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

Nature Protocols volume 6, pages 296–313 (2011)Cite this article

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Abstract

In this paper, 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 comprises 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 granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 d of culture with cytokines, depending on the cell type.

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Figure 1: Schematic diagram of the protocol used to obtain multipotent lin−CD34+CD43+CD45+ progenitors from human pluripotent stem cells.
Figure 2: Directed differentiation of lin−CD34+CD43+CD45+ progenitors into mature myelomonocytic cells.
Figure 3: Timing for directed differentiation of mature myelomonocytic cells from human pluripotent stem cells.
Figure 4: Morphological changes in OP9 cells associated with a loss of hematopoiesis-inductive properties.

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Acknowledgements

This study is supported by funds from the National Heart, Lung, and Blood Institute (R01 HL081962, R21 HL085223) and by National Institutes of Health grant P51RR000167 to the National Primate Research Center, University of Wisconsin, Madison. We thank T. Nakano for providing OP9 cells.

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Authors and Affiliations

  1. National Primate Research Center, University of Wisconsin Graduate School, Madison, Wisconsin, USA

    Kyung-Dal Choi, Maxim Vodyanik & Igor I Slukvin

  2. Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, USA

    Igor I Slukvin

Authors
  1. Kyung-Dal Choi
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  2. Maxim Vodyanik
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  3. Igor I Slukvin
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Contributions

K.-D.C. designed and performed the experiments, analyzed the data and wrote the paper. M.V. designed and performed the experiments. I.I.S. supervised the project, designed the experiments, analyzed data and wrote the paper.

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Correspondence to Igor I Slukvin.

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I.I.S. owns stock and is a scientific founder of Cellular Dynamics International.

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Choi, KD., Vodyanik, M. & Slukvin, I. Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc 6, 296–313 (2011). https://doi.org/10.1038/nprot.2010.184

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