endocrine pancreas and type 1 diabetes
The Signature Series Event “Advancements in CellularTherapies and Regenerative Medicine for Digestive auspices of the Society that was fully dedicated to the application of stem cell and tissue engineering technologies to digestive diseases.The symposium convened opinion leaders from three continents and seven countries, with a common interest in developing cell therapy platforms and regenerative medicine (RM) technologies for clinical application in liver diseases and diabetes, as well as illnesses affecting the digestive tract. The event represented an opportunity to share knowledge and experience and promote understanding of the supporting technologies and potential target populations, with the overarching goal of enabling the clinical implementation of promising cell- and RM-based therapies.This article succinctly reports the topics that were discussed and the debate generated as a result.
Diseases” was held as a pre-meeting of the 25th International Society for Cellular Therapy annual congress in London, United Kingdom, May 3, 2017. This was the first workshop organized under the by more than 5 years worldwide, mainly due to a significant decrease of mortality for cardiovascular diseases and cancer (Figure 1, upper panel). By contrast, improvement of management of digestive diseases, including colorectal cancer, accounts for an average increase of life expectancy from 1990 to 2015 of only 0.1 year. In addition, the prevalence of a number of disorders affecting the digestive system, mostly those characterized by a chronic inflammatory process, has undergone a significant increase not only because a growing number of patients are now correctly diagnosed but also by virtue of a real spread in the general population.
Among the illnesses showing an increased frequency are inflammatory bowel diseases, including Crohn disease (CD) and ulcerative colitis, and other autoimmune conditions affecting the intestine, such as celiac disease; the liver, such as autoimmune hepatitis ; and the pancreas, with autoimmune pancreatitis and type I diabetes. These disorders are expected to reach epidemic levels in the near future and, as their peak of incidence is in the young adult age-group with consequent impairment of patient’s work productivity, the social and economic impact may be disastrous. Moreover, recent pooled mortality rate for patients on the waiting list has recently achieved a dramatic 20%, which represents a urgent call for action. In this scenario, new treatment strategies aimed at either replacing dysfunctional livers or preventing progression of chronic liver disease toward its end stage are eagerly awaited.
The Signature Series Event was opened by Professor Ciacci,who contextualized the global impact of digestive diseases. According to. in the past 25 years,human life expectancy has increased data demonstrate that the mortality rate of diseases such as inflammatory bowel disease is on the rise (Figure 1, lower panel). Specifically, Jess et al. found that from 1982 to 2010, mortality was increased by approximately 10% in patients with ulcerative colitis and by 50% among those suffering from CD compared with the general population. Altogether, this information reveals that currently available therapeutic options are unsatisfactory and that, sadly, a definitive cure is still on the too-distant horizon.The complex, and in most cases, still unclear, interplay among genetic, epigenetic and environmental factors that underlies and triggers the majority of these illnesses represents the biggest challenge on the way to a definitive cure.
At the same time, vis-à-vis inadequate treatment tools, common sense suggests that this apparently inextricable complexity may not be resolved with a“magic bullet” like a pill or an intravenous drug; rather, alternative cellular or RM-based therapeutic tools leveraging the ability of cells to repair and regenerate dysfunctional tissues with the ultimate goal of restoring function should be tested. Notwithstanding the therapeutic potential, as of October 31, 2017, among all the clinical trials registered on the National Institutes of Health Clinical Trial Database, only a small proportion was aimed at applying mesenchymal stromal cells (MSCs) and RM in digestive diseases.Specifically,those using MSCs included 55 for liver diseases, 77 for gastrointestinal diseases, 4 for pancreas diseases and 58 for diabetes, whereas those applying RM were 6 for liver diseases, for gastrointestinal diseases, for pancreas diseases and 15 for diabetes.
Therefore, the aim of this workshop was to bring together the leading experts in clinical applications of MSCs and RM in digestive diseases to overcome this view by sharing ideas and methodologies and identifying a number of key challenges, the solution for which will accelerate the advent of cellular therapy and tissue engineering as a bedside reality. A synopsis of the presentations followed by a compilation of current key questions and recommendations follows.The full program of the meeting and list of participants is available in the online supplementary data for this article.
In the liver, a number of triggers, such as obesity, alcohol, viruses, drugs and chemicals, result in cirrhosis, one of the most prevalent conditions worldwide. Currently, liver transplantation is the only therapeutic option for end-stage liver disease, although application is limited by inadequate organ supply, high morbidity and cost. As a consequence of the progressively increasing gap between available organs and patients in need for a new liver, the The ROSAmT/mG is a cell membrane-targeted, twocolor fluorescent Cre-reporter allele. Prior to Crerecombination, cell membrane–localized tdTomato fluorescence expression is widespread in cells/tissues. Cre-recombinase expressing cells (and future cell lineages derived from these cells) have cell membrane– localized enhanced green fluorescent protein (EGFP) fluorescence expression replacing the red expression.
Costaining of EGFP with cell-specific markers confirmed preferential transduction of approximately 30% of stellate cells in normal and 20% of myofibroblasts in carbon tetrachloride–induced fibrotic livers of BALB/c mice. The percentage of in vivo generated induced hepatocytes among the total hepatocyte population ranged from 0.2% to 1.2% following viral transduction,whereas control mice did not show any reprogrammed cells. Importantly, when the adenovirus vector was injected into uninjured mice, no induced hepatocytes were detected. Induced hepatocytes recovered from livers showed stable reprograming as determined by absence of exogenous transcription factors, normal proliferative capacity after partial hepatectomy and chromosomal integrity. Functional improvement was evidenced by the ability of recovered induced hepatocytes to secrete albumin, synthesize urea and store glycogen, as well as by the presence of glycerides and lipids and by cytochrome activity.
