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Yu Y.,Rochester College | Yu Y.,Nanjing Medical University | Liu H.,Rochester College | Liu H.,Liver Failure Diagnosis and Treatment Center | And 5 more authors.
Stem Cell Research

Maturation of induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs) has been proposed to address the shortage of human hepatocytes for therapeutic applications. The purpose of this study was to evaluate hiPSCs, HLCs and hepatocytes, all of human origin, in terms of performance metrics of relevance to cell therapies. hiPSCs were differentiated to HLCs in vitro using an established four-stage approach. We observed that hiPSCs had low oxygen consumption and possessed small, immature mitochondria located around the nucleus. With maturation to HLCs, mitochondria showed characteristic changes in morphology, ultrastructure, and gene expression. These changes in mitochondria included elongated morphology, swollen cristae, dense matrices, cytoplasmic migration, increased expression of mitochondrial DNA transcription and replication-related genes, and increased oxygen consumption. Following differentiation, HLCs expressed characteristic hepatocyte proteins including albumin and hepatocyte nuclear factor 4-alpha, and intrinsic functions including cytochrome P450 metabolism. But HLCs also expressed high levels of alpha fetoprotein, suggesting a persistent immature phenotype or inability to turn off early stage genes. Furthermore, the levels of albumin production, urea production, cytochrome P450 activity, and mitochondrial function of HLCs were significantly lower than primary human hepatocytes. Conclusion: - hiPSCs offer an unlimited source of human HLCs. However, reduced functionality of HLCs compared to primary human hepatocytes limits their usefulness in clinical practice. Novel techniques are needed to complete differentiation of hiPSCs to mature hepatocytes. © 2012 Elsevier B.V. Source

Liu H.,Rochester College | Liu H.,Liver Failure Diagnosis and Treatment Center | Yu Y.,Rochester College | Yu Y.,Nanjing Medical University | And 6 more authors.
Cell Transplantation

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids. © 2014 Cognizant Comm. Corp. Source

Liu H.-L.,Liver Failure Diagnosis and Treatment Center | You S.-L.,Liver Failure Diagnosis and Treatment Center | Li C.,Liver Failure Diagnosis and Treatment Center | Liu W.-S.,Liver Failure Diagnosis and Treatment Center | And 7 more authors.
Medical Journal of Chinese People's Liberation Army

Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium (SFM). Spheroids were then maintained in rocking culture at 37°C(control condition), or cold stored at 4°Cfor 24 or 48 hours in four different cold storage solutions: SFM alone; SFM+1mmol/L deferoxamine (Def); SFM+1|xmol/L cyclosporin A (CsA); and SFM+1mmol/L Def+1|xmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def was significantly higher than that stored in SFM or SFM+CsA(P<0.05). The production of albumin was in low level in SFM, SFM+Def, SFM+CsA and SFM+Def+CsA groups, and there was no difference between them (P>0.05). Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases. © 2014, People's Military Medical Press. All rights reserved. Source

Ji D.,Liver Fibrosis Diagnosis and Treatment Center | Shao Q.,Liver Fibrosis Diagnosis and Treatment Center | Han P.,Tumor Radiotherapy Center | Li F.,Liver Fibrosis Diagnosis and Treatment Center | And 6 more authors.

Objective: To investigate the frequency and determinants of liver stiffness measurement (LSM) failure by means of FibroScan in "real-life" Chinese patients. Methods: A total of 38,464 "real-life" Chinese patients in 302 military hospital of China through the whole year of 2013, including asymptomatic carrier, chronic hepatitis B, chronic hepatitis C, liver cirrhosis (LC), alcoholic liver disease, autoimmune liver disease, hepatocellular carcinoma (HCC) and other, were enrolled, their clinical and biological parameters were retrospectively investigated. Liver fibrosis was evaluated by FibroScan detection. S probe (for children with height less than 1.20 m) and M probe (for adults) were used. LSM failure defined as zero valid shots (unsuccessful LSM), or the ratio of the interquartile range to the median of 10 measurements (IQR/M) greater than 0.30 plus median LSM greater or equal to 7.1 kPa (unreliable LSM). Results: LSM failure occurred in 3.34% of all examinations (1286 patients out of 38,464), among them, there were 958 cases (2.49%) with unsuccessful LSM, and 328 patients (0.85%) with unreliable LSM. Statistical analyses showed that LSM failure was independently associated with body mass index (BMI) greater than 30 kg/m2, female sex, age greater than 50 years, intercostal spaces (IS) less than 9 mm, decompensated liver cirrhosis and HCC patients. There were no significant differences among other diseases. By changing another skilled operator, success was achieved on 301 cases out of 1286, which reduced the failure rate to 2.56%, the decrease was significant (P<0.0001). Conclusions: The principal reasons of LSM failure are ascites, obesity and narrow of IS. The failure rates of HCC, decompensated LC, elder or female patients are higher. These results emphasize the need for adequate operator training, technological improvements and optimal criteria for specific patient subpopulations. © 2014 Ji et al. Source

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