Generation of functional hepatocytes by forward programming with nuclear receptors

Tomaz, R. A.; Zacharis, E. D.; Bachinger, F.; Wurmser, A.; Yamamoto, D.; Petrus-Reurer, S.; Morell, C. M.; Dziedzicka, D.; Wesley, B. T.; Geti, I.; Segeritz, C.-P.; de Brito, M. C.; Chhatriwala, M.; Ortmann, D.; Saeb-Parsy, K.; Vallier, L.

doi:10.1101/2022.06.23.497371

bioRxiv · 10.1101/2022.06.23.497371

Generation of functional hepatocytes by forward programming with nuclear receptors

Tomaz, R. A.·Zacharis, E. D.·Bachinger, F.·Wurmser, A.·Yamamoto, D.·Petrus-Reurer, S.·Morell, C. M.·Dziedzicka, D.·Wesley, B. T.·Geti, I.·Segeritz, C.-P.·de Brito, M. C.·Chhatriwala, M.·Ortmann, D.·Saeb-Parsy, K.·Vallier, L.

View original source ↗Export metadata ↓

Abstract

Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSC) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of 3 hepatocyte nuclear factors (HNF1A, HNF6 and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.

Explore related subjects

developmental biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Tomaz, R. A., Zacharis, E. D., Bachinger, F., Wurmser, A., Yamamoto, D., Petrus-Reurer, S., Morell, C. M., Dziedzicka, D., Wesley, B. T., Geti, I., Segeritz, C.-P., de Brito, M. C., Chhatriwala, M., Ortmann, D., Saeb-Parsy, K., Vallier, L.. 2022-06-26. Generation of functional hepatocytes by forward programming with nuclear receptors. https://doi.org/10.1101/2022.06.23.497371

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Prenatal THC exposure alters metabolic gene expression and KEAP1 regulation in the mouse placental labyrinth

Introduction: Prenatal cannabinoid exposure may influence fetal development through changes in placental biology, yet its effects on metabolic gene regulation remain poorly understood. We examined the effects of {Delta}9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on the mouse placental labyrinth zone. Methods: Pregnant C57BL/6JRj mice received THC (5 mg/kg), CBD (5 mg/kg), or vehicle daily by intraperitoneal injection from gestational day 5 to 17.5. Targeted NanoString profiling and pathway analysis were combined with RT-qPCR and protein measurements. Results: THC exposure reduced glutaminase (Gls), Kelch-like ECH-associated protein 1 (Keap1), and FMS-like tyrosine kinase 3 (Flt3) expression (false discovery rate = 0.0904 for each). Pathway analysis identified transcriptional changes in the nine-gene arginine-metabolism set (false discovery rate = 0.0527). No genes or pathways met the significance criteria following CBD exposure. RT-qPCR confirmed lower Keap1 expression with sexes pooled and identified lower Gls and Flt3 expression in male samples. KEAP1 protein abundance increased despite lower Keap1 mRNA, while nuclear factor erythroid 2-related factor 2 localization and heme oxygenase 1 (Hmox1) expression showed no significant differences. Discussion: These findings identify amino acid metabolic gene expression and KEAP1 regulation as complementary components of the placental response to prenatal THC exposure, connecting transcriptional changes with protein regulation within the placental exchange region.

developmental biology↗

bioRxiv2026

Mutually Repressive Transcription Factors Drive Intestinal Maturation for Weaning

In mammals, rapid neonatal growth depends on specialized intestinal enterocytes that absorb nutrients from milk. These cells exhibit distinct functions, morphologies, and gene expression programs that support neonatal nutrition before transitioning to an adult state at weaning. However, the transcriptional mechanisms that establish neonatal enterocyte identity and coordinate their maturation remain poorly understood. Here we show that the transcription factors BLIMP1 and CASZ1 form a central switch in the timing of enterocyte maturation. While BLIMP1 marks and promotes neonatal enterocytes, CASZ1 is expressed in adult enterocytes and is required for the proper timing of their emergence. Genetic analysis indicates that these neonatal and adult factors are mutually repressive, forming a genetic switch underlying weaning. This regulatory mechanism also controls glucocorticoid-induced maturation, a process relevant to both normal weaning and the clinical use of corticosteroids to promote epithelial maturation in preterm infants. We show that corticosteroids act directly on the intestinal epithelium and require CASZ1 to induce maturation. Together, these findings reveal a transcriptional mechanism that coordinates intestinal epithelial identity with the changing nutritional demands of early life and mediates the response to maturation-promoting hormones.

developmental biology↗

bioRxiv2026

Functional Data Clustering of Filopodia Dynamics

Notch signaling plays a significant role in tissue patterning. During patterning of the fruit fly dorsal thorax peripheral nervous system, basal filopodia extend the spatial range of cell-cell communication, yet it is still unclear how Notch is activated in long range signaling via filopodia interaction. Filopodia undergo dynamic length changes during their lifetime, which could supply the mechanical pulling force required for notch activation. However, their dynamics are often characterized using individual scalar values such as maximum length and extension rate which could obscure more intricate dynamics. Our hypothesis is that signaling filopodia exhibit distinct and recurrent temporal patterns which can be identified by a consideration of their complete lifetime trajectories. Our work analyzes 60 filopodia trajectories at the basal surface of the fly dorsal thorax during patterning. Length profiles were smoothed using cubic B-splines and clustered by first reducing them to their dominant functional principal component followed by the application of various clustering algorithms. We found that 94.29% of the overall trajectory variation is explained by the first four functional principal components which account for variations in maximal length, extension and retraction rate, duration of stationary phase and length fluctuation frequency. Five recurrent temporal profiles (dynamic extension modes) with varying trajectory shapes and physical attributes, were identified using trimmed K-means. These findings demonstrate that whereas the major source of structural variation in filopodia profiles remains the length, extension rate and retraction rate, nontrivial combinations of these create distinct dynamic modes of extension which provide new insight into their length profiles. Our work thus offers a quantitative framework for future research connecting filopodia dynamic extension modes to productive Notch activation.

developmental biology↗