Australasian Science Magazine

Immunofluorescent staining of a colony of pancreatic stem cells showing the expression of insulin (green), the nucleus (blue) and the BrdU nuclear marker of proliferating cells (red).

By Ilia Banakh

The transplantation of insulin-producing cells has been limited by a shortage of donor tissue. Could pancreatic stem cells offer a way forward for the treatment of diabetes?

Type 1 diabetes is the end result of an auto­immune response that destroys insulin-producing beta cells in the pancreas. The subsequent deficiency of insulin prevents the body’s cells from taking glucose from the bloodstream for use as an energy source. This, in turn, results in hyperglycaemia – a state of high glucose levels in the blood.

Long-term exposure to hyperglycaemia is life-threatening. It causes tissue damage, affecting blood vessels and nerves. Type 1 diabetes increases the risk of heart disease and stroke, and triggers blindness and kidney failure in adults.

Loss of insulin production requires treatment with insulin injections, but this rarely controls blood glucose adequately. While this issue can be overcome by transplanting pancreatic islet cells, the high cost, need for toxic immunosuppressive drugs and shortage of donor tissue limit the implementation of this treatment. A further setback is that few transplant recipients have achieved independence from insulin injections.

Therefore there is a need to generate renewable sources of insulin-producing cells. Alternative sources of beta cells, such as embryonic and adult stem cells, have been considered for this role.

Embryonic stem cells are self-renewing and have the capacity to generate all types of adult cells. Already, insulin-expressing cells have been generated from mouse and human embryonic stem cells.

In the past decade the technology of embryonic stem cell cultivation has progressed, with improved synthesis of insulin, reversal of hyperglycaemia in mouse models of Type 1 diabetes and now a focus on the removal of tumour-forming cells from the transplanted cells. Further advances such as large-scale cell expansion and 3D scaffolding cell cultivation were published at the end of 2012.

However, there remains limited access to human embryonic stem cells. The ethical issues of embryo destruction and objections to embryonic tissue use for research and therapeutic purposes, compounded by potential tumour formation after transplantation and the possible need for anti-rejection treatment, weigh against the application of embryonic stem cells for beta cell regeneration.

Adult stem cells are another source of beta cells. Their differentiation potential is more restricted than embryonic stem cells, but they are capable of turning into mature cells. Importantly, the ethical issues troubling embryonic stem cell research do not apply to adult stem cells.

Our research has focused on pancreatic adult stem cells due to the central role of the pancreas in Type 1 diabetes, as well as evidence that beta cells in the adult pancreas can regenerate in response to physiological and pathological pressures.

Pancreatic stem cells are more differentiated than embryonic stem cells, so inducing these cells to become beta cells will require fewer manipulations. We recently reported that adult stem cells from the mouse pancreas can be induced to divide and differentiate into endocrine cells that secrete insulin in response to glucose.

From previous pancreatic regeneration studies we hypothesised that pancreatic stem cells will increase in frequency and function after pancreatic injury, such as the destruction of beta cells through diabetes or the surgical removal of some of the pancreas.

We isolated stem cells from the pancreas of mice at different ages and compared them with the rest of the cell population. At 1 week of age the stem cells comprised about 1% of the total pancreas, and had a higher frequency of colony-forming cells. However, stem cell numbers and the frequency of colony-forming cells decreased with age, suggesting that these stem cells contribute to the growth of the pancreas in the developing mouse.

We then assessed these stem cell features in the pancreas under stress in four mouse models of Type 1 diabetes. In all four models, diabetic mice had more stem cells than their non-diabetic siblings. Additional tests in two of these models revealed that the colony frequency of the stem cell population was greater in diabetic mice. These results suggested that beta cell destruction led to a response from the pancreatic stem cells.

Partial surgical removal of the pancreas of mice that did not have high blood glucose had the same effect. This suggested that hyperglycaemia was not the cause of the stem cell changes; other mechanisms were involved in this response.

The preliminary results led us to test the capacity of pancreatic stem cells to synthesise and secrete insulin. Since these cells did not produce insulin when they were isolated, we tried to stimulate them to produce insulin.

Freshly isolated stem cells were cultured in the presence of growth factors that stimulated their proliferation. Once colonies were formed, another group of growth factors was used to induce their differentiation. At the end of 3 weeks the cells stained positive for insulin. When stimulated with glucose, the cells secreted insulin in a dose-dependent manner.

Now the insulin-secreting cells had to be tested in vivo.

Stem cell-derived colonies were transplanted inside a sealed chamber positioned around the major artery in the groin of diabetic immune-deficient mice. Their diabetic state ensured the presence of regenerative signals, and immune deficiency prevented the rejection of transplanted cells.

Two weeks later the chamber contents were removed. They contained insulin-positive cells.

This experiment revealed that transplanted cells are capable of maintaining insulin production even in the presence of hyperglycaemia. More importantly, this result placed pancreatic stem cells forward for tests of their capacity to reverse diabetes and maintain normal blood glucose levels.

Our findings described a small cell population in the adult pancreas, enriched for stem cells. These cells expanded in response to beta cell injury and had the potential to become insulin-secreting cells capable of maintaining this phenotype in a diabetic environment.

The next step in this work would involve large-scale proliferation to generate adequate quantities of insulin-producing cells that can be transplanted with the aim of reversing hyperglycaemia. This would be first tested in mice and then larger animal models of Type 1 diabetes.

Recently published methods for the expansion of human embryonic stem cells can be employed for pancreatic stem cells. Additionally, the latest technology in human embryonic stem cell 3D cultivation can be transferred to mouse stem cell research. Implementation of these novel methods will reveal the extent to which pancreatic stem cells can supply beta cells as an alternative to multiple daily injections with insulin.

Ilia Banakh is a research scientist at the Walter and Eliza Hall Institute for Medical Research.