**3. hESC-derived primitive and definitive hematopoietic cells**

represses Gata2, c-Kit, c-Myb, and c-Myc, responding for the proliferation of progenitors in

**AML1/Runx1.** Homozygous mutations of AML1/Runx1 did not interfere normal morpho‐ genesis and YS-derived erythropoiesis, but completely inhibit FL hematopoiesis, leading to the death of embryo around E12.5. The same mutation in ES cells do not influence their differentiation potential into primitive erythroid cells in vitro while stop occurence of any definitive myeloid or erythroid progenitors in both the YS and FL after injected to blastocysts to produce chimeric animals. Above proofs support the key role of AML1 and AML1-regulated

**c-Myb.** c-Myb is highly expressed in immature hematopoietic cells and its expression is downregulated as they become more differentiated [67, 68]. c-Myb controls self-renewal and differentiation of adult HSCs and its disruption seriously depletes the HSC pool and inhibits

**EKLF.** EKLF is zero or very weakly expressed in hematopoietic stem cells and multipoten‐ tial myeloid progenitors, while arise since more matured stages and play a role all the time later for RBC differentiation. Its expression is up-regulated when myoloid and erythriod progenitors were committed to the erythroid lineage while down-regulated when differen‐ tiate toward megakaryopoiesis [70]. During the global expansion of erythroid gene expression in primitive and definitive lineages, EKLF also plays a direct role in globin switching. EKLF is weakly expressed during embryonic and fetal development, which led to a low expression of adult β-globin, Bcl11a and a high one of γ-globin. While in adults, EKLF is highly expressed in definitive RBCs that results in high levels of adult β-globin and Bcl11a expression, and represses γ-globin expression [71]. Finally, EKLF stop the cell cycle of RBCs at the terminal maturation [72]. EKLF/KLF1 mutations will change the RBC

**Rbtn2.** Rbtn2 is a nuclear protein expressed in erythroid lineage in vivo, which is essential for erythroid development in mice. The homozygous mutation of rbtn2 inhibits YS erythropoiesis and leads to embryonic lethality around E10.5. YS tissue from homozygous mutant mice and double-mutant ES cells could not process erythroid development in in vitro differentiation system, showing a key role for Rbtn2 in erythroid differentiation, which is high related to

**EPO/EPOR.** Erythropoietin is a glycoprotein produced primarily by kidney and is the principal factor to regulate RBC production, mainly functioning on erythroid progenitors within the FL and adult BM [76]. The erythroid progenitors before BFU-E stage and RBC after late basophilic erythroblast stage are not responsive to EPO. Proliferation in CFU-E stage could highly responsive to EPO and this response is very transient. The affinity between erythro‐ poietin and its receptor (EPO-R) and their concentration decide strength of such response during the erythropoiesis. EPO-R signaling pathway is necessary for both primitive and

earlier stages of hematopoiesis [65].

the definitive hematopoiesis [69].

phenotypes or even lead to disease. [73, 74]

GATA-1 [75]

definitive erythropoiesis [77, 78].

target genes to all lineages in definitive hematopoiesis [10, 66]

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

The first hESC line was established by Thomson's group in 1998 [1] and then the first hiPSC line by Yamanaka's group in 2007 [2]. Both cells provide possibility to uncover various normal or diseased mechanisms in early human development. By in vitro differentiation system, factors controlling the primitive and definitive hematopoiesis could be investigated in detail using the method of embryoid body (EB) forming [79], or co-culture with hematopoietic nichederived stromal cell lines [3-6, 80, 81]. Among them, method of EBs could obtain large quantity of blood cells that were not well matured, mimicking the primitive hematopoietic cells [82, 83]. Then, co-culture with OP9 cells was applied to promote the differentiation based on EB method and large-scale production of mature erythrocytes with some definitive properties could be obtained [84, 85]. Although OP9 co-culture system could obtain robust growth of matured blood cells, it is clearly not a natural process and could not be used as proper model to elucidate the natural mechanism controlling early human hematopoiesis. The stromal cells isolated from early hematopoietic niches, such as AGM region, FL and late-stage fetal BM, should be more reasonable candidates to support the in vitro differentiation of hESC/hiPSCderived hematopoietic stem/progenitor cells. The Lako's group compared the differences among several in vitro differentiation systems based on stromal cell co-culture and optimized their culture conditions. Their result suggested that AGM-derived cell line was most proper for the hematopoiesis differentiation of ESC. [86]

