The maintenance of genome integrity in stem cells (SCs) is critical for preventing cancer formation and cellular senescence. The immortal strand hypothesis postulates that SCs protect their genome by keeping the same DNA strand throughout life by asymmetrical cell divisions, thus avoiding accumulation of mutations that can arise during DNA replication. The in vivo relevance of this model remains to date a matter of intense debate. In this study, we revisited this long-standing hypothesis, by analyzing how multipotent hair follicle (HF) SCs segregate their DNA strands during morphogenesis, skin homeostasis and SC activation. We used three different in vivo approaches to determine how HF SCs segregate their DNA strand during cell divisions. Double labeling studies using pulse-chase experiments during morphogenesis and the first adult hair cycle showed that HF SCs incorporate two different nucleotide analogs, contradictory to the immortal strand hypothesis. The co-segregation of DNA and chromatin labeling during pulse-chase experiments demonstrated that label retention in HF SCs is rather a mark of relative quiescence. Moreover, DNA labeling of adult SCs, similarly to labeling during morphogenesis, also resulted in label retention in HF SCs, indicating that chromosome segregation occurs randomly in most of these cells. Altogether, our results demonstrate that DNA strand segregation occurs randomly in the majority of HF SCs during development, tissue homeostasis and following SC activation.

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Author contributions: P.S.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing; A.C.: Collection and assembly of data, Data analysis and interpretation, Manuscript writing; C.B.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Panagiota A. Sotiropoulou and Aurélie Candi contributed equally to this work.

Adult multipotent stromal cells (MSCs), also known as mesenchymal stem cells, represent an important source of cells for the repair of a number of damaged tissues. Both bone marrow (BM)-derived and amniotic MSCs expressed detectable surface levels of two (TRAIL-R2 and TRAIL-R4) out of four transmembrane TRAIL receptors. Although the best-characterized activity of TRAIL-R2 is the transduction of apoptotic signals, neither recombinant TRAIL (rTRAIL) nor infection with an adenovirus expressing TRAIL (Ad-TRAIL) induced cytotoxic effects on MSCs. Moreover, while rTRAIL did not affect proliferation or differentiation of MSCs along the osteogenic and adipogenic lineages, it significantly promoted the migration of human MSCs in range of concentrations comparable to that of soluble TRAIL in human plasma (100 pg/ml). Since rTRAIL induced the rapid phosphorylation of ERK1/2 in MSC cultures, and pretreatment with pharmacological inhibitors of the ERK1/2 pathway efficiently counteracted the rTRAIL-induced human MSC migration, these data indicate that ERK1/2 is involved in mediating the ability of rTRAIL to stimulate MSC migration. Taking into consideration that the soluble factors able to induce MSC migration have not been extensively characterized, our current data indicate that the TRAIL/TRAIL-R system might play an important role in the biology of MSCs.

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Author contributions: P.S.: Conception and design, assembly of data, data analysis and interpretation, manuscript writing; E.M., F.C., A.P.B., F.A., D.M., F.D, M.Grazia di Iasio and D.C.: Carrying out of experiments, collection and assembly of data, data analysis and interpretation; G.P.B.: Provision of study material, data analysis and interpretation; G.Z.: Conception and design, financial support, manuscript writing, final approval of manuscript.

Background The administration of therapeutic cell types, such as stem and progenitor cells, has gained much interest for the limitation or repair of tissue damage caused by a variety of insults. However, it is still uncertain whether the morphological and functional benefits are mediated predominantly via cell differentiation or paracrine mechanisms. Here, we assessed the extent and mechanisms of adipose tissue-derived stromal /stem cell (ASC)-dependent tissue repair in the context of acute myocardial infarction.

Methods and Results Human ASCs in saline or carrier alone were injected into the peri-infarct region in athymic rats following left anterior descending (LAD) coronary artery ligation. Cardiac function and structure was evaluated by serial echocardiography and histology. ASC-treated rats consistently exhibited better cardiac function, by all measures, than control rats 1 month following LAD occlusion. Left ventricular (LV) ejection fraction and fractional shortening was improved in the ASC group, while LV remodeling and dilation was limited in the ASC group compared to the saline control group. Anterior wall thinning was also attenuated by ASC treatment; and post-mortem histological analysis demonstrated reduced fibrosis in ASC-treated hearts, as well as increased peri-infarct density of both arterioles and nerve sprouts. Human ASCs were persistent at one month in the peri-infarct region, but these were not observed to exhibit significant cardiomyocyte differentiation.

Conclusion Human ASCs preserve heart function and augments local angiogenesis and cardiac nerve sprouting following myocardial infarction predominantly by the provision of beneficial trophic factors.

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Author contributions: L.C.: Conception and design, Collection and assembly of data; Data analysis and interpretation; Manuscript drafting; B.H.J.: Conception and design; Data analysis and interpretation; Manuscript revision; T.G.C.: Collection and assembly of data; J.T.: Collection and assembly of data; M.C.F.: Collection and assembly of data; P.-S.C.: Data analysis and interpretation. Manuscript revision; K.L.M.: Manuscript revision; Data analysis and interpretation; Financial support; Final approval of manuscript.

Background: Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells, but are also multipotent and secrete angiogenic growth factors such as hepatocyte growth factor (HGF) under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied.

Methods and Results: We focused on the role of ASCs in the process of adipose tissue injury and repair, and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemiareperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived BrdU-positive cells were shown to be ASCs (CD31–CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented post-injury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF.

Conclusions: FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs.

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Author contributions: H.S.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; H.E.: Collection and/or assembly of data; T.S.: Collection and/or assembly of data; K.I.: Collection and/or assembly of data; N.A.: Collection and/or assembly of data; H.K.: Collection and/or assembly of data; S.N.: Data analysis and interpretation; I.M.: Data analysis and interpretation; K.G.: Data analysis and interpretation; K.Y.: Conception and design, Financial support, Administrative support, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing.

Vascular endothelial growth factor (VEGF) and it receptors (VEGFR) regulate the development of hemogenic mesoderm. Oxygen concentration-mediated activation of hypoxia-inducible factor targets such as VEGF may serve as the molecular link between the microenvironment and mesoderm-derived blood and endothelial cell specification. We used controlledoxygen microenvironments to manipulate the generation of hemogenic mesoderm and its derivatives from embryonic stem cells. Our studies revealed a novel role for soluble VEGFR-1 (sFlt-1) in modulating hemogenic mesoderm fate between hematopoietic and endothelial cells. Parallel measurements of VEGF and VEGFRs demonstrated that sFlt-1 regulates Flk-1 activation in both a developmental-stage and oxygendependent manner. Early transient Flk-1 signaling occurred in hypoxia due to low levels of sFlt-1 and high levels of VEGF, yielding VEGF-dependent generation of hemogenic mesoderm. Sustained (or delayed) Flk-1 activation preferentially yielded hemogenic mesodermderived endothelial cells. In contrast, delayed (sFlt-1 – mediated) inhibition of Flk-1 signaling resulted in hemogenic mesoderm-derived blood progenitor cells. Ex vivo analyses of primary mouse embryo-derived cells, and analysis of transgenic mice secreting a Flt-1- Fc fusion protein, support a hypothesis whereby microenvironmentally-regulated blood and endothelial tissue specification, is enabled by the temporally variant control of the levels of Flk-1 activation.

