Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background and Objectives@#Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold greatpromise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. @*Methods@#and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances(xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. @*Conclusions@#To the best of our knowledge, this is the first study to show that the electrophysiological function andglycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Immunohistochemical Study of the Endocrine Cells in the Pancreas of the Carp,Cyprinus carpio (Cyprinidae)

The regional distribution and relative frequency of some endocrine cells in the pancreas of the carp, Cyprinus carpio Linnaeus, belonging to the family Cyprinidae in the order Cypriniformes, were observed using specific mammalian antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP) by peroxidase antiperoxidase (PAP) method. The pancreas was divided into four regions (principal and secondary islets, exocrine and pancreatic duct regions). In addition, the pancreatic islet regions were further subdivided into three regions (central, mantle and peripheral regions) and the pancreatic duct regions were subdivided into two regions (epithelial and subepithelial regions). Spherical to spindle or occasionally round to oval shaped immunoreactive (IR) cells were demonstrated in the pancreatic islets, exocrine and pancreatic duct. In the principal islet regions, some cells were also detected in the other regions, most of insulin- and somatostatin-IR cells were located in the central regions, and glucagon- and hPP-IR cells were situated in the peripheral regions. In this regions, insulin-IR cells were most predominant cell types and then, glucagon, somatostatin and hPP in that order. In the secondary islet regions, the regional distribution and relative frequency of these four types of endocrine cells were quite similar to those of the principal islets except for cell clusters consisted of hPP-IR cells that were situated in the peripheral to mantle regions. In the pancreatic duct regions, all four major pancreatic endocrine cells were demonstrated in the inter-epithelial cells and/or basal regions of the epithelial linning. In addition, cell clusters composed of numerous insulin-, moderate glucagon- and somatostatin-IR cells of low frequency were also observed in the subepithelial regions of the pancreatic duct. In the exocrine regions, insulin-, glucagon-, somatostatin- and hPP-IR cells were located in the inter-acinus regions with rare, a few, moderate and moderate frequencies, respectively. In conclusion, the regional distribution and relative frequency of four major pancreatic endocrine cells, insulin-, glucagon-, somatostatin- and hPP-IR cells, in the pancreas of the carp showed general patterns which were observed in other stomachless teleost. However, some species- dependent different distributional patterns and/or relative frequencies were also demonstrated.

An immunohistochemical study on the pancreatic islets cells of the Mongolian gerbils, Meriones unguiculatus

In order to study the regional distribution and relative frequency of the immunoreactive endocrine cells in the pancreatic islets of the Mongolian gerbil, pancreatic sections of Meriones unguiculatus were immunostained using an immunohistochemical (PAP) method with four types of specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (PP). The pancreatic islets were subdivided into three portions (central region, mantle zone and peripheral region) according to their composition of immunoreactive cells. Spherical to spindle shaped insulin, glucagon, somatostatin and PP-immunoreactive cells were observed in this study. Insulin-immunoreactive cells were present in the central regions with high frequency, and a few of these cells were also demonstrated in the mantle zones. Glucagon-immunoreactive cells were mainly restricted to the mantle zones. However, rare examples were found in the peripheral regions. As for the glucagon-immunoreactive cells, somatostatin-immunoreactive cells were detected in the mantle zones and peripheral regions with moderate and rare frequencies, respectively. PP-immunoreactive cells were found in the mantle zones and peripheral regions with rare and moderate frequencies, respectively. In the mantle and the peripheral regions, cytoplasmic process of glucagon-, somatostatin- and PP-immunoreactive cells were intermingled. In conclusion, the regional distribution of endocrine cells in the pancreatic islets of Mongolian gerbil was found to be similar to that of other mammals, especially other rodents, except for the topographical different distribution of somatostatin which differs that of other rodents.

Comparative study of endocrine cells in the principal pancreatic islets of two teleosts, Silurus asotus (Siluridae) and Siniperca scherzeri (Centropomidae)

The regional distribution and relative frequency of some endocrine cells in the principal pancreatic islets of two teleosts, Silurus asotus Linne (Siluridae) and Siniperca scherzeri Steindachner (Centropomidae), which have similar feeding habits, were observed using specific antisera against insulin, glucagon, somatostatin and bovine pancreatic polypeptide (bovine PP) using the peroxidase antiperoxidase (PAP) method. Spherical to spindle shaped cells were demonstrated in the principal pancreatic islets in both species of teleost fishes. However, they were not detected in the exocrine portions nor the pancreatic ducts. Insulin-immunoreactive cells were located in the central regions of the principal pancreatic islets at high frequency in both species. Glucagonimmunoreactive cells were restricted to the peripheral regions of the principal pancreatic islets in both species. They formed a mantle zone in the peripheral regions of Silurus asotus with moderate frequency, and occupied a narrower mantle zone in Siniperca scherzeri with moderate frequency. In addition, glucagonimmunoreactive cell cores were also found in the peripheral zone of some principal pancreatic islets of Siniperca scherzeri. Somatostatin-immunoreactive cells were dispersed in the central zone of the principal pancreatic islets of Silurus asotus with moderate frequency, but were located in the peripheral regions with low frequency in Siniperca scherzeri. Bovine PPimmunoreactive cells were found in the peripheral region and the mantle zone of the principal pancreatic islets with low and rare frequency, respectively in both species. In conclusion, the regional distribution and relative frequency of endocrine cells in the principal pancreatic islets of Silurus asotus showed general patterns similar to those of other teleostean fishes. But, some speciesdependent distributional patterns and/or relative frequencies, particularly in glucagon-, somatostatin- and bovine PP-immunoreactive cells, were detected in the principal pancreatic islets of Siniperca scherzeri