Robust protein-based engineering of hepatocyte-like cells from human mesenchymal stem cells.

Background: Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs).

Methods: MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4).

Development of a protein-based system for transient epigenetic repression of immune checkpoint molecule and enhancement of antitumour activity of natural killer cells.

Background: Immune checkpoint blockade (ICB) therapy improved the prognosis of cancer patients, but general administration of ICBs occasionally induces side effects that include immune-related adverse events and tumour hyper-progression. Here, we established a protein-based system, by which endogenous expression of IC molecule in natural killer (NK) cells was transiently repressed on enhancement of their antitumour activity.

Methods: A protein-based genome modulator (GM) system is composed of a transcription activator-like effector (TALE), DNA methyltransferase and a newly identified potent cell-penetrating peptide with nuclear-trafficking property named NTP.

Identification of a cell-penetrating peptide applicable to a protein-based transcription activator-like effector expression system for cell engineering.

Cellular reprogramming is a promising technology in regenerative medicine, but most studies have been performed by using expression vectors. For future clinical applications, it is necessary to establish a system in which cell engineering can be manipulated without any risk of damaging the genome. Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11).

Deregulation of miR-92a expression is implicated in hepatocellular carcinoma development.

MicroRNAs (miRNAs) belong to a class of the endogenously expressed non-coding small RNAs which primarily function as gene regulators. Growing evidence suggests that miRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. The miR-17-92 cluster especially is markedly overexpressed in several cancers, and is associated with the cancer development and progression.

Engineered FADD Induces Apoptosis via an Artificial Death-Inducing Signaling Complex (DISC).

An engineered Fas-associated death domain protein (FADD), 2DEDplusE-made previously by fusing the tandem DEDs of FADD to the E protein of lambda phage-greatly enhances apoptosis-inducing activity in adherent cells in vitro. To investigate the mechanism of apoptosis-inducing activity of this engineered FADD, we compared the apoptosis-inducing activity of various other engineered FADDs. The tandem DED of 2DEDplusE contributed most to the enhancement of apoptosis, and the E protein contributed moderately.

Revival of apoptotic cells that display early-stage dynamic membrane blebbing.

The critical point at which apoptosis becomes irreversible and how cells attain an anti-apoptotic state remain unknown. Here, we report that apoptotic cells undergoing early-stage dynamic membrane blebbing revive. We examined this phenomenon in cell lines that stably express 2DED2DD, a modified FADD produced by fusing the tandem death effector domains (DEDs) and tandem death domains (DDs).

Modifications enhance the apoptosis-inducing activity of FADD.

The ability to enhance apoptosis-inducing activity in specific cells, despite the presence of cellular antiapoptotic proteins, would allow the removal of target cells from a cell population. Here, we show that modification of Fas-associated protein with death domain (FADD) by fusing the tandem death effector domains (DED) of FADD to the E protein of lambda phage, a head coat protein with self-assembly activity, greatly increases the apoptosis-inducing activity of FADD in both adherent NIH3T3 and HEK293 cells. Induction of apoptosis in cell lines that stably express modified FADD (2DEDplusE) resulted in rapid blebbing, and most cells detached from the flask within 5 h.