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Author contributions: K.A.P.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.H.G.: conception and design, collection and/or assembly of data, manuscript writing; S.M.D.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; K.C.: provision of study material, BL-CFC assay assistance; A.N.: provision of study material; P.W.Z.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

Symptoms of Parkinson's disease have been improved by transplantation of fetal dopamine neurons recovered from aborted fetal tissue, but tissue recovery is difficult. Human embryonic stem cells may provide unlimited cells for transplantation if they can be converted to dopamine neurons and survive transplantation into brain. We have found that the BMP-antagonist Noggin increased the number of dopamine neurons generated in vitro from human and mouse embryonic stem cells differentiated on mouse PA6 stromal cells. Noggin effects were seen with either early (for mouse, days 0 to 7, and human, days 0 to 9) or continuous treatment. After transplant into cyclosporin-immunosuppressed rats, human dopamine neurons improved apomorphine circling in direct relation to the number of surviving dopamine neurons which was five-fold greater after Noggin treatment than with control human embryonic stem cell transplants differentiated only on PA6 cells. We conclude that Noggin promotes dopamine neuron differentiation and survival from human and mouse embryonic stem cells.

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Author contributions: S.C.: Conception and design, financial support, collection of data, data analysis, manuscript writing, final approval of manuscript; Y.M.L.: Collection of data, data analysis, final approval of manuscript; W.Z.: Collection of data, data analysis, final approval of manuscript; C.R.F.: Conception and design, financial support, administrative support, provision of study material, data analysis, manuscript writing, final approval of manuscript.

Recent reports have challenged the clonality of the neurosphere assay in assessing neural stem cell (NSC) numbers quantitatively. We tested the clonality of the neurosphere assay by culturing mixtures of differently labeled neural cells, watching single neural cells proliferate using video microscopy, and by encapsulating single NSCs and their progeny. The neurosphere assay was clonal when using primary cells plated at 10 cells/µl or less, however when using passaged NSC's the spheres were only clonal if plated at 1 cells/µl. Most important, moving the plates during the growth phase (to look at cultures microscopically) greatly increased the incidence of non-clonal colonies. To ensure clonal sphere formation, and investigate non-autonomous effects on clonal sphere formation frequencies, single NSCs were encapsulated in agarose and proliferated as clonal free-floating spheres. We demonstrate that clonal neurospheres can be grown by avoiding movement-induced aggregation, by single cell tracking and by encapsulation of single cells.

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Author contributions: B.C.-T.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; K.P.: Conception and design, Collection and/or assembly of data; I.B.: Collection and/or assembly of data; P.K.: Conception and design, Collection and/or assembly of data; C.M.: Conception and design, Financial Support, Data analysis and interpretation, Final approval of manuscript; P.Z.: Conception and design, Financial Support, Data analysis and interpretation, Final approval of manuscript; D.van der Kooy: Conception and design, Financial Support, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Reactivation of Oct4 gene expression occurs within two days of fusion of somatic cells with pluripotent stem cells and within nine days of post-infection of four transcription factors. We sought to determine whether somatic genome reprogramming is completed by the onset of Oct4 reactivation. The complex regulation of the reactivation of inactive X chromosome (Xi) serves as a model for studying reprogramming of chromatin domains. A time-course analysis of the DNA methylation, gene expression, and X inactivation-specific transcript (Xist)/Tsix RNA FISH revealed that expression of pluripotency- and tissue-specific marker genes was reset to the level of pluripotent stem cells within two days of fusion, while reprogramming of Xist/reactivation of Xi took at least nine days. We found that trichostatin A, which normally activates gene expression, results in down-regulation of Xist. This is due to activation of Dnmt3a and Tsix, two negative regulators of Xist. Moreover, delayed reprogramming of Xist/reactivation of inactive X chromosome after cell fusion was accelerated by DNA methylation and histone deacetylation of Xist that follow up-regulation of Dnmt3a and Tsix.

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Author contributions: J.T.D.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; D.W.H.: Collection and/or assembly of data, Data analysis and interpretation; L.G.: Data analysis and interpretation; I.S.-K.: Provision of study material or patients; M.S.: Collection and/or assembly of data; H.R.S.: Conception and design, Financial support, Manuscript writing, Final approval of manuscript.

Oct4, Sox2 and Nanog are key components of a core transcriptional regulatory network that control the ability of embryonic stem cell to differentiate into all cell types. Here we show that Zfp281, a zinc finger tanscription factor, is a key component of the network and that it is required to maintain pluripotency. Zfp281 was shown to directly activate Nanog expression by binding to a site in the promoter in very close proximity to Oct4 and Sox2 binding sites. We present data showing that Zfp281 physically interacts with Oct4, Sox2, and Nanog. Chromatin immunoprecipitation experiments identified 2,417 genes that are direct targets for regulation by Zfp281, including several transcription factors that are known regulators of pluripotency, such as Oct4, Sox2, and Nanog. Gene expression microarray analysis indicated that some Zfp281 target genes were activated while others were repressed upon knockdown of Zfp281. The identification of both activation and repressive domains within Zfp281, suggests that this transcription factor plays bifunctional roles in regulating gene expression within the network.

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Author contributions: Z.-X.W.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing; C.H.-L.T.: Conception and design, Collection and assembly of data; C.M.-Y.C.: Collection and assembly of data; C.C.: Collection and assembly of data; M.R.: Collection and assembly of data; G.K.: Data analysis and interpretation; T.B.A.: Collection and assembly of data; K.-Y.W.: Collection and assembly of data, Data analysis and interpretation; L.W.S.: Conception and design, Data analysis, Manuscript writing.

Mesenchymal stem cells (MSCs) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/β-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled Related Protein-1 (sFRP1), a modulator of Wnt/Fz pathway, could modify MSC capacities endowing MSC to increase vessel maturation. In engraftment model, we show that murine bone marrow derived MSCs induced a beneficial vascular effect through a direct cellular contribution onto vascular cells. MSCs quickly organized into primitive immature vessel tubes connected to host circulation which organization preceded host endothelial (EC) and smooth muscle cell (SMC) recruitment to lately form mature neo vessel. MSCs sustained neo vessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neo-vessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased PDGF-BB expression in MSC, enhanced β-catenin dependent cell-cell contacts between MSCs themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neo vessels and vessel maturation through a GSK-3β dependent pathway. sFRP1-overexpressing MSCs compared to control-MSCs were well elongated and in a closer contact with the vascular wall, events required to achieve an organized mature vessel wall. We propose that genetically modifying MSCs to overexpress sFRP1 may be potentially effective to promote therapeutic angiogenesis/arteriogenesis processes.

