Induction Of Dedifferentiated Fat Cells Into Pacemaking Cells

Cardiac rhythm disorders such as sick sinus syndrome (SSS) or atrioventricular nodalblock (AVB) are a serious and life threatening condition, requiring implantation ofelectronic pacemakers in over300,000people annually in the US. While highly successful,these devices also have significant shortcomings. As a result, numerous laboratoriesworldwide are developing biological pacemakers as a replacement or complement toelectronic pacemaker.Various approaches in the field of gene therapy, cell therapy and tissue engineeredhave been developed by different groups and this combined effort makes it increasinglyrealistic that this therapy will eventually find its way to clinical application. However, astable, safe and long-term expression system is needed in gene therapy. Cell therapy andtissue engineered are alternative approaches that can overcome some of the shortcomingsof gene therapy. Cell therapyapproaches and tissue engineered have explored the “forcing”of embryonic stem cells，iPS cells, cardiac derived cells to evolve along cardiac (andspecifically pacemaker) cell lines, but all of these cells have some shortcomings. Inconclusion, it is critical to seek an ideal seeding cell for creating biological pacemaker.Attention has been focused on DFAT cells that might be useful in tissue engineering.Mature adipocytes are the most abundant cell type in adipose tissue. It has been shown thatthe mature adipocytes isolated from fat tissue can be dedifferentiated into fibroblast-likecells with an in vitro dedifferentiation strategy, known as ceiling culture. In vitrodifferentiation analysis revealed that DFAT cells could differentiate into adipocytes,chondrocytes, osteoblasts, myocytes, and so on under appropriate culture conditions. Inaddition, clonally expanded mouse DFAT cells showed the ability to differentiate intocardiomyocytes. Jumabay et al. have reported that mouse white adipocytes, isolated fromsubcutaneous fat, can be de-differentiated into a homogeneous population of DFAT cells in20%FBS, which undergoes spontaneous differentiation into autorhythmic cardiacmyocyteswith slow diastolic depolarization. However it is difficult to develop biological pacemakersbecause the heart of mouse was to small, and whether there was pacing function in thespontaneous contraction cells or not. Here we investigated whether mature adipocyte-derived dedifferentiated fat (DFAT)cells could differentiate to pacemaker-like cells in vitro induced by5-AZA or coculturedwith sinoatrail node cells. The objective is to provide a new source of cardiac pacemakingcells, which could proliferate quickly, readily received and no immunogenicity for theconstruction of biological pacemaker.Part1. Isolation, culture, and identification of dedifferentiated fat cellsObjective: To isolate, culture, identify DFAT cells and harvest cells which can beinduced into pacemaker-like cells.Methods：Subcutaneous adipose tissue was removed from SD rat, and was digestedusing collagenase solution, after filtration the floating top layer containing unilocularadipocytes was collected and cultured in ceiling cultures. After7days, the medium wasremoved, and the flasks were inverted. DFAT cells were stained with CD13, CD29, CD31,CD34and CD44by immunofluorescence. We also examined the multilineagedifferentiation potential of DFAT cells. Morphological characteristics of DFAT cells wereinvestigated by light and transmission electron microscope.Result：During the ceiling culture, the cells attached and flattened to the upper surfaceof the flasks followed by conversion to fibroblast-like dedifferentiated fat (DFAT) cells.DFAT cells were uniformly positive for CD13, CD29, CD44, but negative for CD31andCD34. In vitro differentiation analysis revealed that DFAT cells could differentiate intoadipocytes and osteoblasts under appropriate culture conditions. TEM showed there weremany Golgi and endoplasmic reticulum in DFAT cells.Conclusion：Isolation and culture of DFAT cells were succeeded.Part2. Differentiation of DFAT cells into pacemaker-like cells induced by5-AZAObjective: To investigate the possibility of DFAT cells induced into pacemaker cellsby5-azacytidine.Methods: DFAT cells were induced by5-azacytidine. Morphological characteristicsof DFAT cells were investigated by TEM (transmission electron microscope, TEM).TotalRNA was extracted from cells after induced by5-AZA, cardiac specific genes weredetected by reverse transcription-polymerase chain reaction (RT-PCR).Immunofluorescence detection of some related proteins were done. The DFAT cells wereplanted in resting neonatal rat cardiomyocytes. Result: Cell morphology of DFAT cells by5-AZA changed apparently and becomelarger, the majority showed long spindle-shaped. TEM showed there were manysarcomeres and intercellular junctions in DFAT cells induced by coculture with sinatrialnode cells.Cardiac genes Nkx2.5and GATA-4were up-regulated in week1and week2,and down-regulated in week3after induced by5-AZA. HCN, Cx, and Tn mRNAexpressions were increased. DFAT cells were uniformly positive for Tn, HCN, Cx. Therewas no spontaneous contraction when induced DFAT cells palnted in resting neonatal ratcardiomyocytes.Conclusion: DFAT cells induced by5-AZA cannot differentiate into typicalpacemaker cells, but gained expression of pacemaker cells specific genes and proteins.Part3. Differentiation of DFAT cells into pacemaker-like cells by inducedby coculture with sinus node cellsObjective: To investigate the possibility of DFAT cells induced into pacemaker cellsby cocultured with sinatrial node cellsMethods: Transwell chambers seeded with neonatal rat sinoatrial node cells wereinserted into6-well, which was seed with DFAT cells. Morphological characteristics ofDFAT cells were investigated by TEM.The expression of related molecules was detectedby immunofluorescent and PCR at week1, week2and week3. Immunofluorescencedetection of some related proteins were done. The DFAT cells were planted in restingneonatal rat cardiomycytes.Result: Cocultured DFAT cells were become larger and showed long spindle-shaped.TEM showed there were many sarcomeres and intercellular junctions in DFAT cellsinduced by coculture with sinatrial node cells. Cardiac genes Nkx2.5and GATA-4wereup-regulated in week1and week2, and down-regulated in week3after induced. HCN, Cx,and Tn mRNA expressions were increased. Expression of HCN, Cx, and Tn was positiveby immunofluorescence staining. Pulse of resting neonatal rat cardiomyocytes reappearedwhen cocultured with induced DFAT cells.Conclusion: DFAT cells cocultured with sinatrial node cells can not differentiate intotypical pacemaker cells with automaticity. However, resting neonatal rat cardiomyocytescan be derived by these cells in vitro.