Investigators report on what they claim is a fast and efficient method that allows induced pluripotent stem cells (iPSCs) to be derived from fibroblasts in stirred suspension bioreactors (SSBs) rather than adherent cultures. The technique’s developers, at the University of Calgary’s department of medicine, claim their method allows the production of iPSCs that exhibit multineage differentiation in vitro and in vivo and contribute to the germline in chimeric mice.
Derek E. Rancourt, Ph.D., and colleagues, suggest suspension reprogramming could address a major current bottleneck in iPSC production and enable efficient, reproducible, and large-scale generation of cells for both research and potentially clinical applications. They describe their approach in Nature Methods in a paper titled “Derivation of iPSCs in stirred suspension bioreactors.”
Reprogramming fibroblasts into iPSCs is currently a lengthy and inefficient process in which cells are maintained, fed, and passaged in adherent cultures, as fibroblasts are substrate-dependent. In contrast to adherent culturing used to generate iPSCs from fibroblasts, SSBs provide a more homogenous environment for expanding both embryonic stem cells and iPSCs as aggregates, with even small, laboratory-scale SSBs effectively enabling the expansion of billions of cells in just a few days.
The Calgary team investigated whether SSBs could also represent a suitable environment for deriving iPSCs from mouse embryonic fibroblasts (MEFs) without the need to resort to adherent culturing. They first transduced MEFs taken from 129/Sv strains using retroviral vectors encoding the reprogramming factors Oct4, Sox2, Klf4, and c-Myc and then two days later transferred the cells to a 100 ml, 100 rpm SSB.
Aggregates that resembled ESC aggregates cultured in SSBs developed after three days and expressed alkaline phosphatase from day five. Growing aggregates were passaged every four days from day 10 onward. Within 16 days, 90–100% of the resulting suspension culture iPSCs (SiPSCs) expressed pluripotency markers including Rex1.
Encouragingly, iPSCs could also be generated in the SSBs when the MEFs were transduced with vectors carrying Oct4, Sox2, and KLlf4 but without c-Myc. Although fewer iPSCs were generated when c-Myc was omitted, 10 million iPSCs could still be produced in two weeks. Moreover, on transfer to adherent culture the three-factor SiPSCs formed ESC-like colonies that stained positively for the pluripotency markers. And when the SiPSC colonies were dissociated into single cells and cultured on agar, they generated cystic embryoid bodies that differentiated spontaneously into all three germ layers when transferred to gelatin plates. The cells could also be directed to differentiate into bone and cartilage lines.
Importantly, when the three-factor siPSCs were injected into C57BL/6 mouse blastocysts, they contributed to chimeric mice as evidenced by variations in coat color patterns, and a number of the chimeras transmitted the iPSC-originated coat color through the germline to their offspring.
The authors conclude that “combined with new methods of reprogramming that do not use integrating constructs, SiPSC technology has the potential to accelerate and standardize iPSC research for basic and clinical applications.”