Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

Abstract

Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells (DCs), Langerhans cells (LCs), macrophages, and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 days of culture, and an additional 2 days are needed to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 days of culture with cytokines, depending on the cell type.

Transient CD15-positive endothelial phenotype in the human placenta correlates with physiological and pathological fetoplacental immaturity

“…Mouse monoclonal IgG against human CD34 (Ready-to-Use, DAKO, clone QBEnd 10; antibodies reacts with human hematopoietic progenitor cells, including myeloid and lymphoid progenitors, labels capillaries of most tissues [22]). Mouse monoclonal IgG against human CD45 (Ready-to-Use, DAKO, clones 2B11 and PD7/26; interacts with all hematopoietic cells from HSCs to mature blood cells [23]). For negative controls, the primary antibodies were substituted with the PBS.…”

Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141+XCR1+ dendritic cells

“…Although conventional DC have been differentiated previously from human iPS cells, 14,15 the reliance on animal products and the mouse OP-9 stromal cell line precludes their downstream clinical application. We therefore directed the differentiation of C19 towards the DC lineage by adapting protocols we had established previously for the differentiation of DC from human ES cells in the absence of animal products with the sole exception of matrigel, required for the routine passage of undifferentiated ES cells and iPS cells.…”

Role of SOX17 in hematopoietic development from human embryonic stem cells

“…Pre-HPCs then mature into CD34 ϩ CD43 ϩ CD45 ϩ HPCs that express CD45, a marker antigen specific to hematopoietic cells ( Figure 1A). 20,21 We improved the conventional culture system to efficiently induce HPCs in EB culture by modifying cytokine conditions and adding an inhibitor of TGF-␤ signaling ( Figure 1B). In our culture system, the expression of hematopoietic regulator genes such as RUNX1 and SCL/TAL1 increased in EBs after day 4 of culture accompanied by the decrease in expression of early mesodermal marker genes such as Brachyury ( Figure 1C).…”