Microfluidic reprogramming to pluripotency of human somatic cells

Abstract

Induced pluripotent stem cells (iPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and intensive workload of the reprogramming process. Here, we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 days using microfluidics. We successfully downscaled an 8day protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160±20 in a single 27-mm 2 microfluidic chamber) 15 days after seeding ~1500 cells per independent chamber and under xeno-free defined conditions. Only ~20-µL medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization due to the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold cost reduction of raw material compared to a standard multiwell culture condition. This system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.

The transcriptional regulator ZNF398 mediates pluripotency and epithelial character downstream of TGF-beta in human PSCs

“…Reprogramming. All reprogramming experiments were performed in microfluidics in hypoxia conditions (37°C, 5% CO 2 , 5% O 2 ) 48 . The protocol for reprogramming experiments was optimised to transfect siRNA in order to test the requirement of ZNF398 for reprogramming.…”

“…Cells were transfected daily at 6 p.m. and fresh medium was given daily at 9 a.m. siRNAs were transfected at a final concentration of 20 nM at day 1, day 3 and day 5 (see Supplementary Table 1 for sequences of the siRNAs used) together with mRNAs. The dose of mRNAs transfected was gradually increased according to cell proliferation rate and transfection-induced cell mortality 48 .…”

“…We decided to test the function of ZNF398 in an orthogonal system, the induction of pluripotency from somatic cells. Reprogramming from somatic cells, such as fibroblasts, requires an early mesenchymal to epithelial transition (MET) followed by the activation of endogenous pluripotency factors 23,47,48 . We noticed that ZNF398 is expressed in human fibroblasts ( Supplementary Fig.…”

“…8a), raising the possibility that ZNF398 promotes acquisition of epithelial character and pluripotency from early stages of reprogramming. We reprogrammed human fibroblasts by delivery of mRNAs encoding for either OSKMNL 23,47,48 (OCT4, SOX2, KLF4, MYC, NANOG, LIN28A) or OSKM, in combination with siRNAs, allowing to test the requirement of endogenous ZNF398 for reprogramming ( Fig. 8a).…”

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

“…Notwithstanding the highly justified initial enthusiasm associated with the laudable discoveries that led to the development of the above method(s), it does appear today that the associated risks and complexities, as well as the time-consuming nature of the methods, make it necessary for us to search for a more efficient, economical, risk-free and ethically acceptable method of inducing either pluripotency or multi-potency (Brouwer et al, 2016;Borgohain et al, 2018;Kogut et al, 2018;Apostolou and Stadtfeld 2018;Badieyan and Evans;2019;Shivashankar 2019;Gagliano et al, 2019;Haake et al, 2019).…”