**3. Methods and procols to study haematopoitic development during es cell differentiation**

ES cell differentiation provides a relatively easy and accessible system to study early embry‐ onic haematopoiesis. Using well-defined protocols, it is possible to effectively study the events happening *in vivo* using this *in vitro* approach. This experimental system was shown to recapitulate the early *in vivo* events of development of the haematopoietic system.

#### **3.1. Mouse embryonic fibroblasts (MEFs)**

There are several methods to keep ES cells undifferentiated. One of them consists of growing ES cells on mouse embryonic fibroblasts (MEFs). Before working with ES cells, it is recom‐ mended to prepare a good stock of MEFs to be used as feeder cell layer. For that, wild type ICR or DR4 (resistant to four drugs) [75] MEFs are harvested from E14.5 embryos and cultured in Iscove's modified Dulbecco medium (IMDM, Lonza) supplemented with 50 µg/ml penicil‐ lin-streptomicin (Gibco), 2mM L-Glutamine, 10% of FCS (PAA Laboratories) and 1,5x10-4 monothioglycerol (MTG, Sigma) under low oxygen conditions. When amplified, MEFs are harvested (TrypLE, Invitrogen) and irradiated at 30Gy to stop the cells proliferation. The cells should be frozen at around 1 million cells per ml of IMDM supplemented with 50% FCS and 10% of dimethyl sulfoxide (DMSO). Cells should be stored at-80 0 C. Thawed MEFs should be replated in one six-well plate previously coated with gelatine and let to adhere to the plastic wells overnight. Upon microscopic examination, MEFs should cover the entire surface of the cell-culture dish and be ready to be seeded with ES cells.

## **3.2. ES cell culture**

**Figure 2. Molecular regulation of early embryonic haematopoiesis.** The stages of blood development where the

**3. Methods and procols to study haematopoitic development during es cell**

ES cell differentiation provides a relatively easy and accessible system to study early embry‐ onic haematopoiesis. Using well-defined protocols, it is possible to effectively study the events happening *in vivo* using this *in vitro* approach. This experimental system was shown to

There are several methods to keep ES cells undifferentiated. One of them consists of growing ES cells on mouse embryonic fibroblasts (MEFs). Before working with ES cells, it is recom‐ mended to prepare a good stock of MEFs to be used as feeder cell layer. For that, wild type ICR or DR4 (resistant to four drugs) [75] MEFs are harvested from E14.5 embryos and cultured in Iscove's modified Dulbecco medium (IMDM, Lonza) supplemented with 50 µg/ml penicil‐

recapitulate the early *in vivo* events of development of the haematopoietic system.

function of the different genes is critical are indicated.

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

**3.1. Mouse embryonic fibroblasts (MEFs)**

**differentiation**

ES cells are cultured on irradiated MEFs in a media constituted of Dulbeco's modified Eagle Medium (DMEM, Gibco) supplemented with 50 µg/ml penicillin-streptomycin, 2mM L-Glutamine, 15% FCS (PAA Laboratories), 2% Leukaemia Inhibitory Factor (LIF) (conditioned medium from LIF-generating cell line, see [76]) or 50 units of recombinant ESGRO LIF/ml (Millipore) and 1,5x104 MTG (Sigma). Leukaemia inhibitory factor (LIF) – is a cytokine inhibiting differentiation. ES cells, when cultured on MEFs feeder cell layer in the presence of LIF remain undifferentiated. Upon microscopic observation they form tightly associated clusters of cells that are bright and shiny in appearance (Fig. 3A).

#### **3.3. Generation of embryoid bodies**

Embryoid bodies (EBs) are three-dimensional structures spontaneously generated by ES cells during differentiation. They contain precursors for the three primary germ layers ectoderm, endoderm and mesoderm. Two passages on gelatine are performed to remove the MEFs that would hamper ES cells differentiation. The first passage is performed in DMEM-ES media (described above), whereas for the second passage DMEM is replaced with IMDM. The ES cells are then harvested by trypsinisation and seeded into liquid cultures in non-tissue culture Petri dishes (Sterilin) in differentiation medium containing: IMDM supplemented with 15% FCS serum selected for differentiation (PAA Laboratories), 2mM L-Glutamine, 180 µg/ml transferring (Roche), 25 µg/ml Ascorbic Acid (AA, Sigma) and 4,6x10-4 MTG. The density of cell seeding should be adjusted in function of the day at which the cultures will be harvested, varying from 1,5x104 cells/ml (for day 4-6) up to 3,0 x 104 cells/ml (for days 2.5-3.5). 10-20ml of "Differentiation medium" should be used per one Petri dish.

