Peter Sartipy

Long-Term Chronic Toxicity Testing Using Human Pluripotent Stem Cell–Derived Hepatocytes

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

Identification of novel biomarkers for doxorubicin-induced toxicity in human cardiomyocytes derived from pluripotent stem cells

Doxorubicin is a chemotherapeutic agent indicated for the treatment of a variety of cancer types, including leukaemia, lymphomas, and many solid tumours. The use of doxorubicin is, however, associated with severe cardiotoxicity, often resulting in early discontinuation of the treatment. Importantly, the toxic symptoms can occur several years after the termination of the doxorubicin administration. In this study, the toxic effects of doxorubicin exposure have been investigated in cardiomyocytes derived from human embryonic stem cells (hESC). The cells were exposed to different concentrations of doxorubicin for up to 2 days, followed by a 12 day recovery period. Notably, the cell morphology was altered during drug treatment and the cells showed a reduced contractile ability, most prominent at the highest concentration of doxorubicin at the later time points. A general cytotoxic response measured as Lactate dehydrogenase leakage was observed after 2 days’ exposure compared to the vehicle control, but this response was absent during the recovery period. A similar dose-dependant pattern was observed for the release of cardiac specific troponin T (cTnT) after 1 day and 2 days of treatment with doxorubicin. Global transcriptional profiles in the cells revealed clusters of genes that were differentially expressed during doxorubicin exposure, a pattern that in some cases was sustained even throughout the recovery period, suggesting that these genes could be used as sensitive biomarkers for doxorubicin-induced toxicity in human cardiomyocytes. The results from this study show that cTnT release can be used as a measurement of acute cardiotoxicity due to doxorubicin. However, for the late onset of doxorubicin-induced cardiomyopathy, cTnT release might not be the most optimal biomarker. As an alternative, some of the genes that we identified as differentially expressed after doxorubicin exposure could serve as more relevant biomarkers, and may also help to explain the cellular mechanisms behind the late onset apoptosis associated with doxorubicin-induced cardiomyopathy.

Microarray Analysis of Undifferentiated and Differentiated Human Pluripotent Stem Cells

Over the last decade, a tremendous progress has been made regarding our understanding of the molecular program involved in early human development. The main reason behind these advancements can be ascribed to the successful isolation of human embryonic stem cell (hESC) lines in the late 1990’s. Based on their fundamental properties of pluripotency and unlimited proliferation, these unique cells have provided the possibility to study early human developmental processes in vitro. However, there are many obstacles to overcome before the potential of these cells can be fully realized. One important issue is to increase the understanding about the gene regulatory mechanisms that control the differentiation of hESCs. A wide variety of tools and technologies have been used to manipulate and study basic hESC characteristics and functions. Furthermore, the parallel analysis of functional derivatives of hESCs has provided important insights into the mechanisms that govern their differentiation into specific cell lineages. Global transcriptional changes in cells and tissues can be studied using molecular techniques such as DNA microarray, EST-enumeration, MPSS profiling, and SAGE. The results from such experiments provide a snapshot of the status of the cells under study. This approach has proven well suited for characterization of the “stemness” state of hESCs, but also for the identification of crucial pathways involved in their differentiation. Large scale gene expression databases have been generated using various hESC lines and technical platforms, and subsequently the information has been analyzed using different bioinformatic approaches. A discrete set of genes has been identified which are highly expressed in hESCs, and these genes are considered to be involved in preserving the pluripotency and self-renewal capacity of the undifferentiated cells. Furthermore, several studies have focused on characterizing the molecular signature of specific differentiation processes. Again, global expression analysis has proven to be a very suitable tool since novel important mechanisms can be revealed in the context of such experiments. More recently, the possibility to analyze also the global expression profile of microRNA (miRNA) has been realized, and microarray based platforms designed specifically for the detection of miRNA species are now available. The concurrent analysis of the global mRNA and miRNA expression profiles of hESCs and their differentiated progenies are anticipated to provide additional insights into the regulatory pathways which are active in the cells in the undifferentiated and differentiated states. In the present chapter, we will

Employment of the Triple Helix concept for development of regenerative medicine applications based on human pluripotent stem cells

Using human pluripotent stem cells as a source to generate differentiated progenies for regenerative medicine applications has attracted substantial interest during recent years. Having the capability to produce large quantities of human cells that can replace damaged tissue due to disease or injury opens novel avenues for relieving symptoms and also potentially offers cures for many severe human diseases. Although tremendous advancements have been made, there is still much research and development left before human pluripotent stem cell derived products can be made available for cell therapy applications. In order to speed up the development processes, we argue strongly in favor of cross-disciplinary collaborative efforts which have many advantages, especially in a relatively new field such as regenerative medicine based on human pluripotent stem cells. In this review, we aim to illustrate how some of the hurdles for bringing human pluripotent stem cell derivatives from bench-to-bed can be effectively addressed through the establishment of collaborative programs involving academic institutions, biotech industries, and pharmaceutical companies. By taking advantage of the strengths from each organization, innovation and productivity can be maximized from a resource perspective and thus, the chances of successfully bringing novel regenerative medicine treatment options to patients increase.