Maintenance Of Stem Cell Pool Research Articles

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells

IntroductionThe maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). MethodsThe hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. ResultsThe c-Kit–silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1–positive cells was inhibited when we silenced c-Kit in hDPSCs. ConclusionsThese results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.

Endothelial-Initiated Crosstalk Regulates Dental Pulp Stem Cell Self-Renewal

Interactions with the microenvironment modulate the fate of stem cells in perivascular niches in tissues (e.g., bone) and organs (e.g., liver). However, the functional relevance of the molecular crosstalk between endothelial cells and stem cells within the perivascular niche in dental pulps is unclear. Here, we tested the hypothesis that endothelial cell–initiated signaling is necessary to maintain self-renewal of dental pulp stem cells. Confocal microscopy showed that ALDH1high and Bmi-1high stem cells are preferentially localized in close proximity to blood vessels in physiological human dental pulps. Secondary orosphere assays revealed that endothelial cell–derived factors (e.g., interleukin-6 [IL-6]) promote self-renewal of dental pulp stem cells cultured in low-attachment conditions. Mechanistic studies demonstrated that endothelial cell–derived IL-6 activates IL-6R (IL-6 Receptor) and signal transducer and activator of transcription 3 (STAT3) signaling and induces expression of Bmi-1 (master regulator of stem cell self-renewal) in dental pulp stem cells. Transplantation of dental pulp stem cells stably transduced with small hairpin RNA (shRNA)–STAT3 into immunodeficient mice revealed a decrease in the number of blood vessels surrounded by ALDH1high or Bmi-1high cells (perivascular niches) compared to tissues formed upon transplantation of vector control stem cells. And finally, in vitro capillary sprouting assays revealed that inhibition of IL-6 or STAT3 signaling decreases the vasculogenic potential of dental pulp stem cells. Collectively, these data demonstrate that endothelial cell–derived IL-6 enhances the self-renewal of dental pulp stem cells via STAT3 signaling and induction of Bmi-1. These data suggest that a crosstalk between endothelial cells and stem cells within the perivascular niche is required for the maintenance of stem cell pools in dental pulps.

POLTERGEIST and POLTERGEIST-LIKE1 are essential for the maintenance of post-embryonic shoot and root apical meristems as revealed by a partial loss-of-function mutant allele of pll1 in Arabidopsis.

POLTERGEIST (POL) and POL-LIKE1 (PLL1) encoding related protein phosphatase 2Cs are essential for the establishment of both shoot and root meristems during embryogenesis. As the strong pol pll1 are seedling-lethal due to the lack of hypocotyl vasculature, the roles of POL/PLL1 for the post-embryonic development is difficult to be assessed. To prepare a weak pol pll1 double mutant that are able to produce post-embryonic organs. Several T-DNA insertion mutants of pll1 were crossed to pol-6 for the preparation of weak pol pll1. To understand the epistatic interactions between POL/PLL1 and CLAVATAs, the phenotypes of clvs pol pll1 were assessed and the expressionpatterns of stem cell markers were examined in pol pll1. POLpro:PLL1-GFP expression was examined during the embryogenesis with confocal microscopy. We isolated a pll1-3 (S544N)allele and prepared a weak pol-6 pll1-3. About 5% of pol-6 pll1-3 seedlings continued the post-embryonic development displaying short roots with reduced root meristem, wuschel-like adventitious phyllotaxis, and defective flowers lacking carpel. The clv1, clv2, and clv3 phenotypes led by enlarged shoot meristems were almost completely suppressed in the pol-6 pll1-3. POL/PLL1 were required for the indeterminate floral organ development displayed by agamous. PLL1-GFP asymmetrically localized in the shootward sides of columella cells and increased the size of distal root meristem region by enhancing the WUS-RELATED HOMEOBOX5 expression suggesting that PLL1 might provide the stem cells and progenies with proper positional information for the asymmetric cell divisions. Together, POL/PLL1 are required for the maintenance of stem cell pools for the post-embryonic development in Arabidopsis.