By performing two passages on gelatine and removing feeder cell layer and LIF, ES cells become primed for differentiation and formation of three-dimensional embryoid bodies in liquid culture (Figure 3B). Early EBs contain precursors for the three primary germ layers. By day 7, hemoglobinisation can be observed as red areas present within the EBs (Figure 3C). This system is versatile and allows to access and study in details several subsequent stages of blood development such as the emergence of haemangioblast, production of blast colonies and the development of primitive and definitive blood precursors (Figure 4).

**Figure 4. The ES/EB differentiation system.** Upon differentiation of ES cells, three-dimensional structures, called em‐ bryoid bodies (EBs) are formed. Sorting EBs at day 2.5-3.5 for the expression of FLK1 enrich for BL-CFC, the *in vitro* equivalent of the haemangioblast. Upon culture, the BL-CFCs generate blast colonies that contain precursors for hae‐ matopoietic, endothelial and vascular smooth muscle cells. Day 4-6 EBs contain haematopoietic progenitors that can

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There are two alternative approaches to study the development of blast colonies; liquid culture on gelatine or semi-solid culture in methylcellulose. Liquid culture on gelatine facilitates harvesting of the cells for flow cytometry analysis or time-lapse imaging techniques. Alterna‐ tively, the semi-solid culture is a clonogenic assay allowing the growth of of individual blast colonies and their quantification. Individual blast colonies can then further be isolated and their haematopoietic potential assessed. To perform blast colony culture, EBs are harvested between day 2.5 and 3.5 and the cells are enriched for haemangioblast by sorting for the expression of FLK1, the receptor 2 for VEGF. Isolated FLK1 positive cells are then replated on gelatinised cell culture plates at the density of 8,5 x 104 cell/10cm2 in a medium containing IMDM supplemented with 10% FCS, 2mM L-Glutamine mM, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 15% endothelial cell line-D4T conditioned medium [11], 10

give rise to various primitive and definitive haematopoietic colonies.

**3.4. Blast colonies**

**Figure 3. Morphology of ES cells and embryoid bodies (EBs).** A) Typical appearance of ES cell in culture. Cells form bright, shiny adherent colonies and are cultured on a layer of MEFs feeder cell layer. B) Typical appearance of EBs in culture. These three-dimensional structures are formed during ES cell differentiation and contain precursors for three primary germ cell layers. C) Embryoid bodies at day 7 of differentiation containing haemoglobin (indicated with aster‐ isks). Scale bar 300µm.

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**Figure 4. The ES/EB differentiation system.** Upon differentiation of ES cells, three-dimensional structures, called em‐ bryoid bodies (EBs) are formed. Sorting EBs at day 2.5-3.5 for the expression of FLK1 enrich for BL-CFC, the *in vitro* equivalent of the haemangioblast. Upon culture, the BL-CFCs generate blast colonies that contain precursors for hae‐ matopoietic, endothelial and vascular smooth muscle cells. Day 4-6 EBs contain haematopoietic progenitors that can give rise to various primitive and definitive haematopoietic colonies.

#### **3.4. Blast colonies**

**Figure 3. Morphology of ES cells and embryoid bodies (EBs).** A) Typical appearance of ES cell in culture. Cells form bright, shiny adherent colonies and are cultured on a layer of MEFs feeder cell layer. B) Typical appearance of EBs in culture. These three-dimensional structures are formed during ES cell differentiation and contain precursors for three primary germ cell layers. C) Embryoid bodies at day 7 of differentiation containing haemoglobin (indicated with aster‐

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

isks). Scale bar 300µm.

