6J, GFP- recipient mice with GFP+ marrow demonstrated GFP+ donor origin esophageal and intestinal, mucin+ cell foci indicating that the bone marrow had bilineage squamous and mucin differentiation capacity. ESCs differentiated to cytokeratin+ squamous cells in vitro, and did not form detectable hematopoietic colonies compared to bone marrow Sca1-lin-Thy1low cells from the same mice. Bone marrow from GFP+ male mice were transplanted in a direct dose-response fashion into the esophagus of irradiated female C57BL/6J, GFP- mice. ESCs recovered from these mice, using the technique of Goddell, demonstrated serial transfer to the esophagus of second generation irradiated female C57BL/6J, GFP- mice. ESCs, isolated in greater numbers from male C57BL/6J mice and injected with HA-MnSOD-PL, were Sca1-lin-Thy1+ and HA+. Conclusion: The population of Sca1-lin-Thy1+ ESCs is found in the adult mouse esophagus. These cells are transplantable to irradiated recipient esophagus and form squamous colonies of donor origin. BMT to TBI, boost irradiated mice produces donor origin esophageal and intestinal foci in recipients. ESCs isolated from BMT recipients demonstrate serial transfer capacity to the esophagus of second generation recipient mice. Thus, the bone marrow is a source of stem cells for repopulation of the esophagus and small intestine. These ESCs were detectably transduced by HA-MnSOD-PL following intraesophageal injection, and irradiated mice receiving HA-MnSOD-PL demonstrated greater numbers of recoverable ESCs and greater survival, suggesting that ESCs are possible targets of MnSOD-PL radioprotective gene therapy.