Notch Signaling Regulates Motor Neuron Differentiation of Human Embryonic Stem Cells

In the pMN domain of the spinal cord, Notch signaling regulates the balance between motor neuron differentiation and maintenance of the progenitor state for later oligodendrocyte differentiation. Here, we sought to study the role of Notch signaling in regulation of the switch from the pMN progenitor state to differentiated motor neurons in a human model system. Human embryonic stem cells (hESCs) were directed to differentiate to pMN‐like progenitor cells by the inductive action of retinoic acid and a Shh agonist, purmorphamine. We found that the expression of the Notch signaling effector Hes5 was induced in hESC‐derived pMN‐like progenitors and remained highly expressed when they were cultured under conditions favoring motor neuron differentiation. Inhibition of Notch signaling by a γ‐secretase inhibitor in the differentiating pMN‐like progenitor cells decreased Hes5 expression and enhanced the differentiation toward motor neurons. Conversely, over‐expression of Hes5 in pMN‐like progenitor cells during the differentiation interfered with retinoic acid‐ and purmorphamine‐induced motor neuron differentiation and inhibited the emergence of motor neurons. Inhibition of Notch signaling had a permissive rather than an inductive effect on motor neuron differentiation. Our results indicate that Notch signaling has a regulatory role in the switch from the pMN progenitor to the differentiated motor neuron state. Inhibition of Notch signaling can be harnessed to enhance the differentiation of hESCs toward motor neurons. Stem Cells 2015;33:403–415Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110586/1/stem1873.pd

Systemically transplanted mesenchymal stem cells induce vascular-like structure formation in a rat model of vaginal injury.

The beneficial effect of mesenchymal stem cells (MSCs) on wound healing is mostly attributed to a trophic effect that promotes angiogenesis. Whether MSCs can contribute to the formation of new blood vessels by direct differentiation is still controversial. Pelvic floor dysfunction (PFD) is a group of disorders that negatively affect the quality of women's lives. Traditional vaginal surgical repair provides disappointing anatomical outcome. Stem cell transplantation may be used to supplement surgery and improve its outcome. Here we aimed to examine the engraftment, survival, differentiation and angiogenic effect of transplanted MSCs in a vaginal injury rat model. MSCs were obtained from the bone marrow of Sprague Drawley (SD) rats, expanded and characterized in vitro. The MSCs expressed CD90 and CD29, did not express CD45, CD34, CD11b and CD31 and could differentiate into osteogenic, chondrogenic and adipogenic lineages. Cells were labeled with either PKH-26 or GFP and transplanted systemically or locally to female SD rats, just after a standardized vaginal incision was made. Engraftment after local transplantation was less efficient at all-time points compared to systemic administration. In the systemically transplanted animal group, MSCs migrated to the injury site and were present in the healed vagina for at least 30 days. Both systemic and local MSCs transplantation promoted host angiogenesis. Systemically transplanted MSCs created new vascular-like structures by direct differentiation into endothelium. These findings pave the way to further studies of the potential role of MSCs transplantation in improving surgical outcome in women with PFD

Axin-mediated CKI phosphorylation of β-catenin at Ser 45: a molecular switch for the Wnt pathway

The Wnt pathway controls numerous developmental processes via the β-catenin–TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of β-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic β-catenin associates with APC and axin and is continuously phosphorylated by GSK-3β, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3β from phosphorylating β-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of β-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces β-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate β-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3β and is both necessary and sufficient to initiate the β-catenin phosphorylation–degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway

Pseudosubstrate regulation of the SCF(β-TrCP) ubiquitin ligase by hnRNP-U

β-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(β-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IκBα and β-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIκBα peptide, or by a specific point mutation in the E3RS WD region, indicating an E3–substrate-type interaction. However, unlike pIκBα, which is targeted by SCF(β-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(β-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase