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2001 - LONG-TERM EX VIVO EXPANSION OF FUNCTIONAL HEMATOPOIETIC STEM CELLS
Experimental Hematology 2019;76 (Suppl): S42−S50
Short Talk Presentations 2000 - MONOLINEAGE ORIGIN OF RELAPSE FOLLOWING MULTILINEAGE DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIA Ross Dickins1, Steven Ngo1, Ethan Oxley1, Margherita Ghisi1, Mark McKenzie2, Maximilian Garwood1, Swathy Jayakrishnan1, Olivia Susanto1, Helen Mitchell1, Michael Hickey1, Andrew Perkins1, Benjamin Kile1, Ross Dickins1 1 Monash University, Melbourne, Australia; 2Walter and Eliza Hall Institute, Melbourne, Australia
transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.
Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite nearcomplete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.
2001 - LONG-TERM EX VIVO EXPANSION OF FUNCTIONAL HEMATOPOIETIC STEM CELLS Adam Wilkinson1, Reiko Ishida2, Misako Kikuchi2 , Kazuhiro Sudo3 , Maiko Morita2, Ralph Valentine Crisostomo1, Ryo Yamamoto1 , Kyle Loh1, Yukio Nakamura3, Motoo Watanabe2, Hiromitsu Nakauchi1, Satoshi Yamazaki2 1 Stanford University, Stanford, United States; 2 University of Tokyo, Tokyo, Japan; 3RIKEN BioResource Center, Tsukuba, Japan The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a »900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC
2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL Robert Signer, Miriama Kruta, Mary Jean Sunshine, Yunpeng Fu, Lorena Hidalgo San Jose UC San Diego, La Jolla, United States The inability to maintain and expand hematopoietic stem cells (HSCs) in culture represents a major barrier to their expanded use in cell-based therapies. We recently discovered that HSCs exhibit low protein synthesis in vivo, regardless of whether they are quiescent or undergoing self-renewing divisions. In the present study, we determined that cultured HSCs rapidly upregulated genes that promote translation and exhibited a »2000% increase in protein synthesis. Increased protein synthesis overwhelmed protein quality control systems within HSCs, caused an imbalance in protein homeostasis, and was associated with nuclear translocation of Hsf1. Hsf1 is the master regulator of the heat shock pathway, and induces transcription of heat shock proteins that coordinate proteotoxic stress response to maintain protein homeostasis. Inactive Hsf1 is typically sequestered in the cytoplasm, and is rarely seen in the nucleus of HSCs in vivo. Genetic deletion of Hsf1 exacerbated HSC depletion in vitro, but had little effect on HSC function in vivo. These data indicated that Hsf1 promotes ex vivo HSC maintenance, and raised the possibility that increasing Hsf1 activation could enhance HSC self-renewal. To test this, we developed a new serum- and stroma-free platform to culture purified HSCs. In 10-day cultures initiated with just 10 purified HSCs, small molecules that promoted Hsf1 nuclear translocation supported extensive proliferation and complete retention of long-term multilineage reconstituting activity in serial transplantation assays. The positive effect of these small molecules on HSC growth was completely ablated in
Short Talk Presentations 2000 - MONOLINEAGE ORIGIN OF RELAPSE FOLLOWING MULTILINEAGE DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIA Ross Dickins1, Steven Ngo1, Ethan Oxley1, Margherita Ghisi1, Mark McKenzie2, Maximilian Garwood1, Swathy Jayakrishnan1, Olivia Susanto1, Helen Mitchell1, Michael Hickey1, Andrew Perkins1, Benjamin Kile1, Ross Dickins1 1 Monash University, Melbourne, Australia; 2Walter and Eliza Hall Institute, Melbourne, Australia
transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.
Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite nearcomplete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.
2001 - LONG-TERM EX VIVO EXPANSION OF FUNCTIONAL HEMATOPOIETIC STEM CELLS Adam Wilkinson1, Reiko Ishida2, Misako Kikuchi2 , Kazuhiro Sudo3 , Maiko Morita2, Ralph Valentine Crisostomo1, Ryo Yamamoto1 , Kyle Loh1, Yukio Nakamura3, Motoo Watanabe2, Hiromitsu Nakauchi1, Satoshi Yamazaki2 1 Stanford University, Stanford, United States; 2 University of Tokyo, Tokyo, Japan; 3RIKEN BioResource Center, Tsukuba, Japan The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a »900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC
2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL Robert Signer, Miriama Kruta, Mary Jean Sunshine, Yunpeng Fu, Lorena Hidalgo San Jose UC San Diego, La Jolla, United States The inability to maintain and expand hematopoietic stem cells (HSCs) in culture represents a major barrier to their expanded use in cell-based therapies. We recently discovered that HSCs exhibit low protein synthesis in vivo, regardless of whether they are quiescent or undergoing self-renewing divisions. In the present study, we determined that cultured HSCs rapidly upregulated genes that promote translation and exhibited a »2000% increase in protein synthesis. Increased protein synthesis overwhelmed protein quality control systems within HSCs, caused an imbalance in protein homeostasis, and was associated with nuclear translocation of Hsf1. Hsf1 is the master regulator of the heat shock pathway, and induces transcription of heat shock proteins that coordinate proteotoxic stress response to maintain protein homeostasis. Inactive Hsf1 is typically sequestered in the cytoplasm, and is rarely seen in the nucleus of HSCs in vivo. Genetic deletion of Hsf1 exacerbated HSC depletion in vitro, but had little effect on HSC function in vivo. These data indicated that Hsf1 promotes ex vivo HSC maintenance, and raised the possibility that increasing Hsf1 activation could enhance HSC self-renewal. To test this, we developed a new serum- and stroma-free platform to culture purified HSCs. In 10-day cultures initiated with just 10 purified HSCs, small molecules that promoted Hsf1 nuclear translocation supported extensive proliferation and complete retention of long-term multilineage reconstituting activity in serial transplantation assays. The positive effect of these small molecules on HSC growth was completely ablated in