Y S Chung1, B Choi, C H D Kwon, J W Joh, S J Kim. 1. Transplantation Research Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea.
Abstract
BACKGROUND: It has been shown that the AFT024 stromal cell line sustains the engraftment capacity of human hematopoietic progenitor cells (HPCs) in vitro. However, the process by which AFT024 cell line maintains human HPCs is a more primitive state ex vivo remains unclear. METHODS: Human umbilical cord blood (UCB)-derived fluorescent activated cell sorter (FACS)-purified CD34(+) CD38(-)hsc/HPCs were cultured with cytokines on hpdi (0.4 micron pore size) coated with irradiated AFT024 cells. The HSC/HPC and AFT024 cells contacted each other through 0.4 micron pores on HPDI membranes; the irradiated AFT024 cells could not migrate through the HPDI to contaminate the HSC/HPC. The frequency of CD34(+)Lin(-) cells was determined as HSCs/HPCs using flow cytometry. To evaluate their engraftment potential in vivo, the co-cultured cells were assayed as Long Term Culture-Initiating Cells (LTC-IC). To understand the process whereby AFT024 cells govern enhanced engraftment, we employed Western blot analysis for histone modifications. RESULTS: There was a 30-fold increase in frequency of CD34(+)Lin(-) cells in co-cultures on HPDI coated on the outer bottom surface with irradiated AFT024 cells and cytokines in contrast to 6-fold among controls. Total colonies from LTC-IC increased approximately 1.5-fold among cells cultured with AFT024, compared with controls. More importantly, cells co-cultured with AFT024 showed a more primitive state with over-methylated h3k4 (Me-H3K4), under-methylated h3k9 (Di-Me-H3K4), and over-acetylated h4 (Ac-H4) compared with controls. CONCLUSION: Our results suggested that co-culture of the AFT024 cell line with HPDI maintained hematopoietic progenitors as a more primitive state through histone modification.
BACKGROUND: It has been shown that the AFT024 stromal cell line sustains the engraftment capacity of human hematopoietic progenitor cells (HPCs) in vitro. However, the process by which AFT024 cell line maintains human HPCs is a more primitive state ex vivo remains unclear. METHODS:Human umbilical cord blood (UCB)-derived fluorescent activated cell sorter (FACS)-purified CD34(+) CD38(-)hsc/HPCs were cultured with cytokines on hpdi (0.4 micron pore size) coated with irradiated AFT024 cells. The HSC/HPC and AFT024 cells contacted each other through 0.4 micron pores on HPDI membranes; the irradiated AFT024 cells could not migrate through the HPDI to contaminate the HSC/HPC. The frequency of CD34(+)Lin(-) cells was determined as HSCs/HPCs using flow cytometry. To evaluate their engraftment potential in vivo, the co-cultured cells were assayed as Long Term Culture-Initiating Cells (LTC-IC). To understand the process whereby AFT024 cells govern enhanced engraftment, we employed Western blot analysis for histone modifications. RESULTS: There was a 30-fold increase in frequency of CD34(+)Lin(-) cells in co-cultures on HPDI coated on the outer bottom surface with irradiated AFT024 cells and cytokines in contrast to 6-fold among controls. Total colonies from LTC-IC increased approximately 1.5-fold among cells cultured with AFT024, compared with controls. More importantly, cells co-cultured with AFT024 showed a more primitive state with over-methylated h3k4 (Me-H3K4), under-methylated h3k9 (Di-Me-H3K4), and over-acetylated h4 (Ac-H4) compared with controls. CONCLUSION: Our results suggested that co-culture of the AFT024 cell line with HPDI maintained hematopoietic progenitors as a more primitive state through histone modification.