Based on in vitro differentiation systems, researchers tried to make clear the details of early human hematopoiesis using hESCs as a model. Keller's group found two distinct types of hemangioblasts during hESCs differentiation culture, one could give rise to primitive eryth‐ roid, macrophages and endothelial cells, while the other one generated only primitive erythroid and endothelial cells [87]. Their work provided the first evidence to prove the existence of hemangioblasts derived from hESCs in vitro. The follow-up work showed that under the control of growth factors and hematopoietic cytokines the hematopoiesis and myelopoiesis will happen during the later stage; and common bi-potent progenitor capable of generating erythroid and megakaryocytic cells could be observed, mimicking the process in vivo [88, 89]. Through the analysis of hemoglobin components, an erythroid maturation could be observed by the ratio of β-globin expression during hESC differentiation culture, which seems a maturation switch but not lineage switch [90]. Above researches indicated that the in vitro hESC differentiation system could reflect the in vivo hematopoietic process during the early human embryonic development.

Slukvin's group applied PO9 co-culture system to explore the detail pathway from hESCs to definitive hematopoiesis. They firstly characterized a population of differentiating hemato‐ poietic cells defined by the expression of CD43, which is distinct to endothelial and mesen‐ chymal cells. Then they defined the erythro-megakaryocytic progenitors (CD34+CD43+CD235a+CD41a+/-CD45-) and multi-potent lymphohematopoietic progenitors (CD34+CD43+CD235a-CD41a-CD45-) in later stage, which replicated the beginning of definitive hematopoiesis in some degree [91, 92]. The human myelomonocytic cells could also be generated from expansion and differentiation of pluripotent stem cell-derived linCD34+CD43+CD45+ progenitors. All the defined population above could be detected during the in vitro differentiation for different hESCs or hiPSCs lines [92].

**3.1. hESC-derived erythrocytes**

tion and release oxygen [5].

production by this method.

**3.2. hESC-derived HSCs**

definition.

done by several other groups using OP9 system [95, 96]

Research on hESC-derived erythrocytes is one of the hot points because of the fact that development of erythrocytes shows distinct trait between primitive and definitive waves. More important is that the matured RBCs have no nucleus, which could avoid the exogenous gene interference when clinically applied to treat patients. Much labor has been donated to optimize the method of in vitro differentiation of RBCs from hESCs. For example, Lu's group firstly use EBs formation and then co-culture with OP9 to obtain a high yield of erythrocytes with some efficiency of enucleation. However, these erythrocytes were still immature because only 16% of these hESC-derived erythrocytes expressed β-globin [84]. Similar work has been

Human Embryonic Stem Cell-Derived Primitive and Definitive Hematopoiesis

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

97

In our laboratory, we have recently established efficient blood cell-yeilding systems by coculture of hESC/hiPSCs with murine AGM and FL stromal cells (Figure 3). In the co-culture, undifferentiated hESC/hiPSC colonies grow up and differentiate firstly to a mesoderm-like structure, then to hematopoietic progenitor cells on days 10 to 14. In the second suspension culture, these hESC/hiPSC-derived hematopoietic progenitors are further induced to some specific blood cell lineages, such as erythrocytes, mast cells, and eosinophils, etc. [3]. By a clonetracing method, we gained concrete results that hESC-derived erythrocytes kept continuously progressing toward maturation over a time course. The expression of β-globin vs ε-globin showed a typical switching pattern, mimicking the normal development of human erythro‐ cytes. On day 12 of the co-culture with murine FL-derived stromal cells, hESC-derived erythrocytes (BFU-E) express β-globin at about 60%, but up-regulated to almost 100% with additional 6 days of culture. These matured hESC-derived erythrocytes can undergo enuclea‐

