Revision as of 17:01, 17 October 2014

“Transgenic artificial β-cells”

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A cell reprogramming approach to regenerate pancreasβ-cells

Background

Wouldn’t it be awesome if we could generate more β-cells in vivo which are lacking in the patients with type I diabetes ? Is it possible that we could generate some “artificial β-cells” within the body of the patients? If this is possible, these new “artificial β-cells” would serve as new “langerhans' islet” within the body of patients, which may potentially have the ability to ameliorate hyperglycaemia in type I diabetes patients.

The idea of cellular reprogramming or lineage reprogramming is not novel and has been shown to be theoretically doable by different studies. [1,2] During the past decade, several studies showed that adult skin cells could be reprogrammed into induced pluripotent stem (iPS) cells by a combination of key transcription factors. [3,4]

Figure 1. An illustration of the concept of cell reprogramming by introducing key transcription factors. When key transcription factors of A cell line are introduced into B cell line, it is possible that B cell line could exhibit certain properties that would otherwise only exists in A cell line.

To reprogram adult cells into β-cells, key transcription factors that are expressed along the process islet differentiation and formation need to be identified. Fortunately, this important knowledge has been collected by previous studies. Two transcription factors, PDX-1 and MafA, have been identified to be very crucial for the development and normal function of β-cells, including the expression and secretion of insulin. [5,6,7] And a pioneering study has shown that the reprogramming of pancreatic exocrine cells to (induced) β-cells using these transcription factor feasible. [8]

In our project, we tried to use the adeno-associated virus (AAV), as the vehicle to deliver our transcription factors to 293T cells. We tried to use inverted phase contrast microscope to monitor the morphological change of the cells and used western blot to detect the insulin expression induced by exogenous transcription factors.

Experiment Design

We got the mouse PDX-1 and MafA gene from plasmids provided by Addgene (Plasmid 19411: pAd PdxI-I-nGFP, Plasmid 19412: pAd MafA-I-nGFP). We cloned these genes into the pAAV-MCS plasmid so that they are able to be delivered by adeno-associated virus(AAV), as shown in Figure 2a.

We also tried to transfect the pAd PdxI-I-nGFP and pAd MafA-I-nGFP plasmids directly into 293T cells and used western blot to detect the insulin expression, as shown in Figure 2b. This may serve as a short cut to determine whether our idea of cell reprogramming would work or not.

Results and Discussion

Due to the late arrival of insulin antibody, we are not able to show any results of the western blot from the transfected 293T cells. But we have tested the efficiency of the antibody using the non-transfected cells and the purified insulin proteins. From Figure 3., we can see the background of this antibody is low, which means that it has a good specificity. And comparing the two lanes on the right, insulin A chain seems to be better detected when reducing agent is added, probably because the breakage of the disulfide bond released and exposed A chain.

Figure 2. Test of the insulin antibody. Non-transfected 293T , 3T3 cell lysate and purified insulin protein were subjected to western blot using insulin A chain antibody. DTT was used as a reducing agent.

Reference

[1] Hochedlinger, Konrad, and Rudolf Jaenisch. "Nuclear reprogramming and pluripotency." Nature 441.7097 (2006): 1061-1067.
[2] Orkin, Stuart H., and Leonard I. Zon. "Hematopoiesis: an evolving paradigm for stem cell biology." Cell 132.4 (2008): 631-644.
[3] Takahashi, Kazutoshi, and Shinya Yamanaka. "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors."Cell 126.4 (2006): 663-676.
[4] Takahashi, Kazutoshi, et al. "Induction of pluripotent stem cells from adult human fibroblasts by defined factors." cell 131.5 (2007): 861-872.
[5] Kataoka, Kohsuke, et al. "MafA is a glucose-regulated and pancreatic β-cell-specific transcriptional activator for the insulin gene." Journal of Biological Chemistry 277.51 (2002): 49903-49910.
[6] Kaneto, Hideaki, et al. "A crucial role of MafA as a novel therapeutic target for diabetes." Journal of Biological Chemistry 280.15 (2005): 15047-15052.
[7] Ferber, Sarah, et al. "Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia." Nature medicine 6.5 (2000): 568-572.
[8] Zhou, Qiao, et al. "In vivo reprogramming of adult pancreatic exocrine cells to &bgr;-cells." nature 455.7213 (2008): 627-632.