Establishment of parthenogenetic embryonic stem cells and differentiation of pluripotent stem cells into cardiac lineage in mouse

Abstract

EXP. 1 aimed to establish embryonic stem (ES) cell lines from parthenogenetic embryos and examine pluripotency differences among the parthenogenetic ES (pES) cell lines. We are able to report the successful establishment of four pluripotent pES cell lines from blastocysts of parthenogenetic origin. Four pES cell lines (pES#1-4) exhibited a typical ES cell morphology and expression of key pluripotency markers (ALP, Oct-4, Nanog and SSEA-1). Three of the four pES cell lines (pES#1-3) exhibited a higher efficiency towards endo-mesoderm differentiation than pES#4. Differentiation towards cardiac cells resulted in all cell lines 33-100% of spontaneous beating cell clusters/well. In conclusion, our results have demonstrated that there are major differences among pES lines in their differentiation ability in vitro. EXP. 2 aimed to enable large-scale culture of ES-derived cells for cardiac differentiation, we developed a scalable bioprocess that directs embryoid body (EB) formation in a fully controlled STLV (slow turning lateral vessel, Synthecon, Inc, Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. We investigated the effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation. Our results showed that 3x105 cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture are the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method. EXP. 3 aimed to compare the efficiency of EB formation and cardiac differentiation by using STLV bioreactor to static suspension culture (SSC) and hanging drop (HD) condition. After three days culturing, a 4-fold improvement in the yield of EB formation/ml and a 6-fold enhancement in total cell yield of EBs in STLV conditions vs. SSC conditions were detected. On the other hand, a nearly 10-fold diminishment in total cell yield of free cell which were not incorporation into EBs in STLV condition vs. SSC was observed. Overall, STLV conditions produced more uniform EBs than SSC and HD. During cardiac differentiation, EBs cultured in STLV showed the highest distribution of cardiac troponin T (cTnT). RT-PCR assay demonstrated that EBs cultured in STLV and HD expressed more cardiac markers (Nkx2.5 Tnnt2 Nppa and Myh6) compared with SSC condition. Hence, EBs culture in STLV provides a technological platform for the large-scale generation of ES cell-derived cells and differentiation into cardiomyocytes. EXP. 4 aimed to characterizes EB formation and subsequent cardiomyocyte differentiation of mouse iPS cells using STLV bioreactor compared to HD. EBs derived from STLV are homogenous in size similar with HD methods. Similar gene expression patterns were observed in both differentiation systems with cardiomyocyte markers by using RT-PCR. Moreover, the percentage of beating cardiomyocytes and the area of cTnT were higher in EBs derived from STLV bioreactor than HD culture. Our study describes, for the first time, a strategy for scalable differentiation of iPS cells into cardiomyocytes in STLV bioreactor culture system.