Mammalian numb-interacting protein 1/dual oxidase maturation factor 1 directs neuronal fate in stem cells.

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFß3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFß3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures.

Application of stem cells in bone repair.

Bone has the ability to repair minor injuries through remodeling. However, when the host source of osteoprogenitors is compromised at the defect site, one effective treatment may be cell-based therapy, as it replenishes the area of bone loss with cells possessing osteogenic potential. This review is a concise comparison of different types of stem cells that have the potential to be used in tissue-engineered scaffolds for bone repair. The clinical use of mesenchymal stem or stromal cells isolated from the bone marrow for treating various diseases has been well documented. However, the scarcity of these cells prompts the search for alternative sources of multipotential cells such as amniotic fluid stem cells and umbilical cord perivascular cells. Embryonic stem cells are another controversial source of cells with osteogenic potential. These cells have the ability to differentiate into all cell types of the adult body. Issues such as the use of human embryos and the risk of contamination from animal-derived culture components continue to prevent the therapeutic use of ESCs. As a result, abundant research has been carried out to design defined culture conditions for culturing ESCs, and alternative strategies such as the generation of induced pluripotent stem cells are being developed to eliminate the need for using embryos for cell derivation. In addition to the cell source, the ability to control stem cell differentiation into functional bone and the choice of biomaterial are also paramount objectives that are being examined in research and clinical trials.