Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous
The pursuit of the derivation of pluripotent stem cells (PSC) from livestock species has been one of the major goals for the agribiotech industry. Although some reports demonstrate that it is possible to maintain primary isolates of Embryonic Stem Cell (ECS) in cattle for extended periods of time, a truly, well-characterized cell line is yet to be obtained [
Subsequent reports have demonstrated that it is feasible to obtain putative biPSC [
One of the principal challenges with generation of iPSCs, especially for species other than mouse and human, is the cues necessary for the culture system to maintain the cells after being in a pluripotent, autorenewal state.
Culture conditions affect the pluripotential of PSC and could even revert cells to a more primitive and undifferentiated state [
Furthermore, it is also possible to inhibit ERK pharmacologically. Its inhibition sustains pluripotency and self-renewal even in the absence of LIF, feeder layers, and serum [
Finally, 5-azacytidine (5-AzaC) and 5-aza-2′-deoxycytidine (Decitabine) are irreversible inhibitors of DNA methyl transferases (DNMTs) that, at low doses, induce hypomethylation, avoiding cytotoxicity [
To induce pluripotency in adult bovine fibroblasts, we used ectopic expression of Yamanaka’s factors: Oct4, SOX2, KLF4, and c-MYC, in addition to NANOG; the latter is required for stable reprogramming in bovine species [
Cell lines at passages 24–26 were cultured under defined conditions corresponding to the eight treatments summarized in Table
Summary of the different treatments used to evaluate the effect of pathway inhibition on gene expression profiles. 5-Azacytidine (5-AzaC), glycogen synthase kinase-3 (GSK-3), extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and DNA methyltransferases (DNMTs)
GSK3 | ERK/MAPK | DNMT (Di) | GSK3 + ERK/MAPK (2i) | GSK3 + DNMT | ERK/MAPK + DNMT | 2i + Di | Control | |
---|---|---|---|---|---|---|---|---|
Bio | 1 |
|
|
1 |
1 |
|
1 |
|
SC1 |
|
0.5 |
|
0.5 |
|
0.5 |
0.5 |
|
5-AzaC |
|
|
5 |
|
5 |
5 |
0.5 |
|
For immunostaining, cells were fixed with 100% ice-cold ethanol for 10 min and then stained as described previously [
Standard RT-PCR reactions were performed using GoTaq Green Master Mix. qPCR reactions were performed using Power SYBR Green PCR Master Mix (Invitrogen). Analyses were performed in duplicate (technical replicate). mRNA relative fold change values were calculated as the
After 21 days in culture colonies appeared (Figures
Isolation of biPSC colonies. (a) Initial morphology changes in the original plate were noticeable after two weeks of culture. (b) Well-defined colonies growing on the fibroblast-like background. Large colonies showing a dome shaped (c-d) or flat morphology (e-f).
All noticeable colonies observed were expanded. Morphologically, all colonies obtained fell in one of two categories, based on their morphology, both easily identifiable from the fibroblast-like background. The first is described as large colonies dome shaped (Figures
The second category appears more close to what has been described for human ESC and murine EpiSC. They were flat well-spread colonies with regular edges and without signs of differentiation. Boundaries between cells were not easily identifiable (Figures
From the colonies isolated, one clone was selected randomly, from each individual line, for further characterization, for a total count of three cell lines.
First, the expression of the pluripotent markers OCT4 and NANOG was confirmed by immunofluorescence analysis of all colonies. The colonies were also positives for SSEA-1 and SSEA-4 along with TRA-1-60. The expressions of SSEA-3 and TRA-1-81 were also evaluated and found to be absent (Figure
Pluripotency markers immunostaining at early passage. Cells at passage 12 were positive for the nuclear markers OCT4 and NANOG and the surface markers SSEA-1, SSEA-4, and TRA-1-60. None of the colonies were positive for SSEA-3 or TRA-1-81.
The endogenous and exogenous expression of the core set of reprogramming factors
Endogenous and exogenous gene expression before and after differentiation. (a) Gene expression by RT-PCR of the exogenous (exo) and endogenous (endo) genes ONSKcM used during reprogramming and REX-1. Plasmids are used as control. (b) Cells were differentiated by EBs and a set of different genes representative of all three germ cell layers were evaluated by RT-PCR.
Differentiating embryoid bodies from the three cell lines was evaluated for the expression of the genes
Finally, the
biPSC differentiation
Next, we evaluated different culture conditions able to sustain the endogenous expression of factors related to pluripotency. Previously it has been described that the inhibition of the different pathways increases the rate of isolation of ESC in mouse and human. In this vein, two different cell lines at passages 25 and 28 (
There were no apparent changes in terms of gross morphology between treatments. However, the expression of the ESC markers was upregulated with two inhibitors 2i and 2i combined with DNMTs inhibition (Di). In the case of
Effect of inhibitors on the expression endogenous genes. REX (a), NANOG (b), OCT4 (c), SOX2 (d), c-Myc (e), and KLF4 (f) genes. Results expressed as the mean of fold change
Among the factors used for reprogramming,
A common practice during the isolation and generation of PSC from species different to human and mouse is the combined use of two growth factors, bFGB and LIF, in order to provide both signals required to maintain the naïve and primed states represented by mouse and human ESC, respectively [
Cells were maintained for up to eight passages under either LIF or bFGF stimulus. The combination of both LIF and bFGF was used as a control. After eight passages cells did not show any apparent changes in gross morphology with any differences observed when compared with the control (data no showed).
However, when the levels of RNA expression of the endogenous and exogenous
Effect of LIF and bFGF on endogenous genes: REX-1 (a), NANOG (b), OCT4 (c), and SOX2 (d) genes. Results expressed as the mean of fold change
The second transcription factor affected by the use of different growth factors during culture was endogenous
The effect of LIF on
We demonstrated that, even after induced cell reprogramming, the gene expression profile is malleable by modification of the cell culture conditions. Using simultaneous inhibition of GSK-3, ERK1, and Raps-GAP it is possible to increase the expression of at least four genes of the core transcription complex responsible for pluripotency. Levels of
Using similar inhibitors it has been widely reported that both reprogramming and ESC isolation are augmented with the highest RE and isolation success, respectively [
Finally, we evaluated the use of LIF and bFGF on long-term culture and gene expression of biPSC. These two factors are believed to sustain one or the other of the two characteristic states: naïve and primed, respectively. Under LIF stimulus alone the levels of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors want to thank Mr. David Fajardo for his help in organizing the figures. Luis Fernando Malaver-Ortega was a recipient of an Australian Postgraduate Award (APA) and was supported by a DAIRY FUTURES CRC scholarship. The Victorian Government’s Operational Infrastructure Support Program supported this research.