The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells

Reference:

Mao, G, Chen G, Bai H, et al. "The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells." Biomaterials, 2008: 1706-1714.

Link: http://www.ncbi.nlm.nih.gov/pubmed/19135250

Introduction/Motivation:
Diabetes mellitis Type 1 affects million of people world wide. Diabetes Type 1 is the result of autoimmune destruction of beta cells which produce insulin within the pancreas. Insulin, along with glucagon, is a hormone that maintains the body's blood glucose levels. Two treatments for diabetes Type 1 which have shown promise is beta cell replacement therapy via islet transplantation and the use of xenografts. However, there is a lack of suitable donor tissues for beta cell replacement and the use of xenografts comes along with the risk of zoonotic infections. To overcome these issues the use of islet cells derived from adult or embryonic stem (ES) cells has been explored. Human ES cells are self-renewing and able to differentiate into all three germ layers in vitro. It has been reported in previous studies that mouse and human ES cells can spontaneously or be induced to differentiate into islet-like cells in vitro and that transplantation of these cells into streptozotocin-induced diabetic mice could correct their hyperglycaemia.

In the paper by Mao G, et al they attempt to improve upon the protocol described by Lumelsky et al. to differentiate human embryonic stem cells into islet-like cells by using exendin-4 instead of nicotinamide. Both exendin-4 and nicotinamide stimulate the differentiation and proliferation of beta cells. In the 5 stage protocol previously reported by Lumelsky et al. they were successful in inducing the differentiation of insulin-producing cells from human ES cells by selection of nestin-positive cells. The kidney capsule and spleen have been two common areas used for cell transplantation, however these sites may be unsuitable for clinical application. The study conducted by Mao G, et al. also wanted to see whether the use of a PLGA scaffold seeded with islet-like cells would prove to be a better vehicle for transplantation of the islet-like cells versus transplantation under the kidney capsule.

Summary of Methods & Results:
Undifferentiated human ES cells were grown on a feeder layer of mouse embryonic fibroblasts. Upon embryoid body formation the cells were moved to a serum-free medium (ITSF) in which many other cell types die to increase the proportion of nestin positive cells. Islet-like cell clusters appeared at stage 5 of the protocol after bFGF was withdrawn and exendin-4 added to promote beta cell differentiation.

Immunoflurescence staining showed that the cells coexpressed insulin and c-peptide ( a byproduct created when insulin is produced). RT-PCR was used to confirm that pacreatic beta cell specific genes were present. Double immunofluorescence staining showed that the islet-like cells coexpressed several pancreatic markers including: insulin, nestin, glucagon, PDX-1, c-peptide, somatostatin and pancreatic polypeptide. The expression of pancreatic markers within the islet-like cells suggest that these cells resemble immature pancreatic endocrine cells.

The insulin levels of the islet-like cells in vitro were measured using a chemiluminescent immunoassay in which the amounts were normalized by measuring total intracellular protein using BCA protein assay system. To determine if the cell clusters use physiological signaling pathways to regulate insulin release the effect of several agonists and an antagonist of insulin secretion were examined. Figure 3 shows the amount of insulin secreted from the islet-like cells under high glucose conditions and in the presence of agonist and antagonists of insulin secretion.

Figure 3. A)The effect of KCL, tolbutamide, IBMX and nifedipine on insulin secretion in response to 25mM gluclose. B) The effect of KCL on insulin secretion.

The amount of insulin released form the islet-like cells increased significantly with the addition of KCL under high glucose conditions. However, there was no significant effect on insulin secretion from the islet-like cells in the presence of any of the other agonist or antagonist or with the addition of KCL under low glucose conditions.

The blood glucose levels were measured in control mice (diabetic mice which did not receive cells), cell transplanted mice (mice who received islet-like cell injections under the kidney capsule) and cell-scaffold complex transplanted mice and the results shown in Figure 5 below.

Figure 5. Effects on blood glucose levels by transplanted islet-like cells in diabetic mice at different times.

Mice which received islet-like cells transplanted under the kidney capsule had lower 6 h fasted blood glucose levels. By comparing the 6 h fasted glucose levels in the control mice and cell transplanted mice there is some improvement in glucose levels in the cell transplanted mice. However, no improvement in randomly fed mice blood glucose levels is shown in the above results except only during the 6th and 7th week in which there is a decrease in glucose levels. In mice who received the PLGA scaffold seeded with islet-like cells there is an improvement in the 6 h fasted blood glucose levels in comparison to the control mice, however the randomly fed levels in the control mice and the cell-scaffold transplanted mice remained similar. After graft removal the 6 h fasted blood glucose levels returned to hyperglycemic conditions. Mao G, et al. concluded from these results that scaffolds seeded with islet-like cells derived from human ES cells could reverse fasted hyperglycemia in diabetic mice.

Shortfalls & Comments:
One issue with this paper is that in Figure 3 they do not show any control for insulin secretion without glucose stimulation. It would be interesting to compare the amount of insulin being secreted from the cells with no stimulation. Additionally, in the same figure they obtained results which showed that insulin secretion was slightly greater when the cells were in the presence of the antagonist nifedipine. Nifedipine is a blocker of L-type calcium channels which are present in beta cells, so one would expect that insulin secretion would be lower with the addition of nifedipine. The study conducted by Lumelskly et al. from which this paper adopted its protocol to differentiate human ES cells into islet-like cells reported results that showed inhibition of insulin secretion with the addition of nifedipine in the presence of high glucose.

Furthermore, the islet-like cells do not behave in a glucose dependent manner. In figure 3b it shows that basal insulin secretion is greater when stimulated with low glucose concentration and lower when stimulated by high glucose concentration indicating that these cells do not respond to insulin like normal islet like cells would. This paper speculates the lack of glucose dependency on insulin may be due to the high concentration of glucose in the culture media.

Another issue is in figure 5, they do no show any control for glucose levels in healthy non-diabetic mice and the glucose levels only decrease in fasted mice.

I think that this paper was successful in demonstrating that islet-cell transplantation using biodegradable scaffolds may be a better alternative for cell transplantation when compared to subcutaneous injections under the kidney capsule. Also, they showed that modifying the protocol given previously by Lumelsky et al. by using exendin-4 successfully enhanced the formation of islet-like cell clusters, however, the cells did not behave in a glucose dependent manner as the cells in the Lumelsky et al. paper did. So possibly using a combination of exendin-4 and nicotinamide along with reducing the concentration of glucose in the media will improve the amount of insulin being secreted from the cells in response to glucose. While the induction of human ES cells into islet like cells looks to be a promising treatment for diabetes type 1 in the future, further research still needs to be done to improve the efficiency of differentiating the human ES cells into insulin-producing cells that behave in a manner mimicking pancreatic endocrine beta cells.