Effects And Mechanisms Of The Generation Of Hematopoietic Cells From Mouse Pluripotent Stem Cells Involving The Three Dimensional Self-assembling Peptide Hydrogel

Background:Hematopoietic stem cell(HSC)owns the ability of self-renewal and multipotential differentiation.Recently,HSC transplantation is a well established life-saving therapy used to treat various diseases including leukemia,lymphoma,immune system disorders,multiple myeloma and bone marrow failure syndrome.Currently,although allograft and autologous HSC transplantation has been widely applied in clinic,it still faces many problems,such as the number of HSC for the source,the human leukocyte antigen matching donor and graft-versus-host disease(GVHD).Pluripotent stem cell(PSC)including embryonic stem cell(ESC)and induced pluripotent stem cell(iPSC),with in vitro self-renewal and multilineage differentiation potential,it can provide everfount HSC for transplantation.In addtion,in vitro generation of HSC from PSC obtained from the patient via somatic cell reprogramming technology can be used as a model to explore the causes of disease,providing a cell-based drug screening platform at the same time.Although researchers have been exploring hematopoietic differentiation approach of PSC to produce transplantable HSC in the past decades,there has not been an effective method for enerating functional HSC from PSC.At present,the mainly ways to HSC differentiation of PSC focus on:(1)using mouse or human stromal cells to induce the hematopoietic differentiation of PSC;(2)using embryonic bodies(EBs)to induce hematopoietic differentiation of PSC;(3)combining the two methods;(4)teratoma method.At the same time,cytokines and transcription factors are combined with the above methods.However,there are still some key problems to be solved in the field of studying the hematopoietic differentiating of PSC:(1)the efficiency of induced differentiation is not high,(2)the generated HSC is difficult to obtain long-term hematopoietic reconstitution potential.Study on PSC hematopoietic differentiation focused on 2D induced environment,in fact,3D environment can better simulate the three-dimensional tissue structure in natural environment compared with two-dimensional condition.In the process of embryonic hematopoiesis in vivo,cells not only contact with each other,also need contact with each other various adhesion molecules and collagen in extracellular matrix environment,and The extracellular matrix environment plays an important role in cell proliferation,differentiation,maintenance and tissue morphology.Now,researchers are increasingly committed to developing new biomaterials that simulate the three-dimensional structure and extracellular matrix biological functions to regulate the biological functions of stem cells.In the process of embryonic hematopoiesis,hematopoietic microenvironment such as aorta-gonads-mesonephros(AGM),fetal liver(FL)and bone marrow stromal cells promote the differentiation of PSC into HSC and the HSC maintenance[34].AGM-S3 stromal cell derived from AGM was reported to promote the differentiation of human and mouse PSC into HSC and expand the HSC.At the same time,AGM-S3 that combined with various cytokines can promote differentiation of human PSC into adult nucleated red blood cell[35,36].AFT024 stromal cells derived from murine FL can maintain mouse long term hematopoietic reconstitution potential of HSC[37].Human ESC co-cultured with OP9,S17 and stromal cells derived from fetal liver can differentiated into hematopoietic cells,but the low differentiation efficiency and the HSC has no low engraftment potential[38].This study intended to establish a three-dimensional hematopoietic differentiation system using 3D biological materials,combining with exogenous growth factors that promote HSC proliferation and hematopoietic differentiation of PSC.In addition,we used the stromal cells within the hematopoietic microenvironment to optimize our three-dimensional differentiation system to obtain long-term reconstitution HSC of PSC,providing the molecular regulatory mechanism platform for in vitro HSC differentiation of PSC,establishing the experimental basis for promoting the transformation of HSC derived of PSC into clinical application.Part1 Generation of hematopoietic cells from mouse pluripotent stem cells in a 3D culture system of self-assembling peptide hydrogelObjective:In vitro inducing the production of HSC from PSC can serve as an effective alternative to bone marrow transplantation,and will not cause graft-versus-host disease(GVHD).At present,many methods have been used to evaluate differentiation of PSC into HSC,but low efficiency differentiation and incomplete HSC function limit the further study of hematopoietic differentiation.We aim to establish a better simulation of 3D hematopoietic differentiation environment as embryonic development niches in vivo,promoting the production of functional HSC from PSC.And the complexity of 3D induced system is difficult to be realized via 2D culture conditions.Methods:Firstly,scanning electron microscope was used to observe the structure characteristics of 3D self-assembled peptide hydrogel.To explore the generation of hematopoietic cells from PSC using 3D self assembling peptide hydrogel combined with hematopoietic related cytokines.Flow cytometry assay was used to detect the expression level of ckit,CD41 and CD45 surface markers.Fluorescence quantitative PCR experiment was to detect the mRNA expression level of pluripotent related gene Oct3/4,endothelial gene CDH5 and hematopoietic related genes Gatal and HOXA9.Immunofluorescence assay was used to detect the expression of hematopoietic marker CD41 and CD45.CFU assay was used to analyze the hematopoietic lineage differentiation of HSC and progenitor cells from 3D induction system.6-8 weeks NOD-SCID mice were used to evaluate the in vivo reconstitution potential of hematopoietic cells derived from 3D induction system.Results:Using 3D self assembling peptide hydrogel combined with cytokines can induce PSC