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BackgroundHematopoietic progenitor cells are a multipotent cell type with the ability to differentiate into various blood lineages. Using directed differentiation following the correct developmental pathway, iCell® Hematopoietic Progenitor Cells 2.0 are induced pluripotent stem cell (iPSC)-derived CD34+/CD43+ cells capable of producing progeny of myeloid, erythroid, and lymphoid lineages. iCell Hematopoietic Progenitor Cells 2.0 are a consistent source of a readily available and easy to use intermediate cell type that can serve as a gateway to immunology.MethodsiCell Hematopoietic Progenitor Cells 2.0 can be used directly out of thaw or plated and maintained in culture for extended periods of time. Cell identity was confirmed by flow cytometry of specific markers such as CD34, CD43, CD45, and CD31. Colony Forming Units were assessed and quantified by methylcellulose gel to establish multipotency. Downstream directed differentiation was performed for multiple cell types, including macrophages, microglia, dendritic cells, natural killer cells, mesenchymal stem cells, and erythrocytes.ResultsHematopoietic markers such as CD34, CD43, CD45, and CD31 were expressed at high levels (>90%) consistently across lots. These multipotent cells demonstrate consistent CFU activity down the myeloid, granulocyte, erythroid, and megakaryocyte lineages as measured by Colony Forming Unit counts. Differentiation of this intermediate cell type was performed to generate CD68+ macrophages, IBA+ microglia, CD53+ NK cells, CD71+ erythroid cells, and CD105+ mesenchymal stem cells. Some of these products were characterized further in functional assays.ConclusionsCD34+/CD43+ iPSC-derived hematopoietic progenitor cells provide a constant, reproducible source of biologically relevant material for use in immunology and oncology. iCell Hematopoietic Progenitor Cells 2.0 are multipotent and able to generate multiple blood cell lineages. Functionally, iCell Hematopoietic Progenitor Cells 2.0 display consistent, quantifiable CFU potential with the capacity to generate downstream cells, such as macrophages, NK cells, and erythrocytes. This overall profile of iCell Hematopoietic Progenitor Cells 2.0 demonstrates its suitability as a continuing source of human multipotent hematopoietic cells.

BackgroundMacrophages modulate immune activity through cytokine and chemokine release in response to stimuli. These cytokines signal tissue and immune cells to elicit a pro- or anti-inflammatory response. iCell® Macrophages 2.0, which are derived from human induced pluripotent stem cells (iPSC), are functionally naïve and offer an effective, dynamic response to stimulation for a biologically relevant model of human macrophage function.MethodsiCell Macrophages 2.0 can be used directly out of thaw or plated and maintained for up to 14 days in culture. To measure cytokine release, cells were plated and stimulated with pro-inflammatory (LPS, IFNγ) or anti-inflammatory (IL-4, IL-13) stimuli across multiple doses and at different timepoints. Supernatants were collected and cytokines quantified by different immunoassay technologies, including Luminex, ELISA, Lumit, or HTRF. For phagocytosis, iCell Macrophages 2.0 were plated for 3 days, then incubated with pHrodo-labeled bioparticles and monitored for uptake. Antibody-dependent cellular phagocytosis (ADCP) assays were run with stimulated macrophages to measure the potency of Rituximab for CD20+ Raji target cells. iCell Macrophages 2.0 were cultured with isogenic iCell Hepatocytes 2.0, iCell Sensory Neurons, or iCell Cardiomyocytes2 to evaluate the functional effects of macrophage stimulation on complex culture systems.ResultsiCell Macrophages 2.0 express macrophage surface markers such as CD68, CD11c, and CD11b. These full function macrophages can be polarized toward a pro- or anti-inflammatory state, with cytokine release comparable to human primary macrophages for IL-6, TNFα, IL-10, and CCL18. This response can be measured within hours in both mRNA and secreted protein and can be detected at stimulant concentrations below 100pg/ml. Phagocytosis is readily measurable in both short- and long-term culture. iCell Macrophages 2.0 have a functional ADCP response to antibody-labeled target cells with a larger dynamic range than primary monocyte-derived macrophages. Proinflammatory iCell Macrophages 2.0 are able to sensitize hepatocytes to immune-mediated hepatotoxic compounds, while sensory neurons and cardiomyocytes have altered functional outputs to polarized macrophages.ConclusionsHuman iPSC-derived macrophages provide a constant, reproducible source of biologically relevant material. iCell Macrophages 2.0 are in a naïve functional state, able to respond to pro- or anti-inflammatory stimuli. Functionally, cytokine release and phagocytosis assays can be performed anytime from thaw up to 14 days in culture with consistent, reliable lot-to-lot performance. These cells are compatible with complex culture conditions across numerous tissue types. The functional profile of iCell Macrophages 2.0 demonstrates their suitability as a continuing source of human macrophages.