As PcG proteins and miRNAs are usually

As PcG proteins and miRNAs are usually co-expressed in many cell types or cancer tissues, such as keratinocytes (Eckert et al., 2011; Yi et al., 2008), hematopoietic stem leptomycin b (Takamatsu-Ichihara and Kitabayashi, 2016), hepatocellular carcinoma (Yonemitsu et al., 2009), lung cancer (Chen et al., 2015; Jin et al., 2013), breast cancer (Ru et al., 2011; Yu et al., 2012), and prostate cancer (Viticchie et al., 2011), it will be of interest to elucidate the complicated regulatory network involving multiple epigenetic factors that are responsible for determining cell fate and balancing the proliferation and differentiation of different cell types in future studies. The detailed regulatory network involving PcG proteins and other epigenetic factors that are responsible for altered NSPC behaviors will provide critical insights into the cellular control of NSPC proliferation and fate choices, and might lead us to find new therapeutic strategies for the treatment of neurological diseases.

Experimental Procedures

Author Contributions

Acknowledgments
This work was supported by grants from the National Science Foundation of China (grant nos. 31571043 to C.M.L. and 81571212 to Z.Q.T.), National Science and Technology Major Project (grant no. 2016YFA0101402 to C.M.L.), State Key Laboratory of Stem Cell and Reproductive Biology, and the Hundred Talents Program of CAS.

Introduction
Neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) in the walls of the lateral ventricles of many adult mammals. This neurogenic niche is composed of NSCs (B1 astrocytes) that divide slowly to give rise to transit-amplifying cells (C cells), which in turn generate neuroblasts (A cells) that migrate tangentially to the olfactory bulb (Alvarez-Buylla et al., 2001; Lois and Alvarez-Buylla, 1994). B1 cells are characterized by their highly polarized morphology, which presents a thin apical process that contacts the lateral ventricle (LV) and cerebrospinal fluid (CSF). Moreover, they also exhibit a basal process ending on blood vessels (Doetsch et al., 2002; Mirzadeh et al., 2008; Tavazoie et al., 2008). The apical surface of B1 cells is surrounded by large apical surfaces of ependymal cells in a pinwheel configuration (Mirzadeh et al., 2008). NSCs cells can exist as quiescent/slowly dividing (qNSCs) or activated/dividing (aNSCs) primary progenitors. It has been suggested that these two populations represent two functionally distinct types of NSCs which differ in their cell-cycle status and molecular properties (Codega et al., 2014; Llorens-Bobadilla et al., 2015; Mich et al., 2014; Morshead et al., 1994). aNSCs maintain the expression of glial fibrillary acidic protein (GFAP), CD133, epidermal growth factor receptor (EGFR), and Nestin, while qNSCs preserve the expression of GFAP, CD133, but not EGFR and Nestin. Furthermore, qNSCs do not express proliferation markers and survive infusion of cytosine-β-D-arabinofuranoside (Ara-C), which eliminates the aNSC population (Codega et al., 2014; Doetsch et al., 1999; Morshead et al., 1994; Pastrana et al., 2009). Recently, it has been suggested that qNSCs have an embryonic origin; pre-B1 cells are produced during mid-fetal development (embryonic day 13.5 [E13.5] to E15.5), remaining relatively quiescent until reactivated postnatally (Fuentealba et al., 2015; Furutachi et al., 2015).
The maintenance of quiescence is thought to be directly co-related with the regulation of gene expression, which can be observed as large heterochromatic regions likely corresponding to silenced genes (Capelson and Corces, 2012). Previously, it has been suggested that a distinctive nuclear morphology is linked to the maintenance of pluripotency (Gorkin et al., 2014; Ito et al., 2014; Sexton and Cavalli, 2013), and possibly associated with quiescence. However, despite NSC chromatin presenting peculiar topographical configurations (Krijger et al., 2016; Peric-Hupkes et al., 2010; Phillips-Cremins et al., 2013), the relationship between chromatin organization and nuclear morphology remains poorly understood. Previous studies have shown that murine and human fetal V-SVZ B cells have irregular nuclei that exhibit unusual nuclear envelope (NE) invaginations (Capilla-Gonzalez et al., 2014; Doetsch et al., 1997; Guerrero-Cazares et al., 2011).