PO-282 SMAR1 transcriptionally repress hTERT and alters the stem cell population in colorectal cancer cells

IntroductionOne of the distinguishing features that set the cancer cell apart is its ability for indefinite proliferation. Cancer progression is accompanied by perturbations in the chromatin structure that results in inappropriate gene expression and genomic instability. Aberrant activation of signalling pathways in cancer cells lead to a cascade of events resulting in ‘replicative immortality’. One of the mechanisms by which cancer cells keep dividing is by the reactivation of enzyme telomerase (hTERT). hTERT reactivation confers tumour cell with various advantages like protection against DNA damaging agents, maintenance of stem cell pool, increased angiogenesis, etc. This cancer stem cell (CSC) population then remains quiescent owing to its radio and chemo-resistant nature and leads to cancer relapse.Material and methodsWestern Blotting, Real time PCR, Co-Immunoprecipitation, Chromatin Immunoprecipitation, Telomerase Repeat Amplification Protocol (TRAP) Assay, Polyps generation in Balb/c mice.Results and discussionsOur study elucidates the role of a Martix Attachment Region Binding Protein (MARBP), SMAR1 as a transcriptional repressor of hTERT. SMAR1 directly interacts with HDAC1/mSin3a co-repressor complex and bind to the hTERT promoter. This binding of SMAR1 brings about Wnt/β-Catenin mediated repression of hTERT. Interestingly we find that occupancy of HDAC1 on hTERT promoter is SMAR1-dependent. The recruitment of repressor complex on the hTERT promoter results in the deacetylation of histones and an increase in the H3K9me2 mark.This helps maintain a repressive environment on the hTERT promoter and alters the occupancy of PolII on the promoter. We also find that knock-down of SMAR1 promotes CD133+ CSC’s and SMAR1 over-expression inhibits CD133+ CSC’s. Moreover, CD133high and CD133low populations exhibit differential SMAR1 and hTERT expression. We generated polyps in Balb/c mice using azoxymethane (AOM)/dextran sodium sulphate (DSS). When the levels of SMAR1 and hTERT were assessed; polyps expressed lower levels of SMAR1 and elevated hTERT as compared to the adjacent non-cancerous tissue.ConclusionWe show that SMAR1 transcriptionally repress hTERT. Given that hTERT reactivation is central for tumour development, progression and cancer stem cell maintenance, this regulation by SMAR1 represents an important mechanism of tumour suppression and inhibition of cancer stem cell trait.

Function of Polycomb repressive complexes in stem cells

Stem cells are unique cell populations identified in a variety of normal tissues and some cancers. Maintenance of stem cell pools is essential for normal development, tissue homeostasis, and tumorigenesis. Recent studies have revealed that Polycomb repressive complexes (PRCs) play a central role in maintaining stem cells by repressing cellular senescence and differentiation. Here, we will review recent findings on dynamic composition of PRC complexes and sub-complexes, how PRCs are recruited to chromatin, and their functional roles in maintaining self-renewal of stem cells. Furthermore, we will discuss how PRCs, CpG islands (CGIs), the INK4A/ARF/INK4B locus, and developmental genes form a hierarchical regulatory axis that is utilized by a variety of stem cells to maintain their self-renewal and identities.

