Increasing Proliferation While Controlling Cancer Formation in Induced Multipotent Progenitor

Increasing proliferation while controlling cancer formation in induced multipotent progenitor cells

Reid Thomson

Introduction:

The liver is an extremely important major organ with more than 100 different identified functions from detoxification to storage of vitamin A and glycogen to being the first site of hematopoiesis in the embryo. The liver, aside from its significance to our health and survival, is one of the few organs in adult humans that remains capable of partial regeneration of lost or damaged tissue. The breakthrough discovery of mouse embryonic stem cells in 1981 and the advent of induced pluripotent stem cells (iPSCs) from fibroblast cells in 2006, both yielded glimpses of the plausible treatment options. By increasing the potency of a group of cells it makes them more likely to be able to integrate into different tissues and assume the host tissues’ roles. The success with iPSCs has been limited and thus far they have not reached a thousandth of the level proliferation that adult hepatocytes do when transplanted to liver tissue (Liu, 2011 and Basma, 2009). There is some unknown mechanism or mechanisms blocking iPSC proliferation post-transplant. There are multiple cell types in the liver, so a cell with at least multipotent/progenitor properties would be required for non-specific repair of liver tissue. New processes have allowed the normal process of iPSC from fibroblast to be “cut short”. By altering the reprogramming process, induced multipotent progenitor cells (iMPCs) can be generated that have less overall potential to become differing cell types than iPSCs, but possess all the required potency to form the liver components (Zhu, 2014). The iMPCs can proliferate much more readily than iPSCs in vivo and the time required to generate them from fibroblasts is significantly less than iPSCs (Szabo, 2010).

One concern to be addressed is the total availability of iMPCs due to their low rate of replication in vitro. This can be potentially be solved by increasing the rate at which the culture proliferates by adding genes that increase division or growth rates. In this study we will add an SV40 T antigen encoding gene that will be stably expressed that is flanked by Cre LoxP sites so that before it is transplanted the SV40 T antigen can be removed (Orban, 1992 and Smith, 2011). A retrovirus will be used as the vector for transmission because the desired genes will be directly integrated into the chromosomes. The second concern is that with increased proliferation in cells where their potency has been increased, there is a correlation with increased cancer formation (Ohnishi, 2014). To address this, a ‘kill switch’ is added to cells using an adenovirus as a vector. A caspase-1 encoding gene will be inserted that, when stimulated to dimerize, will induce apoptosis with chemical inducers of dimerization (CID) (Shariat, 2001). The apoptosis induction should be specific to cells with the additional caspase-1 encoded, which should allow any potential cancer formation to be destroyed in a targeted manner (Shariat, 2001).

The ultimate goal is to observe whether or not a proto-oncogene such as SV40 T antigen can increase the rate of iMPC proliferation in the culturing process, be effectively removed with the Cre LoxP system, and then possess a last-resort ‘death switch’ specific to the iMPCs in the case where they do become cancerous.

Experimental Design:

iMPC creation and sequence loading:

First the retrovirus must be loaded with the appropriate genes (Sinn, 2005 and Yu, 1986). The human fibroblast must be transduced using the retrovirus to express OCT4, SOX2, KLF4, and antibiotic resistance as a reporter, as per the process of creating iPSCs (Takahashi, 2006). Controls with equivalent amount of DNA but no encoded genes are created. The other sequence-loading techniques in the experiment are also applied at this stage (see below). The transduced colonies then are plated with selective media for a couple of days to ensure successful transduction of all desired sequences. They are then replated into a medium that supports their reprogramming into endoderm. The medium will contain CHIR99021, which aids in reprogramming fibroblasts, as well as other growth factors such as DLPC (dilauroyl phosphatidylinositol), NaB (histone deacetylase inhibitor), Par (lysine-specific demethylase inhibitor), and RG108 (DNA methyltransferase inhibitor) (Zhu, 2014). SOX17 and FOXA2 can be the positive detection signals to detect if reprogramming to endoderm has succeeded (Zhu, 2014). Adult hepatocytes are grown as well in culture, but are not altered using in any of the same ways aside from equal culture time.