Our results showed that the in vitro differentiation from hESCs to matured hematopoietic cells is a progressive process if proper co-culture condition provided. Functionally matured blood cells similar to those from the definitive hematopoiesis could be obtained in large-scale

The most challenging task for research on hESC/hiPSC-derived hematopoiesis is to obtain the real HSC from in vitro differentiation system [97]. Although hESC/hiPSC-derived hemato‐ poietic cells that could be engrafted in immune compromised mice have been reported [86, 98-102], effort to obtain real HSCs from hESCs has largely been done in vain during past years. In most experiments, the engraftment rate was very low and mostly restricted to the myeloid lineage, and it was ambiguous if these engrafed cells were derived from real HSCs. The coculture with S17 could induce hESCs to HSC-like properties with low capacity of RBC potential [103]. If a modified cell line generated from mAGM (AM20.1B4) was used the RBC activity of HSC will be increased much [86]. But these hESC-derived HSC-like cells could not repopulate in NOD-SCID mice, representing they were not true definitive HSCs that satisfy the functional

By different culture conditions, nearly all of the blood lineages could be obtained from hESCs according to the experience from murine ESC protocols, which pave the way to the clinic application [93]. Jame's group also identified a wave of hemogenic endothelial development during the transition from endothelial to hematopoietic cell [94], reflecting the classic property of definitive hematopoiesis. Their result showed that the definitive hematopoiesis could be researched in vitro differentiation system and most progenitor population could be obtained in such a system.

Although research on the early development of human hematopoiesis using human embryo is rigorously restricted by ethics, so far accumulated data have clearly demonstrated that in vitro hESC-derived hematopoiesis is more or less similar to the events happening in murine fetal development (Figure 2). Thus, research on hESC-hematopoiesis should contribute greatly to understand the developmental controls of human early hematopoiesis. Fig. 2. Comparison of mouse model and in vitro hESC/hiPSC-derived hematopoiesis

**Figure 2.** Comparison of mouse model and in vitro hESC/hiPSC-derived hematopoiesis

#### **3.1. hESC-derived erythrocytes**

CD34+CD43+CD45+ progenitors. All the defined population above could be detected during

By different culture conditions, nearly all of the blood lineages could be obtained from hESCs according to the experience from murine ESC protocols, which pave the way to the clinic application [93]. Jame's group also identified a wave of hemogenic endothelial development during the transition from endothelial to hematopoietic cell [94], reflecting the classic property of definitive hematopoiesis. Their result showed that the definitive hematopoiesis could be researched in vitro differentiation system and most progenitor population could be obtained

Although research on the early development of human hematopoiesis using human embryo is rigorously restricted by ethics, so far accumulated data have clearly demonstrated that in vitro hESC-derived hematopoiesis is more or less similar to the events happening in murine fetal development (Figure 2). Thus, research on hESC-hematopoiesis should contribute greatly

Fig. 2. Comparison of mouse model and in vitro hESC/hiPSC-derived hematopoiesis

Fetal Liver

Bone Marrow

the in vitro differentiation for different hESCs or hiPSCs lines [92].

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

to understand the developmental controls of human early hematopoiesis.

Yolk Sac AGM

Primitive Hematopoiesis (E.7.5~ ) Definitive Hematopoiesis (E10.5~)

Hemangioblasts Hemogenic Endothelial Cells

Various Hematopoietic Cells

Cells Blood

Hemangioblasts Hemogenic Endothelial Progenitor

(Definitive Hematopoiesis)

(M Kennedy & G Keller, 2006) (H Maria & M Lako, 2008; ) ; (F Ma & K Tsuji, 2008); (KD Choi & II. Slukvin, 2012)

(AL Medvinsky & EA Dzierzak 1996) ; (F Ma & K Tsuji, 2008 ) (J Palis & MC Yoder, 2001)

Co-culture of hESC-derived cells with hematopoietic niche-stromal cells

HSC

Endothelial

Stromal cells

Hemogenic endothelium

Hematopoietic stem/progenitor cells

in such a system.