Proteinase 3 Is Expressed in Stem Cells and Regulates Bone Marrow Hematopoiesis

Previously published data from our lab identified proteinase 3 (PR3) as a key protein in regulating spontaneous death of neutrophils. Our lab showed that PR3 could cleave caspase 3 in a caspase 9-independent manner in ageing neutrophils (Loison et al. JCI. 2014). Meanwhile, the maintenance of stem cell pool in the bone marrow and the development of mature blood cells are tightly regulated processes. In an effort to understand the hematopoietic consequences of absence of PR3, we analyzed stem and progenitor cell compartments in wild-type (WT) and PR3 knock-out (PR3-/-) mice.Of note, PR3 is thought to be a neutrophil-specific serine protease. Surprisingly, we observed PR3 expression in sorted Lin- Sca-1+ c-Kit+ (LSK) cells by gene expression analysis at mRNA level as well as Western blotting at protein level. Conventional flow cytometry methods also confirmed this expression.While bone marrow cellularity was similar between WT and PR3-/- mice, conventional flow cytometry studies showed that bone marrow from PR3-/- mice contained a higher frequency of LSK and Lin- Sca-1- c-Kit+ (LK) cells. Colony forming cell assays also suggested that both bone marrow cells and splenocytes from PR3-/- mice contained a higher number of progenitor cells compared to their WT counterparts. Histological analysis of bone marrow smears revealed that bone marrow from PR3-/- mice had a higher number of immature myeloid cells and a reduced number of mature leukoyctes and lymphocytes.Next, we asked if the enhanced stem and progenitor cell compartments in PR3-/- mice were functional in vivo. Toward this aim, we performed competitive reconstitution experiments using total bone marrow cells as well as sublethal irradiation experiments. PR3-/- donor-derived cells gave rise to a higher number of cells in the recipient mice compared to WT donor-derived cells in competitive reconstitution experiments. Also, after WT and PR3-/- mice were irradiated with 4 Gy, peripheral blood cell counts recovered faster in PR3-/- mice. Similarly, when mice were irradiated with 6 Gy, PR3-/- mice had a higher survival rate. These data suggest that enhanced stem and progenitor cell compartments in PR3-/- mice are functional. To understand if the enhanced hematopoietic activity in PR3-/- mice is an intrinsic feature of stem cells, another set of competitive reconstitution experiments were performed using equal numbers of sorted LSK cells. Again, PR3-/- donor-derived cells were more dominant than WT donor-derived cells in the recipient mice.Then, we wanted to understand the underlying mechanism. The rate of proliferation and senescence in LSK cells were analyzed by BrdU incorporation studies and C12FDG staining, respectively. No difference in proliferation or senescence was observed between WT and PR3-/- mice. We also looked at the apoptosis rate of LSK cells in vivo and in vitro. Untreated LSK cells from WT and PR3-/- mice exhibited similar levels of viability both in vivo and in vitro. However, when bone marrow macrophages responsible for clearance of apoptotic cells were depleted using clodronate liposomes, PR3-/- LSK cells exhibit enhanced viability compared to WT LSK cells as evidenced by annexin V and 7-AAD staining. Using a fluorogenic substrate of caspase 3, we also observed that the enhanced viability seen in PR3-/- LSK cells after depletion of macrophages is due to a reduction in caspase 3 activation.Additionally, we asked whether LSK cells from PR3-/- mice represent features associated with stem cells from aged mice such as an increase in DNA damage accumulation and reduced polarity. Immunofluorescent microscopy studies suggested that long-term hematopoietic stem cells from PR3-/- mice exhibit higher levels of p-gH2AX foci, a marker of DNA damage, and a reduced polarized distribution of a-tubulin. Finally, we saw a reduced lifespan in PR3-/- mice. These data indicate that PR3 regulates bone marrow hematopoiesis by contributing to the maintenance of a healthy stem cell pool in the bone marrow. DisclosuresNo relevant conflicts of interest to declare.

Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation.

BackgroundThe Sox genes, a family of transcription factors characterized by the presence of a high mobility group (HMG) box domain, are among the central groups of developmental regulators in the animal kingdom. They are indispensable in progenitor cell fate determination, and various Sox family members are involved in managing the critical balance between stem cells and differentiating cells. There are 20 mammalian Sox genes that are divided into five major groups (B, C, D, E, and F). True Sox genes have been identified in all animal lineages but not outside Metazoa, indicating that this gene family arose at the origin of the animals. Whole-genome sequencing of the lobate ctenophore Mnemiopsis leidyi allowed us to examine the full complement and expression of the Sox gene family in this early-branching animal lineage.ResultsOur phylogenetic analyses of the Sox gene family were generally in agreement with previous studies and placed five of the six Mnemiopsis Sox genes into one of the major Sox groups: SoxB (MleSox1), SoxC (MleSox2), SoxE (MleSox3, MleSox4), and SoxF (MleSox5), with one unclassified gene (MleSox6). We investigated the expression of five out of six Mnemiopsis Sox genes during early development. Expression patterns determined through in situ hybridization generally revealed spatially restricted Sox expression patterns in somatic cells within zones of cell proliferation, as determined by EdU staining. These zones were located in the apical sense organ, upper tentacle bulbs, and developing comb rows in Mnemiopsis, and coincide with similar zones identified in the cydippid ctenophore Pleurobrachia.ConclusionsOur results are consistent with the established role of multiple Sox genes in the maintenance of stem cell pools. Both similarities and differences in juvenile cydippid stage expression patterns between Mnemiopsis Sox genes and their orthologs from Pleurobrachia highlight the importance of using multiple species to characterize the evolution of development within a given phylum. In light of recent phylogenetic evidence that Ctenophora is the earliest-branching animal lineage, our results are consistent with the hypothesis that the ancient primary function of Sox family genes was to regulate the maintenance of stem cells and function in cell fate determination.

