Differentiation Of Human Embryonic Stem Cells Into Hematopoietic Cells

Currently , blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important means for cellular therapy. These methods are widely used in the treatment of incurable hematological disorder, genetic diseases, infectious diseases and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. So people try hard to obtain more safe, effective and economic resource of blood cells. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of preimplantation embryos possess indefinite proliferative capacity in vitro, and maintain pluripotency as well. They also have the capacity to differentiate into all cell types.Recently, the potential of hESCs to differentiate into hematopoietic cells has been reported in several studies, suggesting clinical applications in cellular therapy, such as blood transfusion or hematopoietic stem cell transplantation, and treatment for hematological disorder. Currently, the major sources of transplantable HSCs for clinical therapy are human bone marrow (BM), mobilized peripheral blood and umbilical cord blood (CB), but their availability for clinical use is limited by both quantity and compatibility. Pluripotent hESCs may provide an alternative.With the repid development of biology research, pluripotent human embryonic stem cells (hESCs) may provide a unique tool for hematopoietic transplantation and the study of human embryonic hematopoiesis.Currently, various methods including cytokinses administration and co-culture with stromal cells have been used to promote the hematopoietic differentiation of embryonic stem cells (ESCs). Although hESCs are competent for development into hematopoietic cell fate, the cellular and molecular mechanisms that control this process are very poorly understood and yield heterogeneous outcomes. Furthermore, the risk of mouse-related disease, high cost of cytokines and low differentiation efficiency greatly limit the clinical applications. In the present study, we first demonstrate the role for PGE2 in regulating hematopoietic differentiation from hESCs, then we establish a novel method which can induce hESCs differentiated into hematopoietic lineages safely, efficiently, and economicly.1. Prostaglandin E2 promotes hematopoietic development from hESCsRecent studies have suggested that PGE2 and the prostaglandin pathway are essential for hematopoietic stem cell growth, survival and development. These findings have led to a greater interest in the possible role of the prostaglandin pathway in human hematopoietic development. Here we evaluated the effects of PGE2 on hematopoietic differentiation of hESCs. The induced cells from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by RT-PCR, flow cytometry, colony-forming, assays and Wright-Giemsa staining. Our results demonstrated that the hematopoietic colonies increased in a dose-depentent manner after exposure to PGE2. The expression of Oct-4 and hematopoiesis-inductive transcription factors and hematoendothelial specific genes, including Brachyury, CD34, SCL, GATA-1, Runx1, vWF and VE-cad were examined with RT-PCR. PGE2 exposure also altered the morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with the higher frequencies of CD34+ and CD45+ cells. Furthermore, PGE2 supplement increased the activation of p-Smad1/5 and enhanced the expression of Smad4 compared to the non-treated counterpart. When the effect of PGE2 was blocked by its specific inhibitor indomethacin, p-Smad1/5 and Smad4 expression declined correlatively. This research may improve our knowledge of stem cell regulation, which hopefully will lead to improved stem cell-based therapeutic options.2. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cellsDevelopment of hematopoietic system in human embryo involves several anatomical sites including the yolk sac hematopoiesis occurring from 4 to 6 weeks, the fetal liver hematopoiesis occurring from 6 to 22 weeks, and the bone marrow hematopoiesis occurring during the remainder of life. Different hemopoietic microenvironment plays important roles in the different stages. In the mode of hematopoietic differentiation from hESCs, the selection of microenvironment is an important part. The fetal liver is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated. So we suppose that the fetal liver microenvironment could promote the hematopoietic differentiation from hESCs. On the other hand, the most important cytokine regulator of erythropoiesis is erythropoietin (EPO). It acts on erythroid progenitor cells, stimulates proliferation, promotes differentiation, and prevents apoptosis. The main EPO production site is the liver in the fetus and the kidney in the adult. As a conclusion, the combination of fetal liver cells and EPO must improve the erythropoietic differentiation. It was also shown that direct secreted cytokines from stromal cells exert their function better than medium supplement. Based on these understanding, a transgenic stromal line was built.We have developed a novel method to promote the differentiation of hESCs toward hematopoietic lineages. The human fetal liver stromal cell (hFLSCs) expressing EPO were established using lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce hESCs differentiate into hematopoietic cells without exogenous cytokines.In this experiment, hESCs were cultured in low cell-attachment dishes to obtain EBs. While the EBs were induced into hematopoietic cells by different inducing systems including hFLSCs-CM, hFLSCs-CM+EPO, and EPO/hFLSCs-CM after treated by BMP4. Then the expression of CD34 was analyzed by flow cytometry. For EBs treated with hFLSCs-CM CD34+ cells peaked to 10.57% on day 12. For the EBs treated with hFLSCs-CM+ EPO, the number of CD34+ cells reached the maximum of 13.1% at day 12. For the EBs treated with EPO/hFLSCs-CM, CD34+ cells peaked to 22.74% at 12 days of culture. These results demonstrated that EPO/hFLSCs-CM treated EBs could yield more hematopoietic cells than other groups. In addition, the genotypic expression was characterized by realtime RT-PCR. Among these different inducing systems, EPO/hFLSCs-CM had the highest hematopoitic gene expression level. The features of hESCs-derived erythroid cells were characterized by morphology, flow cytometry, wright-Giemsa staining, 3'3-diaminobenzidine staining, and RT-PCR. The results showed that the induced cells showed the feature of hematopoietic cells. Among the inducing systems, the EPO/hFLSCs-CM could promote hematopoietic differentiation of hESCs, especially into erythrocytes. Furthermore, the hESC-drived erythroid cells were also able to function as oxygen carriers, and exhibited an oxygen dissociation pattern similar to human CB. In summary, our studies support an expected role for PGE2 in regulating hematopoietic differentiation from hESCs. We demonstrate that PGE2 treatment, together with OP9 stromal cell co-culture, promotes hematopoietic development from hESCs. Furthermore, we established the transgenic human fetal liver stromal cells (EPO/hFLSCs) that stably express EPO gene and used the conditioned medium to induce erythropoietic differentiation of hESCs. Furthermore, this method can avoid the mouse-related disease and cut down the cost of experiment. The system we developed will provide a reliable alternative for the research in the future therapeutic applications of human embryonic stem cells.