(Primitive hematopoiesis)

hESC-derived Embryoid Body

Primitive Erythroid Cells

hESC-derived hematopoiesis in vitro

Hematopoiesis in murine model

Vessel

**Figure 2.** Comparison of mouse model and in vitro hESC/hiPSC-derived hematopoiesis

Primitive Erythroid Cells and Microphages Research on hESC-derived erythrocytes is one of the hot points because of the fact that development of erythrocytes shows distinct trait between primitive and definitive waves. More important is that the matured RBCs have no nucleus, which could avoid the exogenous gene interference when clinically applied to treat patients. Much labor has been donated to optimize the method of in vitro differentiation of RBCs from hESCs. For example, Lu's group firstly use EBs formation and then co-culture with OP9 to obtain a high yield of erythrocytes with some efficiency of enucleation. However, these erythrocytes were still immature because only 16% of these hESC-derived erythrocytes expressed β-globin [84]. Similar work has been done by several other groups using OP9 system [95, 96]

In our laboratory, we have recently established efficient blood cell-yeilding systems by coculture of hESC/hiPSCs with murine AGM and FL stromal cells (Figure 3). In the co-culture, undifferentiated hESC/hiPSC colonies grow up and differentiate firstly to a mesoderm-like structure, then to hematopoietic progenitor cells on days 10 to 14. In the second suspension culture, these hESC/hiPSC-derived hematopoietic progenitors are further induced to some specific blood cell lineages, such as erythrocytes, mast cells, and eosinophils, etc. [3]. By a clonetracing method, we gained concrete results that hESC-derived erythrocytes kept continuously progressing toward maturation over a time course. The expression of β-globin vs ε-globin showed a typical switching pattern, mimicking the normal development of human erythro‐ cytes. On day 12 of the co-culture with murine FL-derived stromal cells, hESC-derived erythrocytes (BFU-E) express β-globin at about 60%, but up-regulated to almost 100% with additional 6 days of culture. These matured hESC-derived erythrocytes can undergo enuclea‐ tion and release oxygen [5].

Our results showed that the in vitro differentiation from hESCs to matured hematopoietic cells is a progressive process if proper co-culture condition provided. Functionally matured blood cells similar to those from the definitive hematopoiesis could be obtained in large-scale production by this method.

#### **3.2. hESC-derived HSCs**

The most challenging task for research on hESC/hiPSC-derived hematopoiesis is to obtain the real HSC from in vitro differentiation system [97]. Although hESC/hiPSC-derived hemato‐ poietic cells that could be engrafted in immune compromised mice have been reported [86, 98-102], effort to obtain real HSCs from hESCs has largely been done in vain during past years. In most experiments, the engraftment rate was very low and mostly restricted to the myeloid lineage, and it was ambiguous if these engrafed cells were derived from real HSCs. The coculture with S17 could induce hESCs to HSC-like properties with low capacity of RBC potential [103]. If a modified cell line generated from mAGM (AM20.1B4) was used the RBC activity of HSC will be increased much [86]. But these hESC-derived HSC-like cells could not repopulate in NOD-SCID mice, representing they were not true definitive HSCs that satisfy the functional definition.

differentiation system based on ESCs. Some differentiation models identified a progenitor with properties of the hemangioblast [115, 116], indicating that the primitive hematopoiesis derived from ESCs should pass through a hemangioblast stage. Further studies showed that these hemangioblasts might have more potential than hematopoietic and endothelial cells, such as

Human Embryonic Stem Cell-Derived Primitive and Definitive Hematopoiesis

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

99

hESC-derived hemangioblasts have also been identified in differentiation cultures [87]. These hESC-derived hemangioblasts were defined by the expression of KDR and could generate clonal cells sharing both hematopoietic and vascular potential. There were two distinct types of hESC-derived hemangioblasts: one gave rise to primitive erythroid cells, macrophages, and endothelial cells and the other generated only the primitive erythroid population and endo‐ thelial cells. This finding provided evidence that hESC-derived hematopoiesis mimicked the

