Experimental Study Of Hematopoietic Potential Of Mouse Placenta

BackgroundHematopoiesis is a process that hematopoietic stem cells(HSCs) in the hematopoietic tissues expanse,differentiate,mature and give rise to all kinds of blood cells.The so-called HSCs are a type of primitive ceils which exist in the hematopoietic tissues, develop and differentiate into a variety of blood cells.They are also called totipotentiai hematopoietic stem cells(THSC).Under the induction of certain hematopoietic microenviroment and some factors,THSC can proliferate and differentiate into pluripotential lymphoid stem cells and pluripotential myeloid stem cell(PMDC).The former can differentiate and development into functional lymphocytes,while the latter can develop into CFU-GM,BFU-E,and CFU-GEMM, which will further give rise to white blood cells,red blood cells and platelets. Therefore,on the aspect of hematopoiesis,hematopoietic ceils can be typically classified into three types:hematopoietic stem cells,hematopoietic progenitor cells (HPC) and recognized precursors.The process of hematopoiesis is a dynamic balance of HSCs and HPCs expansion,differentiation and blood cells formation.However,the storage,release and distribution of mature blood cells in the body are not included in this process.HSCs and HPCs play a major role in the hematopoiesis.During the development of embryonic hematopoietic system,the mesenchymal tissues of mesoderm first differentiate into hemangioblast,and then HSCs and angioblast.The first HSC is found in the extraembryonic blood island of yolk sack and paraaortic splanchnopleure/aortagonad mesonephros(PAS/AGM).With the establishment of intra and extra embryonic circulation,HSCs were seeded into liver and spleen,and lastly into bone marrow to maintain the hematopoiesis during the lift time.The study on the development of embryonic hematopoiesis has been carried on for over a century.In mammal embryos,yolk sac,liver,thymus have been thought to be hematopoietic organs.Recently,several studies indicated that placenta is also a hematopoietic organ,which plays an important role on the embryonic stage.However, at present,it is still controversial about the hematopoiesis of palcenta.In our studies, various empirical methods were used to detect the hematopoietic ability of mouse placenta and try to identify whether the placenta possesses the hematopoietic activity and what the characteristics of placental hematopoiesis are.PARTⅠISOLATION AND IDENTIFICATION OF MONONUCLEAR CELLS IN MOUSE PLACENTAObjectiveClear all the fetal and maternal blood out of the mouse placenta,isolate and purify the proper hematopoietic stem/progenitor cells(HS/PCs) in the placenta,and explore if the mouse placenta possesses the ability of hematopoiesis.Methods1.Develop the technique of placenta flushing to clear the fetal blood contained in the placenta.Pregnant females of E12.5 were killed and the uterine horns were removed. The embryos(including the fetus and the placenta) were harvested.Under a stereomicroscope,the umbilical artery and vein were identified by the direction of blood flow.A fine needle(BD 29 G) was inserted into the umbilical artery with the tip pointing to the placenta.Heparin saline(concentration 0.5 mg/ml,290000 IU/ml) was used as flushing fluid and injected into the umbilical artery slowly(0.1ml/min) to flush the embryonic vessel system of the placenta.The flushed embryonic blood was collected as control.Then the placentas were separated from the maternal decidua, umbilical vessels and remnants of the yolk sac.Wash the placenta repetitively,until the palcenta turned pale,to remove the maternal blood.2.The paraffin sections of the flushed placenta were prepared.After HE stain,the sections were observed whether there is residued blood cells in the placental tissue.3.The placentas were mechanically dissected free and drawn through a 16 G needle and incubated with 0.1%collagenase in 10%fetal calf serum(FCS) / phosphate-buffered saline(PBS) for 30 minutes at 37℃and trypsin for 10 minutes at 37℃.Terminate the digestion and centrifugate.Then the cells were resuspensensed with 1 ml IMDM for the subsequent isolation of the mononuclear cells by centrifugation.4.Then the Percoll fluids of different densities(1.1.g/ml,1.080g/ml,1.055g/ml) were added into the centrifuge tube one by one from higher density to less,and then the cell suspensions were gently placed to the utmost upper layer.After centrifugalization of 25 minutes with 2000 rpm,the cells residing between the top layer and the middle layer were harbored and resuspensed with 1 ml IMDM to prepare the mononuclear cells suspension.5.Make cell film preparations and stain with Giemsa.Under microscope,observe if the obtained cells were mononuclear.6.Use CFU-S assay to identify if the extracted mononuclear cells from the placenta were hematopoietic stem cells.Results1.We flushed each placenta with 0.2 ml flushing fluid.After flushing,the fetal circulation blood was all cleared out of the placenta.2.After the combined enzymatic digestion and density gradient centrifugation with Percoll fluids,the cell film preparations showed that the obtained isolated cells were mononuclear and small lymphocyte-like,with a diameter of 7～10μm.3.The isolated mononuclear cell suspensions were injected via the tail vein after the recipient mice were irradiated lethally.CFU-Ss were detected in the spleens of recipient mice.After the sections of the spleen were stained with HE,the CFU-Ss were confirmed under microscope.ConclusionPlacenta flushing is effective to clear the fetal and maternal blood in the placenta. Continuous enzymatic digestion with collagenase and trypsin and Percoll density gradient centrifugation method were effective to isolate the mononuclear cells of the placenta.CFU-S assay demonstrated that these mononuclear cells harbored