Speaker
Description
The emergence of human induced stem cells (hiPSCs) circumvents the ethical controversy of human embryonic pluripotent stem cells(hPSCs), introducing great feasibility in stem cell research. There are various methods to trigger the cellular reprogramming process, including viruses, plasmids, episomal plasmids, RNAs, proteins, and small molecules. mRNA-based reprogramming has up to 4% efficiency (0.01%~0.1% generally), and iPSCs colonies emerge faster without any genome integration; Moreover, mRNA can be easily produced by any biological laboratory, which increases the flexibility of this strategy. The effective delivery of these pluripotency-related transcription factors is critical for successful reprogramming. Thus, we employ our expertise in microfluidics in mRNA reprogramming, enabling large-scale and high-efficiency production of high-quality hiPSCs. By implementing the reprogramming procedure inside a confined microfluidic chamber (27 mm2-channel, only 1500 fibroblasts required, and 20μL medium consumption daily), we find that in 15 days the yield of hiPSCs has elevated >50 folds compared to the standard wells. Also, the low number of cells required makes reprogramming feasible even when there is limited availability of biological samples. The volume of the system (~5 μL/channel) reduces the cost of reagents >100-fold with a considerable effect on the feasibility of reprogramming a large cohort of patients’ cells to study pathology. For example, high-passage fibroblasts from Alzheimer's patients (AD, ~70 years old) have been converted into iPSCs in our microfluidic chips. AD is a neurodegenerative disease that induces progressive dementia in elder people. Unclear pathology of AD hampers our prevention at a young age. However, AD-iPSCs allow us to build the disease models, studying its pathology from the early pluripotent stage with a specific genetic and epigenetic mutation. Disease-specific iPSCs also help to test the efficacy of current drugs and the development of new medicine.
In terms of obtaining patient cells, fibroblasts(FB) are from biopsies, which bring invasive damage to the patients, especially to kids, thus peripheral blood monocyte cells(PBMCs) are an ideal alternative cell supply. Blood collection is less invasive, also PBMCs carry fewer gene mutations. Blood outgrowth endothelial cells (BOECs) isolated from PBMCs are an optimal cell source to produce iPSCs. They are an adherent cell population compared to T, B, or other circulating cells in the blood. Besides, BOECs are highly proliferative and less resistant to exogenous mRNA transfections. BOEC-iPSCs start to appear fastly in our microfluidic chips on day 12 when only daily mRNA transfection is performed. Furthermore, instead of verifying the pluripotency of AD-iPSCs, DMD-iPSCs, and BOEC-iPSCs via 2D differentiation, we use the 3D organoids technique to fully characterize their pluripotency and differentiation ability. Our organoids harbor a group of self-renewing FB-iPSCs or BOECs-iPSCs, resembling similar organ physiological conditions in vivo, and are proven capable of all 3 germ layer differentiation.
Various types of patient cells have been efficiently reprogrammed into iPSCs in our microfluidic chips by mRNA. It is both time and cost-saving, facilitating the disease pathology study and regenerative medicine research.
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