IntroductionHuman mesenchymal stem cells (hMSCs) from bone marrow are characterized by their ability of self-renewal paired with the capacity to differentiate into diverse mesodermal cell types such as osteoblasts, chondrocytes, and adipocytes. 1,2 Moreover, hMSCs were shown to give rise to cells beyond the germ layers with visceral mesoderm, neuroectoderm, or endoderm characteristics. [2][3][4] Additional functions have been reported for hMSCs in providing cytokine and growth factor support for the expansion of hematopoetic 5 and embryonic stem cells, 6 or by playing an immunomodulatory role. 7 One of the most remarkable but least understood findings is the ability of hMSCs to migrate from bone marrow or peripheral blood into damaged tissues. Transplantation experiments in animals and patients demonstrated that mesenchymal stem cells migrate to sites of injury, where they enhance wound healing, 8 support tissue regeneration following myocardial infarction, 9 home to and promote the restoration of bone marrow microenvironment after damage by myeloablative chemotherapy, 10 or help to overcome the molecular defect in children with osteogenesis imperfecta. 11 Another interesting observation is that systemically delivered hMSCs are mobilized to and integrate into tumor tissue. 12 Taken together, these exciting features have rendered hMSCs a promising tool for tissue engineering 13 as well as multiple cell and gene therapy strategies. [14][15][16] Detailed studies have demonstrated that homing of hematopoetic stem cells from blood into bone marrow or their mobilization from bone marrow into blood and tissues is mainly controlled by cytokines/chemokines, adhesion molecules, and proteolytic enzymes. [17][18][19] However, little is known about the molecular mechanisms regulating cell movement and relocalization in hMSCs.A key requirement for cells to reach distant target sites is the ability to traverse the protein fibers of the extracellular matrix (ECM) which is present between cells of all tissue types. 20 Basement membranes represent a specialized form of the ECM that separate epithelium or endothelium from stroma by a dense layer of ECM. To overcome these matrix barriers, migrating cells require specific proteolytic enzymes. Besides some serine-and cysteineproteinases, in particular the matrix metalloproteinases (MMPs) consisting of more than 24 zinc-dependent endopeptidases, are capable of degrading ECM components. Consequently, MMPs are found to be involved in various physiologic and pathologic processes. 21 The 2 gelatinases, MMP-2 and MMP-9, preferentially cleave denatured collagens (gelatin), laminin, and collagen type IV as the major constituent of basement membranes. 20,21 Biosynthesis and activity of the gelatinases are associated with the invasive capacity of various cell types such as leukocytes, endothelial cells, and metastasizing tumor cells. 22-24 MMP-2 and MMP-9 are secreted from the cells as latent zymogens which are rapidly complexed by their specific endogenous inhibitors, the tissue inhibitor of ...

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. A major challenge for the translation of neuronal reprogramming into therapy is whether the adult human brain contains cell populations amenable to direct somatic cell conversion. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks can be reprogrammed into neuronal cells by retrovirus-mediated coexpression of the transcription factors Sox2 and Mash1. These induced neuronal cells acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons, indicating their capability of integrating into neural networks. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue-nonspecific alkaline phosphatase promoter corroborated the pericytic origin of the reprogrammed cells. Our results raise the possibility of functional conversion of endogenous cells in the adult human brain to induced neuronal fates.

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is ~19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on "the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)--fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.The microtubules of the mitotic spindle are in some form of assembly steady-state with a soluble pool of tubulin subunits (1-3), but the actual pathways of tubulin exchange are unresolved (4-6). The rate of tubulin exchange with nonkinetochore spindle and astral microtubules appears surprisingly rapid (7-9). At metaphase and early anaphase, the assembly of spindle and astral microtubules is near steady state; since the amount of polymer is approximately constant, the rate of tubulin association must equal the rate of dissociation. If polymerization is blocked at metaphase by abrupt treatment with high concentrations of colchicine or colchicine-like drugs, nonkinetochore microtubule depolymerization follows exponential kinetics with a half-time of 6.5 s. This half-time is equivalent to an initial tubulin dissociation rate of 992 dimers/s/microtubule, if the initial average length of nonkinetochore spindle microtubules is 5.5 #m, half the distance between the chromosom...

The regulative network conducting adult stem cells in endogenous tissue repair is of prime interest for understanding organ regeneration as well as preventing degenerative and malignant diseases. One major signal transduction pathway which is involved in the control of these (patho)physiological processes is the Wnt pathway. Recent results obtained in our laboratories showed for the first time that canonical Wnt signaling is critically involved in the control of the migration/invasion behaviour of human mesenchymal stem cells (hMSC). In the first part of this review, we describe that the regenerative state is closely linked to the activation of the Wnt pathway. Central hallmarks of activated stem cells are recapitulated in a similar way also in cancer metastasis, where the acquisition of an invasive cancer stem cell phenotype is associated with the induction of Wnt-mediated epithelial to mesenchymal transition (EMT). In the second part, the influence of proinflammatory cytokines such as transforming growth factor (TGF-)beta1, interleukin (Il-)1beta, and tumor necrosis factor (TNF-)alpha is discussed with regard to the invasive characteristics of hMSC. In this context, special attention has been paid on the role of matrix metalloproteinases (MMPs), such as MMP-2, MMP-9 and membrane type 1 (MT1)-MMP, as well as on the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. Putative cross-talks between different signal transduction pathways that may amplify the invasive capacity of this stem cell population are also discussed. Finally, the consequences towards future drug-mediated therapeutical modifications of Wnt signaling in stem cells and tumor cells are highlighted.