OBJECTIVE: Augmentation of the number of cord blood (CB) hematopoietic stem cells (HSC) present in a unit is required before it can be considered as an alternative graft for hematopoietic reconstitution for adult patients. In order to further optimize strategies to augment HSC numbers, we examined whether expansion of HSC mediated by epigenetic mechanisms remains permissive to external environmental cues. MATERIALS AND METHODS: The chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) were used to ameliorate epigenetic alteration of CB cells during ex vivo culture by adding various cytokines. After culture, CD34(+)CD90(+) cell numbers, their division history, in vitro clonogenic potential, and in vivo hematopoietic reconstitution potential and frequency were determined. RESULTS: 5azaD/TSA-treated, CD34(+)CD90(+) cells were greatly influenced in terms of their degree of expansion, clonogenic potential, cell-division rate, and transplantability by the combination of cytokines used in culture. Furthermore, our current results verify that the sequential addition of 5azaD followed by TSA is crucial for expansion of HSC. We demonstrate that following 5azaD/TSA treatment, the rate of CD34(+)CD90(+) cell division is also dependent on the cytokine cocktail and that this is associated with functional changes, including alteration of in vitro clonogenic potential and in vivo reconstitution potential. CONCLUSIONS: Our studies indicate there are interactions between intrinsic factors influenced by epigenetic mechanisms and external environmental signals in the regulation of HSC expansion. Epigenetic influences on HSC can be accentuated by environmental factors. Regulation of the rate of divisions may be a critical determinant for the maintenance of HSC functional potency during ex vivo expansion.
OBJECTIVE: Augmentation of the number of cord blood (CB) hematopoietic stem cells (HSC) present in a unit is required before it can be considered as an alternative graft for hematopoietic reconstitution for adult patients. In order to further optimize strategies to augment HSC numbers, we examined whether expansion of HSC mediated by epigenetic mechanisms remains permissive to external environmental cues. MATERIALS AND METHODS: The chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) were used to ameliorate epigenetic alteration of CB cells during ex vivo culture by adding various cytokines. After culture, CD34(+)CD90(+) cell numbers, their division history, in vitro clonogenic potential, and in vivo hematopoietic reconstitution potential and frequency were determined. RESULTS: 5azaD/TSA-treated, CD34(+)CD90(+) cells were greatly influenced in terms of their degree of expansion, clonogenic potential, cell-division rate, and transplantability by the combination of cytokines used in culture. Furthermore, our current results verify that the sequential addition of 5azaD followed by TSA is crucial for expansion of HSC. We demonstrate that following 5azaD/TSA treatment, the rate of CD34(+)CD90(+) cell division is also dependent on the cytokine cocktail and that this is associated with functional changes, including alteration of in vitro clonogenic potential and in vivo reconstitution potential. CONCLUSIONS: Our studies indicate there are interactions between intrinsic factors influenced by epigenetic mechanisms and external environmental signals in the regulation of HSC expansion. Epigenetic influences on HSC can be accentuated by environmental factors. Regulation of the rate of divisions may be a critical determinant for the maintenance of HSC functional potency during ex vivo expansion.
Authors: A Peled; I Petit; O Kollet; M Magid; T Ponomaryov; T Byk; A Nagler; H Ben-Hur; A Many; L Shultz; O Lider; R Alon; D Zipori; T Lapidot Journal: Science Date: 1999-02-05 Impact factor: 47.728
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