Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells

Abstract

Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.

Figure 1

Hematopoietic development in flk-1 mutant EBs. (A) Day-10 EBs derived from wild-type, flk-1 (+/−), and flk-1 (−/−) ES cells, showing hemoglobinized EBs for all ES cell clones. (B) Proportion of hemoglobinized EBs scored on day 10 after plating in methylcellulose. (C) The numbers of erythroid colonies in secondary methylcellulose culture of flk-1 (+/+), flk-1 (+/−), and flk-1 (−/−) EB cells. Numbers of erythroid colonies were determined by replating disaggregated day 7 EB cells in methylcellulose culture containing Epo (open column) and Epo/SLF (closed column). BFU-E, erythroid burst-forming units. (D) A time course of myeloid–erythroid colony assays of EB-derived hematopoietic progenitors. The number of cell colony-forming units (CFU-Cs; the y-axis) is calculated per 5 × 10⁵ EB input cells. d4, etc., day 4, etc. The black and vertically striped bars represent flk-1 (+/−) cultures without or with exogenously added mVEGF, respectively. The white and speckled bars represent flk-1 (−/−) cultures without or with exogenously added mVEGF, respectively. Both genotypes generated approximately the same proportions of BFU-E and granulocyte–erythrocyte–macrophage–megakaryocyte, granulocyte–macrophage, macrophage, and granulocyte colony-forming units.

Figure 2

Impaired hematopoiesis by flk-1-null cells in OP9 cocultures. (A) Photomicrographs of wild-type, flk-1 (+/−), and flk-1 (−/−) ES cells 2, 5, 8, and 12 days after hematopoietic induction by coculturing with OP9 cells. (×20.) Each culture was fixed with glutaraldehyde and stained for β-gal activity on the day as indicated. Undifferentiated colonies (day 2), differentiated mesodermal colonies (day 5), and hematopoietic clusters (day 8/day 12) are observed in wild-type and flk-1 (+/−) cultures. Strong β-gal expression was detected in mesodermal colonies in both flk-1 (+/−) and flk-1 −/− cultures. No hematopoietic clusters are observed in flk-1 (−/−) cultures. (B and C) The numbers of mesodermal colonies (B) and hematopoietic clusters (C) of wild-type, flk-1 (+/−), and flk-1 (−/−) ES cells on OP9 stromal cells. Mesodermal colonies and hematopoietic clusters were counted on days 5 and day 10, respectively. No hematopoietic clusters are scored from flk-1 (−/−) ES cells. (D) A time course of myeloid–erythroid colony assays of ES-OP9-derived hematopoietic progenitors. The number of CFU-Cs (the y-axis) is calculated per 5 × 10⁵ day 5 or 10 OP9 input cells. The black and vertically striped bars represent flk-1 (+/−) cultures without or with exogenously added mVEGF, respectively. The white and speckled bars represent flk-1 (−/−) cultures without or with exogenously added mVEGF, respectively. No obvious differences in colony types were apparent between flk-1 (+/−) and flk-1 (−/−) cultures.

Figure 3

A time course of myeloid–erythroid colony assays derived from switch cultures. ES cells differentiated as EBs for 2, 4, or 6 days, then were passaged onto OP9 stromal cells for 5 days, then replated onto OP9 cells for an additional 5 days. Colony assays were performed on the day 10 OP9 cultures. The number of CFU-Cs (the y-axis) is calculated per 5 × 10⁵ day 10 OP9 input cells. The black and white bars represent flk-1 (+/−) and flk-1 (−/−) cultures, respectively. No obvious differences in colony types were apparent between flk-1 (+/−) and flk-1 (−/−) cultures.

Figure 4

Expression of various differentiation marker genes was examined by reverse transcription–PCR of EB cultures (A) and ES-OP9 cocultures (B). Number of days in culture is given at the top. Amplified hypoxanthine phosphoribosyltransferase (HPRT) is shown as a positive control. (A) No differences in gene expression between genotypes were observed in EB cultures. (B) Embryonic (βH1) and adult (β^major) globin, and Epo receptor (EpoR) transcripts were diminished in flk-1 (−/−)-derived cells compared with flk-1 +/−-derived cells in the OP9 system. c-kit was down-regulated by day 5, whereas scl was not expressed in flk-1 (−/−) cultures. vegf was constitutively expressed in these cultures, whereas flk-1 and flk-1/lacZ expression was induced upon ES differentiation.

Figure 5

β-gal and CD31 expression in flk-1 (+/−) and flk-1 (−/−) EBs. Photomicrographs of day 8 after dispase attached EB cultures stained for β-gal activity with FDG (A and C) and CD31 expression stained with phycoerythrin-conjugated anti-CD31 (B and D). (×4.) flk-1 +/− EBs form a vascular endothelial network expressing β-gal (A) and CD31 (B). Although flk-1 −/− ES cells differentiate into β-gal-positive cells (C), few (arrows) also express CD31 (D), and none exhibit an endothelial-like morphology.