This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
Human Fibroblast Reprogramming to Naïve Induced Pluripotent Stem Cells in Microfluidics
Nicola Elvassore
University of Padua
Corresponding Author
Graziano Martello
University of Padua
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Induced pluripotent stem cells (iPSCs) are generated by expression of transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naïve iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here we report a detailed protocol for the generation of human naïve iPSCs (niPSCs) from less than 1000 primary human somatic cells without stable genetic manipulation by delivery of modified messenger RNAs with microfluidics. Expression of OSKM in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal, and display transcriptional, epigenetic and metabolic features indicative of the naïve state. Specifically, we describe the production of microfluidics chips, the reprogramming of somatic cells into niPSCs and their expansion out of the microfluidics chips. Our microfluidics approach will allow robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.
Keywords
reprogramming; microfluidics, mmRNAs, naive pluripotency
Figure 1
Figure 2
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 10 Jan, 2019
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
Human Fibroblast Reprogramming to Naïve Induced Pluripotent Stem Cells in Microfluidics
Nicola Elvassore
University of Padua
Corresponding Author
Graziano Martello
University of Padua
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 10 Jan, 2019
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Induced pluripotent stem cells (iPSCs) are generated by expression of transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naïve iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here we report a detailed protocol for the generation of human naïve iPSCs (niPSCs) from less than 1000 primary human somatic cells without stable genetic manipulation by delivery of modified messenger RNAs with microfluidics. Expression of OSKM in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal, and display transcriptional, epigenetic and metabolic features indicative of the naïve state. Specifically, we describe the production of microfluidics chips, the reprogramming of somatic cells into niPSCs and their expansion out of the microfluidics chips. Our microfluidics approach will allow robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.
Figure 1
Figure 2