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Author contributions: P.D.: Data analysis and interpretation, Conception and design, Collection and assembly of data, Manuscript writing; B.D., N.F.T., L.L., P.O., C.M.: Data analysis and interpretation; D.D.: Collection and assembly of data; J.-M.T.: Manuscript writing; C.D.: Conception and design, Manuscript writing.

Glioblastomas, the most aggressive primary brain tumors, occur almost exclusively in adult patients. Neural precursor cells (NPCs) are anti-tumorigenic in mice, as they can migrate to glioblastomas and induce tumor cell death. Here, we show that the anti-tumor effect of NPCs is age-dependently controlled by cell proliferation in the subventricular zone (SVZ) and that NPCs accumulating at a glioblastoma are diverted from their normal migratory path to the olfactory bulb. Experimentally induced cortical glioblastomas resulted in decreased subventricular proliferation in adult (postnatal day 90), but not in young (postnatal day 30) mice. Adult mice supplied less NPCs to glioblastomas and had larger tumors than young mice. Apart from the difference in proliferation, there was neither a change in cell number and death-rate in the SVZ nor in angiogenesis and immune cell-density in the tumors. The ability to kill glioblastomas was similar in NPCs isolated from young and adult mice. The proliferative response of NPCs to glioblastomas depended on the expression of D-type cyclins. In young mice, NPCs express the cyclins D1 and D2, but the expression of cyclin D1 is lost during aging and in adult NPCs only cyclin D2 remains. In young and adult cyclin D2-deficient mice we observed a reduced supply of NPCs to glioblastomas and the generation of larger tumors, as compared to wild-type mice. We conclude that cyclin D1 and D2 are non-redundant for the antitumor response of subventricular NPCs. Loss of a single D-type cyclin results in a smaller pool of proliferating NPCs, lower number of NPCs migrating to the tumor and reduced anti-tumor activity.

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Author contributions: J.-H.W.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; M.S.: Financial support, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; B.E.: Provision of study material, Data analysis and interpretation; D.S.M.: Collection and/or assembly of data, Data analysis and interpretation; K.G.: Collection and/or assembly of data, Data analysis and interpretation; E.N.: Collection and/or assembly of data; K.Y.: Provision of study material, Data analysis and interpretation, Manuscript writing; B.K.: Data analysis and interpretation, Manuscript writing; G.K.: Conception and design, Data analysis and interpretation, Manuscript writing; W.U.: Provision of study material, Data analysis and interpretation, Manuscript writing; L.K.: Provision of study material, Data analysis and interpretation, Manuscript writing; H.K.: Conception and design, Financial support, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; R.G.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Joo-Hee Walzlein, Michael Synowitz, and Rainer Glass contributed equally to this work.

Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here, we demonstrate that we can recapitulate the spatio-temporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of ES cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic FGF-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming EGF-responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the GFAP promoter, similar to that observed in the developing brain. Moreover, the rostro-caudal and dorso-ventral spatial identities of the NS/PCs can be successfully regulated by sequentially administering several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease-model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.

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Author contributions: Y.O.: Conception and design, Financial support, Collection and assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; A.M.: Collection and assembly of data, Data analysis and interpretation; T.S.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing; R.E.: Collection and assembly of data, Data analysis and interpretation; A.K.: Collection and assembly of data, Data analysis and interpretation; S.I.: Collection and assembly of data; Y.I.: Provision of study materials; G.S.: Financial support; H.O.: Conception and design, Financial support, Manuscript writing, Final approval of manuscript.

Cell cycling regulation plays a fundamental role in stem cell biology. A balance between quiescence and proliferation of hematopoietic stem cells in interaction with microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. We analyzed the molecular mechanisms by which SDF-1 exhibited a cell cycle promoting effect and interacted with TGF-β negative effects on cell cycle orchestration of human hematopoietic CD34⁺ progenitor cells. We demonstrated that low concentration of SDF-1 modulated the expression of key cell cycle regulators such as cyclins, cyclin dependent kinase inhibitors and TGF-β target genes, confirming its cell cycling promoting effect. We showed that cross-talk between SDF-1- and TGF-β-related signaling pathways involving PI3K/Akt phosphorylation participated in the control of CD34⁺ cell cycling. We evidenced a pivotal role of two downstream effectors of the PI3K/Akt pathway, FoxO3a and mTOR, as connectors in the SDF-1-/TGFβ-induced control of the cycling/quiescence switch and proposed a model integrating a dialogue between the two molecules in cell cycle progression. Our data bring new lights on the signaling pathways involved in the SDF-1 cell cycle promoting activity and suggest that the balance between SDF-1- and TGF-β-activated pathways is critical for the regulation of hematopoietic progenitor cycle status.

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Author contributions: A.C.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; C.D.: Provision of study material or patients, Collection and/or assembly of data, Data analysis and interpretation; E.R.: Collection and/or assembly of data, Data analysis and interpretation; D.C.: Collection and/or assembly of data, Data analysis and interpretation; B.G.: Provision of study material or patients, Collection and/or assembly of data; L.B.: Provision of study material or patients; A.B.-G.: Provision of study material or patients, Manuscript writing, Final approval of manuscript; O.P.-L.: Provision of study material or patients; G.U.: Manuscript writing, Final approval of manuscript; L.A.: Provision of study material or patients; M.-F.B.: Provision of study material or patients, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; J.-J.L.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; M.-C.Le Bousse-Kerdilés: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Jean-Jacques Lataillade and Marie-Caroline Le Bousse-Kerdilès contributed equally to this work.

Prostate cancer (CaP) metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity and there is mounting evidence that the urokinase-type plasminogen (uPA) system is causatively involved in the progression of CaP. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the uPA antagonist amino-terminal fragment (hATF) in order to impair osteolytic CaP cells progression in bone and to repair bone lesions. Bioluminescence imaging (BLI) revealed that both primary MSCs (pMSCs) and MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) inhibited significantly intra-tibial PC-3-Luc growth following co-injection in SCID mice. Furthermore, micro-computed tomography (µCT) imaging of vascular network clearly demonstrated a significant decrease of tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected and area of new bone formation was evidenced in 60% of animals. Altogether, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential.

