University of Twente Student Theses
Differentiation of hPSC derived embryoid bodies to atrial and ventricular CMs in a trapping system on a chip : Defining requirements for asymetric differentiation on chip
Wesselink, Britt Anna Maria (2022) Differentiation of hPSC derived embryoid bodies to atrial and ventricular CMs in a trapping system on a chip : Defining requirements for asymetric differentiation on chip.
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| Abstract: | Human pluripotent stem cells (hPSCs)-derived cardiomyocytes (CMs) have an advantage in studying regenerative medicine, disease modeling, drug screening, and early cardiomyogenesis. Ideal platforms should consist of both atrial and ventricular tissues due to the unique chamber-specific defects and drug-induced myopathies present in the atria and ventricles. BMS-753 is an essential signaling molecule in the differentiation of hPSCs towards atrial cardiomyocytes. Traditional culture plates make it impossible to asymmetrically differentiate atrial cardiomyocytes (AMs) and ventricular cardiomyocytes (VMs) due to diffusion of this signaling molecule. The main goal of this project is to determine the biological limitations of moving the embryoid body (EB) differentiation from standard well plate culture to a microfluidic device which can in future enable asymmetric differentiation to AMs and VMs. First, replication of the RAR-α Schwach et al.’s protocol was performed to rule out systematic errors. By performing flow cytometry analysis the generated AMs and VMs were characterized for determining the percentage of NKX2.5eGFP and COUP-TFIImCherry and fluorescence imaging is done to examine the NKX2.5eGFP and COUP-TFIImCherry expression. Based on the results, the following observations were made in regard to asymmetric differentiation on a chip. The student LAF in ML-1 is not suited for EB differentiation. A better alternative is the BIC ML-2 lab because pipettes are better calibrated and incubators are more stable. Second, EB growth over time using different initial cell seeding densities was measured. Cell seeding density does not have a lot of impact on the EB size, while it does have an influence on the percentages of VMs and AMs. Third, the transition in AM and VM induction using different BMS concentrations was tested. A mixed population is obtained using 0.5 and 1 µM BMS. Finally, three chip designs were designed, fabricated and tested by culturing EBs in the device. The trapping systems shown here partially worked for hPSCs differentiation towards VMs as could be seen by the proof-of-concept. All in all, some requirements were defined for differentiation of hPSCs derived EBs towards asymmetric differentiation of AMs and VMs in a microfluidic system. For the future, a model is presented that should be able to maintain a gradient of BMS-753 while trapping an embryoid body. |
| Item Type: | Essay (Master) |
| Faculty: | TNW: Science and Technology |
| Subject: | 42 biology, 52 mechanical engineering |
| Programme: | Biomedical Engineering MSc (66226) |
| Link to this item: | https://purl.utwente.nl/essays/93011 |
| Export this item as: | BibTeX EndNote HTML Citation Reference Manager |
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