INTERNATIONAL CONGRESS ON THE NEW FRONTIERS OF RESEARCH ON CELLULAR AND MOLECULAR THERAPY FOR TYPE 1 DIABETES MELLITUS: “STEM CELLS-DERIVED BETA CELLS”. BOSTON, USA.
Riccardo Calafiore, Diabetes Research Foundation ETS and University of Perugia
At the end of April last year, 2023, in Boston (USA), after the dark years of COVID, the World Congress on pluripotent stem cell precursors capable of producing insulin was held. The theme, addressed by leading international experts in the field, including myself, who presented the data from our Laboratory, University of Perugia and Diabetes Research Foundation, focused on the current state of this advanced research on cellular transplants for the hopeful radical cure of type 1 diabetes mellitus (T1D).
Today, this terrible disease, which usually, though not necessarily, occurs in adolescence or young adulthood, can only rely on a single therapy consisting of the administration of exogenous insulin, in the form of multiple subcutaneous injections every day, or alternatively insulin mini-pumps, in an attempt to maintain blood glucose levels within a range as close as possible to normal conditions. In T1D, as it is known, an immune-mediated auto-destruction of Beta cells, abrogates insulin production: this crucial hormone regulates, under physiological conditions, blood glucose levels whose values range from 70 to 140 mg/dl under any circumstances, both in fasting and after meals. Obviously, after the destruction of Beta cells during the course of the disease, insulin production ceases, leading to uncontrolled hyperglycemia. To avoid acute, and sometimes fatal, complications resulting from hyperglycemia, the only available resource is the use of injectable insulin therapy. This is indeed a life-saving medication, indispensable for the survival of patients with T1D. However, apart from the daunting task of continuously measuring blood glucose levels (despite the help of recent glucose sensor models) to determine the units of insulin to administer to the patient to regulate blood glucose, especially at meal times, daily multi-injection insulin therapy exposes to the risk of even severe hypoglycemia. By the way, insulin therapy may limit, but never abolish, the risk for developing the serious chronic complications of diabetic disease (retinopathy, kidney failure, cardiovascular disease, peripheral neuropathy), which can be disabling or fatal for the affected patients.
An alternative to insulin therapy, and we are approaching the theme of the Congress, aimed at the radical solution for the problems posed by T1D, lies on the possibility of replacing the Beta pancreatic cells destroyed by the disease with healthy cells isolated from the pancreas of healthy donors (Beta cells in the normal pancreas are part of larger structures, called Langerhans islets, aggregates of endocrine cells that represent 1-2% of pancreatic mass). In studies initiated in the 1980s and subsequently refined in recent times, it has been shown that intrahepatic transplantation of Langerhans islets extracted from donor pancreases can restore normal blood glucose values. However, the international casuistry officially communicated so far is very limited, since there are at least two significant obstacles to the application of this technology on a large scale:
1) the need to subject the transplanted patient to life-long pharmacological immunosuppressive therapy (with all the associated risks for toxicity to various organs and systems);
2) the limited availability of donor pancreases, a problem also shared for other organs such as the heart, liver, etc. The possibility of enveloping the islets of Langerhans within microcapsules fabricated with natural polymers (alginic acid) derived from brown seaweeds has shown, in studies conducted and published by our Laboratory, in a first international pilot study, to effectively counteract the immune rejection of islet transplants. However, a main problem remains the critical mass of islets available for transplantation, which may be insufficient, if inadequate, to reverse hyperglycemia in T1D patients.
Hence, and this was the theme of the Congress, the idea of using virtually inexhaustible sources of cells capable of synthesizing and secreting insulin, namely the use of stem cells. The stem cell, during embryonic development, has pluripotency characteristics, i.e., it can give rise to all 200 types of cells present in our body. Hence the idea of developing technologies capable of exploiting this potential to produce Beta-like cells, i.e., cells similar to normal pancreatic Beta cells, capable of producing insulin in an almost unlimited manner. During the Congress, the various types of stem cells potentially capable of generating Beta cell precursors were reviewed. A first possible approach is to use pluripotent cells from human embryos and condition their differentiation towards Beta-like cells. But even in this case, the Harvard group that first undertook this research direction, upon demonstrating that it is indeed possible from a pluripotent embryonic cell to derive a Beta-like cell capable of secreting insulin, through sophisticated methods of molecular differentiation, found itself faced with limitations in the performance of the neo Beta-like cells. Not only problems of immune rejection, but also flaws of the differentiation process. Moreover, in many countries of the Western World, including ours, the use of human embryos is not allowed by law. The problem of immune rejection has been addressed both with classic immunosuppressive drugs and with the use of immunoisolating membranes, capable of preventing contact between encapsulated cells and the host immune system.