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Author contributions: V.F.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; D.N.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; C.Bouquet: Provision of study material or patients, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; P.O.: Data analysis and interpretation, Final approval of manuscript; R.V.: Data analysis and interpretation, Final approval of manuscript; F.A.: Conception and design, Manuscript writing, Final approval of manuscript; P.L.-P.: Conception and design, Manuscript writing, Final approval of manuscript; C.Bouffi: Data analysis and interpretation, Final approval of manuscript; H.D.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; C.X.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; M.P.: Final approval of manuscript; R.M.: Conception and design, Data analysis and interpretation, Final approval of manuscript; E.S.: Conception and design, Manuscript writing, Final approval of manuscript; C.J.: Conception and design, Financial support, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Human embryonic stem cells (hESCs) have recently demonstrated the potential for differentiation into germ-like cells in vitro. This provides a novel model for understanding human germ cell development and human infertility. Mouse embryonic fibroblast (MEF) feeders and basic fibroblast growth factor (bFGF) are two sources of signaling essential for primary culture of germ cells, yet their role has not been examined in the derivation of germ-like cells from hESCs. Here protein and gene expression demonstrate that both MEF feeders and bFGF can significantly enrich germ cell differentiation from hESCs. Under enriched differentiation conditions, flow cytometry analysis proved 69% of cells to be positive for DDX4 and POU5F1 protein expression, consistent with the germ cell lineage. Importantly, removal of bFGF from feeder free cultures resulted in a 50% decrease in POU5F1 and DDX4 positive cells. Quantitative RT-PCR analysis established that bFGF signaling resulted in a up regulation of genes involved in germ cell differentiation with or without feeders, however feeder conditions caused significant up regulation of pre-migratory/migratory (Ifitm3, DAZL, NANOG and POU5F1) and post-migratory genes( PIWIL2, PUM2) along with the meiotic markers SYCP3 and MLH1. After further differentiation, >90% of cells expressed the meiotic proteins SYCP3 and MLH1. This is the fist demonstration that signaling from MEF feeders and bFGF can induce a highly enriched population of germ-like cells derived from hESCs thus providing a critically needed model for further investigation of human germ cell development and signaling.

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Author contributions: F.D.W.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; D.W.M.: Data analysis and interpretation, Manuscript writing; N.L.B.: Data analysis and interpretation, Manuscript writing; K.P.: Collection and/or assembly of data; K.R.R.: Data analysis and interpretation; S.L.S.: Financial support, Provision of study material or patients, Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

A new source of stem cells has recently been isolated from amniotic fluid (AFSC) with significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human AFSC (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury tail vein injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C (SPC). Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kD protein (CC10). These results illustrate the plasticity of hAFSC to respond distinctly to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers depending on the type of injury to recipient lung.

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Author contributions: G.C.: Conception and design, data analysis and interpretation, manuscript writing, assembly of data; L.P.: Conception and design, hAFSC culture; S.S.: Tail vein injection of hAFSC; S.G.: Bioluminescence experiments; C.T.: Real-time PCR experiments; J.L.: Mice preparation and Intratracheal administration of hAFSC; B.D.: Conception and design, data analysis and interpretation; G.T.: Cytospin experiments; S.P.D.L.: Naphthalene injury experiments; S.B.: Manuscript writing, collection and assembly of data; P.M.: Data analysis and interpretation; A.A.: Data interpretation; R.E.D.F.: Collection and assembly of data, financial support; D.W.: Manuscript writing, final approval of manuscript, financial support.

The roles of Notch signaling in cardiac differentiation from murine embryonic stem cells have been well documented. We investigated whether Notch signaling plays a similar role in human embryonic stem cells (hESCs). Although, as previously reported, blocking Notch signaling via the addition of -secretase inhibitor (GSI) alone failed to affect hESC differentiation, we found that GSI plus reduced volume culture medium (GSI/RVCM) accelerated mesodermal differentiation. GSI/RVCM conditions simultaneously suppressed commitment toward neuroectodermal lineages. Furthermore, sustained inhibition of Notch signaling further enhanced differentiation into cardiac mesoderm. Spontaneous beating activity was typically observed from 12 days after initiation of GSI treatment in RVCM. Moreover, hESC-derived cardiomyocytes expressed connexin 43, and possessed spontaneous calcium oscillations and cardiomyocyte beats coupled to neonatal rat cardiomyocytes when co-cultured. These findings strongly suggest a distinct role for Notch signaling in the induction and specification of hESC-derived cardiac mesoderm in vitro.

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Author contributions: J.J.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; S.K.: Conception and design, Data analysis and interpretation, Manuscript writing; J.E.K.: Provision of study material or patients, Collection and/or assembly of data; K.C.: Conception and design, Data analysis and interpretation; Y.Y.K.: Provision of study material or patients, Collection and/or assembly of data; H.S.K.: Provision of study material or patients; S.K.O.: Administrative support, Data analysis and interpretation; E.J.L.: Provision of study material or patients, Data analysis and interpretation; H.C.: Provision of study material or patients; Y.H.S.: Provision of study material or patients; S.Hun Lee: Provision of study material or patients; S.Ho Lee: Provision of study material or patients, Data analysis and interpretation; C.S.S.: Provision of study material or patients, Collection and/or assembly of data; S.H.K.: Financial support, Administrative support; S.Y.M.: Conception and design, Financial support; Y.M.C.: Conception and design, Financial support, Data analysis and interpretation, Final approval of manuscript.

We have tested specific laminin isoforms for ability to serve as substrata for maintaining mouse embryonic stem (ES) cells pluripotent in vitro in the absence of leukemia inhibitory factor (LIF) or any other differentiation inhibitors or feeder cells. Recombinant human laminin-511 alone was sufficient to enable self-renewal of mouse ES cells for up to 169 days (31 passages). Cells cultured on laminin-511 maintained expression of pluripotency markers such as Oct4, Sox2, Tert, UTF1 and Nanog during the entire period, and cells cultured for 95 days (17 passages) were used to generate chimeric mice. Laminin-332 enabled ES cells proliferation, but not pluripotency. In contrast, under the same conditions laminin-111, Matrigel and gelatin caused rapid differentiation, while laminin-411 and poly-Dlysin did not support survival. ES cells form a thin monolayer on LN-511 that strikingly differs from typical dense cluster ES cell morphology. However, expression of pluripotency markers is not affected by morphological changes. The effect was achieved at low ES cell density (<200 cell/mm2). Ability of LN-511 and LN-332 to support ES cell proliferation correlated with increased cell contact area with those adhesive substrata. ES cells interact with LN-511 via β1-integrins, mostly 6β1 and Vβ1.

This is the first demonstration that certain extracellular matrix molecules can support ES cell self-renewal in the absence of differentiation inhibitors and at low cell density. The results suggest that recombinant laminin isoforms can provide a basis for defined surface coating systems for feederfree maintenance of undifferentiated mammalian ES cells in vitro.

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Author contributions: A.D.: Conception and design, provision of material, collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.R.: Conception and design, provision of material, collection and/or assembly of data, data analysis and interpretation, manuscript writing; A.B.: Provision of material; K.T.: Conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, final approval of manuscript.

Anna Domogatskaya and Sergey Rodin contributed equally to this work.

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Author contributions: A.H.: Conception and design, drafting the manuscript, final approval of manuscript; A.S.: Conception and design, drawing figure, manuscript writing and finalizing, approval of manuscript.

