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Hepatocyte Differentiation Metabolism

ABSTRACT: In the first group of samples, we are looking into the different metabolites and their concentrations within our human embryonic stem cells over the course of differentiation. Our goal is to study how the metabolism of stem cells are changing as they undergo this conversion to become hepatocyte-like cells. In the second group of samples labeled HyCell, we would like to understand the concentrations of metabolites within our CHO cells in order to update the parameters for our metabolic model. Included are two samples from different days of a suspension batch culture which may have different lactate production qualities.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Stem Cells

SUBMITTER: Maureen Kachman

PROVIDER: ST000837 | MetabolomicsWorkbench | Wed Aug 02 00:00:00 BST 2017

REPOSITORIES: MetabolomicsWorkbench

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Project description:Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine.

Project description:A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC laden-microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27% higher and the number of CMs per initial hiPSC was 250% higher than for aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.

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Hepatocyte Differentiation Metabolism

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