Protocol for the generation of HLF+ HOXA+ human hematopoietic progenitor cells from pluripotent stem cells

Sherry Li Zheng; Jonas L Fowler; Julie Y Chen; Christopher Li; Elaine Lin; Alana T Nguyen; Angela Chen; George Q Daley; Lay Teng Ang; Kyle M Loh

doi:10.1016/j.xpro.2024.103592

Protocol for the generation of HLF+ HOXA+ human hematopoietic progenitor cells from pluripotent stem cells

STAR Protoc. 2025 Mar 21;6(1):103592. doi: 10.1016/j.xpro.2024.103592. Epub 2025 Jan 24.

Authors

Affiliations

¹ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: slzheng@stanford.edu.
² Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA.
³ Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Belfer Center for Science and International Affairs, Harvard Kennedy School, Cambridge, MA 02138, USA.
⁴ Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
⁵ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA 94305, USA; Department of Urology, Stanford University, Stanford, CA 94305, USA.
⁶ Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: kyleloh@stanford.edu.

Abstract

Hematopoietic stem cells (HSCs) generate blood and immune cells. Here, we present a protocol to differentiate human pluripotent stem cells (hPSCs) into hematopoietic progenitors that express the signature HSC transcription factors HLF, HOXA5, HOXA7, HOXA9, and HOXA10. hPSCs are dissociated, seeded, and then sequentially differentiated into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and hematopoietic progenitors through the sequential addition of defined, serum-free media. This 10-day protocol enables the manufacturing of blood and immune cells in monolayer cultures. For complete details on the use and execution of this protocol, please refer to Fowler and Zheng et al.¹.

Keywords: cell differentiation; developmental biology; stem cells.

MeSH terms

Cell Culture Techniques* / methods
Cell Differentiation
Hematopoietic Stem Cells* / cytology
Hematopoietic Stem Cells* / metabolism
Homeodomain Proteins* / genetics
Homeodomain Proteins* / metabolism
Humans
Pluripotent Stem Cells* / cytology
Pluripotent Stem Cells* / metabolism
Transcription Factors* / metabolism

Substances

Homeodomain Proteins
Transcription Factors
HoxA protein