PIAS adds methyl-bias to HSC-differentiation

Epigenetic modifications of stem cell genome including DNA methylation and histone modifications are critical for the regulation of stem cell gene expression and maintenance of stem cell pool and their differentiation. Although the importance of epigenetic modifications specifically DNA methylation to adult hematopoietic stem cells (HSC) has been established, the identity of specific modulators and precise mechanism of integration of methylation events remain to be uncovered. In this issue, Shuai and colleagues identify the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT1) as a key regulator of DNA methylation of HSC required for their maintenance and lineage commitment (Liu et al, 2014).

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-KithighSca-1+Lineage− (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Plant Stem Cell Signaling Involves Ligand-Dependent Trafficking of the CLAVATA1 Receptor Kinase

Cell numbers in above-ground meristems of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex. CLV3 produced in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 and WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. Overexpression of CLV3 eliminates the stem cells, causing meristem termination, and loss of CLV3 function allows meristem overproliferation. There are many questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype. Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model. This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants.

Development of novel inhibitors targeting HIF-1α towards anticancer drug discovery

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.

The Impact of Developmental Stage of Osteoblasts and Collagen Fibril Matrix Properties On Hematopoietic Stem Cell Function.

Abstract 3638Poster Board III-574Previously we reported the impact of hematopoietic niche (HN) elements, calvaria-derived osteoblasts (OB) and bone marrow derived stromal cells (SC), on murine hematopoietic stem cells (HSC, defined as Lin-sca1+c-kit+ or LSK) cultured in a conventional two-dimensional (2D) format. Interestingly, we found that OB maintained the functional properties of LSK cells including their long-term marrow repopulating potential significantly better than SC thus corroborating the importance of OB in the overall competence of the HN. We also reported that this stem cell function-enhancing activity is induced by the up regulation of Notch-mediated signaling between HSC and OB. However, which specific lineage or developmental stage of OB enhances HSC function is still unknown. In the present study, we evaluated the interactions between HSC and OB at various stages of development. We also examined the impact of physical parameters of the extra cellular matrix, such as stiffness/storage modulus using a more physiologically relevant 3D context consisting of collagen-fibril matrices in which the fibril density and shear storage modulus was systematically varied. OB were harvested from murine calvaria and used directly (fresh OB) or were cultured for 1, 2, or 3 weeks in basic or complete medium (supplemented with ascorbic acid and β-glycerophosphate). A total of 1000 LSK cells were co-cultured for 1 week with either fresh OB or OB cultured for 1, 2, or 3 weeks. Cultures were examined for hematopoietic cell number increase, colony forming unit (CFU) expansion, LSK phenotype, and plating efficiency. In addition, OB function was analyzed by measuring alkaline phosphatase activity and calcium deposition/mineralization. Surprisingly, total hematopoietic cell number on day7 was significantly higher in cultures containing fresh OB (34.5±3.3×105) compared to OB cultured for 1 week (12.4±4.0 ×105, p=0.05), 2 weeks (9.8±4.6 ×105, p=0.03), or 3 weeks (7.8±3.4 ×105, p=0.02). Total CFU fold expansion was also significantly elevated in fresh OB co-cultures (149.6±45.7) vs OB cultured for 1 week (62.4±18.7, p=0.04), 2 weeks (51.0±14.5, p=0.01), or 3 weeks (52.4±23, p=0.03). The percentage of Lin-Sca1+ cells was also significantly higher in co-cultures of fresh OB (29.5±3.6%) vs OB cultured for 1 week (5.3±0.9, p=0.02), 2 weeks (0.9±0.2, p=0.008), or 3 weeks (1.0±0.2, p=0.008). Interestingly, alkaline phosphatase activity and calcium deposition were inversely associated with all hematopoietic properties examined. In order to examine the significance of 3D spatial and physical properties of the extra cellular matrix microenvironment, which is absent in traditional culture systems, LSK cells were cultured in both the presence and absence of calvariae-derived OB within 3D engineered collagen type I matrix constructs in an effort to recreate the in vivo niche conditions. Matrix parameters, including collagen fibril density (9-20%) and shear storage modulus (50-800 Pa, G') were systematically varied and quantified. LSK proliferated most rapidly when co-cultured with OB in low fibril density/G' (50 Pa) collagen type I matrices (836.8±74 fold-increase) compared to 200Pa (172.3±26, p=0.007) or 800Pa (128.4±27, p=0.006). Proliferation of LSK cells in matrices not containing OB was minimal. Interestingly, the clonogenicity of cells harvested from 800Pa matrices was nearly 3-fold and 1.5-fold higher than that for cells from 50Pa and 200Pa matrices, respectively. Furthermore, 800Pa matrices maintained the highest percentage of Lin-Sca1+ cells for 7days (15.9±7.3) compared to 200Pa (9.3±3.5) and 50Pa (6.9±1.5) matrices. These data illustrate that collagen matrices with relatively high fibril density/G' suppressed rapid proliferation and expansion and maintained progenitor cell function. Alternatively, low stiffness matrices promoted cell expansion at the apparent expense of differentiation and loss of clonogenic potential of progenitor cells. Collectively these results demonstrate that early stage OB maintains HSC function significantly better than later stage OB. Furthermore, physical properties such as fibril density and shear storage modulus (stiffness) of the surrounding collagen fibril matrix also play a critical role in HSC proliferation and maintenance of stem cell pool. Disclosures:No relevant conflicts of interest to declare.