Such studies, also carried out by other Authors, such as J. Millman and C. Nostro, applied mainly in preclinical animal models such as diabetic mice, proved the principle that the path could be correct, albeit with significant limitations. The same Harvard group, merged into a large Biotechnology Company, Vertex Inc., presented the results of the first two patients with T1D treated with embryonic cells becoming Beta-like cells, inside artificial biomembranes and subjected to pharmacological immunosuppression, one of which discontinued insulin therapy after 270 days from transplantation. The same group is also using induced pluripotent stem cells to overcome the ethical problems posed by the use of human embryonic cells. Briefly, a Japanese scientist, N. Yamanaka, discovered in 2006 (and for this discovery he received the Nobel Prize for Medicine in 2012) a method to make a normal and differentiated cell (for example a skin or blood cell) pluripotent, i.e., effectively reversing it into an embryonic state, by inoculating stemness genes (e.g., Nanog, Oct 4, etc.). In this way, a normal cell, made pluripotent, can be reconditioned to become a Beta-like cell (or all the other possible cells of the organism), capable of producing insulin. Studies with induced pluripotent human cells (iPSC) have been presented by various Authors, limited to rodent animal models. Our group also presented experimental data in mice with iPSC embodied within a new prototype of microcapsules. These have been shown to represent an effective way to counteract transplant rejection, but also to restrain/eliminate the uncontrolled expansion of iPSC towards undifferentiated, potentially tumorigenic elements, and finally to promote, within a favorable 3D microenvironment, the maturation of stem cells towards the desired cell type (in our case Beta-like cells).
The “PROs” of iPSCs, as reiterated at the Congress, certainly lie on the possibility of producing differentiated cells such as, in our case, Beta-like cells, in an unlimited manner, capable of fulfilling the physiological tasks they are normally assigned, although iPSC differentiation protocols still need to be perfected.
The “CONs” of iPSCs concern:
1) the relevant expenses for their production and maintenance and the extremely long time for their maturation before use;
2) the need to prevent immune rejection through pharmacological immunosuppression and the use of artificial immunoisolating membranes (e.g., microcapsules or other devices);
3) the possibility that such cells may take “wrong paths” and give rise to neoplastic tissues (although the capsules would control this problem).
Another type of stem cell was discussed at the Congress, this time not embryonic but adult, which appeared to be more manageable and potentially very useful in solving the problem of the radical cure of T1D. These are the so-called adult mesenchymal stem cells (MSCs) found in many tissues of the body, such as adipose tissue, bone marrow, and postpartum umbilical cord, to name the most well-known sources. Since they are adult stem cells, MSCs do not pose ethical problems. MSCs retain powerful immune-regulatory activities, thanks to the molecules they produce, capable of controlling immune responses (let’s not forget that T1D is an autoimmune disease) in various human disorders. Furthermore, although being adult stem cells (and therefore not pluripotent but multipotent), MSCs can theoretically differentiate, in the presence of specific and sophisticated stimuli, towards the definitive endoderm, the embryonic sheet that will give rise among others, also to pancreatic cells and therefore Beta cells. Experimental studies in rodents (NOD mouse) with spontaneous autoimmune T1D, extremely similar to the human counterpart, have shown that if implanted early after the development of T1D, MSCs can arrest the auto-destruction process, allowing still healthy Beta cells to repair their lesions and eventually resume insulin production. A type of MSC particularly favorable in this regard is represented by cells harvested from the Wharton’s jelly of the umbilical cord (which notoriously after birth is discarded as biological waste).
These cells are physiologically located in a privileged position, namely the maternal-fetal interface, which during pregnancy monitors and counteracts the possible passage of foreign antigens between mother and fetus, safeguarding the well-being of the new organism. Precisely for this reason, MSCs from the human umbilical cord, which are easily separated, cultured, and replicated in vitro, and can be frozen and thawed at will, represent an ideal source of stem cells to be used in T1D. Furthermore, MSCs also possess a pro-regenerative capacity of other structures such as skin and subcutaneous tissue, and could be usefully employed in the treatment of deep ulcers typical of the diabetic foot, a serious complication of the disease, increasingly frequent. In our Laboratory, we have developed a method for the separation, purification, and cryopreservation of MSCs from the postpartum umbilical cord which, after microencapsulation, we have transplanted into diabetic NOD mice and others with deep skin lesions obtaining excellent results in both cases. Some Chinese Authors, and recently also Scandinavians, have infused MSCs intravenously into patients with T1D with results that currently are under evaluation.
In summary, during the two-day duration of the Congress, all the main aspects of cellular and molecular therapy, using both conventional cells (islets of Langerhans) and stem cells, as an alternative to daily insulin injections, for T1D were reviewed. Certainly, those with stem cells are still experimental approaches, which are only recently entering human application, but which are nevertheless part of and partly mixed with the older strand of islets of Langerhans transplants, recently back in the spotlight for the possibility of separating these cell complexes from non-heart beating cadaver pancreas. It is increasingly evident that stem cells and islets of Langerhans represent that frontier for radical cellular therapy for T1D, possibly capable of overcoming the limitations of conventional insulin therapy, hopefully in the not too far future.