Objective: Our aims were to derivate horse ES cell lines by repeated passage of ICM cells and assess HLIF and bFGF effects on maintenance of their pluripotenciesMaterials and Methods: A basic medium was prepared for the culture of ICMs and horse embryonic stem cells using KODMEM supplemented with 10% FBS, 0.1mM Non-essential Amino Acids, 2mM L-glutamine, 1% Insulin-Transferrin-Selenium, 100mg/ml of streptomycin, 100 units/ml of penicillin and 0.1mM 2b- mercaptoethanol on γ-irradiated MEFs. ICM cells were recovered mechanically from day-7 blastocysts and cultured in basic medium on feeder cells. The culture medium was changed every day after first passage and the passage was performed by mechanical dissociation with a needle every 6-8 days until passage 15. The putative horse ESCs were cultured in the basic medium supplemented with human leukemia inhibitory factor (HLIF 40ng/ml) only, basic fibroblastic growth factor (bFGF 4ng/ml) only, or bFGF plus HLIF with MEF feeder cells. Colony morphology was evaluated, continuously. Finally, for determination of cell pluripotency, the hESCs were analyzed for markers of pluripotency. Immunostaning of the putative horse ESCs was done for Oct4, SSEA-1, GCTM-2, TRA1-60 and TRA1-81.Results: Derived ICMs, from day-7 blastocysts, were cultured with basic medium on MEF feeder cell monolayer. They grew as flat colonies including cells with different morphology (P0). Different morphological cells were transferred for next passages separately (P1). Cells in basic medium only, and basic medium added LIF could keep colony form similar to each other until passage 15. Although the cells in basic medium containing bFGF could produce some colonies in early passages, they could not maintain their morphology in later passages. Those colonies which were plated in basic medium added HLIF and bFGF did not grow after a few passages. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct4, SSEA-1, GCTM-2, TRA1- 60 and TRA1-81. These results indicated that those cell colonies were not completely purified embryonic stem cells, but some of them were still pluripotent.Conclusion: Our study show the primary horse ESCs are able to self-renew when they are cultured in the basic medium on γ-irradiated MEFs. These results also indicated that cell colonies were not completely purified embryonic stem cells, but some of them were still pluripotent. ESC-like cell morphology of horse putative ESCs were well maintained in the basic medium supplemented with or without HLIF. This result suggests that HLIF is neither prerequisite nor negative for maintenance of horse ESCs; bFGF seem to be negative for maintenance of horse ECSs. The combination of LIF and bFGF is unable to improve the culture condition. Thus, the new factors need to be investigated further to maintain horse ESCs in purified population.

Background: Leukemia Inhibitory Factor (LIF) is largely used in stem cell researches for the maintenance of Embryonic Stem Cells(ESCs) in a pluripotent state. However, the relatively high costs of LIF is a potential limit of such researches. Objectives: The aim of this study was prokaryotic expression and purification of the recombinant hexahistidine-tagged human LIF(His6-HLIF) fusion protein and assessment of its ability to maintain a pluripotent or undifferentiated state of ESCs. Methods: Encoding the DNA sequence of mature HLIF was codon optimized for expression in Escherichia coli and chemically synthesizedand cloned in the expression vector pET-28a (+). Immobilized Metal Affinity Chromatography (IMAC) was performed topurify the recombinant His6-HLIF. Then, His6-HLIF was tested for its ability to maintain mESC by comparison with commercial LIF asa control. Results: The yield for the recombinant His6-HLIF was assessed to be approximately 1. 7 mg from one liter of culture. There were nostatistically significant differences in expression of two pluripotency gene markers, oct-4 and Nanog, between mESCs treated withHis6-HLIF and those with commercial HLIF (P = 0. 09 and P = 0. 13, respectively). Besides, morphological characteristics (round cellularmorphology) were similar between them. Conclusions: Collectively, the findings showed that the ability of the recombinant His6-HLIF protein in maintaining pluripotentstate of ESCs was comparable to commercial HLIF, providing evidence that the presence of the N-terminal hexahistidin tag does notinfluence biological activities of HLIF.

Objective: Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (HLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state.Materials and Methods: In this experimental study, we cloned HLIF into the pENTR-D/TOPO entry vector by a TOPO reaction. Next, HLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-HLIF fusion protein.Results: This straightforward method produced a biologically active recombinant HLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble HLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro.Conclusion: Our results showed no significant differences in function between laboratory produced and commercialized HLIF.

Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (HLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus HLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse ESCs were well maintained in the basic medium supplemented with or without HLIF. This result suggested that HLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs and the combination of HLIF and bFGF was unable to improve the culture condition.