Stemming out of a new PML era?

The promyelocytic leukaemia protein PML is a growth and tumour suppressor inactivated in acute promyelocytic leukaemia (APL). Recent evidence indicates that PML plays a tumour-suppressive role in cancer of multiple histological origins. However, it is only very recently that PML growth-suppressive functions have been implicated in regulating physiological processes and tissue homoeostasis. In particular, it has been shown that PML is one of the key cell-cycle regulators controlling stem cell function in multiple tissues, from the blood to the brain. As a consequence, PML loss has an impact on tissue development and maintenance of stem cell pools. In addition, new data suggest that PML regulates self-renewal in cancer stem cells. Finally, the oncogenic fusion protein PML/RARalpha, contrary to the conventional view, appears to hijack growth-suppressive pathways to promote transformation of haematopoietic stem cells and to maintain the APL stem cell niche. Overall, these findings not only represent a change in paradigm in the field of PML/APL research, but also contribute to the understanding of fundamental mechanisms underlying stem cell function in vivo. The main objective of this review is to critically discuss the very recent literature on the role of PML in stem cells and tumour-initiating cells. Ultimately, it aims to propose new avenues of investigation.

Autocrine Fibroblast Growth Factor 2 Signaling Is Critical for Self‐Renewal of Human Multipotent Adipose‐Derived Stem Cells

Adipose tissue-derived stem cells offer tremendous potential for regenerative medicine. However, characterization of their self-renewal ability has not been performed yet, although it is a crucial feature for in vitro expansion of undifferentiated cells and in vivo maintenance of stem cell pools. We have undertaken the identification of molecular events that are involved in in vitro self-renewal of human multipotent adipose-derived stem (hMADS) cells from young donors, by assessing their proliferation rate, their ability to grow at the single-cell level (clonogenicity), and their differentiation potential. As hMADS cells are propagated in culture, cell morphology changes dramatically, concomitantly to a progressive decrease in proliferation, clonogenicity, and differentiation potential. This decrease is associated with a decrease in fibroblast growth factor 2 (FGF2) expression and can be circumvented by chronic treatment with exogenous FGF2. Moreover, analysis of FGF2 secretion revealed that it is exported to hMADS cell surface without being released into the culture medium, suggesting a strictly autocrine loop. Indeed, treatment of FGF2-expressing hMADS cells with PD173074, a specific FGF receptor inhibitor, decreases dramatically their clonogenicity and differentiation potential. Thus, hMADS cells express a functional autocrine FGF loop that allows maintenance of their self-renewal ability in vitro. Finally, inhibition of mitogen-activated protein kinase kinase 1 reduces the clonogenic potential of hMADS cells but does not affect their differentiation potential, indicating that the extracellular signal-related kinases 1/2 signaling pathway is partly involved in FGF2-mediated self-renewal. Together, our data clearly identify the key function of FGF2 in the maintenance of self-renewal of adipose tissue-derived stem cells.