Different to hemangioblasts from YS, the concept of "hemogenic endothelial" come from the research of AGM, which is in the ventral wall of the aorta and buds off HSCs [13]. The molecular control of hemogenic endothelium is different to that of hemangioblasts. For example, Runx1 was indispensable for the hematopoiesis originated from hemogenic endothelia, but not hemangioblasts [119, 120]. And the hemogenic endothelium did not originate from heman‐ gioblasts but presumptive mesoangioblasts, which could express endothelial-specific genes and ultimately express HSC-associated markers [121]. Since hemogenic endothelium is closely related to definitive hematopoiesis and has been regarded as the necessary stage for generation of HSCs, the details of its potential and developmental process to produce HSCs should be

In some in vitro differentiation systems, hESC-derived CD34+ hemogenic progenitor cells could also be detected with the endothelium potential [3, 126]. In an OP-9 co-culture system, hESC-derived hemogenic endothelium progenitors (HEPs) were identified pinpoint by VEcadherin+CD73-CD235a/CD43- intermediate phenotype, which arise at the post primitive streak stage of differentiation directly from a hematovascular mesodermal precursor (KDR +APLNR+PDGFRalow/-). These HEPs differ from non-HEPs (VE-cadherin+CD73+) and early hematopoietic cells (VEcadherin+CD235a+CD41a−) [124]. This subtle finding may provide clue

**3.6. Switching mechanisms controlling primitive hematopoiesis to definitive one**

The origin of two waves of hematopoiesis was distinct in the sight of embryogenesis by the research for Xenopus [127], mice [128] and human [54]. However, the reciprocal transplant test in Xenopus embryo proved that the hematopoietic progenitor cell in VBI(corresponding to YS) and DLP(corresponding to AGM) could change their potential according to microenvironment of the graft site [129]. Similarly, blast-like cells derived from murine ESCs could differentiate to both primitive and definitive lineages [130]. In our study by a clonal tracing method, hESCderived erythrocytes showed the primitive properties at the early stage and progressively

normal development pattern of human earliest stage of hematopoietic commitment.

smooth muscle [117, 118].

**3.5. Hemogenic endothelium**

finely elucidated [122-125].

to facilitate generation of HSCs from hESCs (Figure 4).

**Figure 3.** Co-culture of hESC/hiPSCs with murine AGM or FL stromal cells

#### **3.3. hESC-derived lymphocytes**

A recent report showed that when co-cultured with OP9-DL1 cells, the hESC-derived hema‐ topoietic progenitor cells that formed endothelium-lined cell clumps could be induced into Tlineage cells [104]. These hESC-derived T cells expressed sequential surface markers from Tlymphoid progenitors (CD34+CD7+) to matured T cells (double positive CD4+CD8+and finally maturedCD3+CD1-CD27+). The T-lineage cell production provided concrete evidence that hESC-derived hematopoiesis endowed with definitive property. The functional matured hESC-derived NK cells could also be generated by sequential co-culture on different feeder cells [105, 106]. But the detail of HMC molecular and globulin class-switching during this process has still not been elucidated.

#### **3.4. Hemangioblasts**

The blood islands region in YS consists of clusters of primitive erythroblasts surrounded by mature endothelial cells. The close development relationship between hematopoietic and endothelial cells in such a region indicated that these lineages ought to share a common progenitor named as the hemangioblast [16, 33, 107]. This concept was provided nearly one century ago [108,109] while it has been circumstantially supported by the latest molecular genetic and embryological proofs [110-114]. More direct evidence came from the in vitro differentiation system based on ESCs. Some differentiation models identified a progenitor with properties of the hemangioblast [115, 116], indicating that the primitive hematopoiesis derived from ESCs should pass through a hemangioblast stage. Further studies showed that these hemangioblasts might have more potential than hematopoietic and endothelial cells, such as smooth muscle [117, 118].

