Papers by Alina Ostrowska
Frontiers in Medicine
Acute hepatic failure is associated with high morbidity and mortality for which the only definiti... more Acute hepatic failure is associated with high morbidity and mortality for which the only definitive therapy is liver transplantation. Some fraction of those who undergo emergency transplantation have been shown to recover native liver function when transplanted with an auxiliary hepatic graft that leaves part of the native liver intact. Thus, transplantation could have been averted with the development and use of some form of hepatic support. The costs of developing and testing liver support systems could be dramatically reduced by the availability of a reliable large animal model of hepatic failure with a large therapeutic window that allows the assessment of efficacy and timing of intervention. Non-lethal forms of hepatic injury were examined in combination with liver-directed radiation in non-human primates (NHPs) to develop a model of acute hepatic failure that mimics the human condition. Porcine hepatocyte transplantation was then tested as a potential therapy for acute hepatic...
Hepatology Communications
The only definitive therapy for end‐stage liver disease is whole‐organ transplantation. The succe... more The only definitive therapy for end‐stage liver disease is whole‐organ transplantation. The success of this intervention is severely limited by the complexity of the surgery, the cost of patient care, the need for long‐term immunosuppression, and the shortage of donor organs. In rodents and humans, end‐stage degeneration of hepatocyte function is associated with disruption of the liver‐specific transcriptional network and a nearly complete loss of promoter P1‐driven hepatocyte nuclear factor 4‐alpha (P1‐HNF4α) activity. Re‐expression of HNF4α2, the predominant P1‐HNF4α, reinstates the transcriptional network, normalizes the genes important for hepatocyte function, and reverses liver failure in rodents. In this study, we tested the effectiveness of supplementary expression of human HNF4α2 messenger RNA (mRNA) in primary human hepatocytes isolated from explanted livers of patients who underwent transplant for end‐stage irreversibly decompensated liver failure (Child‐Pugh B, C) resulting from alcohol‐mediated cirrhosis and nonalcoholic steatohepatitis. Re‐expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver‐specific protein expression. End‐stage liver disease in humans is associated with both loss of P1‐HNF4α expression and failure of its localization to the nucleus. We found that while HNF4α2 re‐expression increased the amount of P1‐HNF4α protein in hepatocytes, it did not alter the ability of hepatocytes to localize P1‐HNF4α to their nuclei. Conclusion: Re‐expression of HNF4α2 mRNA in livers of patients with end‐stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation. The efficacy of this strategy may be enhanced by discovering the cause for loss of nuclear P1‐HNF4α localization in end‐stage cirrhosis, a process not found in rodent studies.
Hepatology Communications, 2022
The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the f... more The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well-defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC-derived hepatocytes (iHep) and iPSC-derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte-specific and cholangiocyte-specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat-containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole-cell patch-clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases.
Organogenesis, 2021
The prevalence of end-stage liver disease (ESLD) in the US is increasing at an alarming rate. It ... more The prevalence of end-stage liver disease (ESLD) in the US is increasing at an alarming rate. It can be caused by several factors; however, one of the most common routes begins with nonalcoholic fatty liver disease (NAFLD). ESLD is diagnosed by the presence of irreversible damage to the liver. Currently, the only definitive treatment for ESLD is orthotopic liver transplantation (OLT). Nevertheless, OLT is limited due to a shortage of donor livers. Several promising alternative treatment options are under investigation. Researchers have focused on the effect of liver-enriched transcription factors (LETFs) on disease progression. Specifically, hepatocyte nuclear factor 4-alpha (HNF4α) has been reported to reset the liver transcription network and possibly play a role in the regression of fibrosis and cirrhosis. In this review, we describe the function of HNF4α, along with its regulation at various levels. In addition, we summarize the role of HNF4α in ESLD and its potential as a thera...
Organogenesis, 2021
ABSTRACT The use of primary human hepatocytes has been hampered by limited availability of adequa... more ABSTRACT The use of primary human hepatocytes has been hampered by limited availability of adequate numbers of fresh and viable cells due to the ongoing shortage of liver donors. Thus, there is no surplus of healthy organs from which freshly isolated cells can be prepared when needed. However, primary hepatocytes can be successfully isolated from explanted liver specimens obtained from patients receiving orthotopic liver transplantation for decompensated liver cirrhosis or for metabolic liver disease without end-stage liver disease and are a valuable resource for the pharmaceutical industry research. This review focuses on the isolation, characterization and cryopreservation of hepatocytes derived from therapeutically resected livers with various hepatic diseases.
