Creating induced pluripotent cells

The science of cultured meat is occupied with misunderstanding and over estimation. The most essential building blocks, the cells from which tissue can be made, are missing or are not satisfying the criteria for sustainable expansion. Currently to obtain animal cells from farmed species, one has to visit a slaughterhouse or step on a fishing boat to isolate these cells themselves. While some methods and protocols from human and mouse cell culture may apply to agricultural cellular materials, it has become clear that most are not. This is evidenced by the fact that established protocols for creating human and mouse embryonic stem cells have not succeeded in establishing ungulate embryonic stem cell lines (Gandolfi, 2012). The most important criteria for such a cell line includes: immortality, high proliferation ability, surface independence, serum independence, and tissue-forming ability.

Induced pluripotent stem cells (iPSCs) are generated by inducing the expression of pluripotency-related genes such as Oct4, Sox2, Klf4, and c-Myc in differentiated somatic cells (Takahashi and Yamanaka, 2006). Since the original report in mice (Takahashi et al., 2007), iPSCs have been generated from humans and several other species.

From a biomedical perspective, iPSCs offer several advantages over embryonic stem cells (ESCs) because they can be generated without using embryos. iPSCs can proliferate without limit and yet maintain the potential to generate derivatives of all three germ layers. These properties make them interesting candidates for cellular agriculture. Furthermore, optimization of medium composition has been developed and surface independent growth (Amit, 2011) and serum free growth medium is available (Chen, Guokai 2011). Today, the creation of iPSC can be achieved without the use of a viral component that integrates into the host genome. Instead episomal vectors containing the reprogramming factors are used.

The episomal vectors have the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone that delivers the reprogramming genes, Oct4, Sox2, Nanog, Lin28, L-Myc, Klf4, and SV40LT. This system has been demonstrated successful and uses a defined medium. High transfection efficiency due to oriP/EBNA-1-mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection. In addition, the removal of episomal vectors from the iPSCs can be accomplished by cell culture without any additional manipulation, due to the silencing of the viral promoter driving EBNA-1 expression and the loss of the episomes at a rate of ~5% per cell cycle due to defects in vector synthesis and partitioning (Fontes, 2013).

The most commonly used method is the transfer of naked DNA into cells via liposome-based methods or via electroporation. Electroporation is an instrument-based method that uses high voltage electric shock to introduce DNA into cells for transient or stable expression. The high voltage disrupts areas of the phospholipid bilayer of the cell membrane resulting in the formation of temporary aqueous pores. The electric potential across the membrane simultaneously rises allowing the charged DNA molecule to be driven across the membrane through the pores in a directional manner. As DNA  flows through the pores, the cell membrane discharges, the phospholipid bilayer reassemble to quickly close the temporary pores. Neon™ transfection system is a next generation electroporation-based transfection system. It differs from the traditional electroporation systems in using a pipette tip instead of the standard cuvette as the electroporation chamber. The electrode in the pipette tip is designed to produce a uniform electric  filed to result in cell toxicity with higher transfection efficiency of diverse cell types (Ying   Liu, 2013).

Using episomal reprogramming, we are aiming to develop a iPS cell bank that contains serum and feeder free iPS cells from a broad range of animals that are used as food like: cow, pig, chicken, tuna, salmon, lobster.

Figure 1. Schematic model of integration-free human iPSC generation. Episomal plasmids carrying reprogramming factors are transfected into cells and. Resistant selection is kept for a week. After 14 days, iPSC colonies are visible. Individual colonies are expanded and ready for characterization. At this time, no evidence of plasmid integration is found.

References

Gandolfi, F., Pennarossa, G., Maffei, S., & Brevini, T. A. L. (2012). Why is it so difficult to derive pluripotent stem cells in domestic ungulates?. Reproduction in Domestic Animals, 47(s5), 11-17.

Liu, Y., Judd, K., & Lakshmipathy, U. (2013). Stable transfection using episomal vectors to create modified human embryonic stem cells. Pluripotent Stem Cells: Methods and Protocols, 263-272.

Yamanaka, S., & Takahashi, K. (2006). Induction of pluripotent stem cells from mouse fibroblast cultures. Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 51(15), 2346.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. cell, 131(5), 861-872.

Amit, M., Laevsky, I., Miropolsky, Y., Shariki, K., Peri, M., & Itskovitz-Eldor, J. (2011). Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nature protocols, 6(5), 572-579.

Chen, G., Gulbranson, D. R., Hou, Z., Bolin, J. M., Ruotti, V., Probasco, M. D., … & Wagner, R. (2011). Chemically defined conditions for human iPSC derivation and culture. Nature methods, 8(5), 424-429.

Fontes, A., MacArthur, C. C., Lieu, P. T., & Vemuri, M. C. (2013). Generation of human-induced pluripotent stem cells (hiPSCs) using episomal vectors on defined Essential 8™ Medium conditions. Pluripotent Stem Cells: Methods and Protocols, 57-72.

Liu, Y., Judd, K., & Lakshmipathy, U. (2013). Stable transfection using episomal vectors to create modified human embryonic stem cells. Pluripotent Stem Cells: Methods and Protocols, 263-272.