hESC-derived hemangioblasts have also been identified in differentiation cultures [87]. These hESC-derived hemangioblasts were defined by the expression of KDR and could generate clonal cells sharing both hematopoietic and vascular potential. There were two distinct types of hESC-derived hemangioblasts: one gave rise to primitive erythroid cells, macrophages, and endothelial cells and the other generated only the primitive erythroid population and endo‐ thelial cells. This finding provided evidence that hESC-derived hematopoiesis mimicked the normal development pattern of human earliest stage of hematopoietic commitment.

#### **3.5. Hemogenic endothelium**

**3.3. hESC-derived lymphocytes**

**Figure 3.** Co-culture of hESC/hiPSCs with murine AGM or FL stromal cells

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

process has still not been elucidated.

**3.4. Hemangioblasts**

A recent report showed that when co-cultured with OP9-DL1 cells, the hESC-derived hema‐ topoietic progenitor cells that formed endothelium-lined cell clumps could be induced into Tlineage cells [104]. These hESC-derived T cells expressed sequential surface markers from Tlymphoid progenitors (CD34+CD7+) to matured T cells (double positive CD4+CD8+and finally maturedCD3+CD1-CD27+). The T-lineage cell production provided concrete evidence that hESC-derived hematopoiesis endowed with definitive property. The functional matured hESC-derived NK cells could also be generated by sequential co-culture on different feeder cells [105, 106]. But the detail of HMC molecular and globulin class-switching during this

The blood islands region in YS consists of clusters of primitive erythroblasts surrounded by mature endothelial cells. The close development relationship between hematopoietic and endothelial cells in such a region indicated that these lineages ought to share a common progenitor named as the hemangioblast [16, 33, 107]. This concept was provided nearly one century ago [108,109] while it has been circumstantially supported by the latest molecular genetic and embryological proofs [110-114]. More direct evidence came from the in vitro

Different to hemangioblasts from YS, the concept of "hemogenic endothelial" come from the research of AGM, which is in the ventral wall of the aorta and buds off HSCs [13]. The molecular control of hemogenic endothelium is different to that of hemangioblasts. For example, Runx1 was indispensable for the hematopoiesis originated from hemogenic endothelia, but not hemangioblasts [119, 120]. And the hemogenic endothelium did not originate from heman‐ gioblasts but presumptive mesoangioblasts, which could express endothelial-specific genes and ultimately express HSC-associated markers [121]. Since hemogenic endothelium is closely related to definitive hematopoiesis and has been regarded as the necessary stage for generation of HSCs, the details of its potential and developmental process to produce HSCs should be finely elucidated [122-125].

In some in vitro differentiation systems, hESC-derived CD34+ hemogenic progenitor cells could also be detected with the endothelium potential [3, 126]. In an OP-9 co-culture system, hESC-derived hemogenic endothelium progenitors (HEPs) were identified pinpoint by VEcadherin+CD73-CD235a/CD43- intermediate phenotype, which arise at the post primitive streak stage of differentiation directly from a hematovascular mesodermal precursor (KDR +APLNR+PDGFRalow/-). These HEPs differ from non-HEPs (VE-cadherin+CD73+) and early hematopoietic cells (VEcadherin+CD235a+CD41a−) [124]. This subtle finding may provide clue to facilitate generation of HSCs from hESCs (Figure 4).