ag1478 It seems that D R

It seems that D1R improves HSC transplantation likely through affecting both HSCs and their niches. Activation of Notch signaling in HSCs by D1R bound to ECs might promote HSC expansion principally through their enhanced proliferation, and decreased apoptosis might also contribute to the ex vivo expansion to certain extent. The gene profiling experiment indicated that D1R stimulated the expressions of a group of stemness-related genes in HSCs, consistent with a role of forced Notch activation in promoting HSC expansion. In addition to the direct effects on HSCs, D1R might also promote HSC engraftment through influencing, structurally and/or functionally, stem cell niches. Ex vivo, D1R promotes the formation of adhesive structures between HSCs and HUVECs, whose survival is necessary for the expansion of HSCs. D1R significantly promoted the recovery of the structure and organization of BM SECs after radiation, consistent with the role of Notch signaling in regulating EC proliferation and differentiation (Dou et al., 2008). In addition to BM, the application of D1R also leads to increased hematopoiesis in the liver and spleen, which are hematopoietic tissues in rodents. D1R could bind to ECs in BM as well as in the liver and spleen, and modulates their activity in supporting HSCs. D1R might also modulate HSCs to increase their homing to hematopoietic tissues. Notch signaling regulates the ag1478 of CXCR4, a critical receptor for HSC homing (Kelly et al., 2010; Delaney et al., 2010a; Blank et al., 2008). Moreover, adhesion molecules such as some members of the integrin family are critically involved in HSC homing to BM niches (Wagers et al., 2002). Indeed, ag1478 the mRNA levels of the αv, α5, α6, and β1 integrins were upregulated in the presence of mD1R compared with that of the controls (supplementary Fig. S10). It is possible that the interaction between HSCs and ECs mediated by Notch signaling helped the homing and proliferation of HSCs in the hematopoietic tissues including BM, spleen, and liver of mice treated with D1R, because Notch signaling has been demonstrated to crosstalk with many important signaling pathways involved in stem cell niches (Blank et al., 2008).

Authorship and disclosures

Competing interests

Introduction
Directed cell migration (chemotaxis) towards a stimulus is a well defined function of many mammalian and non-mammalian cells and is vital throughout embryonic and postnatal life (Petrie et al., 2009). A key example is the homing or migration of hematopoietic stem/progenitor cells (HSPCs) to specific microenvironmental niches, where their fate is determined (Bianco, 2011; Lawal and Calvi, 2011; Mazo et al., 2011; Mercier et al., 2011; Nagasawa et al., 2011; Calderón and Boehm, 2012; Park et al., 2012) or mobilization from these niches using small molecule strategies or in disease states (Kolonin and Simmons, 2009; Shiozawa and Taichman, 2010; Mohty and Ho, 2011; Psaila et al., 2012). Importantly, in the clinical setting, prior manipulation or expansion of HSPCs can compromise or enhance their homing or migratory capacities and this can affect transplant outcomes (Aljitawi, 2012). This is particularly pertinent for cord blood where HSPC content is limited, engraftment and hematological reconstitution are delayed compared to bone marrow or mobilized peripheral blood, one cord blood unit will engraft in preference to another in double cord blood transplants, and expansion/manipulation ex vivo prior to transplant is used to reduce delayed engraftment (Dahlberg et al., 2011; Nagasawa et al., 2011; Petropoulou and Rocha, 2011; Watt, 2011; Aljitawi, 2012; Broxmeyer, 2012; Christopherson et al., 2012; Csaszar et al., 2012; Ramirez et al., 2012).
The CXC chemokine, CXCL12, is a key chemo-attractant for HSPC homing to bone marrow, also regulating HSPC motility, homing to, and retention, survival, and proliferation in this niche (Peled et al., 1999; Dar et al., 2006; Watt and Forde, 2008; Sharma et al., 2011; Bonig and Papayannopoulou, 2013). The cognate receptors for CXCL12 are CXCR4 and CXCR7, although the latter is poorly expressed on human HSPCs (Hartmann et al., 2008; Sun et al., 2010). However, where expressed on other cells, CXCR7 is thought to act as a decoy receptor or co-receptor for CXCR4 (Naumann et al., 2010; Sun et al., 2010). CXCL12/CXCR4 deficient mice demonstrate defects in hematopoietic, immune, circulatory and central nervous systems (Zou et al., 1998; reviewed in Watt and Forde, 2008). Co-operation and cross talk between CXCL12/CXCR4, other receptors/proteins, and signaling molecules are thought to fine tune cellular responses and/or specificity for microenvironmental niches (Forde et al., 2007; Christopherson et al., 2012; Schiraldi et al., 2012).

br Molecular changes associated with

Molecular changes associated with THZ2 withdrawal have been described, but molecular mechanisms of terminal differentiation remain largely uncharacterized
The transition to the terminally differentiated phenotype is accompanied by down-regulation of cell cycle factors and up-regulation of cell cycle inhibitors (Soonpaa et al., 1996; Mollova et al., 2013; Adler and Costabel, 1975; Brooks et al., 1998; Poolman and Brooks, 1998). Cell cycle activators such as the Cdk/cyclin complex, Myc, E2F transcription factors were demonstrated to be repressed. Negative cell cycle regulators are increased, such as p21, p27, retinoblastoma protein, and cyclin-dependent kinase inhibitors (Pasumarthi and Field, 2002; Li et al., 1998; Flink et al., 1998). However, the functional relevance of these associations has not been fully characterized. Decreased cardiomyocyte cell cycle activity was shown to be associated with the increase of the tissue oxygen tension upon birth, leading to increased cardiomyocyte DNA damage (Puente et al., 2014).