There are two alternative approaches to study the development of blast colonies; liquid culture on gelatine or semi-solid culture in methylcellulose. Liquid culture on gelatine facilitates harvesting of the cells for flow cytometry analysis or time-lapse imaging techniques. Alterna‐ tively, the semi-solid culture is a clonogenic assay allowing the growth of of individual blast colonies and their quantification. Individual blast colonies can then further be isolated and their haematopoietic potential assessed. To perform blast colony culture, EBs are harvested between day 2.5 and 3.5 and the cells are enriched for haemangioblast by sorting for the expression of FLK1, the receptor 2 for VEGF. Isolated FLK1 positive cells are then replated on gelatinised cell culture plates at the density of 8,5 x 104 cell/10cm2 in a medium containing IMDM supplemented with 10% FCS, 2mM L-Glutamine mM, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 15% endothelial cell line-D4T conditioned medium [11], 10 ng/ml interleukin 6 (IL-6) (Peprotech) and 5 ng/ml vascular endothelial growth factor (VEGF, Peprotech). For semi-solid cultures, FLK1 positive cells are seeded in 35 mm x 10 mm (BD Falcon) dishes at a density of 1.5 x 104 cells/ml in IMDM medium supplemented with 10% FCS, 2mM L-Glutamine mM, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 15% endothelial cell line-D4T conditioned medium [11], 10 ng/ml interleukin 6 (IL-6) (Peprotech) and 5 ng/ml vascular endothelial growth factor (VEGF, Peprotech). The medium is additionally supplemented with 10 g/L methylcellulose (dissolved in IMDM, Alfa-Aesar). Blast colonies are scored 3-4 days after replating. Plating 3.0 x 104 cells per 1 ml of semi-solid medium should result in the formation of around 300-400 of blast colonies.

It was recently shown that the haemangioblast progenitor can also generate vascular smooth muscle cells in addition to endothelial and haematopoietic cells [14, 77, 78]. Accordingly, Yamashita and co-workers also established that FLK1 positive cells can differentiate towards endothelial and mural cells [78]. Smooth muscle cells appear, under the microscope as large flat adherent cells. These cells express smooth muscle specific genes such as smooth muscle actin α, transgelin, calponin, and smooth muscle myosin heavy chain [79]. A typical immunostaining of α-smooth muscle actin is presented in figure 6A. We have recently analysed in more detail the generation of smooth muscle cells and showed that these cells are largely generated

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**Figure 6. The differentiation of ES cells towards smooth muscle cells.** Representative image of smooth muscle cells stained with SMAα-Cy3 antibody. These cells also carry a transgenic BAC containing H2B-VENUS cDNA under the

During blast colony development *in vitro*, tight clusters of adherent cells that represent haemogenic endothelium are observed after around two days. This population of cells corresponds to an intermediate stage in the formation of blood cells from the haemangioblast. It was recently shown that these cells express TIE2 and VE-cadherin (markers of endothelial cells), c-KIT (expressed by both haematopoietic and endothelial cells) and are negative for CD41 (first haematopoietic marker) expression [20]. To isolate haemogenic endothelium, cells in day 2 liquid blast cultures are harvested and FACS sorted for the expression of these markers [20]. It is also possible to isolate a more advanced haemogenic endothelium cell population, positive for the expression of CD41 [70]. The cells are next plated onto gelatin-coated plates at

Glutamine, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 10 ng/ml Oncos‐ tatin M (R&D Systems) and 1 ng/ml bFGF (Peprotech). Cells are grown in standard culture

in IMDM medium supplemented with 10% FCS, 2 mM L-

control of the α-smooth muscle actin transcriptional regulatory elements. Scale bar 100µm.

**3.5. Culture of haemogenic endothelium**

a density of 1.2 x 105 cells/cm2

independently from the haemogenic endothelium [80].

Sorting EBs between day 2.5 and 3.5 for the expression of FLK1 enrich for blast colony forming cell (BL-CFCs) [11, 13]. During the formation of blast colonies, several distinct morphological stages can be observed. First, there is the formation of tight clusters of adherent cells and later, single round cells emerging from these tight clusters. These cells then proliferate (Figure 5A). Alongside these morphological changes, there is first an upregulation of endothelial markers, such as TIE2 and VE-Cadherin during the formation of the tight clusters. Later the expression of these endothelial markers is gradually downregulated whereas the expression of haema‐ topoietic markers (such as CD41 and then CD45) is upregulated (Figure 5B). This correlates with the emergence of round floating–haematopoietic-cells.

**Figure 5. The development of the blast colony during four days of differentiation**. A) Microscopic image showing representative blast colonies. Scale bar 100µm. B) Schematic representation of the different cell surface markers ex‐ pressed on cells during blast colony differentiation.

It was recently shown that the haemangioblast progenitor can also generate vascular smooth muscle cells in addition to endothelial and haematopoietic cells [14, 77, 78]. Accordingly, Yamashita and co-workers also established that FLK1 positive cells can differentiate towards endothelial and mural cells [78]. Smooth muscle cells appear, under the microscope as large flat adherent cells. These cells express smooth muscle specific genes such as smooth muscle actin α, transgelin, calponin, and smooth muscle myosin heavy chain [79]. A typical immunostaining of α-smooth muscle actin is presented in figure 6A. We have recently analysed in more detail the generation of smooth muscle cells and showed that these cells are largely generated independently from the haemogenic endothelium [80].

