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| Status |
Public on Apr 10, 2016 |
| Title |
High-efficiency cellular reprogramming with microfluidics |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-β and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (μ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.
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| Overall design |
We performed cell reprogramming of human foreskin BJ fibroblasts, seeded on a feeder layer of inactivated human newborn foreskin fibroblasts NuFF-RQs, by modified mRNA (mmRNA) daily transfections of OCT4, SOX2, KLF4,c-MYC, NANOG, LIN28, and nuclear GFP for 18 days. The process was performed in parallel in 6-well plates and within a microfluidic system. 48 hours after last reprogramming mmRNA transfection, 4 freshly derived colonies of comparable size were selected both from the microfluidic system and from a parallel reprogramming experiment in well. After a 24 h conditioning in StemMACS Brew XF medium, each colony was split in two halves: one was stopped at passage p0 and used for total RNA extraction, the other one was further expanded in feeder-free conditions for 3 passages (p3) in well before total RNA extraction from another sectioned colony of comparable size. Thus, the overall study includes: (i) N=4 p0 hiPS colonies reprogrammed in well, (ii) N=4 p0 hiPS colonies reprogrammed in microfluidics, (iii) N=4 p3 hiPS colonies (the same as in (i)) expanded in well, (iv) p3 hiPS colonies (the same as in (ii)) expanded in well.
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| Citation(s) |
27088312 |
| Submission date |
Jul 17, 2014 |
| Last update date |
Jan 09, 2018 |
| Contact name |
Camilla Luni |
| Organization name |
University of Bologna
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| Department |
DICAM
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| Street address |
Via Terracini 28
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| City |
Bologna |
| ZIP/Postal code |
40131 |
| Country |
Italy |
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| Platforms (1) |
| GPL17077 |
Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray 039381 (Probe Name version) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA255510 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE59534_RAW.tar |
197.9 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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