Peripheral blood stem cells (PBSCs), usually mobilized with G-CSF alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Owing to marked inter-individual variation in humans, we developed a robust non-human primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) to standard G-CSF and the combination of G-CSF and pegylated MGDF (pegMGDF) to G-CSF plus Stem Cell Factor (SCF) by measuring the levels of CD34⁺ cells, colony forming cells (CFCs) and SCID repopulating cells (SRCs) before and after. PegG-CSF mobilized CD34⁺ cells, colony forming cells (CFCs) and SCID repopulating cells (SRCs) to similar levels compared with 5 days of standard G-CSF. The combination of GCSF+ pegMGDF mobilized progenitors to similar levels as G-CSF+SCF but greater than standard G-CSF for CD34⁺ cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily GCSF and the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies.

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Author contributions: S.L.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; K.C.: Provision of study material or patients, collection and/or assembly of data; F.B.: Conception and design, collection and/or assembly of data; R.M.-W.: Statistical Analysis; J.E.R.: Conception and design, manuscript writing, final approval of manuscript.

The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. OCT4 plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture, and for colonization activity following cell transplantation, using lentiviral-mediated shRNA expression to knock down OCT4 in an in vitro model for SSCs (germline stem or GS cells). Expression of PLZF, a factor known to be required for SSC self renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, pre-meiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state, and RA triggers spermatogonial differentiation through the direct or indirect down-regulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells.

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Author contributions: C.D.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.A.: Collection and/or assembly of data; L.M.: Collection and/or assembly of data; B.D.: Collection and/or assembly of data; D.G.: Financial support; M.P.: Financial support, manuscript writing, experimental design.

Rat umbilical cord matrix (RUCM) cells are stem-cell-like cells and have been shown to reduce neuronal loss in the selectively vulnerable brain regions after cardiac arrest (CA). Here, we investigate whether this protection is mediated by the RUCM cells' modulation of the post-ischemia inflammation responses, which have long been implicated as a secondary mechanism of injury following ischemia. Brain sections were examined immunohistochemically for GFAP, vimentin, and nestin as markers for astroglia and reactive astrogliosis, Ricinus Communis Agglutinin-1 (RCA-1) as a marker for microglia, and Ki67 as a marker for cell proliferation. Rats were randomly assigned to six experimental groups: (1) 8-min CA without treatment, (2) 8-min CA pretreated with culture medium injection, (3) 8-min CA pretreated with RUCM cells, (4) sham-operated CA, (5) medium injection without CA, and (6) RUCM cell transplantation without CA. Groups 1-3 have significantly higher Ki67⁺ cell counts and higher GFAP⁺ immunoreactivity in the hippocampal CA1 region compared to groups 4-6, irrespective of treatment. Groups 1 and 2 have highly elevated GFAP⁺, vimentin⁺, and nestin⁺ immunoreactivity, indicating reactive astrogliosis. Strikingly, RUCM cell treatment nearly completely inhibited the appearance of vimentin⁺ and greatly reduced nestin⁺ reactive astrocytes. RUCM cell treatment also greatly reduced RCA-1 expression, which is found to strongly correlate with the neuronal loss in the CA1 region. Our study indicates that treatment with stem-cell-like RUCM cells modulates the inflammatory response to global ischemia and renders neuronal protection by preventing permanent damage to the selectively vulnerable astrocytes in the CA1 region.

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Author contributions: A.H.: Data collection, analysis, and intepretation; manuscript/figure preparation; R.D.: Data collection; tissue preparation for histology; stem cell culturing; S.J.: Data collection; cardiac arrest and resuscutation; stem cell culturing; Y.X.: Conception and design; financial support; provision of study material; data interpretation; manuscript writing; final approval of manuscript.

The ability of stem cells to differentiate into specified lineages in the appropriate locations is vital to morphogenesis and adult tissue regeneration. While soluble signals are important regulators of patterned differentiation, here we show that gradients of mechanical forces can also drive patterning of lineages. In the presence of soluble factors permitting osteogenic and adipogenic differentiation, human mesenchymal stem cells at the edge of multicellular islands differentiate into the osteogenic lineage, while those in the center became adipocytes. Interestingly, changing the shape of the multicellular sheet modulated the locations of osteogenic versus adipogenic differentiation. Measuring traction forces revealed gradients of stress that preceded and mirrored the patterns of differentiation, where regions of high stress resulted in osteogenesis while stem cells in regions of low stress differentiated to adipocytes. Inhibiting cytoskeletal tension suppressed the relative degree of osteogenesis versus adipogenesis, and this spatial patterning of differentiation was also present in threedimensional multicellular clusters. These findings demonstrate a role for mechanical forces in linking multicellular organization to spatial differentials of cell differentiation, and represent an important guiding principle in tissue patterning that could be exploited in stem cell-based therapies.

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Author contributions: S.A.R.: Conception and design, administrative support, provision of study materials, collection and assembly of data, data analysis and interpretation, and manuscript writing; C.S.C.: Conception and design, financial support, administrative support, provision of study materials, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Human embryonic stem cells (hESCs) are often cocultured on mitotically-inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multi-layered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we show that hESCs under these conditions primarily express proteins belonging to epitheliumrelated cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions (EMT), indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation.

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Author contributions: D.V.H.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.R.B.: Conception and design, collection and/or assembly of data, data analysis and interpretation; W.D.: Collection and/or assembly of data, data analysis and interpretation; D.W.-V.O.: Collection and/or assembly of data; A.J.H.: Financial support, final approval of manuscript; J.K.: Financial support, final approval of manuscript; C.L.M.: Conception and design, financial support, data analysis and interpretation, final approval of manuscript.

Dennis Van Hoof and Stefan R. Braam contributed equally to this work.

Success in islet transplantation-based therapies for type 1 diabetes mellitus and an extreme shortage of pancreatic islets has motivated recent efforts to develop renewable sources of islet-replacement tissue. Although pancreatic progenitor cells hold a promising potential, only a few attempts have been made at the prospective isolation of pancreatic stem/progenitor cells, due to the lack of specific markers and the development of effective cell culture methods. We found that prominin1 (also known as CD133) recognized the undifferentiated epithelial cells, while PDGFRβ was expressed on the mesenchymal cells in the mouse embryonic pancreas.We then developed an isolation method for putative stem/progenitor cells by flow cytometric cell sorting and characterized their differentiation potential to pancreatic tissue using both an in vitro and in vivo protocol. Flow cytometry and the subsequent RT-PCR and microarray analysis revealed pancreatic epithelial progenitor cells to be highly enriched in prominin1^highPDGFRβ- cell population. During in vivo differentiation, these cell populations were able to differentiate into endocrine, exocrine, and ductal tissues, including the formation of insulin-producing cell cluster. We established the prospective isolation of putative pancreatic epithelial progenitor cells by sorting for prominin1 and PDGFRβ. Since this strategy is based on the cell surface markers common to human and rodents, these findings may lead to the development of new strategies to derive transplantable islet-replacement tissues from human pancreatic stem/progenitor cells.

