Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

The development and magnitude of thermotolerance during chronic hyperthermia in murine granulocyte-macrophage progenitors: II.

We have previously reported that murine granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during chronic hyperthermia at temperatures of 40 to 42 degrees C. However, a differential profile of intrinsic thermal response and, in particular, the capability of developing thermotolerance during chronic heating was identified between CFU-GM and macrophage colony-forming units (CFU-M) stimulated respectively, by lung conditioned medium (LCM) and L929 cell conditioned medium (CCM). Nucleated marrow cells treated in vitro were cultured in McCoy's 5A medium plus 15% fetal bovine serum (FBS) in semisolid agar with 10% of CCM. Two different treatment protocols were used in this study to determine the kinetics of thermotolerance in CFU-M: (1) nucleated marrow from mouse tibia and femur were chronically heated in vitro at temperatures of 40, 41 and 42 degrees C (up to 480 min) or (2) nucleated marrow cells were heated over a period of 90 min stepwise from 37 to 42 degrees C, at a heating rate of 0.056 degrees C/min, before exposure to 42 degrees C. The amount of thermotolerance developed was analysed at various times after chronic incubation at 40-42 degrees C by a challenge with 15 min at 44 degrees C. In contrast to CFU-GM, the surviving fraction of CFU-M heated with 15 min at 44 degrees C did not increase during chronic hyperthermia at 40 degrees C for up to 480 min indicating failure to develop thermotolerance. However, CFU-M were able to develop thermotolerance during prolonged incubation at 41 and 42 degrees C, although to a much less extent than observed in CFU-GM. In other words, there was much less development of thermotolerance in murine CFU-M compared to that in CFU-GM. Furthermore, a slow temperature transit from 37 to 42 degrees C over 90 min before exposure to 42 degrees C induced CFU-M to develop thermotolerance. The thermotolerance ratio (TTR, the ratio of the surviving fraction at maximum tolerance versus normotolerance) increased from a maximum of 3.5 after 180 min at 42 degrees C (no warm-up) to a maximum of 4.1 after 60 min at 42 degrees C when the cells received a slow warm-up to 42 degrees C. This implies that in the murine bone marrow granulocyte/macrophage lineage, CFU-M does not normally develop thermotolerance during hyperthermia and that the colony forming unit-granulocyte (CFU-G) and CFU-GM play a more critical role than CFU-M in the initiation and promotion of thermotolerance during chronic hyperthermia. However, in a situation that simulates the slow heat-up used clinically in wholebody hyperthermia, e.g., the 90 min slow warm-up from 37 to 42 degrees C, stimulated CFU-M to develop greater thermotolerance more rapidly than during rapid heating.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

PURPOSE: Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. METHODS AND MATERIALS: Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. RESULTS: The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h. Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C. Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy). By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively. CONCLUSION: These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C. The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

The development of thermotolerance in bone marrow CFU-S during chronic hyperthermia.

The purpose of this investigation was to study the response of the hematopoietic stem cell, spleen colony-forming unit (CFU-S), to hyperthermia. We have shown that CFU-S can acquire a transient resistance to further heating (thermotolerance). Hyperthermia was applied in vitro to nucleated bone marrow cells in McCoy's 5A medium plus 15% fetal bovine serum. Day-10 CFU-S (CFU-S10) were detected as spleen colonies after inoculation into the tail vein of irradiated (450 cGy plus 4 h plus 400 cGy) Balb/c male mice. Thermotolerance development was detected with a "step-up" heating protocol consisting of heating for various times at 42 degrees C followed immediately with a thermal challenge of 26 min at 44 degrees C. The inverse of the slopes of the heat "dose-response" curves (D degree +/- SE) of the normotolerant CFU-S heated to 42 degrees, 42.5 degrees, 43 degrees, 43.5 degrees, and 44 degrees C were 108 +/- 13, 54 +/- 8, 25 +/- 1, 17 +/- 2, and 12 +/- 5 min, respectively. A plot of the slopes of the heat "dose-response" relationships versus the inverse of the absolute temperature (Arrhenius plot) showed an inflection at approximately 43 degrees C. Analysis of the regression coefficient above and below the inflection point (Arrhenius analysis) yielded inactivation enthalpies (+/- SE) of 598 +/- 130 kJ/mol (143 +/- 31 kcal/mol) and 1205 +/- 171 kJ/mol (288 +/- 41 kcal/mol), respectively. The difference in inactivation enthalpy indicates a change in mechanism in the thermal inactivation of CFU-S above and below 43 degrees C, possibly due to thermotolerance development during exposure to temperatures less than 43 degrees C. Prolonged incubation at 42 degrees C for up to 180 min with a step-up to 44 degrees C for 26 min showed that CFU-S survival increased rapidly from 0.25 (26 min at 44 degrees C) to 0.52 within 10 min. The thermotolerance ratio (TTR, ratio of the surviving fraction of the maximum thermotolerant cells to that of the normotolerant cells) was 2.1. Both the higher inactivation enthalpy for exposures less than 43 degrees C and the rapid increase in survival during the "step-up" heating experiments at 42 degrees C demonstrate that CFU-S can develop thermotolerance during prolonged hyperthermia. These results suggest that thermotolerance can influence the thermal response of pluripotent bone marrow stem cells heated during whole-body or local-regional clinical hyperthermia protocols.