**1. Introduction**

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

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 development of research on hESC/hiPSC-derived hematopoiesis.

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

matured erythrocytes and other functional blood cells could be harvested. In this chapter we will discuss the latest progress in this research field along with our recent discoveries. We will emphasize on the origin, evolution and the development of both primitive and definitive hematopoietic waves, especially those derived from hESCs in vitro systems. The critical problems need to be solved and the research prospects of this field will also be addressed at the end of the chapter.

of primitive erythroid progenitors and definitive HPCs in YS of Ncx1-/- and WT mice through E9.5, while the P-Sp region in Ncx1-/- mice lacks primitive erythroblasts and definitive hematopoietic progenitor cells (HPCs) from E8.25 to E9.5 [17]. So it is reasonable to believe that primitive erythroblasts and nearly all definitive HPCs seeded the fetal liver after E9.5 are generated from the YS between E7.0-E9.5 and are re-distributed into the embryo proper via the systemic circulation [17]. The definitive hematopoiesis may originate from primitive hematopoiesis during embryonic development and migrating HSCs come from the murine YS, which seed the liver and initiate hematopoiesis on 10.0 day postcoitus (dpc). But whether YS cells isolated before day 11.0 dpc possess any long-term repopulating HSC activity remains controversy. Yoder et al proved that donor day 9.0 dpc YS cells could establish long-term hematopoietic system in conditioned newborns, but not in adult recipients [19]. When these early YS cells were co-cultured for 4 days with AGM-S3, an AGM region-derived stromal cell line, they obtained such capacity of reconstitution [20]. All the evidence so far agree that YS itself couldn't provide long-term repopulating HSC activity while the hematopoietic cells originate from YS could own this activity through proper "education" by definitive hemato‐

Human Embryonic Stem Cell-Derived Primitive and Definitive Hematopoiesis

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

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**AGM.** Following the onset of circulation at E8.5, hematopoietic progenitors rapidly move within the embryo. Determining the anatomical origins of definitive hematopoiesis is a complicated and controversial topic. It is now widely recognized that main source of definitive hematopoiesis originates from the AGM region [8]. Using irradiated adult mice as the recipients, Müller et al reported that LTR-HSC first appeared in the AGM region at 10 dpc and expanded in 11 dpc AGM region [13]. Medvinsky et al then demonstrated that, at day 10 in gestation, hematopoietic stem cells initiate autonomously and exclusively within the AGM region under in vitro organ culture condition [8]. All these findings suggested that the AGM region at 10 to 11 dpc provides a microenvironment suitable for generation of LTR-HSC. Xu et al obtained a stromal cell line derived from the AGM region of 10.5 dpc mouse embryo (AGM-S3) that could support the growth and proliferation of hematopoietic progenitor/stem cells from adult mouse bone marrow and human cord blood without additional cytokines [21]. The same research group found that no definitive hematopoiesis-derived colony-forming cells were generated from YS and P-Sp cells at 8.5 dpc before co-cultured with AGM-S3 cells. However after 4-day co-culture of 8.5 dpc YS and P-Sp cells with AGM-S3, spleen colonyforming cells and HSCs capable of reconstituting definitive hematopoiesis in adult mice simultaneously appeared [20]. It is proposed that precursors that had the potential to generate definitive HSCs appear in both extra-embryonic (YS) and intra-embryonic (P-Sp) region, the latter providing microenvironment to support the definitive hematopoiesis from both

**Fetal liver and bone marrow.** At 9 dpc, the liver rudiment begins to form an evagination of gut into the septum transversum. The liver does not generate hematopoietic cells de novo but is instead colonized at late E9 by hematopoietic cells generated in other tissues [22; 23]. The first erythroblasts are visible in the liver at 9 dpc. From 10 dpc onwards, the erythroid lineage begins to develop definitive characteristics. Myeloid CFU-Cs appears in the fetal liver at 9 dpc and macrophages and B cells are present at 10-11 dpc [24]. Although most of these differenti‐

poiesis niches or the stromal cell line derived from them [20, 21].

precursors.
