Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5

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   Fig. 1.

   Generation of the Stat5-inducible ES cell line. (a) Integration of pLoxStat5CA into the LoxP site on the X chromosome of Ainv15 ES cells places the cDNA for Stat5CA under the control of the tetracycline response element. Recombination between the chromosomal and plasmid LoxP sites, denoted by χ, is mediated by transient expression of Cre recombinase. Reconstitution of neo gene function by the promoter-ATG sequence 5′ to the loxP site allows for selection of successful integration events. rtTA, reverse tetracycline transactivator. (b) Exposure of iStat5CA ES cells to doxycycline, but not the parental cell line Ainv15, results in the bandshift of a probe containing Stat5 binding sites. Arrowhead denotes the Stat5-specific bandshift.

   
   
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   Fig. 2.

   Effects of Stat5 signaling during EB differentiation. EBs were grown for 6 days, either exposed or not exposed to doxycycline from day 4 to day 6. (a) CFC assay: day-6 EB cells were disaggregated and plated into methylcellulose suspension culture with hematopoietic cytokines. Filled bars denote colony number from doxycycline-treated EBs; open bars denote colony number from untreated EBs. Standard errors for three independent experiments are shown. (n = 3 for each bar; P < 0.05 for combined CFCs.) (b) Apoptosis assay: day-6 EB cells were disaggregated and stained with annexin V (y axis) to label apoptotic cells and anti-CD41 (x axis) to label hematopoietic cells. The percentage of cells falling into single- and double-positive quadrants is shown. (c) Hematopoietic compartment quantitation: day-6 EB cells were disaggregated and stained with antibodies to c-Kit (y axis) and CD41 (x axis). The percentage of cells falling within the double-positive rectangular gate is shown.

   
   
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   Fig. 3.

   Stat5 signaling promotes blast cell outgrowth. (a) Cumulative cell number from OP9 stromal cell cocultures initiated by 2 × 10⁵ cells. (b) Stat5 DNA-binding activity was seen in OP9 cocultures of iStat5CA day-6 EB cells grown in the presence of doxycycline, but not in the residual growth that appeared in the absence of doxycycline. For comparison, the Stat5 bandshift from BaF/3 cells growing exponentially in the presence of IL-3, or BaF/3 cells infected with a MSCV retrovirus expressing Stat5CA are shown. Arrowhead denotes Stat5-specific bandshift product. (c) Cytospin of iStat5CA day-6 EB cells expanded on OP9. Cells were spun onto glass slides and stained with Wright-Giemsa.

   
   
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   Fig. 4.

   Characterization of Stat5-induced blast cells growing on OP9. (a) Surface antigen expression. Day-6 iStat5CA EB cells expanded on OP9 were labeled with the antibodies indicated and analyzed by flow cytometry. Cell number is plotted on the y axis; fluorescence intensity is plotted on the x axis. The first plot, labeled “iso,” is staining by an isotype control, nonspecific antibody. Percentages of cells positive for each marker are indicated. (b) Double staining. The same cells were costained with antibodies against CD41 (x axis) vs. c-Kit, Sca-1, or CD31 (y axis). The percentage of cells falling into each single- and double-positive quadrant is shown. (c) Globin gene expression. RNA was derived from cells expanded on OP9 with Stat5CA induction or with HoxB4-induction. RT-PCR for actin, β-H1 globin, and β-major globin was performed. M represents the molecular mass marker lane.

   
   
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   Fig. 5.

   FACS analysis of bone marrow of a doxycycline-treated recipient mouse. Bone marrow cells were harvested 1 month after transplantation, stained with the indicated antibodies, and subjected to flow cytometry. GFP fluorescence is plotted on the x axis; antibody staining is plotted on the y axis. “iso” represents an isotype control nonspecific antibody. The upper left plot is from a control, uninjected mouse; all others are from the experimental mouse. In each plot, the percentage of double-positive cells is shown in the upper right quadrant. Double positives represent donor cells expressing a given antigen.