Biological Characteristics Of Mesenchymal Stem Cells Derived From Human Fetal Bone Marrow And Liver And Differentiation Of These Cells Into Islet β-like Cells

ObjectivesTo study the basic biological characteristics of the mesenchymal stem cells derived from human fetal bone marrow(hfBM-MSCs) and the mesenchymal stem cells derived from human fetal liver(hfL-MSCs),and to induce hfBM-MSCs and hfL-MSCs to differentiate into islet β-like cells in vitro.MethodsCells were isolated from the aborted fetuses of 12-20 week's gestation.Rinsing the medullary cavity of the four limb bones to form the whole bone marrow suspension,cutting and pipetting the hepatic tissue to obtain the hepatocyte suspension,then collecting the non-parenchyma mesenchymal stem cell through two-step centrifugation,and enriching the karyota through removing the red blood cells by hydroxyethyl starch sedimentation.The cells in suspension were inoculated and cultured,and MSCs purification was done by adhesive screening method(because different kinds of cells have the different adherence to the wall of culture flask).The cell cycle and surface markers of hfL-MSCs and hfBM-MSCs were identified using flow cytometry.The expression of the embryonic stem cell-specific antigens such as AKP, SSEA-4,hTERT and Oct4 were detected with immunostaining or immunofluorescence staining at protein level,and the expression of Oct 4,SSEA-4 and hTERT were also tested by RT-PCR at RNA level.Inducing hfL-MSCs and hfBM-MSCs by routine inducing protocols to differentiate toward cells derived from three germ layers represented by nerve-like cells,muscle-like cells (adipocytes and osteoblastes) and hepatocytes respectively.Karyotype analysis of hfL-MSCs and hfBM-MSCs were made at passage 8 and after recovery from cryopreservation for 6 months. Grafting hfL-MSCs and hfBM-MSCs to the dorsum and renal subcapsule of nude mice to evaluate their oncogenicity.Culturing hfL-MSCs and hfBM-MSCs with K562 cells to observe the effect of hfL-MSCs and hfBM-MSCs on the growth of K562 cells.hfL-MSCs and hfBM-MSCs were induced to differentiate toward isletβcells under various inductive conditions,and the best protocols were finally selected and optimized basing on the results of morphological change,the expression of isletβcells related genes by RT-PCR and islet-specific proteins by immumofluorescent staining.Then,for the islet-like clusters(ILCs) induced by the optimized protocol,scanning and transmission electron microscope were used to show their exterior and interior ultramicroscopic structures.Dithizone(DTZ) staining was performed to identify the Zinc in ILCs.In addition,the quantity of insulin secretion and intracellular insulin were examined by chemiluminescence immunoassay.The test of glucose-simulated insulin release was made to evaluate the function of the ILCs.Western blot was conducted to identify the nature of secretion(insulin,proinsulin or both of them).Finally, the ILCs were implanted into the left renal subcapsular space of diabetic mice.Blood glucose levels were monitored every 2-3 days after implantation.Excising the left kidney of the xenotransplanted diabetic mice which glucose level had restored to normal to detect whether there is a rebound of blood glucose.Harvesting the pancreas tissue and the left kidney of the xenotransplanted diabetic mice for immunohistochemistry.ResultshfBM-MSCs and hfL-MSCs can be isolated and purified from human fetal bone marrow and liver.There are over 90%hfBM-MSCs and hfL-MSCs of passage 3 in G0/G1 phase. hfBM-MSCs and hfL-MSCs expressed adhesion molecules CD29 and CD44,but not antigens of hematopoietic CD15,CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD40, CD80 and CD86.hfBM-MSCs and hfL-MSCs expressed the embryonic stem cell-specific markers,such as Oct 4,SSEA-4 and hTERT at RNA level and were positive for Oct 4,SSEA-4, hTERT and AKP at protein level.Exposure of hfBM-MSCs and hfL-MSCs to routine inductive agents resulted in morphological changes towards nerve-like cells,adipocytes,osteoblasts, muscle-like cells and hepatocyte-like cells,and all these kinds of cells were identified by special staining or RT-PCR.hfBM-MSCs and hfL-MSCs showed normal karyotype at passage 8 and after cryopreservation for six months.hfL-MSCs and hfBM-MSCs were not cancerous after being grafted to nude mice.Co-culture of hfL-MSCs and hfBM-MSCs with K562 cells resulted in the growth inhibition of K562 cells.As for the induction of hfBM-MSCs and hfL-MSCs toward isletβcells,the untreated MSCs were spindle-shaped adherent cells,but during the course of differentiation induced by the optimized protocol,they changed quickly into round and oval shape and gathered more and more islet-like clusters(there were hundreds of clusters in the growth surface of a flask of T25). RT-PCR showed the ILCs expressed islet-related genes including Pdx-1,isl-1,ngn3,insulin and glucagon,and immumofluorescence demonstrated the ILCs were strongly positive for Insulin, C-peptide and Glucagon.The ILCs were stained crimson red by dithizone indicating the high zinc content in the cells.Scanning electron microscope showed there no vesicular prominency on the surface of untreated MSCs,but many vesicular structures of different size appeared on the surface of ILCs.Transmission electron microscope displayed the microvilli on the surface of ILCs and numerous secretory granules of variable size and color in the cytoplasm of ILCs. Chemiluminescence immunoassay demonstrated the ILCs were able to secrete a higher level of immunoreactive insulin into medium,the average level are(210±35.07)μU/mL for ILCs induced from hfBM-MSCs and(106.7±28.21)μU/mL for ILCs induced from hfL-MSCs, respectively.The test of glucose-simulated insulin release showed the ILCs could elevate the insulin secretion upon glucose challenge,and the stimulus index are 4.38±0.32 for ILCs induced from hfBM-MSCs and 4.22±0.27 for ILCs induced from hfL-MSCs,respectively.The results of Western blot showed that most of the protein extraction of ILCs was proinsulin suggesting the ILCs not enough mature.After the ILCs were implanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually,but control animals that did not receive implants exhibited persistent hyperglycemia.When the left kidneys that contain the implanted cells were removed,the hyperglycemia reappeared and the mice died rapidly.Immunohistochemistry staining showed the implanted cells under the kidney capsule were positive for insulin,and there were a few of atrophy islet in the pancreas of the xenotransplanted diabetic mice.ConclusionsThe hfBM-MSCs and hfL-MSCs possess the embryonic stem cell-like biological characteristics and have the ability to differentiate into the derivative cell types of the three embryonic germ layers,moreover,they have a lower immunogenic property and no tumorigenicity,so provide the ideal source for tissue engineering and cellular therapeutics.The hfBM-MSCs and hfL-MSCs can rapidly and easily be induced to differentiate into isletβ-like cells in vitro,but the induced ILCs are not enough mature.In order to exert the function of regulating the blood glucose level,it is necessary for the ILCs to be implanted into diabetic mice for further development in vivo.