#### **3.6. Switching mechanisms controlling primitive hematopoiesis to definitive one**

The origin of two waves of hematopoiesis was distinct in the sight of embryogenesis by the research for Xenopus [127], mice [128] and human [54]. However, the reciprocal transplant test in Xenopus embryo proved that the hematopoietic progenitor cell in VBI(corresponding to YS) and DLP(corresponding to AGM) could change their potential according to microenvironment of the graft site [129]. Similarly, blast-like cells derived from murine ESCs could differentiate to both primitive and definitive lineages [130]. In our study by a clonal tracing method, hESCderived erythrocytes showed the primitive properties at the early stage and progressively

expression profile during such a switch. A thorough transcriptome analysis by deep sequenc‐

Human Embryonic Stem Cell-Derived Primitive and Definitive Hematopoiesis

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

101

The in vitro production of functionally matured blood cells from hESC/hiPSCs has provided an excellent model for both basic research and clinical applications. hESC/hiPSC-derived RBCs are highly fancied because of their unlimited use as the substitute for blood transfusion, not to mention the the utilization of hESC/hiPSC-derived HSCs in transplantation. On the other hand, hiPSCs can provide patient-tailored models for analyzing the pathogenesis of malignant blood diseases and thus develop individual treatment by molecular corrections. Innate immune-related cells differentiated from hESC/hiPSCs will also help us to reveal the initial establishment of human immune system, and to explore drug screening system and cell

However, before the successful application of hESC-derived cellular therapy, there are still many problems needed to be solved. The molecular mechanisms controlling both primitive and definitive hematopoiesis should be clarified at first. The HSC-like cells and functional matured blood cells derived from hESC / hiPSC in vitro must also be proven their activities in

In order to realize above aims, the following efforts need to be done to promote the research

**1.** The latest molecular biological technology and other new technique tool should be applied

Currently, Tetracycline (tc) and Tet repressor (TetR) system (tet-on and off) is one of the most matured and widely applied regulatory systems [133] in organisms ranging from bacteria to mammals. The technique by Transcription activator-like effector nucleases (TALENs [135, 136]) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated (Cas) systems [136, 137] have also been developed. These methods let us be able to do molecular manipulation in genomic scale(named "genome edit") by unlimited times against single cell line without obvious influence to the cell characteristic, thus provide an ideal

**2.** A highly efficient animal-source-free 3D culture system should be developed to search

A big problem that hampers the progress in research on hESC-derived hematopoiesis is a lack of a proper culture system that fully mimics the microenvironment of early development of human hematopoiesis. An ideal culture model should be completely free of any animal source substitutes and to great extent imitate a live structure as done in vivo circumstances. Since HSCs develop in a complicated niche composed of various cell types (endothelial cells, osteoblasts, mesenchymal cells, etc. ), a 3D culture model mixed by several hematopoietic niche

model to investigate the development events in hESC-derived hematopoiesis.

cells should benefit the efficiency of generating true HSCs from hESCs.

vivo. Besides, more efficient culture system free from xenobiotics must be optimized.

ing or gene chip should be useful tools to detect hESC-derived hematopoietic switch.

**4. Concluding remarks and future perspectives**

therapy model for deficiencies of innate immunity.

for the generation of hESC-derived HSCs.

in this field:

in the future research.

**Figure 4.** Development of hemangioblasts and hemogenic endothelia from pluripotent stem cells

turned to be definitive ones along with co-culture on FL cells [5], indicating that there exists a possible switching mechanism controlling the conversion.

However, the molecular mechanism controlling such a switch remains unclear so far. The switch observed in process of hESC-derived hematopoiesis probably is caused by the influence or induction of the definitive hematopoietic niche-derived stromal cells (AGM, FL). The cellular and molecular signals, including the growth factors and adhesion proteins expressed by feeder cells and growth factors added in medium ought to play a key role to activate the switch controlling the definitive hematopoiesis (such as Hedgehog, Notch1 and BMP signal pathways). Among them, HoxB4 had been identified as the key factor to promote such a switch [131]. Many key factors expressed in primitive and/or definitive erythrogenesis could also be tracked in ESC-derived hematopoiesis [94, 132]. Since the hemoglobin switch of erythrocyte could be used as the indicator of premitive and definitive hematopoiesis, the erythrogenesisrelated factors, such as GATA1, GATA2, AML1, SCL, EKLF, ought to be changed their expression profile during such a switch. A thorough transcriptome analysis by deep sequenc‐ ing or gene chip should be useful tools to detect hESC-derived hematopoietic switch.
