- Email: [email protected]
Characterization of a hematopoietic stem cell with engraftment advantage
1498
Abstracts/Experimental Hematology 28 (2000) 1491–1505
pectancy table” published by the Japanese Authority. Results indicted that patients with PA, when appropriately treated, enjoy smooth and uneventful lives. Three laboratory approaches were taken for elucidation of the evidence of apoptosis using fresh untreated bone marrow cells from 6 patients. The apoptotic bodies were not detected in the flowcytometric analysis, and DNA ladder formation was not observed in all specimens. Furthermore, the frequency of apoptotic signals was not increased significantly according to TUNEL method. These observations do not support the apoptosis mechanism as a mode of intramedullar y cell death.
923 A DYNAMIC MODEL OF EX VIVO GRANULOPOIESIS: EFFECTS OF OXYGEN TENSION, pH, AND IL-3 Diane L. Hevehan, Larissa A. Wenning†, E. Terry Papoutsakis, William M. Miller Department of Chemical Engineering, Northwestern University, Evanston, IL, USA; †current address: Merck Research Laboratories, West Point, PA, USA Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Growth factors and other parameters can greatly affect the lineage and maturation stage of a culture. In order to resolve the kinetics and more clearly define the differential effects of O2 tension (pO2), pH, and IL-3 on granulopoiesis, a mathematical model-based approach was undertaken. Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts distributed cell populations by incorporating independent rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. Model simulations of experimental data revealed that (i) pO2 effects are exerted only on the growth rate, (ii) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7.6, the death rate for cells in the early stages of differentiation becomes increasingly significant, and (iii) the absence of IL-3 markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. Integrating experimental data with mathematical modeling can be used to elucidate the mechanisms underlying the regulation of granulopoiesis by pO2, pH, IL-3, and other conditions, and to improve culture strategies.
924 OXYGEN TENSION HAS SIGNIFICANT EFFECTS ON HUMAN MEGAKARYOCYTE MATURATION S. M. Mostafa, E. T. Papoutsakis, and W. M. Miller Department of Chemical Engineering, Northwestern University, Evanston, IL, USA Megakaryocytes (Mks) mature adjacent to bone marrow sinuses and release platelets within the sinusoidal space or in lung capillaries. Since the sites for platelet release have higher levels of
oxygen tension (pO2) compared to the core of the bone marrow where stem and progenitor cells reside, we investigated whether pO2 influences Mk maturation. Mks were generated from CD34⫹ cells under 5% and 20% O2 with TPO, IL-3, and FL (n ⫽ 10). At day 15, CD41⫹ cell expansion in 20% and 5% O2 cultures was 85fold and 31-fold respectively. 20% O2 cultures also had higher levels of polyploid (ⱖ8N) (8-fold higher) and proplatelet-displaying (5-fold higher) Mks. At day 21, 20% O2 cultures had a 5-fold higher number of apoptotic Mks. In contrast, the total number of CFU-Mks and the proportion of large-colony (ⱖ20 Mks)-forming CFU-Mks were higher under 5% O2. Change from 5% to 20% O2 (n ⫽ 4) increased Mk differentiation compared to 5% O2. Change from 20% to 5% O2 (n ⫽ 3) reduced the level of Mk maturation and apoptosis compared to 20% O2. Cultures initiated with CD41⫹ cells showed that pO2 effects on Mks were direct (n ⫽ 3). RT-PCR indicated Mks under 20% O2 had a higher level of NF-E2 mRNA. These culture data may provide insights into in vivo Mk maturation. Decreased Mk differentiation at 5% O2 may explain observations that hypoxia causes thrombocytopenia in animals. Mk maturation and apoptosis followed similar kinetics under different pO2 environments supporting the hypothesis that these two processes are closely linked. The differential effects of pO2 on Mk progenitors versus mature Mks have implications beyond their physiological relevance. Using pO2 as a control parameter in ex vivo cultures one can (1) extend the level of Mk progenitor expansion (5% O2), or (2) generate a high level of non-apoptotic Mks for reinfusion into patients (5% to 20% O2).
925 CHARACTERIZATION OF A HEMATOPOIETIC STEM CELL WITH ENGRAFTMENT ADVANTAGE Jichun Chen, Clinton M. Astle, David E. Harrison The Jackson Laboratory, Bar Harbor, ME, USA In studying hematopoietic stem cell (HSC) function in vivo, we discovered that HSCs from CXB-12/HiaJ (CXB-12) mice have 14 times the total long term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. This was determined using the competitive repopulation assay where bone marrow cells (BMCs) from each RI line donor were mixed with gentically marked competitor BMCs from the (BALBx B6) F1 (F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated F1 recipients. After six months, percentages of donor type erythrocytes and lymphoytes gave a measure of the long term repopulating abilities of the donor RI lines relative to the F1 competitor. When high doses of BMCs were used, CXB-12 cells repopulated 6–12 times better than the F1 competitor cells, while when a low dose of BMCs were used in competitive dilution, CXB-12 donors had 2.4 times the number of HSCs as the F1 competitor, and each CXB-12 HSC repopulated 1.4 times as well. The CXB-12 HSC engraftment advantage is associated with a 50% increase in cobblestone area forming cell frequency and an one fold increase in the number of side population cells revealed by Hoechst 33342 vital dye staining. There was no effect on day 12 spleen colony forming units. The CXB-12 HSC engraftment advantage appears to result from increased HSC proliferation and differentiation abilities which are caused either by a unique recombination of parental genes or by specific mutations in the CXB-12 genome.
