Differentiation Of Mature Eosinophils From Human CD34+ Hematopoietic Stem Cells

Objective:Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are of unlimited proliferation and multi differentiation ability. Many reports have proved their potential to differentiate into various blood cells under in vitro conditions. These hESC/hiPSC-derived blood cells are phenopytically and functionally similar to the counterparts in peripheral blood. They not only provide an abundant source of cells for regenerative medicine research, but also for establishing patient-tailored disease models and ultimately for clinical individual therapy.Human umbilical cord blood hematopoietic stem cells (hUCB-HSCs) have long been used in clinical treatment of a variety of diseases containing hematologic malignancies and autoimmune diseases. They also serve as a conventional source of human HSCs in research. Studies in vitro showed that hUCB-HSC can differentiate into almost all kinds of mature blood cells, such as red blood cells, granulocytes (including eosinophils) and endothelial cells.Eosinophils (Eos) is a kind of granulocytes, originating from HSCs in bone marrow and entering in the peripheral blood after they have been differentiated in bone marrow. Eos consist only 0.5%-5% of peripheral white blood cells. But as a key effector cells mediating inflammation, they conduct hypersensitivities, response to parasite infection and widely regulate the innate immune system.Studies of Eos on inducing differentiation and exploring specific Eos-ralated treatment for allergic diseases are few. Some of the researches reported methods to induce eosinophils from hUCB-HSC, but due to the lack of identification of specific phenotypic molecules, these researches always stay in the methodology stage. Besides, there are no related work reports of inducing hESC specificly differentiate into eosinophils. In view of this, there should be of important theory and practice value to investigate eosinophil differentiation and its regulation mechanism.This study is to induce hESC and hUCB-HSC to differentiate into Eos in vitro conditions, and compare the in vitro induced Eos by the molecular phenotype, morphology and intracellular granule protein expression and other aspects. Our aim is to explore the regulatory pathway of early Eos development and differentiation, and the similarities and differences between hESC-and hUCB-HSC-derived Eos, thus providing critical information in differentiation the maturation processes from different sources of stem cells. We will also establish the models of Eos related diseases by patient-specific iPSC lines and to provide experimental basis for drug screening model.Methods:To harvest large quantity of hematopoietic cells, we use an established hematopoietic stem/progenitor cell inducing system by co-culturing undifferentiated hESC colonies with 13.3Gray X-ray irradiated AGM stromal cells for 14 days. Then the co-cultured cells and cord blood mononuclear cells are sorted by CD34+magnetic beads at the same time. The separated cells are pooled into suspension cultured under the same conditions in vitro. At certain time points, cells were counted, cytospinned, observed morphologically by May-Giemsa staining. The expression of Eos-specific granules are confirmed by immunofluorescence staining while observation of surface molecule expression by flow cytometry weekly.Results:hESC and AGM-S3 co-cultured can generate pluripotent hematopoietic stem/ progenitor cells, and only a few Siglec-8+early Eos developed in the co-culture. However,the suspension culture of co-cultured D14 total cells can produce large quantity of EPO+and Siglec-8+mature Eos with a high purity. The cells in supension culture with different cytokine combinations for 3-4 weeks can obtain different purity of the EPO+ mature Eos cells. Using GM-CSF containning-cocktail of cytokines in serum containing medium at the beginning of suspension culture (the first week) is the best experimental combination, because cells are not only amplified robustly, but the Eos proportion was the highest. With the suspension culture time goes on, the positive rate of expression of EPO and MBP proteins in Eos gradually increased, whereas the positive rate of 2D7 and pro-MBPl expression of basophils (Basophils, Bas) decreased down. It shows that in the process of the differentiation and development of Eos, the early cells undergo a special stage both expressing Eos (EPO, MBP) and Basophil (2D7, ProMBP1) surface markers. Along with the suspension culture, cells are getting more mature and the expression of Eos granule protein significantly increased, while the expression of Bas granule protein decreased, showing a gradual lose of Bas specific granule protein and increase of Eos onse into mature Eos stage. When a comparison of hESC-derived and hUCB-HSC-derived Eos was conducted in the same optimized culture conditions, we found that the two sources of stem cells gave rise to different proliferation and differentiation pattern towards Eos maturation, with hUCB-HSCs producing very low portion of Eos and sustaining a long time in maturation. Further study revealed that the expression of Eos-specific surface marker, Siglec-8, showed a time dependent pattern along with the maturation of hESC-derived Eos, which is consistent with the rate of EPO expression. By multi-color method through the flow cytometry analysis, we found that the expression of CD34,CD43,CD45,CD88 and Siglec-8 also has a certain time-dependent manner, which can be observed in the change of sub-populations.Conclusion:The hematopoietic stem/progenitor cells derived from hESC in in vitro culture can give rise to a large number of Eos with high purity. These hESC-derived Eos share similar surface markers and mature functions as the Eos in peripheral blood. Applying GM-CSF in the early stage of suspension culture favoring of amplification and differentiation of Eos pool is the optimization protocol. The phenomenon of co-expression of Eos and Bas specific granule protein in early stage of Eos differentiation and maturation process mimics the similar developmental pathway with hUCB-HSC-derived Eos, as reported. AS the sole mature eosinophil surface markers,Siglec-8 can be used to observe the early occurrence, development, differentiation and maturation process of hESC-derived Eos. hESCS and hUCBSC induced to differentiate into mature Eos are different in time and efficiency, with hUCBSC requiring a longer time and shwoing a lower efficiency than that of hESCS.