**Figure 6. The differentiation of ES cells towards smooth muscle cells.** Representative image of smooth muscle cells stained with SMAα-Cy3 antibody. These cells also carry a transgenic BAC containing H2B-VENUS cDNA under the control of the α-smooth muscle actin transcriptional regulatory elements. Scale bar 100µm.

#### **3.5. Culture of haemogenic endothelium**

ng/ml interleukin 6 (IL-6) (Peprotech) and 5 ng/ml vascular endothelial growth factor (VEGF, Peprotech). For semi-solid cultures, FLK1 positive cells are seeded in 35 mm x 10 mm (BD

2mM L-Glutamine mM, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 15% endothelial cell line-D4T conditioned medium [11], 10 ng/ml interleukin 6 (IL-6) (Peprotech) and 5 ng/ml vascular endothelial growth factor (VEGF, Peprotech). The medium is additionally supplemented with 10 g/L methylcellulose (dissolved in IMDM, Alfa-Aesar). Blast colonies

Sorting EBs between day 2.5 and 3.5 for the expression of FLK1 enrich for blast colony forming cell (BL-CFCs) [11, 13]. During the formation of blast colonies, several distinct morphological stages can be observed. First, there is the formation of tight clusters of adherent cells and later, single round cells emerging from these tight clusters. These cells then proliferate (Figure 5A). Alongside these morphological changes, there is first an upregulation of endothelial markers, such as TIE2 and VE-Cadherin during the formation of the tight clusters. Later the expression of these endothelial markers is gradually downregulated whereas the expression of haema‐ topoietic markers (such as CD41 and then CD45) is upregulated (Figure 5B). This correlates

**Figure 5. The development of the blast colony during four days of differentiation**. A) Microscopic image showing representative blast colonies. Scale bar 100µm. B) Schematic representation of the different cell surface markers ex‐

cells/ml in IMDM medium supplemented with 10% FCS,

cells per 1 ml of semi-solid medium should

Falcon) dishes at a density of 1.5 x 104

are scored 3-4 days after replating. Plating 3.0 x 104

result in the formation of around 300-400 of blast colonies.

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with the emergence of round floating–haematopoietic-cells.

pressed on cells during blast colony differentiation.

During blast colony development *in vitro*, tight clusters of adherent cells that represent haemogenic endothelium are observed after around two days. This population of cells corresponds to an intermediate stage in the formation of blood cells from the haemangioblast. It was recently shown that these cells express TIE2 and VE-cadherin (markers of endothelial cells), c-KIT (expressed by both haematopoietic and endothelial cells) and are negative for CD41 (first haematopoietic marker) expression [20]. To isolate haemogenic endothelium, cells in day 2 liquid blast cultures are harvested and FACS sorted for the expression of these markers [20]. It is also possible to isolate a more advanced haemogenic endothelium cell population, positive for the expression of CD41 [70]. The cells are next plated onto gelatin-coated plates at a density of 1.2 x 105 cells/cm2 in IMDM medium supplemented with 10% FCS, 2 mM L-Glutamine, 180 µg/ml transferrin, 25 µg/ml ascorbic acid, 4.6 x 10-4 M MTG, 10 ng/ml Oncos‐ tatin M (R&D Systems) and 1 ng/ml bFGF (Peprotech). Cells are grown in standard culture 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 and lose their expression of endothelial markers.

#### **3.6. Haematopoietic colonies assays**

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 (Figure 7).

Although only very succinctly discussed in this chapter, this system is also very amenable to examination of the cell signalling pathways that support the development of normal haema‐ topoiesis [33, 34, 82-84]. Interestingly these pathways are also implicated in leukaemogenesis [85, 86]. Finally with the advent of novel human ES cells or human iPS cells [87], that recapit‐ ulate better the ground state and are easier to work with, and development of new methods facilitating genome editing [88-92], this experimental system is very likely to be instrumental for delivering new advances in our understanding of human haematopoietic development,

**Figure 7.** Examples of haematopoietic colonies obtained during ES cell differentiation A) Primitive erythroid colony – red in colour, compact and relatively small. B) Definitive myeloid colony – bigger in size, white and looser cells. C)

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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).

that is otherwise very difficult to study *in vivo*.

Mixed haematopoietic colony. Scale bar 50µm.

**Acknowledgements**