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Author contributions: Y.H.: Conception and design, Collection and/or assembly of data, Manuscript writing; M.F.: Collection and/or assembly of data, provision of study material or patients; Y.K.: Conception and design, Final approval of manuscript.

Cells isolated from Wharton's Jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew and are multipotent (differentiate into bone, fat, cartilage, etc)in vitro. These properties support the notion that UCMS cells are a member of the MSC family. Here, the immune properties of UCMS cells are characterized in vitro. The overall hypothesis is that UCMS cells possess immune properties that would be permissive to allogeneic transplantation. For example, UCMS cells will suppress of the proliferation of "stimulated’ lymphocytes (immune suppression) and have reduced immunogenicity, e.g., would be poor stimulators of allogeneic lymphocyte proliferation. Hypothesis testing: First, the effect on proliferation of co-culture of mitotically-inactivated human UCMS cells with concanavalin-A stimulated rat splenocytes was assessed in three different assays. Second, the effect of human UCMS cells on one-way and two-way mixed lymphocyte reaction (MLR) assays was determined. Third, the expression of HLA-G was examined in human UCMS cells using RT-PCR, since HLA-G expression conveys immune regulatory properties at the maternal-fetal interface. Fourth, the expression of CD40, CD80 and CD86 was determined by flow cytometry. Fifth, the cytokine expression of UCMS cells was evaluated by focused gene array. The results indicate that human UCMS cells inhibit splenocyte proliferation response to Con-A stimulation, they do not stimulate T-cell proliferation in a one-way MLR, and they inhibit the proliferation of stimulated T-cells in a two-way MLR. Human UCMS cells do not inhibit non-stimulated splenocyte proliferation, suggesting specificity of the response. UCMS cells express mRNA for pan HLA-G. UCMS cells do not express the co-stimulatory surface antigens CD40, CD80 or CD86. UCMS cells express vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), molecules previously implicated in the immune modulation observed in MSCs. Additionally, the array data indicates that UCMS cells make a cytokines and other factors that may support hematopoieisis. Together, these results support previous observations made following xenotransplantation, e.g., no evidence of frank immune rejection of undifferentiated UCMS cells. The results suggest that human UCMS will be tolerated in allogeneic transplantation.

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Author contributions: M.L.W.: Conception and design, assembly of data, data analysis and interpretation, manuscript writing and final approval of manuscript, financial and administrative support; C.K.: Design, assembly of data, data analysis and interpretation, manuscript writing; S.M., K.S., R.J.W.,I.V.: Collection and assembly of data; D.T.: Administrative support, data interpretation.

Abscisic acid (ABA) is a phytohormone involved in fundamental processes in higher plants. Endogenous ABA biosynthesis occurs also in lower Metazoa, in which ABA regulates several physiological functions by activating ADP-ribosyl cyclase (ADPRC) and causing overproduction of the Ca²⁺-mobilising second messenger cyclic ADP-ribose (cADPR), thereby enhancing intracellular Ca²⁺ ([Ca²⁺]_i).

Recently, production and release of ABA have been demonstrated to take place also in human granulocytes, where ABA behaves as a proinflammatory hormone through the same cADPR/[Ca²⁺]_i signalling pathway described in plants and in lower Metazoa. Based on the fact that human mesenchymal stem cells (MSC) express ADPRC activity, we investigated the effects of ABA and of its second messenger cADPR on purified human MSC.

Both ABA and cADPR stimulate the in vitro expansion of MSC, without affecting differentiation. The underlying mechanism involves a signalling cascade triggered by ABA binding to a plasmamembrane receptor and consequent cyclic AMP-mediated activation of ADPRC and of the cADPR/[Ca²⁺]_i system. Moreover, ABA stimulates the following functional activities of MSC: cyclooxygenase 2-catalysed production of PGE₂, release of several cytokines known to mediate the trophic and immunomodulatory properties of MSC and chemokinesis. Remarkably, ABA proved to be produced and released by MSC stimulated by specific growth factors (e.g., bone morphogenetic protein-7), by inflammatory cytokines and by lymphocyte-conditioned medium. These data demonstrate that ABA is an autocrine stimulator of MSC function and suggest that it may participate in the paracrine signalling between MSC, inflammatory/immune cells and hemopoietic progenitors.

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Author contributions: S.S.: conception and design, data analysis and interpretation, manuscript writing, financial support; C.Ferraris: collection and assembly of data, data analysis and interpretation; F.F.: collection and assembly of data, data analysis and interpretation; C.Fresia: collection and assembly of data, data analysis and interpretation; L.G.: collection and assembly of data, data analysis and interpretation; S.B.: collection and assembly of data, data analysis and interpretation; A.P.: collection and assembly of data, data analysis and interpretation; C.U.: collection and assembly of data, data analysis and interpretation; E.M.: collection and assembly of data, data analysis and interpretation; A.S.: collection and assembly of data, data analysis and interpretation; G.B.: provision of study material or patients; E.Z.: conception and design, manuscript writing, financial support; A.D.F.: conception and design, manuscript writing, final approval of manuscript, financial support.

Identification of regulators governing the maintenance of embryonic stem (ES) cells is crucial to the understanding of ES cell biology. We identified a zinc finger protein, Zfp143 as a novel regulator for selfrenewal. Depletion of Zfp143 by RNAi causes loss of self-renewal of ES cells. Chromatin immunoprecipitation and EMSA show the direct binding of Zfp143 to Nanog proximal promoter. Knockdown of Zfp143 or mutation of Zfp143 binding motif significantly down-regulates Nanog proximal promoter activity. Importantly, enforced expression of Nanog is able to rescue the Zfp143 knockdown phenotype, indicating that Nanog is one of the key downstream effectors of Zfp143. More interestingly, we further show that Zfp143 regulates Nanog expression through modulation of Oct4 binding. Coimmunoprecipitation experiments revealed that Zfp143 and Oct4 physically interact with each other. This interaction is important because Oct4 binding to Nanog promoter is promoted by Zfp143. Our study reveals a novel regulator functionally important for the selfrenewal of ES cells and provides new insights into the expanded regulatory circuitry that maintains ES cell pluripotency.

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Author contributions: X.C.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; F.F.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; Y.-C.L.: Provision of study material, Data analysis and interpretation; H.-H.N.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Xi Chen and Fang Fang contributed equally to this work.

Stem cell-based therapies hold therapeutic promise for degenerative motor neuron diseases such as amyotrophic lateral sclerosis and for spinal cord injury. Fetal neural progenitors present less risk of tumor formation than embryonic stem (ES) cells but inefficiently differentiate into motor neurons, in line with their low expression of motor neuron-specific transcription factors and poor response to soluble external factors. To overcome this limitation, we genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1 and Ngn2. Enforced expression of the three factors rendered neural precursors responsive to sonic hedgehog and retinoic acid and directed their differentiation into cholinergic motor neurons that projected axons and formed contacts with co-cultured myotubes. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-specific differentiation markers and projected cholinergic axons in the ventral root. We conclude that genetic engineering can drive the differentiation of fetal neural precursors into motor neurons which efficiently engraft in the spinal cord. The strategy thus holds promise for cell replacement in motor neuron and related diseases.