Journal of Personalized Medicine, 2021
As diet and lifestyle have changed, fatty liver disease (FLD) has become more and more prevalent.... more As diet and lifestyle have changed, fatty liver disease (FLD) has become more and more prevalent. Many genetic risk factors, such as variants of PNPLA3, TM6SF2, GCKR, and MBOAT7, have previously been uncovered via genome wide association studies (GWAS) to be associated with FLD. In 2018, a genetic variant (rs72613567, T > TA) of hydroxysteroid 17-β dehydrogenase family 13 (HSD17B13) was first associated with a lower risk of developing alcoholic liver disease and non-alcoholic fatty liver disease (NAFLD) in minor allele carriers. Other HSD17B13 variants were also later linked with either lower inflammation scores among NAFLD patients or protection against NAFLD (rs6834314, A > G and rs9992651, G > A) respectively. HSD17B13 is a lipid droplet-associated protein, but its function is still ambiguous. Compared to the other genetic variants that increase risk for FLD, HSD17B13 variants serve a protective role, making this gene a potential therapeutic target. However, the mechanis...
Journal of Clinical Investigation
Advances in Tissue Banking, 2002
Hepatology Communications
Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in... more Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post-translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure. (Hepatology Communications 2020;4:859-875).
Arch Toxicol, 2009
The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and... more The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of alpha-amanitin (alpha-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of alpha-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.
Drug Metabolism and Disposition the Biological Fate of Chemicals, Jul 1, 1999
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Transplantation Proceedings
Cell and tissue banking, 2000
There is a pressing need for alternative therapeutic methods effective in the treatment of patien... more There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion techniqu...
Drug metabolism and disposition: the biological fate of chemicals, 1999
Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 ... more Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the sm...
Transplantation proceedings
In vivo (Athens, Greece)
Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical... more Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology. The objective of this study was a comparative evaluation of benzylpenicillin (Bp), acetylcysteine (ACC) and silibinin (Sil) efficacy as antidotes in hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured canine hepatocytes. Cytotoxicity evaluation of cultured cells (MTT assay, extracellular lactate dehydrogenase activity) was performed at 12, 24 and 48 h of exposure to alpha-AMA and/or antidotes. Following 24 and 48 h exposure there was a significant decline of hepatocyte viability and an increase of lactate dehydrogenase activity in groups exposed to alpha-AMA and in groups exposed simultaneously to alpha-AMA and antidotes. Moreover, hepatocyte viability and lactate dehydrogenase activity in all these groups were similar. Administration of studied antidotes without alpha-AMA, was not associated with any adverse effects in hepatoc...
Journal of Stem Cell Research & Therapy, 2011
Scientific Reports, 2013
New small molecules that regulate the step-wise differentiation of human pluripotent stem cells i... more New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients. P arkinson's disease (PD) is a neurodegenerative disorder caused by a progressive degeneration of midbrain dopamine (DA) neurons in the substantia nigra pars compacta. The degeneration of DA neurons causes a gradual dysfunction of the motor system leading to symptoms such as tremor, rigidity, and bradykinesia, among others. There is currently no cure for PD and although treatments such as deep brain stimulation and levodopa can alleviate some of the symptoms, they tend to lose efficacy over time. However, the localized nature of the loss of DA neurons in the substantia nigra (SN) makes cell replacement therapy an attractive approach to treating Parkinson's disease (PD) patients. Implantation of neuronal cells such as neural stem cells (NSCs) and DA neurons have already been shown to improve the motor symptoms in PD animal models 1-5 . In order for stem cell based therapies for PD to become a reality it is critical to be able to generate a homogeneous population of NSCs which will in turn generate functional DA neurons either in situ or in vitro, depending on whether the terminal differentiation occurs in the patient's brain or in culture. In this communication we report the derivation of a high-purity population of DA neurons from human pluripotent stem cells (hPSCs) in a robust and reproducible manner via a stable NSC stage, where the cells can be expanded and cryopreserved, using newly developed chemically directed differentiation methods.
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Papers by Alina Ostrowska