Медицинская иммунология (Sep 2024)
Early markers of induced pluripotent stem cell hematopoietic development
Abstract
The generation of immune cell populations from induced pluripotent stem cells (iPSCs) is a valuable model to study mechanisms that control hematopoietic development; it also is a promising approach to develop immunotherapeutic strategies to treat various diseases, including hereditary, oncological and infectious ones. To date, it has been demonstrated that iPSCs can differentiate into different immune cells, including macrophages, neutrophils, natural killer cells and T cells. However, the protocols suggested so far are experimental, and they require optimization, standardization and scaling. Solution to these tasks requires methods allowing to predict the efficacy of ongoing differentiation at early differentiation stages. Here, we evaluated whether iPSC hematopoietic/myeloid differentiation can be monitored by means of flow cytometry. Human iPSCs were differentiated into hematopoietic/myeloid cells using two protocols previously suggested for the generation of macrophages from iPSCs. The protocols differed by methods used to induce early and late stages of cell differentiation. At early differentiation stages, the protocols differed by approaches used to induce the generation of mesoderm and hemogenic endothelium, i.e., 2D differentiation in the presence of exogenous factors known to promote mesoderm and hemogenic endothelium development (“factor-dependent” protocol) or 3D differentiation in the absence of exogenous cytokines and growth factors (“spontaneous” protocol). At late differentiation stages, the protocols differed by factors added to the cultures to promote hematopoietic/myeloid specification (i.e., SCF, FGF2, IL-6, IL-3 and M-CSF or only IL-3 and M-CSF). At different stages of differentiation, the expressions of antigens known to be expressed by mesoderm, hemogenic endothelium and hematopoietic cells (i.e., CD309, CD34, CD31, CD43 and CD45) were evaluated. At early differentiation stages, the main phenotypic changes observed in cell cultures were an upregulation of the expression of CD309 (a receptor for vascular endothelial growth factor), the appearance of the expression of sialophorin CD43 and the expression of CD34 antigen. In cells cultured in 2D factor-dependent conditions, these changes appeared earlier and were more pronounced as compared with cells cultured in 3D “spontaneous” conditions. The results suggest that CD309 and/or CD43 are valuable markers for an early prediction of the effectiveness of iPSC differentiation into hematopoietic/ myeloid progeny.
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