Induction Of Human Induced Pluripotent Stem Cells Into Hematopoietic Stem/progenitor Cells And The Effects Of Hypoxia

BackgroundHematopoietic stem/progenitor cells can rebuild the function of bone marrowhematopoiesis and immune, and have a significant role in reconstruction the functionof short-term and long-term hematopoietic. Now the hematopoietic stem/progenitorcell transplantation is more from the clinical cord blood, however, due to the limitednumber of cord blood, and the risk of immune rejection, therefore, the usefulness ofiPS cells as an alternative source of hematopoietic precursors that potentially can beused for studies of hematopoietic ontogeny and hematopoietic cell transplantation inhumans.iPS cells represent a unique populationof cells capable of self-renewal anddifferentiation. iPS cells giverise to tissues from all3germ layers, iPS cells arereprogrammed from body cells, therefore it has no immune rejection, ethically canalso be accepted, differentiation of iPS cells into hematopoietic stem/progenitor cellsefficiently in vitro become the focus of everyone’s attention. iPS cells have thepotential to serve as an alternative source of hematopoietic precursors fortransplantation and for the study of hematopoieticcell development.Pluripotent stem cells have been shown to differentiate into hematopoietic stem/progenitor cells, pluripotent stem cells induced differentiate into hematopoietic stemcells can be summarized as three methods, the first, single cytokine induction, thesecond, iPS cocultured with stromal cells, the last, EB (embryoid bodies) cultureinduced differentiation. In this experiment, the system is co-cultured iPS cells withmouse bone marrow mesenchymal cells OP9in vitro.The presence of hypoxia can regulate the expression and distribution of blood -related factors, and thus participate in the follow-up of hematopoietic stem/progenitor cell proliferation regulation. Hypoxia environmental can increase thehematopoietic stem/progenitor cells differentiation into cells faculties yield. Hypoxicenvironment can promote the differentiation of embryonic stem cells into endothelialcells and hematopoietic cells in vitro. However, the role of hypoxia on hematopoieticcells and the corresponding mechanism is unclear.At present the influence of hypoxiaon hematopoietic stem cells is usually use umbilical cord blood CD34+cells as theresearch object, the mechanism of hypoxia in the pluripotent stem cells differentiateinto hematopoietic stem cell is not yet clear.Therefore, this experiment simulating the process of embryonic developmenthypoxic environment, using human iPS cells co-cultured with OP9differentiationsystem in vitro, study the effects of hypoxia on the hematopoietic differentiationefficiency. Provide the functional hematopoietic stem/progenitor cells for clinicalapplication.ObjectiveDifferentiation of human induced pluripotent stem cells intohematopoietic/progenitor stem cell. Observe the function of hypoxia in hematopoieticdifferentiation in vitro.MethodsThe human induced pluripotent stem cells were induced differentiate intohematopoietic stem/progenitor cell by co-culturing with OP9bone marrow stromalcell unde hypoxia5%oxygen and20%oxygen. The expression of hematopoieticstem/progenitor cell surface markers were detected by flow cytometry. The expressionof induced pluripotent stem cells and hematopoietic stem/progenitor cell regulationgenes were measured by real-time PCR. Using immunomagnetic beads sorting CD34+hematopoietic stem/progenitor cell for colony formation assay unde hypoxia5%oxygen and20%oxygen.ResultsWhen induced pluripotent stem cells co-cultured with OP9cells for4days, themorphological changes of induced pluripotent stem cells could be observed.Hematopoietic stem/progenitor cell surface markers CD34and CD43can be detected by flow cytometry after differentiation. Under hypoxia the percent of CD34, CD43,CD31, is increase.The pluripotent marker gene OCT4expression gradually decreasedand blood-related transcription factor Gata-2expression gradually increased, whileRunx-1expression is wavy change, CD34expression gradually increased. Underhypoxia the blood-related genes GATA2and HOXB2showed up regulation.Erythroid colony (CFU-E), granulocyte colony (CFU-G), megakaryocytic colony(CFU-M), granulocyte-megakaryocytic colony (CFU-GM), and mixed colony(CFU-GEMM) were obtained after14days cultures.ConclusionsHuman induced pluripotent stem cells cell can be induced into hematopoieticstem/progenitor cell in vitro by co-culture with OP9cell. Hypoxia can promote thedifferentiation of hematopoietic cells hematopoietic stem/progenitor cells havegreater differentiation potential.