Moreover, in vivo reprograming of myofibroblasts to induced hepatocytes resulted in significant reduction of collagen and hydroxyproline levels, indicating decreased liver fibrosis. This observation highlights the possible collateral benefit of using myofibroblasts as target cells for direct reprograming. Direct reprogramming of hepatic myofibroblasts may not only provide induced hepatocytes to repopulate the damaged liver parenchyma and ultimately restore use of serum-free Kubota’s medium supplemented with 10% dimethyl sulfoxide, 15% human serum albumin and 0.1% hyaluronans. When freshly isolated biliary tree stem cells were cultured in vitro and compared with their cryopreserved counterparts, no differences were noted in terms of self-replication, stemness traits and multi-potency. Just like freshly isolated cells, cryopreserved cells were able to differentiate into functional hepatocytes, cholangiocytes or pancreatic islets and to yield similar capacity to secrete albumin and glucose-inducible insulin.This technology may be expanded to multiple cell types and promises to facilitate the establishment of cell banks with obvious logistic advantages.
Dr. Vincenzo Cardinale presented data from clinical studies that his group at Sapienza University of Rome (Italy) is conducting to prove efficacy of innovative clinical cryopreservation technology and grafting strategies that are expected to fasten the translation of an effective biliary tree stem cell therapy. Although several sources of stem cells including hepatic stem cells, biliary tree stem cells, MSCs, adipose-derived stem cells, umbilical cord cells, amniotic fluid–derived epithelial cells, embryonic stem cells (ESCs) and iPSCs have been investigated for their potential as therapy for chronic liver failure, homing of therapeutic cells to the liver remains a challenge. Although the injection into the hepatic artery showed the greatest percent of engraftment, injection into the liver parenchyma (~10–20%) and the portal vein (<5%) was still consistent with poor engraftment and significant ectopic cell distribution to the vascular beds of other tissues suggesting a high risk of ectopic liver formation. Given that hyaluronic acid is selectively and specifically cleared by the liver and that it has been implicated in various aspects of stem cell therapy optimization, Cardinale’s team hypothesized that it would not only enhance engraftment but also improve the applicability of human biliary tree stem cells to treat liver cirrhosis.
Interestingly, hyaluronic acid coating of human biliary tree stem cells markedly improved viability, colony formation, and population doubling in primary cultures and resulted in higher expression of integrins that are key players and mediators of cell attachment to the extracellular matrix.When hyaluronic acid– coated biliary tree stem cells were transplanted via the spleen into the liver of immunocompromised mice, the engraftment efficiency increased from 3% of uncoated cells to 11%. Notably, hyaluronic acid–coated human biliary tree stem cell transplantation in mice resulted in a 10-fold increase of human albumin gene expression in the liver and in a 2-fold increase of human albumin serum levels with respect to uncoated cells.
Moreover, when other organs were sectioned and stained to track the cells in question, only minimal ectopic cell distribution was detected. Furthermore, because one of the major limitations of cellular therapies is their need for long-term storage, researchers are devising strategies to make this possible.To meet this critical aspect of liver cellular therapies and optimize their sourcing, Cardinale’s group has developed a cryopreservation protocol consisting of a stepwise marrow were responsible, this group performed new gender-mismatched reconstitution experiments, showing that marrow stromal cells, not hematopoietic stem cells, were responsible for bone marrow–derived myofibroblasts in the liver.The same group also published a case series of male patients who had undergone liver transplantations from female donors and who had gone on to require another transplant for cirrhosis.
Explanted liver tissues were analyzed forY chromosome by in situ hybridization together with markers for hepatic stellate cells and myofibroblasts, revealing that 14–45% of myofibroblast were indeed of recipient origin. In contrast to these reports implicating bone marrow stromal cells in fibrosis, Miyata et al. provided evidence of a dominant role for hematopoietic stem cells, and although other groups have not been able to replicate a role for bone marrow–derived stem/ stromal cells in fibrosis, an important contribution of hepatic stellate cells to fibrosis has emerged. Significant heterogeneity in experimental designs, that is, approaches to inducing chronic liver disease, timing, route of administration and animal models have precluded an in-depth understanding of potential MSC-mediated mechanisms of action in this condition.
Despite mixed results, more than 10 clinical trials between 2007 and 2014 using either autologous or allogenic MSCs suggested that they are at least safe in chronic liver disease. The end points of the studies were to evaluate the safety and efficacy of bone marrow and umbilical cord MSC transplantation. The cells were mostly infused intravenously, although two studies reported infusions via the hepatic artery and one in the spleen. Moreover, there was great variation in both the number of cells infused per patient and the frequency of injections among the trials. The results of the studies seemed promising in terms of improvement of liver function and a model for an end-stage liver disease score.
This score is based on objective variables (international normalized ratio and serum creatinine, bilirubin and sodium concentration) and has been validated as a predictor of survival among patients with advanced liver disease. However, for most of the studies discussed by Dr. Haldar, data regarding evaluation of liver histology after cell transplantation is lacking; most studies were underpowered to detect significant differences, controls were either lacking or inadequate and the follow-up period was too short. One of the primary challenges remains the inability to track and monitor the transplanted cells and the absence of standardized transplantation protocols.
Author: RACHELE CICCOCIOPPO , CLAUDIA C. DOS SANTOS , DANIEL C. BAUMGART , GIUSEPPINA C. CANGEMI , VINCENZO CARDINALE, CAROLINA CIACCI, PAOLO DE COPPI , DEBASHIS HALDAR , CATHERINE KLERSY , M. CRISTINA NOSTRO, MICHAEL OTT, LORENZO PIEMONTI, ALICE A. TOMEI , BASAK UYGUN, STEFANIA VETRANO & GIUSEPPE ORLANDO