Differentiation Of Human Embryonic Stem Cells Into Hematopoietic Cells

Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases and immunologic deficiency However, their availability for use is limited by quantity ,capacity of proliferation and the risk of disease. So people hope to obtain more safe and economic resource of blood. With the repaid development of biology research, human embryonic stem cells (ESCs) have been regarded as an alternative source for hematopoietic transplantation and hematopoiesis studies.Now, various means including growth factor administration and co-culture with relevant cells/tissues have been used to encourage the differentiation of embryonic stem cells (ESCs) toward hematopoietic lineages. However, risk of mouse-related disease, low differentiation efficiency of hESCs and high cost of cytokines greatly limit the clinical application. So we hope to establish a new method to solve these problems which can induce hematopoietic differentiation of hESCs safely and efficiently.Development of hematopoietic system in human embryo can be divided into three stages including the first stage of yolk sac hematopoiesis occurring from 4~6 weeks, the second stage of fetal liver hematopoiesis occurring from 6~22 weeks, and the last stage of bone marrow hematopoiesis occurring from 22~birth. Different hemopoietic microenviron- ment plays important roles in different stages. In the mode of hematopoietic development from hES cells, selection of inducing microenvironment is the most important section. Fetal liver is the major site of hematopoiesis, so we speculate the microenvironment of fetal liver could promote the hematopoietic development hESCs. In this experiment, we induced human embryoid bodies (hEBs) into hematopoietic cells by a combination of human fetal liver stromal cells (hFLSCs) feeder and the cell extract from human fetal liver cells. And we compared this method with human fetal liver stromal cells (hFLSCs) feeder system and cytokines/ hFLSCs feeder system aiming for establishing an efficient inducing method.As a first step, we isolated hFLSCs and cell extract and examined the feature of human fetal liver stromal cells by semiquantitative-RT-PCR and flow cytometric analysis. The results showed that cells expressed stromal cells specific markers (CD29 and CD90) and but not hematopoietic markers (CD45 and CD34). In addition, the cells lost their potency to support hematopoietic development with the time of culture going on. Thus we selected the hFLSCs within three generations as the feeders.Then the hES cells were cultured in low cell-binding dishes to form EBs which were induced into hematopoietic cells by different inducing systems including hFLSCs feeder system, cell extract/ hFLSCs feeder system and cytokines system /hFLSCs feeder system after treated by BMP4 . Then, the expression of CD34, and CD 45 was examined by flow cytometry. The time-course hematopoietic differentiation of human EBs was detected in all inducing systems. Without cytokines, hFLSCs feeder demonstrated a limited capacity for hematopoietic differentiation. Only a few CD34+ cells and CD45+ cells could be found during 10 days cultivation (<10%) . However, Cytokine could enhance hFLSCs-mediated hematopoiesis from EBs demonstrated by a frequency of CD34 (from 8.79% to 24.68%), CD45 (from 6.05% to 13.57%). In addition, treating with the cell extract/ hFLSCs feeder could result in a robust hematopoietic differentiation of hEBs with higher frequencies of CD34-positive (32.73%), CD45-positive (27.96%). The hematopoietic progenitor potentials of induced cells were tested by hematopoietic Colony-forming assays. The morphological characterizations were examined by immunofluorescence and Wright-Gie -msa staining. In addition, the genotypic expression was characterized by semiquantit- ative RT PCR. The induced cells showed the feature of hematopoietic cells, expressing hematopoietic specific makers such as SCL, AML1 and GATA-1 and having ability to form the different hematopoietic clusters in Colony-forming assays. Among these different hEBs inducing systems, hFLSCs/extract system had the highest numbers of hematopoietic colonies and the highest hematopoitic gene expression level. In addition, hFLSCs/extract systems yielded predominantly erythroid precursors with more CFU-E colonies than the other kinds of colonies.We further studied the mechanism of erythroid lineage development and hope to establish a system to improve the production of red blood cells from embryonic stem cells/hematopoietic progenitor cells. The production of red blood cells is regulated by some genes expressed in hematopoietic stem cells and some hematopoietic cytokines in microen- vironment. EPO is one of the most important cytokines in the development of red blood cells. Suppressor of cytokine signals-3 (SOCS-3) was first found in 1997, which had been proved a high affinity binding site in EPO receptor regulating EPO negatively. Thus we speculated the down-expression of SOCS-3 could promote erythroid lineage development of hematopoietic stem cells and hESCs. In this experiment, a small interference RNA expression vectors of SOCS-3 were constructed and transfect into K562 cell lines stably by lentiviral system.The efficiency of virus transfection was identified by expression of green fluorescence protein(GFP) analyzed by fluorescence microscope, these K562 cells with the high GFP expression were sorted by fluorescence-activated cell sorting (FACS) according to strong GFP expression. Efficiency of RNA interferencing on SOCS-3 were detected by Real time-PCR and Western-blot. Compared with that in K562 cells transfected with control lentivirus, the SOCS-3 mRNA expression level of cells transfected with SOCS-3 siRNA was only 22.1%. SOCS-3 protein was also down regulated by siRNA. Then, we induced these transfected cells into the erythropoietic cells and found that the K562 cells could differentiate into erythroid lineage cells more easily after silencing of SOCS-3.In summary, three different ways were performed to induce the hematopoietic differentiation of hESCs, and a combination of human fetal liver stromal cells (hFLSCs) feeder and the cell extract from human fetal liver cells was proved to be the most efficient way. In addition this method avoided the mouse–related disease and cut down the cost of experiment. Furthermore, we constructed the small interference RNA expression vectors of SOCS-3 and established an efficient way for erythropoietic development from hematopoi- etic progenitor cells. Our experiments provided a model for analysis of the mechanism involved in the differentiation of hES cells to hematopoietic cells, as well as for understa- nding early development of hematopoiesis and confirmed the important role of SOCS-3 in erythropoietic development and provided a useful new way for production of erythroid lineage cells.