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In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle–promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67and the antiapoptotic protein Bcl-XL. The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle–promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-XL. The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

ß-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (ß0 /ß0, ß0/ß+, ß+ /ß+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free a-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of a-globin that occurs during normal erythropoiesis and to a greater extent in different ß-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free a-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and ß-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, a, ß and ? globin genes expression. To quantify gene expression a mathematical model using calibration data (2-??ct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free a-globin are detectable explaning the lower need of AHSP expression. In ß-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free a-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In ß+/ß+ thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free a-chains excess. In ß0 /ß+ patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in ß0/ß0 patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and ?-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.

β-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (β0/β0, β0/β+, β+/β+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free α-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of α-globin that occurs during normal erythropoiesis and to a greater extent in different β-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free α-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and β-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, α, β and γ globin genes expression. To quantify gene expression a mathematical model using calibration data (2-ΔΔct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free α-globin are detectable explaning the lower need of AHSP expression. In β-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free α-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In β+/β+thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free α-chains excess. In β0/β+patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in β0/β0patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and γ-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.

Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro” human erythropiesis. Human CD34+ from perpheral blood of normal adult subjects were differentiate “in vitro” for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn’t seem to be modulated way, but we didn’t invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.

Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro”human erythropiesis. Human CD34+from perpheral blood of normal adult subjects were differentiate “in vitro”for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn't seem to be modulated way, but we didn't invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.