Table 2 Standard criteria for designing and performing future preclinical studies in vitro and in vivo

- NOD mouse is ideal for studying type 1 diabetes and the characterization of the immunopathology of the disease.

- BioBreeding rat is a suitable model for understanding the genetics of type 1 diabetes [132] and studying of neuropathy-associated diabetes [133].

- Diabetes induction in the murine model is possible via the exposure to certain chemicals, namely streptozotocin and alloxan, and thus offers a useful tool for testing the potentials of therapeutic agents or transplanted cells to reduce glucose level.

- Some disease susceptibility loci in NOD mouse have no marked impact in human disease.

- Various drugs and antibody therapy showed an excellent effect in NOD mice but no effect in the clinical trials [134].

- Induction of diabetes in NOD mice is correlated with microbial infections.

- Induction of diabetes using chemicals in the animal model could show toxic effects to the other organs, such as the kidney, liver, brain, intestine, and reproductive organs.

- Applying humanized mouse model having the components of the human immune system.

- Taking into account the gender-dependent diabetes pathogenicity in animal models.

- Setting up new animal models that recapitulate diabetes pathogenesis in human.

- Maintaining NOD mice under specific pathogen-free environment during diabetes experimentation.

- Considering toxic actions in animal models during the chemical induction of diabetes in vivo. Previous reports showed the occurrence of lymphopenia and high production of T regulatory cells [135].

- For studying type 2 diabetes, the occurrence and the cause of obesity should be considered.

- Studying diabetes complications (neuropathy) need to avoid selecting neuropathy-resistant mouse such as C57BL/6 strain [136].

Stem cell quality

- PSCs could obviate the hurdles of islet application such as lack of donors and weak secretion of insulin post-implantation.

- Application of PSCs allows the understanding of patient-specific disease pathogenicity and also the development of potential therapeutics.

- Generation of iPSCs using integrative or viral-based methods hinders their clinical application in diabetes therapy.

- PSC cultures using undefined or xenogeneic conditions produce cells having unusual characteristics and poor phenotypes, and thus cannot be applied in the clinic.

- Using non-integrative and safe methods for the generation of iPSCs.

- Developing accurate assays for evaluating the quality of iPSCs, such as karyotyping, analysis of the pluripotency markers, and the differentiation capacity.

- Developing efficient methods for heterogeneity and teratoma assays.

- Microbiological assays for the detection of cell contamination, such as mycoplasma test.

- Using defined and xeno-free culture conditions.

Organoid/spheroid culture

- Organoid/spheroid culture allows a detailed understanding of diabetes pathogenicity, molecular mechanisms, and disease model and provides a useful tool for drug screening.

- For organoid culture, Matrigel, collagen-Matrigel, or hydrogels are mainly used as a platform.

- Application of animal-derived ECM such as Matrigel hampers the further application of generated organoids in the clinic.

- Organoid culture is costly and laborious for the large-scale production.

- Designing suitable safe xenogeneic free scaffolds (physical cues) with growth factors (biochemical cues) for the generation of stem cell niche.

- Discovering a cost-effective agents and protocols for efficient organoid culture at the large scale.

- Developing efficient assays for the evaluation of the generated organoids/spheroids prior to their application for disease modeling or drug screening.

Differentiation methods

Various differentiation protocols are developed for the generation of insulin-producing β-like cells from PSCs in either monolayer or 3D culture using a cocktail of various chemicals, growth factors, inhibitors, and cytokines in order to emulate the in vivo system.

- Differentiation protocols depend on agents of high costs.

- Many of the developed protocols are not reproducible.

- The molecular mechanisms of most of the chemicals used in each step of the differentiation method remain unrevealed.

- Characterizing the reproducibility of the current β cell differentiation protocols from PSCs.

- Setting up highly efficient protocols for the generation of mature β cells and their transplantation.

- Culture conditions such as culture media, cell density, ECM, cell-cell, and cell-ECM interactions have an impact on PSC differentiation [68, 137,138,139] and thus should be optimized.

- Characterizing the molecular mechanisms of the factors used in the current differentiation protocols.

Transplantation devices

- Encapsulation devices used for cell transplantation, such as semipermeable capsule or membrane, possess various functions [140]:

 ▪ Avoiding the undesirable host immune reactions against the transplanted cells.

 ▪ Protecting the patient from tumorigenic action of stem cells.

 ▪ Avoiding the loss of viability of the transplanted cells.

 ▪ Maintaining stable insulin secretion

- The encapsulation devices need the application of immune modulating agents.

- Encapsulation devices may provoke the patient’s immune system and ultimately lead to cell death.

- Applying suitable agents with immune modulating functions, summarized previously [140], which protect the transplanted stem cells from rejection.

- Designing an efficient encapsulation device with the following features:

 ▪ Allowing enough blood supply to the encapsulated cells.

 ▪ Having biocompatibility.

 ▪ Avoiding the stimulation of host immune reactions.

 ▪ Permitting the efficient transfer of the secreted insulin to the circulation.