**Acknowledgements**

conditions. Upon culture, at least 1-2% of these cells generate primitive and definitive haema‐ topoietic cells [20]. During this process, the haemogenic endothelial cell population (TIE2+,c-KIT+,CD41-) cells gradually acquire the expression of CD41. The cells then progress further

To evaluate the presence of haematopoietic colonies, EBs should be harvested at day 4,5 or 6 and trypsinised. Cells from the EBs can be then directly used or alternatively sorted for the expression of a marker of haematopoietic progenitors, such as for example CD41. Approxi‐ mately 3.0 x 104 unsorted cells should be plated in 35 mm x 10 mm (BD Falcon) dishes in 1 ml of semisolid medium containing IMDM, 15% plasma derived serum (PDS) (Antech), 10% protein free hybridoma medium (PFHM, Gibco), 2mM L-Glutamine, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG and cytokines such as: 1% c-KIT ligand supernatant, 1% interleukin 3 supernatant (IL-3) (see [76]), 1µg/ml GM-CSF, 1% thrombopoietin condi‐ tioned media, 10 ng/ml IL-6 (Peprotech), 10 ng/ml macrophage colony stimulating factor (M-CSF), 5 ng/ml IL-11 (R&D Systems) and 4 U/ml of Erythropoietin (Ortho-Biotech) and 10 g/L methylcellulose (dissolved in IMDM, Alfa-Aesar). Haematopoietic colonies are assessed and scored based on their morphology. Primitive erythroid colonies are scored at day 5, whereas definitive haematopoietic colonies are usually enumerated 8 days after replating. Morphologic landmarks are used to distinguish the different types of haematopoietic colonies. Haemato‐ poietic progenitors can also be cultured in liquid conditions to allow easier access of the cells for subsequent flow cytometry analysis or cytospin assays. For that cells should be seeded at a density of 2.0 x 106 /ml in ultra low-adherence tissue culture plates (Costar) in the haemato‐ poietic medium described above with methylcellulose being replaced with IDMD medium.

The onset of emergence of primitive erythroid cells is observed within EBs by day 4 of differentiation [81]. Definitive erythroid and macrophage precursors appear shortly after and are followed by mast cells and multilineage precursors [81]. Primitive colonies appear around day 4 of culture. These colonies are round, compact and bright red in colour. By day 6-7 of culture, morphologically distinguishable definitive haematopoietic colonies are detected

In this chapter, we have reviewed recent progress in our understanding of the development of the haematopoietic system. We have emphasized the critical role that the use of ES cells, in particular murine ES cells, has played in these recent advances. ES cells have been instrumental to identify and characterise the elusive haemangioblast. More recently, this model system allowed the merging of the two conflicting theories of the origin of blood cells (haemangioblast and haemogenic endothelium) in a single linear model of development. In addition the precise roles and requirements of many critical regulators of this process have been to a large extent

and lose their expression of endothelial markers.

78 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

**3.6. Haematopoietic colonies assays**

(Figure 7).

**4. Conclusions and future directions**

elucidated using this approach.

We apologize to the many colleagues whose work could not be cited owing to space con‐ straints. Our work is supported by Cancer Research UK (CRUK), Leukaemia and Lymphoma research (LLR) and the Biotechnology and Biological Sciences Research Council (BBSRC).

## **Author details**

Monika Stefanska1,2, Valerie Kouskoff3 and Georges Lacaud1

1 Cancer Research UK Manchester Institute, Stem Cell Biology Group, University of Man‐ chester, Manchester, United Kingdom

[11] Choi, K., et al., *A common precursor for hematopoietic and endothelial cells.* Development,

Embryonic Stem Cell Differentiation – A Model System to Study Embryonic Haematopoiesis

http://dx.doi.org/10.5772/57607

81

[12] Faloon, P., et al., *Basic fibroblast growth factor positively regulates hematopoietic develop‐*

[13] Fehling, H.J., et al., *Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation.* Development, 2003. 130(17): p. 4217-27.

