12 April 2019
Every day we come across innovative, intelligent studies that do not rely on animals to investigate human diseases and that instead find efficient and cost-effective solutions in biomedical research that are human-focused. In this edition of Research Round-up we highlight three papers from scientists who have developed novel human-focused models to mimic the oral mucosa as well as to study diabetes and renal disease.
A model of the oral mucosa
A new study shows that it is now possible to study human oral mucosa using a 3D model created in the lab. According to the authors, this new model is a promising alternative to animal testing and brings new possibilities to the field of regenerative medicine.
In ‘Construction of Vascularized Oral Mucosa Equivalents Using A Layer-By-Layer Cell Coating Technology’ published in Tissue Engineering, a team of researchers from Japan set out to create a 3D model of the human oral mucosa. Their novel approach employed a layer-by-layer cell coating technique to create a lamina propria equivalent – made up of human oral mucosa fibroblasts in extracellular matrix. Keratinised or non-keratinised epithelial cells (commercially available gingival or oral keratinocytes) were seeded onto this construct and in some models, human umbilical vein endothelial cells (HUVEC) were included to create blood vessels. Analysis revealed that the models displayed appropriate features of native human tissue (immunohistochemistry for cytokeratin expression), with increasing transepithelial electrical resistance over time in culture (indicative of the formation of a functional barrier) and, where HUVEC were present, there were tubular structures underlying the epithelium.
Creating accurate, representative 3D models can be a daunting task as it requires technologies that blend the appropriate cell types to allow the reconstruction of the different types of tissues in an organ. The authors claim that the layer-by-layer technique offers an advantage in enabling incorporation of different cell types to the ECM during construction and the inclusion of, for example, fibroblasts, immune cells, salivary gland or even bone, could widen the range of potential applications for these models. The layer-by-layer technique developed by the research group has been efficiently used previously to develop other human tissues, namely skin, liver, and islet, myocardial and peritoneal models. The dermo-epidermis skin model previously developed by the group has density-controlled blood capillaries, and can be used as an in vitro skin model for a variety of tests currently done using animals in some countries.
Although further developments are required, the team believes that the successful creation of this model will now contribute to the development of new drugs and the study of oral mucosa regeneration without animal testing. What is more, using patient-specific or disease-specific cells to create the 3D model will help develop new personalized treatments and be useful for regenerative medicine in oral regeneration.
In another study, researchers present us with human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured “on a dish” that can be used as a model to better understand the relationship between endothelial dysfunction and diabetes. The study was published in the journal Stem Cell Reports with the title Calpain Inhibition Restores Autophagy and Prevents Mitochondrial Fragmentation in a Human iPSC Model of Diabetic Endotheliopathy.
Previous studies have shown that a family of proteins called calpains play an important role in the development of diabetes and have been shown to be abnormally activated in diabetes, where they regulate other proteins, leading to endothelial dysfunction and inflammation, early hallmarks of diabetes. If not under tight control, calpains can induce great damage leading to cell death.
In this study, researchers generated patient-specific material using (iPSCs) to produce cells differentiated into endothelial cells (iPSC-ECs) to understand how uncontrolled calpain activity contributes to diabetes. When EC cultures were subjected to hyperglycemic conditions, calpain activity increased as expected, but ECs also presented high levels of autophagy, high levels of damaged mitochondria and were more susceptible to cell death when submitted to lack of oxygen. However, by inhibiting calpains with MDL 2817, a potent calpain and cathepsin inhibitor, researchers were able to reverse the damage triggered by hyperglycemia – rescuing ECs from cell death.
Altogether, these results show not only that inhibiting calpains can save ECs from damage under diabetic conditions, but also that using human stem cells provides a reliable model to study diabetes that can be used to test potential new drugs and their effect in protecting cells against this disease.
Kidney disease model
In a study titled Tubuloids derived from human adult kidney and urine for personalized disease modeling and published in the journal Nature Biotechnology, a group from the Netherlands have successfully turned human stem cells from urine into kidney surface cells.
In this study, the authors developed a mini-kidney that was basically a set of “tubuloids” that are a simpler version of the urine-producing tubules found in kidneys. These “tubuloids” do not represent a complete human kidney but carry many of its functional and architectural characteristics. The model was tested with the BK virus (a virus that infects the kidneys and for which no cure exists) and revealed that the “tubuloids” were not only affected the same way as an intact kidney in vivo, but also that anti-viral treatment with cidofovir significantly decreased the infection, a very important finding supporting the in vivo functioning of the mini-kidney model. This is an important finding if we consider that infection by BK virus is responsible for the loss of about 10% of donor organs in kidney transplant recipients.
Using tumor cells from patients with Wilms tumor (or nephroblastoma) as the starting material to generate “tubuloids”, researchers observed that the “tubuloid” structures presented the same genetic anomalies and features that characterize this disease in patients.
Notably, the team also used urine from an individual with cystic fibrosis to isolate cells that could be turned into “tubuloids”. Again, the “tubuloids” retained the same genetic anomalies and behaved the same way as the originating cells. Importantly, this experiment suggests that “tubuloids” can be created from cells present in the urine, which may reduce the need for more invasive diagnostic methods such as biopsies or blood samples.
This model allows the study of both healthy and sick human kidney tissues, and the study of virus infection in a case-by-case basis, contributing to the advance of personalized medicine.
The work presented in each of these papers puts us significantly closer to a better understanding of the mechanisms involved in these conditions and to find new drugs to prevent these diseases in the first place or to treat the millions of patients already suffering.