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Cell therapy is one of the promising approaches used against type 1 diabetes. Efficient generation of human embryonic stem cell (hESC)-derived pancreatic progenitors (PPs) is of great importance. Since signaling pathways underlying human pancreas development are not yet fully understood, various differentiation protocols are conducted, each considering variable duration, timing, and concentrations of growth factors and small molecules. Therefore, we compared two PP differentiation protocols in static suspension culture. We tested modified protocols developed by Pagliuca et al. (protocol 1) and Royan researchers (protocol 2) until early PP stage. The morphological changes of hESC aggregates during differentiation, and also gene and protein expression after differentiation, were evaluated. Different morphological structures were formed in each protocol. Quantitative gene expression analysis, flow cytometry, and immunostaining revealed a high level of PDX1 expression on day 13 of Royan's differentiation protocol compared to protocol 1. Our data showed that using protocol 2, cells were further differentiated until day 16, showing higher efficiency of early PPs. Moreover, protocol 2 is able to produce hESCs-PPs in a static suspension culture. Since protocol 2 is inexpensive in terms of media, growth factors, and chemicals, it can be used for massive production of PPs using static and dynamic suspension cultures. [ABSTRACT FROM AUTHOR]

Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES‐derived pancreatic duodenal homeobox 1 (PDX1) + early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. Based on our findings, PO‐derived β cells could be considered a novel strategy to study human β‐cell development, novel therapeutics, and regenerative medicine for diabetes. Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell‐derivatives. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicate that 3D PO developed more vascularization, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. ES‐PP: ES cell‐derived pancreatic progenitors; ES‐MSC: ES cell‐derived mesenchymal stem cells; ES‐EC: ES cell‐derived endothelial cells; GF: growth factor; GFR: growth factor reduced; SM: small molecule; IP Tx: intraperitoneal transplantation [ABSTRACT FROM AUTHOR]

Objective: Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. Materials and Methods: This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. Results: We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. Conclusion: Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates. [ABSTRACT FROM AUTHOR]

Objective: Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks. Materials and Methods: In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein. Results: We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm. Conclusion: Our results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO. [ABSTRACT FROM AUTHOR]