A New Source Of Regenerated Cardiomyocytes-human Placenta Tissue

ObjectiveCardiovascular disease is the one of the leading causes of mortality, many forms of disease result in the progressive loss of myocytes by apoptosis or necrosis, which ultimately can progress to heart failure and death, especially coronary artery disease and myocardial infarction (MI).Therapeutic strategies have traditionally focused on restoring blood flow to myocardium in the periinfarction zone to prevent further ischemia. However, these therapies do nothing to address irreversibly injured myocardium. Heart transplantation can treat heart failure, however, it is restricted by donor sourse and immune rejection. It is reported that there are cardiac progenitor cells (CPCs) in the myocardium, which can not prevent the appearance of cardiac ventricle reconstruction and heart failure. Recently, with the development of stem cells, cardiovascular disease is treated by myocardial regeneration, which has become a new research, the transplanted stem cells are called seeds.At present, cell therapies for heart diseases focus on cell culture and animal model, bone marrow mesenchymal stem cells (BMSCs) and hematopoietic stem cells (HSCs) are frequent in clinical trials, which avoid ethics and statute of embryonic stem cells and immune rejection of auto-adult stem cells. However, there is a few mesenchymal stem cells in the adult bone merrow, the ratio falls to one to ten in every 10,000, furthermore, heart disease is incident to old person whoes red bone merrow become less and less than yellow bone merrow and numbers of BMSCs significantly decrease with age, leading to that patients can not get enough stem cells, so looking for new source stem cells has become a new research. It was reported that there were adherence cells derived from freezed placenta in 2000,recently,more and more research teams have proved that there are a large number of stem cells in the placenta, and these stem cells can differentiate into chondrocyte,osteoblast,adipocyte,neurocyte and myocyte, but there is a few reports about the differentiation into cardiomyocytes in vitro. We isolate the stem cells from human placenta and induce them into cardiomyocytes, to explore the possibility of healing myocardial infarction.Methods1. Isolation and expension of placenta-derived primary cells: picking up the anchoring villus tissue from the placenta and cut the amniotic membrane, put the anchoring villus tissue into PBS which is with 100U/ml Peniillin-streptomycin, incubate in PBS for 10 min, cut the anchoring villus tissue into smaller pieces and seed them onto dishes, add 10%DMEM with 10% fetal bovine serum (FBS), 0.1 mMβ-mercaptoethanol, 2mM L-glutamine and 0.1 mM Non-essential aminoacids (MEM). Cell cultures were maintained at 37℃with a water-saturated atmosphere and 5% CO₂.2. Immunophenotyping of placenta-derived mesenchymal cells (PDMCs): For cell surface Ag phenotyping, culture-expanded PDMCs (passage 3) were analyzed by flow cytometry. To detect surface Ag, aliquots of the PDMCs after treatment with 0.25% trypsin were washed three times with PBS, pH 7.4. For direct assays, cells were immunolabeled with the anti-human Ab CD13,CD14,CD29,CD31,CD44,CD45,CD73,CD90,CD105,CD166,HLA-ABC and HLA-DR.3. Differentiation of cardiomyocyte-like cells in vitro: PDMCs suspensions with a density of 2.5×10⁴ cells /ml were cultured in differentiation medium. After 2 days, the cells aggregates were formed in 20μl/drop of this suspension, and then transferred to suspension culture in 60-mm bacterial petri dishes for an additional 5 days. 7 days old cell aggregates were plated onto 0.1% gelatin-coated 24-well tissue culture plates at a density of 4 to 6 cell aggregates per well, and the differentiation medium was renewed every 2 days. Differentiation medium applied in this study consisted of 0.1 mM β-mercaptoethanol, 2 mM L-glutamine, in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% FBS.4. Percentage of spontaneously beating cardiomyocytes with daily observation, the percent of spontaneously beating cardiomyocytes was determined up to 30 days after plating.5. Immunocytochemistry: The beating areas of cells aggregates were isolated and reseeded into 12-hole plates with cover slips in IMDM supplemented with 20% FBS.After 24h,for immunocytochemistry. The control cells were uninduced PDMCs.6. Immunofluochemistry: The beating areas of cells aggregates were isolated and reseeded into 12-hole plates with cover slips in IMDM supplemented with 20% FBS. After 24h, for immunofluochemistry. The control cells were uninduced PDMCs.7. Reverse transcription-polymerase chain reaction (RT-PCR): collect the cells, abstract RNA, use RT-PCR assay the mRNA, the PCR products were size fractionated by 1.2% agarose gel electrophoresis.Results1. Characterization of placenta-derived cells: After 7 to 12 days inoculation, fibroblast-like cells transferred from placenta isolation, and formed individual colonies. After first passage, the placenta-derived cells were classified into two groups according to the growth and morphologic characteristic. One group could proliferate more than 41 passages, and the other one went into replicative senescence between 15 to 20 passages. The former type had a small and homogeneous morphology, cobblestone-like cells; and the latter type displayed fibroblast-like cells morphology.2. Immunophenotypic characterization of placenta-derived cells: The cells were positive for CD29 and CD105, and were negative for CD13, CD14, CD31, CD44, CD45, CD73, CD90, CD166, HLA-ABC and HLA-DR.3. Differentiation of placenta-derived cells into cardiomyocytes:Our studies showed that both cobblestone-like cells and fibroblast-like cells derived from placenta possessed the potential to differentiate into cardiomyocytes. At 8 to 11 days after plating, some spontaneously beating cardiomyocytes were observed in cells aggregates. There are different appearances of the beating cells aggregates, including clump-like cells aggregates and branch-like cells aggregates. The preliminary beating rate of cardiomyocytes was 40-60 beats/min, and 80-116 beats/min at peak, with the growth and development of the cell aggregates, the beating rate decreased gradually.4. RT-PCR analysis for the expression of cardiomyocyte-specific genes showed that induced cells expressed ANF andβ-MHC. Immunocytochemistry showed that cardiomyocyte-specific proteinα-sarcomeric actin and cTnI were expressed in the beating cells. Cardiomyocyte-specific genes and protein were not expressed in the uninduced PDMCs.Conclusion1. Human placenta is an abundant source of stem cells, which is a new source of regenerated cardiomyocytes.2. The stem cells derived from placenta can be induced to differentiate into cardiomyocytes by "hanging drop" method.In a word, The stem cells derived from placenta can be induced to differentiate into beating cardiomyocytes, the isolation of placental stem cells has no negative effects to the fetus and precursor; the research of it has no ethics and statute, which can be considered as a new source for regeneration medicine.