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Author contributions: D.B.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; S.L.: collection and/or assembly of data, final approval of manuscript; S.B.: collection and/or assembly of data, final approval of manuscript; M.H.: collection and/or assembly of data, final approval of manuscript; G.H.: provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript; J.-M.H.: financial support, administrative support, conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

Aims - Bone marrow stromal cells (BMSCs) contain progenitors capable to participate in postnatal angiogenesis. Hypoxia inducible factors (HIF) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1 and HIF-2 modification in BMSCs, as a tool to improve cell-based angiogenic therapy.

Methods and Results- - BMSCs were retrovirally transduced to express stable forms of HIF-1 and HIF-2. HIF-1 and, to a greater extent, HIF-2 overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis towards SDF-1 was higher in both HIF- expressing BMSCs, enhanced migration towards VEGF was found only following overexpression of HIF-2, supported by a robust expression of its receptor- Flk-1. HIF- expression was associated with up-regulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF- expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2 transduced BMSCs induced a more robust angiogenic response, compared to sham-transduced or HIF-1-transduced BMSCs in the corneal micropocket angiogenesis model.

Conclusions- Our results support the use of HIF- genes, particularly HIF-2, to augment the efficacy of future cell-based therapy.

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Author contributions: J.B.-S.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing; S.S.: Collection and assembly of data, Data analysis and interpretation; G.L.: Collection and assembly of data, Data analysis and interpretation; S.M.-A.: Collection and assembly of data, Data analysis and interpretation; A.Barzelay: Collection and assembly of data; S.P.-C.: Data analysis and interpretation; E.T.: Conception and design, Provision of study material; I.B.: Provision of study material Data analysis and interpretation; A.Barak: Provision of study material Data analysis and interpretation; H.L.-V.: Provision of study material, Data analysis and interpretation; G.K.: Conception and design, Provision of study material, Data analysis and interpretation; J.G.: Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Shulamit Schwartz and Galia Luboshits contributed equally to this work.

Although bone marrow-derived mesenchymal stromal cells (MSCs) may be beneficial in treating heart disease, their ability to transdifferentiate into functional cardiomyocytes remains unclear. Here, bone marrow derived MSCs from adult female transgenic mice expressing GFP under the control of the cardiacspecific -myosin heavy chain promoter were cocultured with male rat embryonic cardiomyocytes (rCMs) for 5–15 days. After 5 days in co-culture, 6.3% of MSCs became GFP⁺ and stained positively for the sarcomeric proteins troponin I and -actinin. The mRNA expression for selected cardiac-specific genes (atrial natriuretic factor, Nkx2.5 and -cardiac actin) in MSCs peaked after 5 days in co-culture and declined thereafter. Despite clear evidence for the expression of cardiac genes, GFP⁺ MSCs did not generate action potentials or display ionic currents typical of cardiomyocytes, suggesting retention of a stromal cell phenotype. Detailed immunophenotyping of GFP⁺ MSCs demonstrated expression of all antigens used to characterize MSCs as well as the acquisition of additional markers of cardiomyocytes with the phenotype: CD45^--CD34⁺-CD73⁺-CD105⁺-CD90⁺-CD44⁺-SDF1⁺-CD134L⁺-collagen type IV⁺-vimentin⁺-troponin T⁺-troponin I⁺--actinin⁺-connexin 43⁺. Although cell fusion between rCMs and MSCs was detectable, the very low frequency (0.7%) could not account for the phenotype of the GFP⁺ MSCs. In conclusion, we have identified a MSC population displaying plasticity towards the cardiomyocyte lineage while retaining mesenchymal stromal cell properties, including a non-excitable electrophysiological phenotype. The demonstration of a MSC population phenotyping of MSCs co-cultured with cardiomyocytes 2 co-expressing cardiac and stromal cell markers may explain conflicting results in the literature and indicates the need to better understand the effects of MSCs on myocardial injury.

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Author contributions: R.AR.: Conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; H.J.: Conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; X.W.: Conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation, final approval of manuscript; S.H.: Conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation, final approval of manuscript; J.N.T.: Conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation, final approval of manuscript; N.G.: Conception and design, final approval of manuscript; S.C.K.: Collection and assembly of data, data analysis and interpretation, final approval of manuscript; T.G.P.: Conception and design, financial support, provision of study materials, data analysis and interpretation, final approval of manuscript; P.H.B.: Conception and design, financial support, provision of study materials, data analysis and interpretation, manuscript writing, final approval of manuscript; A.K.: Conception and design, financial support, provision of study materials, data analysis and interpretation, manuscript writing, final approval of manuscript.

Robert A Rose and Huijie Jiang contributed equally to this work.

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune-suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells (hBMSCs), which express Nanog, Rex-1, Oct-4, and various cell surface antigens, and has multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre-existing Nestin⁺ neural progenitor cells (NPCs) and β-tubulin-III⁺ mature neurons to the site of the graft. Additionally, many cells born during the first 7 days post-implantation proliferated, forming NPCs and neurons, and, to a lesser extent, astrogliosis, forming astrocytes and microglia, by 30 days post-implantation. Although the DPSC graft itself was short-term, it had long-term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of CNTF, VEGF, and FGF for up to 30 days postimplantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the CNS. DPSCs would be a preferable cell source for therapy due to the possibility of a "personalized" stem cell, avoiding the problems associated with host immune rejection.

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Author contributions: A. H.-C.H.: Conception and design, establishment of initial cell line; B.R.S.: Stereotaxic transplantation, histology, data analysis and interpretation, and manuscript writing; P.-H.C.: Collection and assembly of data, data analysis and interpretation; A.W.C.: Conception and design, data analysis and interpretation, manuscript writing and final approval.

Anderson Hsien-Cheng Huang and Brooke R. Snyder contributed equally to this work.

Adipose stromal cells (ASC) are multipotential mesenchymal progenitor cells which are readily induced to undergo adipogenic differentiation, and we have recently demonstrated to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. In this study we addressed the hypothesis that modulation of ASC fate within this perivascular niche can occur via interaction with endothelial cells (EC) which serve to modulate the adipogenic potential of ASC. To this end, we investigated contact as well as paracrine effects of EC on ASC adipogenesis in 2D-co-culture and via conditioned media, and analyzed mutual gene expression changes by real time RT-PCR. A significant decrease in adipogenic differentiation was observed in ASC when co-cultured with EC but not control fibroblasts. This endothelial cell-specific effect was accompanied by increased expression of factors involved in Wnt signaling; most prominently Wnt1, Wnt4 and Wnt10a, well known inhibitors of adipogenesis. Suppression of Wnt1, but not Wnt 10a or scrambled control siRNA in co-cultures partially reversed the endothelial cell effect, thus increasing adipogenic differentiation, suggesting a plausible role of Wnt1 ligand in modulation of adipogenesis by the vasculature. Furthermore, addition of recombinant Wnt ligand or the Wnt signaling agonist inhibited adipogenic differentiation of ASC in the absence of EC. In conclusion, these data define the relationship in adipose tissue between ASC and EC in the perivascular niche, in which the latter act to repress adipogenesis, thereby stabilizing vasculature. It is tempting to speculate that abnormal endothelial function may be associated with pathologic de-repression of adipogenesis.

