**4. Concluding remarks and future perspectives**

turned to be definitive ones along with co-culture on FL cells [5], indicating that there exists a

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

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

possible switching mechanism controlling the conversion.

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

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 therapy model for deficiencies of innate immunity.

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

In order to realize above aims, the following efforts need to be done to promote the research in this field:

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

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

**2.** A highly efficient animal-source-free 3D culture system should be developed to search for the generation of hESC-derived HSCs.

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

**3.** The new concept and theory should be introduced to conduct the research of hESCderived hematopoiesis.

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According to the recent researches by Deng and other group [138, 139], the pluripotency of stem cells is only the balance between several transcription factor groups controlling layer lineage differentiation. Their finding denied the existence of special pluripotent factors and showed that maintenance of ESC/iPSCs may be only because that all the way to any differen‐ tiation direction has been blocked by mutually antagonistic lineage specifiers [140]. Such concept may also help us to uncover the controlling balance by some specifiers in early hematopoiesis, especially for those controlling primitive and definitive ones. Since we always try to obtain more definitive blood cells for clinic application, to discover the key factor leading to definitive hematopoiesis is a challenging task to do. Moreover, how to define and describe the essence of HSCs at molecular level is also an issue need to be re-addressed.