Introduction of SV40 T antigen and LoxP sites:

The human fibroblasts, using retrovirus as a vector, will have the stably expressed SV40 T antigen and its pair of flanking LoxP sites transduced into the genome along with a second antibiotic resistance reporter gene which will also be within the flanking loxP sites. When the colonies are placed on selective media, only those with the appropriate antibiotic resistance, and therefore the SV40 T antigen, will survive. The flanking LoxP sites should allow the specific removal of all intervening sequences (Orban, 1992).

Introduction of genetic ‘kill switch’:

Using adenovirus transduction, Caspase-1 is introduced into the genome with a third antibiotic resistance encoded alongside it. The antibiotic resistance will indicate whether the culture has acquired the caspase-1 gene when placed into selective media.

The different components are introduced separately in different retrovirus instances so that different combinations of induction can be performed as controls. The combinations used would be iMPC with SV40 T antigen and without the ‘kill switch’, iMPC without SV40 T antigen and with the ‘kill switch’, imPC with both SV40T antigen and the ‘kill switch’, and the controls where noncoding nonfunctional DNA is transmitted into the genome by the same vectors.

The iMPCs will then be cultured and amass over several weeks. At this stage, after the desired proliferation has been reached, the proto-oncogene, SV40 T antigen, can be selectively removed using the Cre LoxP system due to LoxP sites flanking the gene. Colonies can be detected by comparing colonies transferred between surfaces with and without the selective media. A subset of mice will not have their SV40 T antigen removed so that any increased oncogenesis can be observed in the transplanted tissue.

Using immune-deficient mice, to prevent immune responses to the human iMPCs, the cells are then transplanted (around 10^6 cells) into the livers where there is some damage. Tyrosinaemia type 1 mice can be used due to the damage caused, but a standardized amount of liver damage can be manually performed as well (Zhu, 2014). Adult hepatocytes are also transplanted to different mice subjects to be used as a comparison for proliferative rates in vivo. The adult hepatocytes are not modified in any way.

After the transplant is complete, allow the mice to recover for a month before recording any follow up data. Human serum albumin is an available quantitative indicator of the amount of cells in a given area due to the fidelity with which it is expressed (Zhu, 2014). By contrasting the amount of human serum albumin produced by the number of cells placed (~10^6) to the amount detected, the amount of proliferation can be inferred. The degree to which the relative increase in human serum albumin exceeds the relative increase produced from adult hepatocytes is an accurate representation of their relative proliferation levels in vivo. Record the human serum albumin levels every month.

Lastly, every month the liver should be checked for cancerous or tumor-like malformations. On any individuals that develop a tumor, the CID should be added and the effects should be observed. Some mice with iMPCs and adult hepatocytes transplanted should be administered with CID to ensure the effects are specific to iMPCs. Apoptosis is expected to occur in all tissues formed of the iMPCs (Shariat, 2001).

Discussion:

The primary objective of this study is to determine whether the proto-oncogene, SV40 T antigen, can be used for increasing the yield of iMPC culturing and whether this gene can be removed effectively before transplantation. The secondary goal is to see whether the potential oncogenic side effects persist despite the Cre LoxP removal of SV40 T antigen, or whether the iMPCs are cancer-prone in general regardless of the SV40 Tantigen. The tertiary goal is to see whether the transduced ‘kill switch’ is effective in selectively destroying the tissue produced with iMPCs to see whether any newly formed tumors can be destroyed.

The expected primary results are that the adult hepatocytes will develop fewer cells than the iMPCs per unit of time when they are developing in cell cultures. The addition of CHIP, other growth factors, and especially the proto-oncogenic gene SV40 T antigen, should causes proliferation in iMPCs to increase far above usual adult hepatocytes proliferation, which is reported to be quite low. Adult hepatocytes are reported to be slower than iPSC generation and iMPCs are reported to be higher than iPSC generation (Zhu, 2014).

