| The biological pacing has become a hot research area in recent years. Biologicalpacing is a regime which replaces or repairs the damaged cardiac pacing and conductionsystem with some kind of cells or tissues with pacing and conduction function. Currently,there are mainly three strategies for biological pacing, including gene therapy,transplantation of stem cells and tissue engineering construction in vitro. In theory, abiological pacemaker produced from the concept of regenerative medicine, will providewith both desired security and self-control properties, and may overcome numerousdisadvantages of those electronic ones. Thus, the biological pacemaker is bound to becomeone of the perfect choices to cure kinds of arrhythmias. Seeding cells is one of the keyissues, so it is very essential to acquire the fact that how stem cells differentiate intopacemaker cells as well as to seek more ideal cell for transplantation and tissueengineering.Nowadays, these specific pacemaker cells might be produced from:(1) tissue cells,such as SAN cells, na ve cardiomyocytes and gene-modified cardiomyocytes;(2)totipotential stem cells that could be induced into pacemaker cells, such as embryonic stemcells (ES) and induced pluripotent stem cells (iPS);(3) somatic multipotent stem cells(SMSC), such as mesenchymal stem cells (MSC) that come from bone marrow, umbilicalcord blood, endometrium, amnion and adipose tissue. Among them, adipose-derived stemcells may be one of the most hopeful cells to be applied extensively due to lots ofadvantages. Furthermore, previous reports have demonstrated that stem cells from brownadipose tissues could convert into spontaneously beating cells. Our preliminary studyshows the spontaneously beating cells are of partial pacing characteristics. However, it isnecessary to further reveal the biological properties and differentiation process of stemcells, as well as the mechanism underline the differentiation.Our work aims to clarify the biological properties of stem cells from brown adiposetissues, further investigate the differentiation process, and characteristics of differentiated cells, and elucidate the possible mechanism of differentiation, and provide theoreticalsupport for its clinical application.Part1. The Biological Properties of BASCsObjective: To clarify the biological properties of BASCs.Methods: BASCs were isolated from interscapular brown adipose tissue of C57BL/6mice aged7-9weeks by enzymolysis and centrifugation. These freshly isolated cells wereexamined surface antigens by flow cytometer (FCM)and induced into tri-lineage cells(adipocytes, osteocytes and chondrocytes) in specific differentiation medium respectively.And corresponding evaluation tests were conducted after2weeks. Later，freshly isolatedBASCs were cultured in regular medium at37℃in5%CO2. Some specific markersexpressed by BASCs were detected by immunofluorescence to further confirm its cellularcomposition after culturing for3days.Results:1. Freshly isolated BASCs expressed both mesenchymal (CD29, CD44, CD90and CD105) and hematopoietic progenitors (CD34and CD45) markers, but excludedcardiac stem markers (c-kit and Sca-1).2. Under conditions for tri-lineage cellsdifferentiation, BASCs could differentiate into adipocytes, osteocytes and chondrocytesrespectively. Interestingly, cardiogenic differentiation was not observed in any of the3groups.3. BASCs highly expressed mesenchymal markers (more than90%) and losthematopoietic progenitors markers after3days’ regular culturing. Moreover, clusters ofc-kit+and Sca-1+cells appeared under regular culturing.Conclusion: Freshly isolated BASCs consisted of mesenchymal stem cells, andexcluded cardiac progenitors. Cultured BASCs could form a preliminarily purified cellpopulation that consisted of mainly mesenchymal stem cells and partially cardiacprogenitor cells in early stage. Part2. Differentiation of BASCs into Pacemaker-likeCellsObjective: To investigate the differentiation process and the BASCs and furtheridentify pacing characteristics and functions of differentiated BASCs.Methods: Freshly isolated BASCs were resuspended in DMEM supplemented with15%fetal bovine serum and incubated at37℃in5%CO2. Firstly, morphologic changeswere recorded during their differentiation. Later, SAN cell-specific structural genes orproteins were tested in well-differentiated cells by real time PCR and Western blot atdifferent time points. And electron microscope was also applied to show theultra-structures of these contracting cells. Intracellular Ca2+transients in the cells wereexamined using confocal microscopy, then electrophysiological studies were performed bythe whole cell confguration of the patch clamp technique under the current-clamp mode.Pharmacological tests were also applied to study the function of adrenergic and cholinergicreceptors.Results: After culturing in15%FBS/DMEM, BASCs could convert intomyotube-like or small round cells with automatic beating activity. qPCR analysis revealedthat the differentiated BASCs expressed structural genes of SAN cells, such as Sr, cTNT,Cx30.2and HCN4. All tested genes presented an increase trend. Western Blot analysisconfirmed a significant accumulation of these above structure proteins. The expressions ofthese SAN cell-specific markers were also detected by immumocytochemistry. Electronmicroscope showed disorganized and half-formed myofibrillar structure of BASCs.Spontaneous as well as provocative Ca2+transients was observed under the confocalmicroscopy, and action potentials were also recorded from these beating cells with typical4-stage automatic depolarization. Adrenergic and cholinergic reagents could significantlyinfluence their beating rates.Conclusion: BASCs underwent a differentiation into contracting cells under regularculture conditions, and these cells could considered as cardiac pacemaker-like cells due tothe exhibiting pacing properties and pharmacological functions. Part3. Role of Tbx18in Differentiation of BASCs intopacemaker-like cellsObjective: To study the role of a T-box transcription factor-Tbx18in thedifferentiation procedure of BASCs and explore its mechanism.Methods: The correlated transcriptional factors in cardiac SAN cells developmentduring the natural differentiation period of BASCs were examined by real time PCR andWestern blot. Then the RNA interference technique (RNAi) was applied to down-regulatethe expression of Tbx18during the whole cultural course. After this treatment,morphological changes and expression difference of correlated transcriptional factors andSAN cell-specific structural proteins were investigated at designed time point (the3rd,8thand13thday).Results: During the differentiation period of BASCs, Tbx18as well as itsdownstream transcriptional factors could be detected on both RNA and protein level(except Nkx2.5). Tbx18could be effectively down-regulated in BASCs by forcedlentivirus transfection, which were termed BASCs[shTbx18]. TheseBASCs[shTbx18]contained much less myotube-like cells, and appeared both dysplasiasand no-contracting compared to control groups. Those downstream transcriptionalfactors-Shox2and Tbx3expression both decreased after the interference ofTbx18.However, the key factor for ventricular lineage cardiomyocytesdifferentiation-Nkx2.5kept no change. And as expected, the reduction in transcript levelsresulted in a decrease in the expression of structural proteins.Conclusion: Tbx18might play a critical role in the differentiation of BASCs intopacemaker-like cells. |
PDF Full Text Request