The combination of living tissue and a synthetic material holds great promise in the areas of tissue engineering and delivery of therapeutics. One of the challenges associated with this emerging field is controlling the behavior of the cells when they are no longer receiving the nutrients and signals that are present under physiological conditions. This study seeks to identify what stresses are placed on tissue in a polymeric environment focusing on encapsulated pancreatic islets.
Encapsulation of pancreatic islets has long been a strategy for treatment of diabetes[1]. A passive encapsulation system acts as a barrier to allow passage of nutrients and waste products, as well as glucose and insulin, while excluding immune cells. A successful barrier system will provide the encapsulated tissue with the nutrients they need while shielding them from the immune system allowing the cells to secrete insulin in response to elevated glucose concentrations. Despite the concerted efforts of numerous research groups, a viable encapsulation system leading to prolonged insulin independence has remained elusive.
This failure is the result of the large number of factors leading to loss of function for the transplanted tissue. Modifications to basic encapsulation systems that seek to address many of these factors individually have shown short-term success but failed to produce a clinically successful system for treatment of human patients. The most obvious factor is immune rejection, but studies comparing survival of encapsulated auto- and allograft tissue showed similar survival time indicating the role of other stresses in loss of function[2]. Some of these stresses include: insufficient diffusion of nutrients and waste products through the capsule resulting in hypoxia and oxidative stress, lack of biocompatibility of the capsule material and the lack of sufficient cell-cell and cell-ECM (extracellular matrix) interactions within the capsule.
EXPERIMENTAL PLAN
The central goal of the work proposed here is to improve the long term function and viability of encapsulated islet cells. The cells of particular interest within the islets are the beta cells which are responsible for the release of insulin. The intracellular signaling pathways that translate extracellular stresses into cell death (with particular attention given to anoikis) will be targeted for genetic modification to block these processes and in turn extend the viability and function of the graft tissue. To observe the role of hypoxia on cell survival, a hypoxia marker was developed to identify cells which have initiated a response to low oxygen.
To test the importance of cell-ECM interactions, islets were infected to overexpress integrin-linked kinase (ILK) and/or Bcl-2 prior to encapsulation in a poly(ethylene glycol) (PEG) matrix. We hypothesized that ILK (an integrin beta1 binding protein capable of phosphorylating the cytoplasmic subunit of b1 integrins) overexpression would prevent beta cell anoikis caused by the lack of physiological cell-ECM interactions within the PEG capsule. Bcl-2 (an integral protein found in the endoplasmic reticulum, nuclear envelope and mitochondria) is able to block apoptosis stimulated by a range of different stresses. Bcl-2 overexpression should also block anoikis but is less specific than ILK in this regard. Previous work using Bcl-2 overexpression in transplanted islets has shown promising results[3].
The encapsulation process isolates the islets from the blood supply which forces oxygen to diffuse a longer distance to reach the cells that compose each islet. To monitor whether encapsulated cells are receiving sufficient oxygen, an adenoviral marker was generated where a red fluorescent protein will be produced when a low oxygen stress response is initiated. Infected islets were encapsulated and exposed to different levels of oxygen to test the efficacy of this marker system.
RESULTS AND DISCUSSION
Initial studies were performed on a cell line (MIN6) to mimic the beta cells present in an islet. Previous work established that dispersed MIN6 cells show low viability in PEG gels after 5 days in culture. MIN6 cells infected to overexpress ILK, Bcl-2 or both showed considerably improved viability under these conditions compared to control transfected cells. Control cells showed 42 ± 9% viability which was similar to the results found for unmodified MIN6 cells encapsulated for 5 days and significantly lower than the experimental groups. After 5 days in the PEG capsule, Bcl-2-infected cells were 76 ± 3% viable, ILK-infected MIN6 cells were 95 ± 3% viable and cells infected with both ILK and Bcl-2 were 88 ± 5% viable. Statistical evaluation showed significant difference between the control cells and all three groups receiving Bcl-2 or ILK (p-values <0.001). The results suggest that anoikis is a cause of cell death observed in encapsulated dispersed MIN6 cells, and that survival may be improved by activating the pathway triggered by interactions of beta1 integrins with the ECM proteins lacking in the PEG hydrogel. We are, in effect, “hotwiring” this pathway to make the cells think they are interacting with the absent ECM matrix.
Similar studies were conducted with encapsulated islets obtained from Balb/c mice. When using the islets, insulin release in response to glucose stimulation was measured instead of viability as function a better metric than viability. Studies have shown a higher release of insulin after 4 weeks of encapsulation for Bcl-2 and ILK-infected islets compared to islets infected with a control virus. Compared to initial insulin release (release on day 1 set as 100%), Bcl-2 islets released 40%, ILK islets released 43% and control islets released 0% after 4 weeks of encapsulation. While 40 and 43% are not ideal values, they are certainly an improvement over the control cells and support the data obtained in the earlier studies.
Testing of the hypoxia marker showed that islets infected with the marker virus exhibited a fluorescent signal after 24 hours in a 1% oxygen environment. Control cells in this hypoxic environment showed no signal as did infected cells placed in a 21% oxygen environment. The intensity of the signal can also be controlled by the amount of virus used for the infection. This system will be used to study the impact of islet size and density as well as implantation site on oxygen diffusion to encapsulated islets.
CONCLUSIONS
The working hypothesis for these studies was that islets encapsulated in PEG networks were exhibiting loss of function due to apoptosis triggered by the absence of proper cell-ECM interactions and perhaps insufficient oxygen supply. Studies using the MIN6 beta cell line have shown that overexpression of signal transduction proteins involved in pro-survival pathways following interactions with ECM proteins can prolong survival in PEG gels. ILK seems to be a promising target for extending function of cell encapsulation systems for treatment of diabetes as well as other disease states. The decrease in insulin release after 4 weeks even in the modified cells indicates that other stresses are present on these cells and has motivated the development of the hypoxia marker. It is hoped that a combinatorial approach that seeks to address the range of stresses placed on encapsulated tissue can lead to longer term function. Since the challenges associated with islet encapsulation are common to many applications of cell-seeded biomaterials, we hope that these results will also be useful in other therapeutic delivery and tissue engineering applications.
REFERENCES:
[1] Lim F and Sun AM. Science; 210, 908 (1980).
[2] De Vos P et al. Diabetologia; 40, 262 (1997).
[3] Contreras JL et al. Transplantation; 71, 1015 (2001).
ACKNOWLEDGEMENTS
The viruses used in these studies were generated with support from the Howard Hughes Medical Institute. The HRE sequences used in the creation of the HRE-DsRed virus were generously donated by Dr Rachel Cowen. We would also like to thank Dr Stephen Langer for his assistance with the construction of the recombinant adenoviruses and Francisco Ramirez-Victorino and Philip Pratt for their assistance with isolating the Balb/c islets for these studies.