The expected secondary results are that the rates of tumor-formation with iMPCs with the SV40T antigen removed via Cre LoxP is equal to that of the adult hepatocytes when transplanted into mice. An increase in cancer formation supports the theory that iMPCs may have a predisposition to being oncogenic and that removal SV40 T antigen is insufficient to prevent this propensity for cancer formation.

The expected tertiary results are that when CID is added to mice with iMPCs that all tissues encoded with caspse-1 will undergo apoptosis and that no other cell lineages will be damaged. If the tissues do not undergo apoptosis then it is possible the gene underwent a mutation, was not transferred into the genome properly, or that another mechanism is blocking the apoptosis-inducing effects of the caspase-1 mechanism. If CID happens to damage other tissues then there is likely an unknown susceptibility in mice tissues proximal to the liver.

Any deviation from these expectations could be due to human error or coincidental error in the protocol. Both the adenovirus and retrovirus steps are sensitive and prone to errors. Retroviral genomic integration can be random and therefore potentially deleterious or even oncogenic. Alternative sequence integration techniques can be attempted if problems arise, such as lentiviral vectors (Sinn, 2005).

Cancer development is associated with genes that increase proliferation of cells. The proto-oncogene in this experimental set-up is only removed from the cell before being transplanted. However, these transplanted cells are derived from a small crop that had many generations of exposure to this proto-oncogene. Therefore it is possible that mutations occurred in regions of the genome not flanked by LoxP sites that induced cancer or at least increased the likelihood of cancer acquisition in the transplant iMPC cells before the SV40 T antigen was removed. Because propensity of acquiring cancer is not detectable and only tumor formation is detectable in the protocol, it is a significant weakness that a high number of mice would have examined for strong significant data on tumor-formation-propensity with iMPC repaired livers. Because there are many different conditions proposed in this experiment and the potentially-low rate of tumor formation, the number of mice examined would be very high.

Other proliferation-increasing proto-oncogenes could be inserted into the cell cultures to increase proliferation in lieu of SV40 T antigen. The partial success or partial failure of the experiment could be due to effects specific to SV40 T antigen. All liver damage that the mice have is unique and could therefore have variable results due to the complexity of in vivo systems.

References

Basma, H. et al. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 136, 990–999 (2009)

Bi, Y., He, Y., Huang, J., Su, Y., Zhu, G., Wang, Y., . . . Tang, N. (2014). Functional characteristics of reversibly immortalized hepatic progenitor cells derived from mouse embryonic liver. Cellular Physiology and Biochemistry, 34(4), 1318-1338. doi:10.1159/000366340

Liu, H., Kim, Y., Sharkis, S., Marchionni, L. & Jang, Y. Y. In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins. Sci. Transl. Med. 3, 82ra39 (2011)

Luo J, Deng ZL, Luo X, Tang N, Song WX, Chen J, Sharff KA, Luu HH, Haydon RC, Kinzler KW, Vogelstein B, He TC: A protocol for rapid generation of recombinant adenoviruses using the adeasy system. Nat Protoc 2007;2:1236-1247.

Miyajima, A., Tanaka, M., & Itoh, T. (2014). Stem/Progenitor cells in liver development, homeostasis, regeneration, and reprogramming. Cell Stem Cell, 14(5), 561-574. doi:10.1016/j.stem.2014.04.010

Ohnishi, K. et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 156, 663–677 (2014)

ORBAN, PC, D. CHUI, and JD MARTH. "Tissue-Specific and Site-Specific Dna Recombination in Transgenic Mice." Proceedings of the National Academy of Sciences of the United States of America 89.15 (1992): 6861-5. Print.

Shariat, S., Desai, S., Song, W., Khan, T., Zhao, J., Nguyen, C., . . . Slawin, K. (2001). Adenovirus-mediated transfer of inducible caspases: A novel "death switch" gene therapeutic approach to prostate cancer. Cancer Research, 61(6), 2562-2571.

Sinn, PL, SL Sauter, and PL McCray. "Gene Therapy Progress and Prospects: Development of Improved Lentiviral and Retroviral Vectors - Design, Biosafety, and Production." Gene therapy 12.14 (2005): 1089-98. Print.