Background One of the main players of immune system and immunological disorders is white blood cells, such as lymphocytes, monocytes, and neutrophils. Although the elucidation of their cellular pathophysiology has been largely based on in vitro studies using patient-derived primary hematopoietic cells, this approach has potential limitations. Patient-derived cells cannot be obtained in unlimited quantities, and their in vitro functions can be affected by in vivo conditions, such as the cytokine milieu or presence of therapeutic agents.
Due to their pluripotency and capacity for self-renewal, human induced pluripotent stem cells (iPSCs) are potential sources of cells for use in drug screening, toxicology testing and the investigation of disease mechanisms 1,2. However, the phenotypic variance often hampers quantitative functional evaluation of iPS-derived progenies. Moreover, directed differentiation into immune cells financially prohibitive and labor-intensive. One of the solution of these issues is to immortalize iPSC-derived progenitor cells and use these cells as a source of functional analysis. Here we took advantage of immortalization of iPSC-derived myeloid progenitors (iPS-MLs) that can differentiate into functional macrophages and dendritic cells.
Objectives To overcome the limitations of iPSC-based study for immunological disorders, we are aiming at establishing a versatile and robust platform for disease modeling and high-throughput compound screening system by establishing iPS-MLs.
Methods Generation of iPS cells from congenital immunological disorder patients was performed as described previously 1. Hematopoietic cell differentiation was performed according to previously described protocols with some modifications 3,4. For establishment of iPS-MLs, lentiviral constructs encoding BMI1, cMYC and MDM2 floating hematopoietic cells differentiated into the monocytic lineage from iPSCs were collected on days 14 or 21. The cells were transfected with the lentiviral vectors encoding BMI1, cMYC and MDM2 and subsequently cultured in the presence of M-CSF and. We differentiated the iPS-MLs into macrophages and used for subsequent analysis.
Results We established iPSCs from patients with congenital immunological dysorders such as autoinflammatory syndrome and immunodeficiency. iPSCs were then immortalized immature monocytic progenitors by introducing three transgenes 5. iPS-MLs expand rapidly and be able to differentiate both macrophages and dendritic cells. By taking advantage of iPS-ML, we constructed phenotypic screening system by evaluating cytokine production or morphological changes in intracellular granules.
Conclusions Our system provides a large number of functional monocytic lineage cells and versatile platform for compound screening for modifying diseases-associated phenotypes. Therefore, it is useful for screening candidate compounds for treatment of congenital immunological disorders associated with monocytic lineage cells.
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Acknowledgement This study was supported by grants from AMED, Japan.
Disclosure of Interest None declared
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