In vitro generation of human hematopoietic cells

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
Human hematopietic stem and progenitor cells


Method keywords
  • human HPCs
  • in vitro differentiation of hematopoietic cells
  • OP9-coculture
  • MS5-coculture
  • ATO system
  • organoid culture
  • FTOC
Scientific area keywords
  • immune deficiency
  • leukemia
  • human hematopoiesis
  • stem cells
  • gene editing
Method description

Better understanding of molecular mechanisms controlling both normal and malignant human hematopoiesis will lead to a more efficient therapy of immune deficiencies and lymphoid leukemias. Therefore, human hematopoietic progenitor cells (HPCs) are differentiated in vitro towards distinct hematopoietic lineages, with or without perturbation conditions such as gene targeting, viral transductions, specific compounds or blocking antibodies. Our lab has a broad expertise in the differentiation of human T cell progenitors, for which 3 different in vitro techniques are available:

  • 1) Fetal thymic organ cultures (FTOCs), using fetal thymic lobes from NOD/SCID mice as 3D-micro environment allowing human HPCs to differentiate towards T cells. ;
  • 2) OP9-coculture system, using OP9 mouse stromal cells with or without specific Notch ligands as a 2D-layer to culture human HPCs on ;
  • 3) Artificial Thymic Organoid (ATO) cultures, using Notch expressing MS5 mouse stromal cells in combination with human HPCs in 3D aggregates.

Furthermore, OP9- and MS5-cocultures are used in order to differentiate HPCs towards myeloid cells (dendritic cells, monocytes, granulocytes), B cells, NK cells and both erythrocytes and megakaryocytes. Differentiation of different cell types is determined using flow cytometry.

Lab equipment
  • Biosafety cabinet level 2 ;
  • Tissue culture incubator ;
  • Centrifuge ;
  • Flow cytometer.
Method status
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential


These techniques allow to study normal or aberrant differentiation of human hematopoietic stem cells in conditions of genetic or other perturbations in vitro. It permits a kinetic and quantitative analysis of human hematopoietic differentiation which is difficult in vivo.


The challenge of in vitro differentiation systems is reproducing the in vivo environment in which different hematopoietic cells arise. Although FTOCs and the ATO system offer a close physiological background, the use of OP9 or MS5 stromal cells also allows us to generate distinct hematopoietic cells resembling their in vivo counterparts. Gene targeting in human HSCs is still inefficient.


More efficient gene targeting in human HSCs is still desired for genetic studies, as well as further modifications that lead to a closer resemblance of the in vivo environment.

References, associated documents and other information


Taghon T et al. Blood 2002; 99(4):1197-204.

Schmitt TM et al. Immunity 2002; 17(6):749-56.

Van de Walle I et al. Blood 2011; 117(17):4449-59.

Seet CS et al. Nat Methods 2017; 14(5):521-530.

Montel-Hagen A et al. Cell Stem Cell 2019; 24(3):376-389.

Associated documents
lab website

Contact person

Tom Taghon


Ghent University (UGent)
Diagnostic Sciences