Establishment of Cardiac Lineage from Rabbit Embryonic and Induced Pluripotent Stem Cells

Abstract

Rabbit is a promising model for studying myocardial infarction as several physiological and pathological conditions are resemble to humans. However, the information on establishment of rabbit pluripotent stem cells (PSC) and differentiation potential especially cardiac differentiation in this species has been limited. This research aimed at establishing and examining in vitro cardiogenesis of rabbit embryonic stem cells (ESC) and rabbit induced pluripotent stem cells (iPSC). In experiment 1, four cell lines of ESC derived from blastocyst stage embryos were cultured and characterized for their pluripotency in terms of gene and immunohistochemistry. They exhibited normal karyotypes (n=44). Embryoid body (EB) formation was conducted to study in vitro differentiation potential. The EB formation induced cell differentiation into three germ layers including mesodermal lineages (Brachyury (BT)) and cardiac progenitor (NKX2.5 and GATA4). Two candidate housekeeping genes (RPL13a and GAPDH) were examined for its stability during 7 days of culture. The results indicated that these two genes were similar stability during EB culture (P>0.05). BT spontaneously upregulated to the highest level on day 2 of EB culture compared with undifferentiated ESC and EB on day 3 (P<0.05). BMP-4 significantly upregulated mesoderm marker BT on day 2 and cardiac progenitor marker NKX2.5 on day 3 and 5 of differentiation comparing with control group (without BMP-4) (P<0.05). It enhanced a number of NKX2.5, FLK1 and cardiac troponin-T positive cells on day 5 and 7, respectively (P<0.05). In conclusion, rabbit ESC retained capability of cardiac differentiation, while BMP-4 significantly promoted cardiac differentiation in terms of gene and protein expressions (P<0.05). In experiment 2, three cell lines rabbit iPSC were generated by reprogramming rabbit embryonic fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). All iPSC cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT3/4 and SSEA-4) essentially described for pluripotency. They demonstrated in vitro differentiation potential by forming embryoid body (EB) and gene and protein expression related three-germ layer. However, ability of individual cell lines and cell numbers at seeding markedly influenced on EB formation. The cell density at 20,000 cells per EB was selected for cardiac differentiation. The differentiated cells expressed cardiac progenitor marker Flk1 (51±1.48%) on day 5. Cardiac troponin-T protein (10.29±1.37%) and other cardiac marker genes (cardiac ryanodine receptors (RyR2), α-actinin and PECAM1) were expressed on day 14. Moreover, cardiac beating areas were observed on day 11 of culture. This study concluded that rabbit iPSC remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted. In conclusion, these studies demonstrated that rabbit iPSC and ESC have capacity to differentiate toward cardiac fate via 3D-structure embryoid body. BMP-4 enhanced gene expressions of cardiac mesoderm BT and cardiac progenitor NKX2.5. BMP-4 treated EB upregulated cardiac progenitor marker positive cells (Flk1) and cardiac marker troponin-T positive when compared with controls. These findings highlight the possibility to generate mature cardiomyocytes from rabbit iPSC and ESC for further use. However, other factors of cardiac differentiation should be further examined in order to improve its efficiency.