differentiation into hematopoietic cells.Flow cytometry results showed that ckit,CD41 and CD45 molecules obtained obvious expression.Fluorescence quantitative PCR detection showed that pluripotent gene Oct3/4 was significantly downregulated,and endothelial cells gene CDH5,hematopoietic related genes Gatal and HOXA9 and surface molecules CD41 and CD45 were upregulated during the process of 3D hematopoietic differentiation.Immunofluorescence detection showed that CD41 and CD45 expressed.CFU assay showed that 3D induction hematopoietic system derived hematopoietic cells owned the ability of differentiation into hematopoietic lineage cells.In vivo animal transplantation experiment showed that mPSCs(CD45.2)could embedded into NOD/SCID mice(CD45.1)with about 3%engraftment efficiency after 3 weeks transplantation.This study demonstrated that we developed the 3D induction approach that could efficiently promoted the hematopoietic differentiation of mPSCs in vitro and obtained the multipotential progenitors that possessed the short-term engraftment potential.Conclusion:3D self assembling peptide hydrogel combined with cytokines can effectively induce the differentiation of mouse PSC into hematopoietic cells which obtain the ability of hematopoietic lineage differentiation in vitro and short-term hematopoietic reconstitution potential in vivo.Part 2 Using OP9 stromal cell to optimize the further hematopoietic differentiation potential of hematopoietic like clones derived from 3D induction systemObjective:OP9 or OP9-DL1 stromal cell that is conducive to promoting hematopoietic differentiation of PSC,at the same time,can promotes in vitro hematopoetic specified differentiation of hematopoietic precursor cells and endothelial cells derived from embryonic development in vivo.In the first chapter,We had established the 3D induction system for mouse PSC differentiating into HSC.The differentiation of each PSC into HSC is not synchronous,and a variety of hematopoietic precursors and the early hematopoietic development cells existed in our 3D induction system.Our study is using OP9 stromal cell to further optimize the 3D induction system in order to enhance hematopoietic differentiation efficiency of PSC and obtain more functional HSC,establishing the basis of studying the mechanism of HSC differentiation of PSC.Methods:OP9 stromal cell combined with cytokines induced the further hematopoietic differentiation of hematopoietic like clones derived from 3D induction system.Flow cytometry analysis was used to detect the expression of surface molecule such as Flk1,ckit,TIE2,CD144,CD41,CD45,Sca-land latest reported CD201 surface molecule expression at different differentiation time point.CFU experiment was done to confirm the hematopoietic lineage cells differentiation potential from hematopoietic cells from the system that co-culturing the OP9 stromal cell with 3D induction system derived hematopoietic like clones.Results:OP9 stromal cell can effectively promotes the differentiation of 3D induction system derived hematopoietic like clone into hematopoietic precursors and hematopoietic stem and progenitor cells.Flow cytometry results showed that ckit,TIE2,CD144,CD41,CD45,Sca-1,CD201 obtained the obvious expression.The flkl expression upregulated from day2 to day5,downregulated from day5 to day8.CFU experiment showed that hematopoietic cells from the system that co-culturing OP9 stromal cell with 3D induction system derived hematopoietic like clone owned the ability of differentiation into hematopoietic lineage.Conclusion:OP9 stromal cell can effectively promotes the differentiation of 3D induction system derived hematopoietic like clones into hematopoietic precursors and hematopoietic stem and progenitor cells,which gets a group of unreported CD201+LSK hematopoietic cells.Part 3 3D culture system mediated by self assembled polypeptide biomaterials enhanced the amplification of mouse hematopoietic stem cell and progenitor cell compared with 2D culture systemObjective:To explore that the 3D induction system mediated by the self assembling peptide biomaterial will do help to provide a good platform for in vitro research of the HSC generation from PSC,we further evaluated that whether the 3D induced system is effective for the expansion of murine fetal liver or bone marrow derived hematopoietic stem cell and progenitor cell compared with 2D culture system or not.laying the foundation for researching the mechanisms of the generation of HSC from PSC via 3D inducement system.Methods:To expand the ckit+ cells derived from bone marrow using 3D self assembling peptide hydrogel biomaterials combined with hematopoietic cytokines and small molecular compounds.Comparing the expansion efficiency within 2D environment under the same condition.Observating the growth condition of ckit+ cells under light microscope.Flow cytometry analysis was used to compare the ratio of Lineage+Sca-1+ckit+(LSK)cells under two different conditions and evaluate the hematopoietic lineage cells in 3D and 2D culture system.CFU assay was used to compare the difference of clone formation ability under two different expansion conditions.Results:Comparing with 2D same culture conditions,3D self assembling peptide hydrogel combined with cytokines and small molecular compounds can better amplify the ckit+ cells and maintain hematopoietic stem cell and progenitor cell.In 3D culture system LSK cells owned the higher ratio compared with 2D under the same condition.CFU assay showed that the total number of CFU in 3D condition is higher than 2D culture system with significantly differences.Conclusion:3D culture system mediated by self assembling peptide biomaterial is more conducive to the maintenance of bone marrow derived hematopoietic stem cell and progenitor cell compared with 2D culture system.Meanwhile,3D culture system is used to the growth of hematopoietic downstream lineage cells.Comparing with 2D,3D system can better simulate the nich of HSC to provide a theoretical basis for exploring the HSC differentiation from PSC.