Abstract
Background: Stem cells have been identified within proliferating infantile haemangioma (IH), the most common tumour of infancy, and have been demonstrated to play a critical role in the rapid proliferation and gradual involution of this tumour. There is accumulating evidence showing that IH is caused by aberrant proliferation and differentiation of a haemogenic endothelium (HE). This HE possesses a functional capacity to undergo primitive erythropoiesis in vitro. Short chain fatty acid (SCFA) derivatives have been shown to stimulate cell proliferation and induce STAT-5 activation in various haematopoietic cell lines.
Aims: The aims of this study were to investigate (1) the activity of the components of JAK-STAT pathway within the three phases of IH development; (2) the haematopoietic capacity of IH in vitro; and (3) the effects of SCFAs, butyric acid and propionic acid, to induce erythroid differentiation of explant-derived cells (IHEDCs) in culture.
Methods: The presence of pSTAT proteins in proliferating, involuted and involuting IH were investigated using 3,3'-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining, 1-DE Western Blotting, and NanoString analysis. Proliferating IH explants were cultured using an in vitro model and the IHEDCs emanating from the explants were harvested. Cell suspension of volume equivalent to 5x105 live cells was plated on Matrigel and incubated in 0.05-1mM butyric acid, RPMI and 0.05-1mM propionic acid, and 0.05M DMSO (positive control) in each of RPMI media only, RPMI enriched with iron and MCDB media. Media was changed daily and cells were extracted and quantified following 24-72 hours in culture. Differentiated IHEDCs were characterised by IF immunocytochemical (ICC) staining with glycophorin-A.
Results: Protein and genomic data reveal the expression of STATs 1, 3 and 5 which are activated in IH, particularly in the proliferative phase, with expression tapering as the lesion involutes. pSTAT3 is expressed most abundantly with pSTAT5 the least abundant. Low concentrations of both butyric acid and propionic acid significantly increased proliferation and differentiation of IHEDCs into blast colonies and the production of bi-concave cells within 72 hours in culture. These enucleated bi-concave cells expressed the erythrocyte-specific marker, glycophorin-A.
Conclusion: The findings of SCFAs promoting proliferation and differentiation of IHEDCs into blast colonies and differentiated erythrocytes reveal a novel role for SCFAs in human haematopoietic differentiation, possibly via pSTAT-5 signalling. IH offers a simple and novel in vitro model for generating haematopoietic precursors and production of human erythrocytes.