Directed Differentiation Of Stem Cells From Brown Adipose Tissue Into Pacemaking Cells

The biological pacing is a promising regime in regenerative medicine to curesevere slow arrhythmia such as sick sinus syndrome. Up to date, there are three majormethods for biological pacing including gene transfection, cell transplantation andtissue engineering construction in vitro. However, gene transfection may have theworries about biological safety and long-time availability in vivo. Cell transplantationand tissue engineering construction both depends on seeding cells. So, it is verycritical to seek an ideal cell source for cell transplantation and tissue engineering.The pacemaking cells might be derived from tissue cells, such as sinoatrial nodecells, naive cardiomyocytes and transfected cardiomyocytes, and various stem cellswhich can be differentiated into pacemaker cells, such as embryonic stem cells(ESCs), induced pluripotent stem cells (iPS) and adult stem cells. Among them adultstem cells may be the most hopeful to be applied in the clinical, while other sources ofcells may have shortcoming like immune rejection, tumor, ethical or security problem.Recently, it is reported that adipose-derived stem cells (ASCs) anddedifferentiated fat (DFAT) cells could be differentiated into beating cardiomyocytesdirectly, while other studies get the contradictory results. After reviewing the previouswork, we think that the differences are due to not only the different culture condition,but also the site of the adipose tissue obtained. Furthermore, it is unknown whetherthe differentiated beating cardiomyocytes are pacemaker cells.The present study was designed to clear the differences between the adiposetissues from interscapular and inguinal region, and also the potential of differentiationinto cardiomyocytes of stem cells aparted from the above two regions. Furthermore,the pacing characteristics of the differentiated beating cardiomyocytes wereinvestigated in order to explore the possibility of biological pacing in the future.Part1. Difference identification of interscapular and inguinaladipose tissueObjective: To investigate the characteristics and differences between interscapular and inguinal fat tissues.Methods: Interscapular and inguinal adipose tissues were obtained from C57mouse. Morphological characteristics of adipose tissues from both sites wereexamined by HE staining and transmission electron microscopy. Tissue RNA fromboth adipose tissues was also extracted for gene expression analysis.Result: With gross observation, interscapular adipose tissue was typical brownadipose tissue (BAT), while inguinal adipose tissue was white adipose tissue (WAT).HE staining showed that there were mainly brown adipocytes in the interscapularadipose tissues with a few scattered white adipocytes in it, and there were mainlywhite adipocytes in inguinal white adipose tissue containing a small amount of brownadipocytes around the lymph nodes. There were dispersed lipid droplets within brownadipocytes, and a large lipid droplet within white adipocytes. The ultrastructureobservation further confirmed the above morphological differences of the two kindsof fat cells. Mitochondria were larger and richer in brown adipocytes than those inwhite adipocytes. The gene expression analysis showed no cardiac gene expression inbrown and white adipose tissues, but some early heart development gene expressed inboth adipose tissues.Conclusion: Interscapular fat tissue is brown adipose tissue with a few of whiteadipocytes, while inguinal fat tissue is white adipose tissue with a few of brownadipocytes around lymph nodes. The two kinds of fat tissues can be distinguishedmorphologically.Part2. Directed differentiation of stem cells from brown adiposetissue into cardiomyocytesObjective: To investigate and compare the ability of stem cells from white andbrown adipose tissue directed differentiation into cardiomyocytes in vitro.Methods: WAT and BAT were taken from the interscapular and inguinal area ofmouse respectively, and were digested with collagenase solution, then filtrated andcentrifugated. The ASCs were cultured with classical routine, the floating top layercontaining mature adipocytes were cultured with ceiling cultures, and differentiateddirectly in20%fetal bovine serum. Immunostaining and gene expression were used toidentify their differentiation into cardiomyocytes.Result: Brown ASCs, but not white ASCs, were differentiated into directlybeating tubular cells or small round cells cultured for1-2weeks, the cells beating at 130/min approximately. Brown ASCs differentiated cardiomyocyte-like cellsexpressing α-actinin, while white ASCs not. They both expressed troponin T, troponinI and connexin40. Brown DFAT cells also were differentiated into muscle tubularcells and gradually formed a beating sheet, while white DFAT cells not yet. HCN4expression positive rate was significantly higher in brown DFAT cells-derived cellsthan that in white DFAT cells-derived cells.Conclusion: Stem cells from brown adipose tissue can directly differentiate intobeating cardiomyocytes in vitro, but white fat adipose tissue.Part3. Identification of characteristics of pacemaking cellsObjective: To investigate whether the beating myocardial cells differentiatedfrom brown ASCs or DFAT cells are pacemaker cells.Methods: HCN4expression of the cells was detected by immunostaining,intracellular calcium imaging and action potentials were recorded to validate theirautomaticity, and pharmacological treatments of the differentiated cells were tested byadrenergic agonists, antagonists or acetylcholine. The ultrastructure of the cells wasobserved by transmission electron microscopy.Result: Brown ASCs differentiated pacemaker-like cells were highly expressedHCN4, and spontaneous calcium transients and spontaneous action potentials wererecorded, which indicated that they had automaticity. Pacemaker-like cells frombrown ASCs and DFAT cells response to ISO, NE and Ach stimulus, and irregularmyofilaments could be seen in both differentiated cells.Conclusion: Cells differentiated from brown ASCs and DFAT cells arepacemaker cells.