Pluripotency of bank vole embryonic cells depends on FGF2 and activin A signaling pathways

Abstract

The objective of this study was to investigate the capability of bank vole (Myodes glareolus) embryonic cells to sustain their pluripotent character during in vitro culture, and to determine the optimal conditions for derivation of embryonic stem (ES) cells. We compared the presence of specific pluripotency (Oct4, Ssea1) and differentiation markers (Gata4 -primitive endoderm marker; Cdx2 -trophectoderm marker) in blastocysts and inner cell mass (ICM) outgrowths obtained from blastocysts of bank vole, and two mouse hybrids F1(C57Bl/6xCBA/H) and F1(C57Bl/6x129/Sv), which differ in the permissiveness of giving rise to ES cells. We found that, in contrast to mouse, the expression of pluripotency markers in the cells of bank vole ICM outgrowths is progressively downregulated and rapidly lost by the 4 th day of culture. This correlates with the appearance of cells expressing Gata4 and Cdx2, indicating differentiation towards primitive endoderm and derivatives of trophectoderm, respectively. We have also shown that heterologous cytokine leukaemia inhibitory factor (LIF) in conjunction with either homologous or heterologous feeder layer is unable to delay differentiation and preserve pluripotency of bank vole embryonic cells. Thus, the conditions optimised for mouse do not support the maintenance of bank vole embryonic cells in the undifferentiated state and do not allow for the isolation of the ES cells. Instead, combination of fibroblast growth factor 2 and activin A allows retention of Oct4 expression in bank vole blastocyst outgrowths during 4-day culture, indicating that signaling pathways operating in human, rather than mouse ES cells, might be involved in the process of self-renewal of bank vole embryonic cells.

Generation of Induced Pluripotent Stem Cells from the Prairie Vole

“…The cocktail 3iR [35] contained inhibitors to GSK3ß (3 µM; CHIR99021, Stemgent) and ERK (1 µM; PD0325901 Stemgent), and a TGFß type I receptor antagonist (0.5 µM; A-83-01, Stemgent). Basic FGF (Invitrogen) and activin-A (Invitrogen) were used at 20 ng/mL each [56].…”

Stem Cells and Calcium Signaling

“…The different signaling pathways activated by these ligands, after integration of their individual messages, lead to a final cellular response: to maintain stem cells in their undifferentiated state, to conduct the cells to apoptosis, or cause them to adopt a route of cell differentiation. As an example of ligands that interact with these receptors there are several growth factors (as fibroblast grow factor 2 – FGF-2) and cytokines (as activin and leukemia inhibitory factor – LIF), among other signaling molecules [15]. …”

“…After binding to its receptors some signaling pathways can be activated, including MAPK, STAT and PI3Ks [15]. Recent evidence indicates that members of the protein complex PCG play key role in normal hematopoiesis, and repress the expression of HOX gene in mice [106]; this HOX gene is an important regulator of differentiation pathways.…”