Attempts for the in vitro differentiation of reconstituting human HSCs from ESCs have been unsuccessful as key factors of HSC specification remain unclear. Enforced HOXB4 expression can enhance haemopoietic differentiation of mouse and human ESCs and generate reconstituting HSCs from mouse ESCs. We have previously shown that HOXB4 can enhance haemopoietic differentiation of mouse ESCs in a paracrine manner. Microarray analysis identified a number of secreted factors upregulated by HOXB4 potentially mediating this paracrine effect. The aim of this study was to assess whether these factors alone and/or in combination can enhance the in vitro haemopoietic differentiation of mouse and human ESCs. We first developed a defined, serum and feeder-free protocol to test the effects of these secreted factors on haemopoietic differentiation. This defined protocol allowed us to compare the haemopoietic potential of mouse ESCs with the recently derived epiblast stem cells (EpiSCs), thought to be comparable to hESCs. Haemopoietic colony forming assay and flow cytometry analysis showed that serum-free conditions generated 8- 10 fold more CD144+CD41+ and c-Kit+CD41+ haemopoietic progenitor cells (HPCs) compared to serum conditions from both ESCs and EpiSCs, with ESCs giving the most significant increase. We then validated the panel of HOXB4 target genes by QRT-PCR and selected those increased in expression by at least 2.5-fold when HOXB4 was activated. We used this defined, serum-free protocol to assess the effects of our panel of secreted factors, FGF17, RSPO3 and APLN, on the haemopoietic differentiation of mouse ESCs. We demonstrated that FGF17 can mediate HOXB4 haemopoietic activity by enhancing the generation of c-Kit+ HPCs. On the other hand increasing concentrations of RSPO3 inhibited haemopoietic development by reducing the numbers of CD41+ and CD41+CD45+ HPCs, while, APLN did not have any effects on the haemopoietic activity of the cells. We finally used the secreted factors in human ESC and iPSC differentiation cultures. We observed differences in the activity of the tested factors not only between species but also between human cell lines. These results suggest that HOXB4 haemopoietic activity is partly mediated by paracrine signalling but more complex cell interactions are probably required for it to fully exert its effects. More importantly, HOXB4 regulatory pathways differ between mouse and human cells stressing the need for careful translation of data between the two species and more detailed analysis of key human haemopoietic factors for the successful generation of reconstituting HSCs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:716614 |
| Date | January 2016 |
| Creators | Kydonaki, Maria |
| Contributors | Forrester, Lesley |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/22056 |
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