Abstracts/Experimental Hematology 28 (2000) 1491–1505
pectancy table” published by the Japanese Authority. Results indicted that patients with PA, when appropriately treated, enjoy smooth and uneventful lives. Three laboratory approaches were taken for elucidation of the evidence of apoptosis using fresh untreated bone marrow cells from 6 patients. The apoptotic bodies were not detected in the flowcytometric analysis, and DNA ladder formation was not observed in all specimens. Furthermore, the frequency of apoptotic signals was not increased significantly according to TUNEL method. These observations do not support the apoptosis mechanism as a mode of intramedullar y cell death.
923 A DYNAMIC MODEL OF EX VIVO GRANULOPOIESIS: EFFECTS OF OXYGEN TENSION, pH, AND IL-3 Diane L. Hevehan, Larissa A. Wenning†, E. Terry Papoutsakis, William M. Miller Department of Chemical Engineering, Northwestern University, Evanston, IL, USA; †current address: Merck Research Laboratories, West Point, PA, USA Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Growth factors and other parameters can greatly affect the lineage and maturation stage of a culture. In order to resolve the kinetics and more clearly define the differential effects of O2 tension (pO2), pH, and IL-3 on granulopoiesis, a mathematical model-based approach was undertaken. Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts distributed cell populations by incorporating independent rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. Model simulations of experimental data revealed that (i) pO2 effects are exerted only on the growth rate, (ii) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7.6, the death rate for cells in the early stages of differentiation becomes increasingly significant, and (iii) the absence of IL-3 markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. Integrating experimental data with mathematical modeling can be used to elucidate the mechanisms underlying the regulation of granulopoiesis by pO2, pH, IL-3, and other conditions, and to improve culture strategies.
924 OXYGEN TENSION HAS SIGNIFICANT EFFECTS ON HUMAN MEGAKARYOCYTE MATURATION S. M. Mostafa, E. T. Papoutsakis, and W. M. Miller Department of Chemical Engineering, Northwestern University, Evanston, IL, USA Megakaryocytes (Mks) mature adjacent to bone marrow sinuses and release platelets within the sinusoidal space or in lung capillaries. Since the sites for platelet release have higher levels of
oxygen tension (pO2) compared to the core of the bone marrow where stem and progenitor cells reside, we investigated whether pO2 influences Mk maturation. Mks were generated from CD34⫹ cells under 5% and 20% O2 with TPO, IL-3, and FL (n ⫽ 10). At day 15, CD41⫹ cell expansion in 20% and 5% O2 cultures was 85fold and 31-fold respectively. 20% O2 cultures also had higher levels of polyploid (ⱖ8N) (8-fold higher) and proplatelet-displaying (5-fold higher) Mks. At day 21, 20% O2 cultures had a 5-fold higher number of apoptotic Mks. In contrast, the total number of CFU-Mks and the proportion of large-colony (ⱖ20 Mks)-forming CFU-Mks were higher under 5% O2. Change from 5% to 20% O2 (n ⫽ 4) increased Mk differentiation compared to 5% O2. Change from 20% to 5% O2 (n ⫽ 3) reduced the level of Mk maturation and apoptosis compared to 20% O2. Cultures initiated with CD41⫹ cells showed that pO2 effects on Mks were direct (n ⫽ 3). RT-PCR indicated Mks under 20% O2 had a higher level of NF-E2 mRNA. These culture data may provide insights into in vivo Mk maturation. Decreased Mk differentiation at 5% O2 may explain observations that hypoxia causes thrombocytopenia in animals. Mk maturation and apoptosis followed similar kinetics under different pO2 environments supporting the hypothesis that these two processes are closely linked. The differential effects of pO2 on Mk progenitors versus mature Mks have implications beyond their physiological relevance. Using pO2 as a control parameter in ex vivo cultures one can (1) extend the level of Mk progenitor expansion (5% O2), or (2) generate a high level of non-apoptotic Mks for reinfusion into patients (5% to 20% O2).
925 CHARACTERIZATION OF A HEMATOPOIETIC STEM CELL WITH ENGRAFTMENT ADVANTAGE Jichun Chen, Clinton M. Astle, David E. Harrison The Jackson Laboratory, Bar Harbor, ME, USA In studying hematopoietic stem cell (HSC) function in vivo, we discovered that HSCs from CXB-12/HiaJ (CXB-12) mice have 14 times the total long term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. This was determined using the competitive repopulation assay where bone marrow cells (BMCs) from each RI line donor were mixed with gentically marked competitor BMCs from the (BALBx B6) F1 (F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated F1 recipients. After six months, percentages of donor type erythrocytes and lymphoytes gave a measure of the long term repopulating abilities of the donor RI lines relative to the F1 competitor. When high doses of BMCs were used, CXB-12 cells repopulated 6–12 times better than the F1 competitor cells, while when a low dose of BMCs were used in competitive dilution, CXB-12 donors had 2.4 times the number of HSCs as the F1 competitor, and each CXB-12 HSC repopulated 1.4 times as well. The CXB-12 HSC engraftment advantage is associated with a 50% increase in cobblestone area forming cell frequency and an one fold increase in the number of side population cells revealed by Hoechst 33342 vital dye staining. There was no effect on day 12 spleen colony forming units. The CXB-12 HSC engraftment advantage appears to result from increased HSC proliferation and differentiation abilities which are caused either by a unique recombination of parental genes or by specific mutations in the CXB-12 genome.