The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and func- IntroductionThe presently accumulated evidence indicates that adult bone marrow (BM) contains at least 2 populations of stem cells: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), responsible for the generation of the BM microenvironment. 1 Intriguingly, several reports have demonstrated the ability of MSCs to differentiate toward derivatives of germ layers other than mesoderm. [2][3][4][5][6] Although it is still unclear whether widely multipotent cells do exist in vivo and if they play a significant role in tissue repair and turnover, the ability to generate in vitro cells that, under defined culture conditions, display a very high developmental plasticity is nonetheless of important clinical relevance.Until now, the most convincing evidence, although debated, 7 of the possibility to grow in culture a population of widely multipotent cells in humans has been obtained only for BM, 8 while a similar feature has been just postulated for other adult human tissues. 9 We therefore planned to verify if human multipotent adult stem cells (hMASCs) could be produced from other adult human organs on top of BM, and we used this latter as a control/reference tissue.By systematically using a highly reproducible method, we were able to grow in culture cell lines from adult human liver, heart, and BM. These cell lines, once cloned at single-cell level, maintained the in vitro properties of parental lines, including the capability to differentiate into morphologically mature and functionally competent cells, even of tissues embryologically not related to the one of origin.Finally, we performed a comparative in vitro analysis on hMASCs originated from the 3 different sources with respect to immunophenotype, growth kinetics, specific transcriptional settings, telomerase activity, and global gene expression profile. Altogether the obtained result...

Abstract-To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of Ϸ3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

BackgroundNiemann Pick C (NPC) disease is a neurovisceral lysosomal storage disorder due to mutations in NPC1 or NPC2 genes, characterized by the accumulation of endocytosed unesterified cholesterol, gangliosides and other lipids within the lysosomes/late endosomes. Even if the neurodegeneration is the main feature of the disease, the analysis of the molecular pathways linking the lipid accumulation and cellular damage in the brain has been challenging due to the limited availability of human neuronal models.ObjectiveThe aim of this study was to develop a human neuronal model of NPC disease by inducing neuronal differentiation of multipotent adult stem cells (MASC) isolated from NPC patients.MethodsStem cells were isolated from 3 NPC patients and 3 controls both from skin biopsies and previously established skin fibroblast cultures. Cells were induced to differentiate along a neuronal fate adapting methods previously described by Beltrami et al, 2007. The surface immunophenotype of stem cells was analyzed by FACS. Stem cell and neuronal markers expression were evaluated by immunofluorescence. Intracellular accumulation of cholesterol and gangliosides were assessed by filipin staining and immunofluorescence, respectively. A morphometric analysis was performed using a Neurite outgrowth image program.ResultsAfter 3 passages in selective medium, MASC isolated either from skin biopsies or previously established skin fibroblast cultures displayed an antigenic pattern characteristic of mesenchymal stem cells and expressed the stem cell markers Oct-4, Nanog, Sox-2 and nestin. A massive lysosomal accumulation of cholesterol was observed only in cells isolated from NPC patients. After the induction of neural differentiation, remarkable morphologic changes were observed and cells became positive to markers of the neuronal lineage NeuN and MAP2. Differentiated cells from NPC patients displayed characteristic features of NPC disease, they showed intracellular accumulation of unesterified cholesterol and GM2 ganglioside and presented morphological differences with respect to cells derived from healthy donors.In conclusion, we generated a human neuronal model of NPC disease through the induction of differentiation of stem cells obtained from patient’s easily accessible sources. The strategy described here may be applied to easily generate human neuronal models of other neurodegenerative diseases.

Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. Methods and results: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

We discuss the problem of local attainability for finite-dimensional nonlinear control systems with quite general assumptions on the target set. Special emphasis is given to control-affine systems with a possibly nontrivial drift term. To this end, we provide some sufficient conditions ensuring local attainability, which involve geometric properties both of the target itself (such as a notion of generalized curvature), and of the Lie algebra associated with the control system. The main technique used is a convenient representation formula for the power expansion of the distance function along the trajectories, made at points sufficiently near to the target set.We notice that Petrov's condition is a first order condition in the sense that it involves only admissible velocities.From another point of view, this condition requires d S to be a sort of Lyapunov function for the system. Indeed, from a geometrical point of view, Petrov's condition states that at every point x of a neighborhood of the target there exists an admissible control u x ∈ U such that the corresponding trajectory points sufficiently toward the target. Moreover, the scalar product between the admissible velocity f (x, u x ) and the gradient of the distance is uniformly bounded away from zero.Petrov's condition is very strong, even if it is weaker than full controllability, which requires that every initial state can be steered to any final state in finite time along admissible trajectories. Moreover, it can be also shown that Petrov's condition is equivalent to the Lipschitz continuity of the minimal time function T up to the boundary of S (see [23]). However, it is also very easy to give simple examples where it fails. For instance, in R 2 take S = {0} and (ẋ(t),ẏ(t)) = (y(t), u(t)), where u : R → [−1, 1] is measurable: Petrov's condition fails on the x-axis.1.3. Higher order condition for pointwise target. When Petrov's condition is not satisfied, i.e., the trajectories of the system do not approach the target at the first order, it is natural to search for higher order conditions, which involve higher order terms in a convenient expansion of the trajectory itself. These conditions will be related to some properties of the Lie algebra generated by the family of vector fields associated with the system (see [15] for a complete introduction).In the early 1960s, Kalman proved the following result. Assume that f is linear, i.e., f (x, u) = Ax + Bu, where A ∈ Mat n×n (R), B ∈ Mat n×m (R) are two constant matrices, and S = {0}. Then the following are equivalent: 1. the system is controllable to the equilibrium point 0, i.e., every point can be steered to the origin in finite time; 2. the matrix (B|AB|A 2 B| . . . |A n−1 B) has full rank (equals n). The second condition above is the celebrated Kalman rank condition, and implies the Hölder continuity of T , with exponent depending on the smallest 0 ≤ k ≤ n − 1 such that the matrixhas full rank.Later, in the 1970s, several generalizations, mainly concerning the case when target set S is an equilibrium po...