[14] Huber, T.L., et al., *Haemangioblast commitment is initiated in the primitive streak of the*

[15] Kennedy, M., et al., *Development of the hemangioblast defines the onset of hematopoiesis in*

[16] Vogeli, K.M., et al., *A common progenitor for haematopoietic and endothelial lineages in the*

[17] Nishikawa, S.I., et al., *Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+cells at a diverging point of endothelial and hemopoietic lineages.* Devel‐

[18] Nishikawa, S.I., et al., *In vitro generation of lymphohematopoietic cells from endothelial*

[19] Jaffredo, T., et al., *Intraaortic hemopoietic cells are derived from endothelial cells during on‐*

[20] Lancrin, C., et al., *The haemangioblast generates haematopoietic cells through a haemogenic*

[21] Zovein, A.C., et al., *Fate tracing reveals the endothelial origin of hematopoietic stem cells.*

[22] Eilken, H.M., S. Nishikawa, and T. Schroeder, *Continuous single-cell imaging of blood generation from haemogenic endothelium.* Nature, 2009. 457(7231): p. 896-900.

[23] Boisset, J.C., et al., *In vivo imaging of haematopoietic cells emerging from the mouse aortic*

[24] Kissa, K. and P. Herbomel, *Blood stem cells emerge from aortic endothelium by a novel*

[25] Bertrand, J.Y., et al., *Haematopoietic stem cells derive directly from aortic endothelium dur‐*

[26] Lam, E.Y., et al., *Live imaging of Runx1 expression in the dorsal aorta tracks the emergence*

[27] Lancrin, C., et al., *Blood cell generation from the hemangioblast.* J Mol Med (Berl), 2010.

*of blood progenitors from endothelial cells.* Blood, 2010. 116(6): p. 909-14.

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*togeny.* Development, 1998. 125(22): p. 4575-83.

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2 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Kraków, Poland

3 Cancer Research UK Manchester Institute, Stem Cell Haematopoiesis Group, University of Manchester, Manchester, United Kingdom

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**Chapter 4**

**Human Embryonic Stem Cell-Derived Primitive and**

It is well believed that human embryonic stem cells (hESCs [1]) and induced pluripotent stem cells (hiPSCs [2]) are of great potential use for tissue substitutes (for example, blood cells) and to cure various congenital disorders. In mammals, hematopoiesis has already been precisely described in murine system but not yet in human. Early development of hematopoietic system can be well defined by a series of waves from primitive hematopoiesis (early embryogenesis) to definitive ones (late fetal stages). In vitro induction of undifferentiated hESC to functionally mature blood cells may mimic the early hematopoietic development during human embryonic and fetal stages. It also provides an ideal model to uncover molecular and cellular mechanisms controlling early development of human hematopoiesis. On the other hand, functionally matured blood cells derived from hESC/hiPSCs are expected to be widely used for clinical cellular therapies. Although almost all kinds of the mature blood cells can be generated from hESCs, there still lacks solid evidence for the generation of reconstituting hematopoietic stem cells (HSCs) from hESC or hiPSC. So far until now, in vitro hESC-derived blood cells possess phenotypical maturity and partial functions while still more or less share embryonic/fetal characteristics, differing greatly from their adult counterparts. This indicated that in vitro culture systems are not perfect enough to exert full mature activities. Lack of knowledge about the molecular and cellular regulations in human early hematopoiesis has handicapped the

Having been focusing on basic and clinical research on hESC/hiPSC-derived functionally mature blood cells for long, our group has established an efficient method to induce large-scale production of multipotential hematopoietic progenitor cells by coculturing hESC/hiPSCs with murine hematopoietic niche-derived stromal cells [3-6]. By this method, large quantity of

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Definitive Hematopoiesis**

Kohichiro Tsuji and Feng Ma

http://dx.doi.org/10.5772/58628

**1. Introduction**

Bo Chen, Bin Mao, Shu Huang, Ya Zhou,

Additional information is available at the end of the chapter

development of research on hESC/hiPSC-derived hematopoiesis.
