Figure 1
Figure 1. Improved radioprotection and chimerism because of culture of KLS cells with BMP4. Murine KLS cells were cultured for 5 days in serum-free medium with SCF+TPO with or without BMP4 and/or TSG or CHD (ST = SCF/TPO; STT = SCF/TPO/TSG; STB = SCF/TPO/BMP4; STBT = SCF/TPO/BMP4 /TSG; STC = SCF/TPO/CHD). (A) Flow cytometry was performed to quantify cells maintaining KLS phenotype after 5 days of culture under different conditions (n = 3). (B) Cell-cycle status was analyzed by propidium iodide staining. The percentage cells in G2/M phase of cell cycle were plotted for the different conditions (n = 3). (C) Survival of mice grafted with the progeny of 200 KLS cells from the different cultures after lethal irradiation. Control denotes mice that did not receive KLS cell progeny. Results were analyzed by log-rank test method for trend and expressed as Kaplan-Meier survival curves (n = 10-18, *P < .01). (D) Donor derived chimerism was determined by FACS in primary recipients, 3 months after the transplantation of either 200 uncultured KLS cells, or of progeny from 200 KLS cells from different culture conditions along with 1 × 106 CD45.2 BM cells. One million total BM cells from primary recipients were transplanted in secondary hosts. Chimerism in secondary recipients was determined after 3 months (n = 8-12; *P < .05). (E) In vitro transwell migration assays was performed to compare the migration potential of the KLS cell progeny (n = 4). (F) PKH-26 labeled KLS cell progeny were allowed to adhere to OP9 cells for 3h. Nonadherent cells were washed and the percentage adherent cells was enumerated and compared for different conditions. In some conditions, a blocking anti-ITGA4 antibody was added in addition to BMP4 (n = 3-4; *P < .05). (G) KLS cell progeny (0.1 × 106) from the different cultures were transplanted intravenously in lethally irradiated mice, and BM harvested after 16 hours. Total BM cells (0.1 × 106) were assayed in methylcellulose colony forming assays and CFU-Cs were enumerated. Percentage homing was determined by comparing total CFU-Cs homed with CFU-Cs injected (n = 6, *P < .05, **P < .01).

Improved radioprotection and chimerism because of culture of KLS cells with BMP4. Murine KLS cells were cultured for 5 days in serum-free medium with SCF+TPO with or without BMP4 and/or TSG or CHD (ST = SCF/TPO; STT = SCF/TPO/TSG; STB = SCF/TPO/BMP4; STBT = SCF/TPO/BMP4 /TSG; STC = SCF/TPO/CHD). (A) Flow cytometry was performed to quantify cells maintaining KLS phenotype after 5 days of culture under different conditions (n = 3). (B) Cell-cycle status was analyzed by propidium iodide staining. The percentage cells in G2/M phase of cell cycle were plotted for the different conditions (n = 3). (C) Survival of mice grafted with the progeny of 200 KLS cells from the different cultures after lethal irradiation. Control denotes mice that did not receive KLS cell progeny. Results were analyzed by log-rank test method for trend and expressed as Kaplan-Meier survival curves (n = 10-18, *P < .01). (D) Donor derived chimerism was determined by FACS in primary recipients, 3 months after the transplantation of either 200 uncultured KLS cells, or of progeny from 200 KLS cells from different culture conditions along with 1 × 106 CD45.2 BM cells. One million total BM cells from primary recipients were transplanted in secondary hosts. Chimerism in secondary recipients was determined after 3 months (n = 8-12; *P < .05). (E) In vitro transwell migration assays was performed to compare the migration potential of the KLS cell progeny (n = 4). (F) PKH-26 labeled KLS cell progeny were allowed to adhere to OP9 cells for 3h. Nonadherent cells were washed and the percentage adherent cells was enumerated and compared for different conditions. In some conditions, a blocking anti-ITGA4 antibody was added in addition to BMP4 (n = 3-4; *P < .05). (G) KLS cell progeny (0.1 × 106) from the different cultures were transplanted intravenously in lethally irradiated mice, and BM harvested after 16 hours. Total BM cells (0.1 × 106) were assayed in methylcellulose colony forming assays and CFU-Cs were enumerated. Percentage homing was determined by comparing total CFU-Cs homed with CFU-Cs injected (n = 6, *P < .05, **P < .01).

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