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Author contributions: G.R.: Collection and/or assembly of data, data analysis and interpretation, conception and design and manuscript writing; D.T.: Collection and/or assembly of data; W.R.: Collection and/or assembly of data; B.J.: Data analysis and interpretation; S.M.-C.: Collection and/or assembly of data; B.V.N.: Provision of study material or patients; E.R.: Data analysis and interpretation; K.M.: Conception and design and manuscript writing; M.C.: Conception and design, and manuscript writing

Keith L March and Matthias Clauss contributed equally to this work.

The mechanisms underlying the regulation of neural stem cell (NSC) renewal and maintenance of their multipotency are still not completely understood. Self-renewal of stem cells in general implies repression of genes that encode for cell lineage differentiation. Enhancer of zeste homolog 2 (Ezh2) is a Polycomb group protein (PcG) involved in stem cell renewal and maintenance by inducing gene silencing via histone methylation and deacetylation. To establish the role of Ezh2 in the maintenance and differentiation of NSCs, we have examined the expression of Ezh2 in NSCs isolated from embryonic (E14) mice during proliferation and differentiation in-vitro. Our results show that Ezh2 is highly expressed in proliferating NSCs. In accordance with its suggested role as transcription repressor, the expression of Ezh2 decreased when the NSCs differentiated into neurons and was completely suppressed during differentiation into astrocytes. Surprisingly, Ezh2 remained highly expressed in NSCs that differentiated into an oligodendrocytic cell lineage, starting from oligodendrocyte precursor cells (OPCs) up to the immature (premyelinating) oligodendrocyte stage. To further establish the role of Ezh2 in NSC differentiation, we silenced and induced over-expression of the Ezh2 gene in NSCs. High levels of Ezh2 in differentiating NSCs appeared to be associated with an increase in oligodendrocytes and a reduction in astrocytes, whereas low levels of Ezh2 led to completely opposite effects. The increase in the number of oligodendrocytes induced by enhanced expression of Ezh2 could be ascribed to stimulation of OPC-proliferation although stimulation of oligodendrocyte-differentiation cannot be excluded.

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Author contributions: F.S.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; R.R.: collection and assembly of data, data analysis and interpretation; N.B.: collection and assembly of data, data analysis and interpretation; V.B.: conception and design, data analysis and interpretation; E.B.: conception and design, data analysis and interpretation, manuscript writing; S.C.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

Mesenchymal progenitor cells (MPCs) exhibited fibroblast-like morphology and are multipotent cells capable of differentiating into various mesenchymal tissues. Although MPCs have been found in adult bone marrow (BM) and umbilical cord blood, there is still controversy as to whether the MPCs are present in adult human blood. To determine whether they are, we cultured mononuclear cells (MNCs) from the pulmonary arterial blood of lung cancer patients. In 94% (29 of 31) of the cases, fibroblasts were expanded ex vivo and were differentiated into an osteogenic lineage or an adipogenic lineage, depending on the specific inducing media. These results indicated that pulmonary arterial blood in the vicinity of lung cancers contains MPCs (PA-MPCs). The cDNA profiles of PA-MPCs, MPCs derived from bone marrow MPC (BM-MPCs), and lung tissue-derived fibroblasts were clustered with a hierarchical classification algorithm. The expression profiles of PA-MPCs (3 cases) and BM-MPCs were clearly separated from those of the tissue-derived fibroblasts, and the profiles of the PA-MPCs from the 2 patients were separated from those of the BM-MPCs.

To identify the source of the PA-MPCs, the MNCs from pulmonary arterial blood were exposed to anti-CD14, anti-CD105, anti-CD3, anti-CD20 antibodies. CD105+ MNCs generated MPCs in 8/8 cases (100%), whereas CD14 +, CD3 +, and CD20 +- mononuclear cells generated MPCs in 3/5 cases (60%), 2/5 cases (40%), and 0/3 cases (0%), respectively. These findings are the first clear proof that CD105 + MNCs fraction in the pulmonary arterial blood of adult lung cancer patients includes MPCs.

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Author contributions: H.C.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; G.I.: Conception and design, Financial support, Provision of study material or patients, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; T.-K.I.: Conception and design, Data analysis and interpretation, Final approval of manuscript; K.A.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; H.S.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; K.N.: Provision of study material or patients, Collection and/or assembly of data, Final approval of manuscript; A.O.: Conception and design, Financial support, Administrative support, Provision of study material or patients, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros (AGM)derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies (hEBs). In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45⁺CD34⁺ cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45⁺CD34^-) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursorsss overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence towards understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and defines the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells (HSCs) from ESC lines.

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Author contributions: J.J.: Collection and/or assembly of data, Data analysis and interpretation; P.M.: Conception and design; K.V.: assistance with experimentation and analysis; M.B.: assistance with experimentation and analysis; K.W.: Conception and design; M.B. Conception and design, Financial support, Manuscript writing, Final approval of manuscript.

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSC) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere ‘merging’ (Singec et al., Nature Methods, 2006; Jessberger et al., Stem Cells, 2007). Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared to conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to elucidate better the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics.

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Author contributions: M.C.: Assembly of data, data analysis and interpretation, manuscript writing ; M.L.: Assembly of data, data analysis and interpretation; S.K.: Assembly of data, data analysis and interpretation; V.T.: Provision of neural stem cells, data analysis and interpretation, manuscript editing ; M.L.: Conception and design, data analysis and interpretation, financial support, manuscript writing.

The development of personalized pluripotent stem cells for research and for possible therapies holds out great hope for patients. Yet such cells will face significant technical and regulatory challenges before they can be used as therapeutic reagents. Here we consider two possible sources of personalized pluripotent stem cells; embryonic stem cells derived from nuclear transfer (NT-ESCs) and induced pluripotent stem cells (iPSCs) derived from direct reprogramming of adult somatic cells. Both sources of personalized pluripotent stem cells face unique regulatory hurdles that are in some ways significantly higher than those facing stem cells derived from embryos produced by fertilization (ESCs). However, the outstanding long-term potential of iPSCs and their relative freedom from the ethical concerns raised by both ESCs and NT-ESCs makes direct reprogramming an exceptionally promising approach to advancing research and providing therapies in the field of regenerative medicine.

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Author contributions: M.L.C.: Conception and design, financial support, administrative support, manuscript writing, final approval of manuscript, other (production of artwork).; M.R.: Conception and design, financial support, manuscript writing, final approval of manuscript.

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs) in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryo fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34 positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long