Specific Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem (ES) Cells with Abortive Ribosome Assembly.

Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes, characterized by erythroid hypoplasia, congenital anomalies and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, RPL35a), which somehow triggers apoptosis of erythroid precursors. There is a marked variation in phenotype among members of the same family and also between subsets of patients with different mutations.

We studied primary and secondary in vitro differentiation of two murine ES gene trap cell lines with mutations in Rps19: S17-10H1, in which Rps19 is disrupted by insertion of the ROSAFARY gene trap vector between exons 2 and 3; and YHC074, in which the pGT0Lxf gene trap vector is inserted between exons 3 and 4 and whose growth is feeder cell-independent. For primary differentiation and generation of embryoid bodies (EBs), the ES cells were cultured in a serum-supplemented methylcellulose-based medium containing stem cell factor (SCF). After 7 days, the cultures were fed with a medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 13 for hematopoietic percentage. Secondary (hematopoietic) differentiation was performed on day 9 EBs. EBs were harvested and disrupted with collagenase, and the disrupted cells were suspended in a serum-supplemented methylcellulose-based medium with SCF, IL-3, IL-6 and epo. Hematopoietic colonies were counted on day 10.

Decreased expression of Rps19 protein was confirmed by Western blot analysis in both S17-10H1 and YHC074 gene trap cell lines. We focused on YHC074 because its growth is feeder-independent, and it expresses approximately 50% of normal Rps19 levels. By polysome analysis, we found a selective reduction in the 40S subunit peak in mutant YHC074 cells as compared to parental controls. By Northern blot assays, we also found a relative increase in the 21S pre-rRNA to 18S rRNA ratio in mutant YHC074 cells. The viability of undifferentiated ES cells was not significantly different from parental control cells in the first 72 hours of culture; however, there was a significantly decreased number of EBs, particularly hematopoietic EBs, following primary differentiation (Fig. 1). Furthermore, when day 9 EBs were induced to secondary (hematopoietic) differentation, there was a significant decrease in the ratio of erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation in mutant YHC074 cells.

In order to confirm these results in an isogenic background, we stably transfected S17-10H1 cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones were found to overexpress Rps19 and were further studied in secondary differentiation experiments. There was a significant decrease in erythroid and myeloid colony formation and in BFU-E size from mutant S17-10H1 cells when compared to the Rps19-overexpressing clone, suggesting a direct relationship between the levels of Rps19 protein and hematopoietic growth and differentiation.

Using two ES cell lines with slightly different Rps19 mutations and genetic backgrounds, we have recapitulated the major DBA erythroid growth and differentiation defect, as well as the defect in ribosome assembly and rRNA processing caused by Rps19 haploinsufficiency.

Fig. 1.

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Decreased efficiency of erythroid body formation in Rps19 mutant cell line. Primary differentiation was performed on Rps19- haploinsufficient and parental ES cell lines. Cultures were scored for total embryoid body (EB) number on day 6 and for hematopoietic percentage on day 13. The Rps19- haploinsufficient line (YHC074) showed a marked reduction in efficiency of EB formation compared with the parental line (E14). This difference was most pronounced in the hematopoietic EB population.

Fig. 1.

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Decreased efficiency of erythroid body formation in Rps19 mutant cell line. Primary differentiation was performed on Rps19- haploinsufficient and parental ES cell lines. Cultures were scored for total embryoid body (EB) number on day 6 and for hematopoietic percentage on day 13. The Rps19- haploinsufficient line (YHC074) showed a marked reduction in efficiency of EB formation compared with the parental line (E14). This difference was most pronounced in the hematopoietic EB population.

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No relevant conflicts of interest to declare.