In Vitro Study On Expression Of Phenotypic Molecules During Early Development Of Erythroid Cells Derived From Human Pluripotent Stem Cells

Objectives:Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the ability of self-renewal, unlimited proliferation and multi-differentiation potential. The establishment of hESCs and hiPSCs has greatly promoted basic research and clinical application of stem cells. The differentiation to mature blood cells from specific lineage cells is an important direction of stem cell research. Mature red blood cells may be the earliest stem cell-derived cellular therapy products to be used in transfusion medicine, because of the fact that mature RBCs are without nuclei, carrying little genetic materials and their long life span in living body. There still are many problems to be solved, such as the low amplification efficiency in vitro culture, the poor maturity, and the uncontrolable enucleation mechanism, as well as no suitable transplantational animal models for hPSC-derived erythroid cells. Before they are implemented successfully for clinical application, much work should be done. These difficulties are mainly due to our poor understanding on the key regulatory mechanism of human erythroid cell development. The process of induction and differentiation of erythroid cells from hPSCs in vitro, which can mimic the early development of human erythropoiesis process during embryogenesis in vivo, provides an ideal tool to study both the regulation mechanism of normal erythropoiesis and the pathogenesis and treatment of abnormal erythropoiesis related diseases.To study subtlety the erythropoiesis process derived from hPSCs in vitro, two problems should be solved first. (1) The accumulated experimental data have shown that there are differences in maturity for hPSC-derived erythroid cells by different inducing methods. Aclear clue hints that tromal cells from hematopoietic niches are essential for generating mature erythroid cells from hPSCs. To establish a natural co-culture method with high efficiency and is necessary to gain mature erythroid cells similar to the natural developmental process. (2) In the culture system to develop hPSCs-derived erythroid cells, the process of both primitive and definitive hematopoiesis are simultaneously existed, In order to distinguish erythroid cells at different developmental stages, a quick, convenient and accurate assay system needs to be established. In the research field of adult stem cells-derived erythroid cells, distinct developmental stages have been successfully distinguished by stage-specific phenotypic molecules, enlightening us to distinguish different developmental stages of hPSC-derived erythroid cells by phenotypic molecules too.Methods:Stromal cell line AGM-S3 isolated from mouse Aorta-Gonad-Mesonephros (AGM) region, which has been proved to be the earliest sites to support definitive hematopoiesis has been established previously. hPSCs are co-cultured with AGM-S3 cell line to generate hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPSCs), and then erythroid cells are differentiated and expanded in suspension culture. Adult stem cell-derived erythroid cells were taken as control. Advanced multi-color flow technology is performed to explore the specific phenotypical expression pattern of hPSC/AGM-S3-derived erythroid cells. By large-scale screening for co-expression of erythroid lineage-specific markers, we found there exist specific subsets of GPA+ CD36-and GPA+ CD34+ at warly developmental stages. Flow cytometry sorting technology was used to precisely isolate the selected cell subsets. Then the cell morphology was observed by MGG stain, the maturity of erythroid cells being evaluated by detecting the hemoglobin components using immunofluorescence staining method, and qRT-PCR to detect the transcriptional level of important hematopoietic and erythroid development related genes.Results:We have established a highly efficient hPSC/AGM-S3 co-culture system, which can produce a large number of erythroid cells with high maturity. By co-cultured for 12 days and then cultured in suspension for 24 days, the number of generated erythroid cells is about 300 folds of the initiated number of H1 cells, in which more than 85% expressed adult hemoglobin β. The rate of hemoglobin beta expressing erythroid cells in our co-culture system is much higher than other laboratory’s reported data.The phenotypic expression pattern of erythroid cells derived from hPSC/AGM-S3 co-culture system was studied. We found that expression patter for CD36 and CD34 on GPA+ cells in co-cultures were distinct from that of adult-stem-cells-derived erythroid cells. According to these clues, further detailed research work is being undertaken.Majority of the erythroid cells in our co-cultures expressed GPA+ CD36- for quite a long period of time (from day 6 to day 18), then became to GPA+CD36low/+ which appeared mostly on day 10+5, and then majority of erythroid cells change rapidly to GPA+CD36- on day 10+9. Different subpopulations of erythroid cells were sorted out by FACS technology on separate days according to the co-eaxpression of GPA and CD36. Sorted erythroid cell subpopulations were investigated further by Hb components, phenotypical molecules and relatively transcriptional levels hematopoietic and erythoid developmental of related genes. We discovered that the series change of CD36 on erythroids was accompanied with losing mesoderm and endothelial cell characteristics, switching primitive hematopoiesis characteristics to definitive hematopoiesis ones and rising the maturity gradually.