the ability to form colonies in spleen,which was the characteristics of HS/PCs,and were the proper HS/PCs of mouse placenta.PARTⅡUTILIZATION OF HS/PCS SURFACE MARKERS TO DETERMINE EXISTENCE,FREQUENCY AND DISTRIBUTION OF HS/PCs IN MOUSE PLACENTAObjectiveExamine the surface markers of the mononuclear cells of placenta with immunofluorescent technique and flow cytometry to determine the existence, frequency and distribution of the proper HS/PCs of mouse palcenta and provide more evidences of placental hematopoiesis.Methods1.The expressions of HS/PCs-specific antigens---CD34,CD117 and Sea-1---were examined in the mononuclear cells of mouse placenta by immunofluorescence to determine the existence of proper HS/PCs in mouse placenta.2.FACS was applied to examine the absolute and relative numbers of CD34,CD117 and Sea-1 positive cells to judge the differentiation condition of proper HS/PCs of mouse placenta.Compare the results between the proper HS/PCs of mouse placenta and the isolated mononuclear cells from the fetal blood in the placenta.3.Placentas of E12.5 were sectioned and immumohistochemically stained with antibodies of CD34,CD117 and Sca-1 to display the distribution of placental proper HS/PCs in the mouse placenta.Results1.The immunohistochemistry showed that the HS/PCs-specific antigens---CD34, CD117 and Sca-1---were expressed by the placental mononuclear cells.The percentages of CD34,CD117 and Sea-1 positive cells in the total cells were 20.1±5.3%,28.5±3.4%,35±8.6%,respectively.These proved thate the placental mononuclear cells were definite HS/PCs.2.FACS showed that the placental HS/PCs expressed HS/PCs-specific antigens,CD34, CD117 and Sca-1.The percentages of the three cell populations in the total purified placental cells were 20.2%,24.6%,and 26.2%respectively.For the mononuclear cells of placental blood cells,the percentages of the three populations were 8.2%,6.3%, and 6.5%respectively.The concentration of positive cells in placental cells was higher than in placental blood.The absolute numbers of positive cells of CD34, CD117 and Sca-1 were 1.4×10~4,1.8×10~4,and 1.8×10~4 respectively in placental mononuclear cells,and were 0.7×10~4,0.5×10~4,and 0.5×10~4 respectively in the mononuclear cells of placental blood.The number of positive cells in the placental mononuclear cells was higher than in the mononuclear cell of placental blood.For the cell subpopulation of CD34~+/Sca-1~+,the percentage in placental mononuclear cells (15.3%) was also higher than in the mononuclear cells of placental blood(5.1%).3.In the E12.5 mouse placenta,CD34~+ cells,CD117~+ cells and Sca-1~+ cells can be found in the palcenta labyrinth.CD117~+ cells can also be observed in the chorionic plate.The expression of Sca-1 was significantly higher than CD34 and CD117.ConclusionPlacental hematopoiesis is further proved with the application of HS/PCs-specific antigens immunohistochemistry,immunofluorescence and flow cytometry.Placental proper HS/PCs exist and distribute regularly in mous placenta. PARTⅢIN VITRO CULTURE OF PLACENTAL MONONUCLEAR CELLS AND DETECTION OF HEMATOPOIESIS-RELATED GENES EXPRESSION TO CONFIRM PLACENTAL HEMATOPOIESISObjectiveConfirm the placental hematopoiesis through in vitro culture of placental mononuclear cells,the observation ofhematopoietic colony forming,and detection of the expression ofhematopoiesis-related genes.Methods1.10~5 purified single cells were plated per 35 mm dish in MethoCult M3434 methylcellulose media.After 14 days of culture,colonies were observed and identified based on their morphology under an inverted microscope.The HPP-CFCs were evalutate.So do the mononuclear cells of placental blood,as positive control.2.RT-PCR was applied to detect the expression of Scl,Runx1,Tel/Etv6,and GATA-2 in the placental mononuclear cells.Results1.Three different types of progenitors-CFU-GEMMs(colonyforming units, granulocytes,erythrocytes,monocytes,macrophages),BFU-Es(burst-forming units, erythroid) and CFU-GMs(colony-forming units,granulocytes,macrophages)-diagnosed from the colony phenotype,were observed on day 14 of culture.The frequencies of placental BFU-Es,CFU-GMs and CFU-GEMMs were higher than that of placental blood.The characteristics of the morphology of placental BFU-Es was multicentered and highly hemoglobinized,while placental blood BFU-Es displayed only weakly hemoglobinized clusters.In the case of CFU-GMs,placental CFU-GMs were dense,medium to large sized,and often actually so large that they seemed to result from the fusion of clusters,whereas placental blood CFU-GMs were small sized.Both placental CFU-GEMMs and placental blood CFU-GEMMs were huge and so qualified more adequately as HPP-CFCs.Compared with placental blood CFU-GEMMs,placental CFU-GEMM appeared larger,denser,and contained more cells,with a very large core of hemoglobinized cells.2.After culturing of 60 days,the colony of HPP-CFCs from both placenta and placental blood exceeded 0.5mm in diameter.When the colonies were scored at 60 days,35 CFU-GEMMs can be obtained from 10~5 replated placental mononuclear cells.By contrast,HPP-CFCs could also be obtained from the mononuclear cells of placental blood and the frequency was about two-thirds of the placenta.Furthermore,the colonies of placental blood were smaller than the placental colonies.3.The expression of hematopoiesis-related genes---Scl,Runx1,Tel/Etv6,and GATA-2---could be detected on the placental mononuclear cells.ConclusionPlacental mononuclear cells harbored the potential to differentiate to CFU-GMs, BFU-Es and CFU-GEMMs.HPP-CFCs activity can also be detected.These indicated that the placental mononuclear cells were HS/PCs and possessed the potential of multipotent differentiation.The placental hematopoiesis was further proved by the hematopoiesis-related genes expression on the placental mononuclear cells.