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 that human naïve iPSCs (niPSCs) can be generated directly 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 free of transgenes, karyotypically normal, and display transcriptional, epigenetic and metabolic features indicative of the naïve state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs could also be generated at low frequency under conventional conditions, our microfluidics approach will allow robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.

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.

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are differently affected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufficient to instruct a functional metabolic zonation during the differentiation of human embryonic stem cells (hESCs) from endoderm toward terminally differentiated hepatocytes, thus mimicking the in vivo situation. For this purpose, a microfluidic device was designed for the generation of a stable oxygen gradient. The oxygen gradient was applied to differentiating hESCs at the pre-hepatoblast stage. The definitive endoderm and hepatic endoderm cells were characterized by the expression of the transcription factor SOX-17 and alpha-fetoprotein (AFP). Immature and mature hepatocytes were characterized by hepatocyte nuclear factor 4-alpha (HNF-4α) and albumin (ALB) expression and also analyzed for cytochrome P450 (CYP3A4) zonation and glycogen accumulation through PAS staining. Metabolic zonated genes expression was assessed through quantitative real time PCR. Application of the oxygen gradient during differentiation induced zonated glycogen storage, which was higher in the hepatocytes grown in high pO2 compared to those grown in low pO2. The mRNA levels of glutamine synthetase (GLUL), beta-catenin (CTNNB) and its direct target cyclin D1 (CCND1) showed significantly higher expression in the cells grown in low pO2 compared to those grown in high pO2. On the contrary, carbamoyl-phosphate synthetase 1 (CPS1), ALB, the proliferative marker ki67 (MKI67) and cyclin A (CCNA) resulted to be significantly higher expressed in cells cultured in high pO2 compared to those cultured in low pO2. These results indicate that the oxygen gradient generated in our device can instruct the establishment of a functional metabolic zonation in differentiating hESCs. The possibility to obtain differentiated hepatocytes in vitro may allow in the future to deepen our knowledge about the physiology/pathology of hepatocytes in relation to the oxygen content.