All the adult stem cells, where do they all come from? An external source for organ-specific stem cell pools

Stem cells can self-renew and maintain the ability to differentiate into mature lineages. Whereas the "stemness" of embryonic stem cells is not discussed, the primitiveness of a stem cell type within adult organisms is not well determined. Data presently available are either inconclusive or controversial regarding two main topics: maintenance or senescente of the adult stem cell pool; and pluripotentiality of the cells. While programmed senescence or apoptosis following uncorrected mutations represent no problem for mature cells, the maintenance of the stem cell pool itself must be assured. Two different mechanisms can be envisaged for that. In the first mechanism, which is generally accepted, stem cells originate during ontogeny along with the organ which they are responsible for, and remain there during all the lifespan of the organism. Several observations derived from recent reports allow the suggestion of a second mechanism. These observations include: organ-specific stem cells are senescent; adult stem cells circulate in the organism; stem cell niches are essential for the existence and function of stem cells; adult stem cells can present lineage markers; embryo-like, pluripotent stem cells are present in adult organisms, as shown by the development of teratomas, tumors composed of derivatives of the three germ layers; and the fact that the gonads may be a reservoir of embryo-like, pluripotent stem cells in adult organisms. The second mechanism for the maintenance of adult stem cells compartments implies a source external to the organ they belong, consisting of pluripotent, embryo-like cells of unrestricted life span, presenting efficient mechanisms for avoiding or correcting mutations and capable to circulate in the organism. According to this model, primitive stem cells exist in a specific organ in adult organisms. They undergo asymmetrical divisions, which originate one "true" stem cell and another one which enters the pool of adult stem cells, circulating through the entire organism. Upon signals liberated by organ-specific niches, this cell becomes activated to express lineage-specific genes, homes to that particular organ and repopulates its stem cell compartment, differentiating thus in what is seen as the organ-specific stem cell. The gonads are the natural candidates for homing the primitive stem cells in adult organisms. The model proposed in this work for the maintenance of organ-specific stem cell pools from an external source, represented by primitive, embryo-like germinal stem cells present in testes and ovaries, may contribute to the more complete understanding of this complex issue.

A Genetic Screen for Zebrafish Mutants Affecting Early Hematopoiesis.

Hematopoiesis, or blood cell formation, starts from hematopoietic stem cells and goes through many rounds of cell proliferation and a number of commitment steps that progressively restrict cells to a particular lineage. The generation and maintenance of stem cell pool and the regulation of lineage commitment are poorly understood. Right now there is only one reported zebrafish mutant, cloche, that has defects in early hematopoiesis. We have undertaken a chemical mutagenesis screen using ENU in the zebrafish to uncover mutants in early hematopoiesis. Haploid embryos from F1 females were screened by RNA in situ hybridization using a stem cell marker, core binding factor ? (cbfb), and a myeloid-specific marker, leukocyte-specific plastin (l-plastin). We have screened 510 F1 females and 47 putative mutants have been identified. Of these, 22 showed decreased expression of l-plastin, 3 for cbfb, 2 for both l-plastin and cbfb and the remainder were mutated for l-plastin and other non-hematopoietic markers. Right now, we have focused our efforts on two mutants.The first mutant line, L32, displayed a blood-less phenotype and showed a significant decrease in the expression of the hemangioblast marker scl. In addition to the marked reduction in the expression of the myeloid specific markers l-plastin, c/ebp1 and mpo, these embryos also showed decreased expression of the erythroid specific marker gata-1. Linkage analysis has placed this mutant to the proximal region of linkage group 12. Currently, we are in the process of testing candidate genes to identify the mutated gene. The second mutant line, L63 also has a blood less phenotype and showed decreased expression of both l-plastin and mpo. Like L32, L63 homozygous embryos have significant developmental defects and die 3 dpf. Low resolution mapping has placed this mutant to LG 8. Through this approach, we hope to identify novel genes that play critical roles in the developmental process regulating early hematopoiesis.