Under homeostatic conditions, cardiomyocyte turnover in the mammalian heart is very low
Turnover is the dropout and generation of new cells without a change of the total number of cells in the organ, i.e. during homeostasis. The number of cardiomyocytes per heart is important when considering the rate of cardiomyocyte turnover and regeneration. Few studies have quantified the number of cardiomyocytes in human hearts (Hsieh et al., 2007; Lesauskaite et al., 2004). Using stereology, we quantified the number of cardiomyocytes in adult humans to be approximately 3.7 billion (n=7), corresponding to 5.6 billion cardiomyocyte nuclei (Mollova et al., 2013). Our results are between the results from another stereology study that quantified the number of cardiomyocyte nuclei in adult humans to be 9.5 billion (n=6 hearts, ref. (Tang et al., 2009)), and a study using biochemical techniques, which showed 2 billion cardiomyocytes (n=30, ref. (Adler and Costabel, 1975)).
Quantification of cardiomyocyte loss is challenging. Apoptosis markers are reported to have variable sensitivity. DNA repair and postmortem enzymatic degradation may yield false-positive results with the terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay (Lesauskaite et al., 2004; Saraste and Pulkki, 2000). The irreversible progression of cell death in cells expressing late stage apoptosis markers has also been called into question in several organs, including the heart (Tang et al., 2012). Thus, there is a lack of consensus for the frequency of cardiomyocyte apoptosis in normal hearts, with estimates varying by more than an order of magnitude (Lesauskaite et al., 2004; Saraste and Pulkki, 2000; Wencker et al., 2003; Chen et al., 2001; Vulapalli et al., 2002).
Cardiomyocyte turnover was quantified with different techniques. The carbon-14 birth dating strategy showed that adult humans turn over approximately 0.5% of cardiomyocytes per year (Bergmann et al., 2009). Our MIMS approach showed a turnover rate of approximately 1% in adult mice (Senyo et al., 2013). Several groups found multinucleation and polyploidy to account for a majority of cell cycle events after the first two weeks of life in murines (Senyo et al., 2013; Malliaras et al., 2013; Walsh et al., 2010). Using immunofluorescence microscopy, we have not been able to detect cardiomyocytes in cytokinesis in adult humans (Mollova et al., 2013). Overall, reported rates of cardiomyocyte turnover in adult mice and humans are converging at approximately 1% or less per year (Senyo et al., 2013; Soonpaa et al., 1996; Mollova et al., 2013; Bergmann et al., 2009).
We have utilized the α-MHC-MerCreMer; Z/EG mice for cell lineage tracing in concert with stable isotope-labeled thymidine to detect cardiomyocytes with a history of cell cycle activity (Senyo et al., 2013). Approximately 80% of preexisting cardiomyocytes were genetically labeled with GFP by injection of 4-hydroxy-tamoxifen. We have labeled with isotope-labeled thymidine for four to ten weeks at different points after birth. We used MIMS analysis of histological sections to visualize isotope-labeled thymidine localization. We identified cardiomyocyte nuclei by staining for cardiac markers (sarcomeric actin) and nuclei (DAPI or PAS histological staining). To analyze multi-nucleated cardiomyocytes, we used fiduciary marks on sections to retrieve corresponding nuclei on adjacent sections. Fluorescent in situ hybridization in at least two sections on either side of the MIMS section was used to determine ploidy. Using this method, we quantified that <1% mononucleated diploid cardiomyocytes per year in adult mice were generated.

br Methods and materials br Results

Methods and materials

Results

Discussion
In vitro differentiation of ESCs closely mimics early embryonic development and can produce large quantity of hematopoietic cells. However, segregation of the EryPs from surrounding buy THZ531 differentiated from mESCs has not been reported. Here, we demonstrated that CD71high could be utilized as a marker to purify EryPs from differentiated ESCs. Although overlapped with markers for hematopoietic progenitors, CD41 and c-kit, the CD71high population was exclusive for myeloid-lymphoid cells which were positive for CD45. In addition, CD71high cells sorted from day 5 EBs appeared to be homogenous, highly enriched for erythroblasts that gave rise about 800 red colonies per 10,000 cells. These data clearly demonstrate that EryPs in the CD71high population are the dominant blood lineage. As previously reported, EryPs differ significantly from EryDs in size, globin expression, and transcriptional regulation (Baron et al., 2012; Fraser, 2013; Palis et al., 2010). The molecular mechanisms underlying these differences are however only partially described because significant amounts of EryPs are required for such mechanistic investigation. Efficient production and segregation of CD71high EryPs from differentiated ESCs will therefore allow us to explore these fundamental questions during erythropoiesis.
Similarly to EryPs from YS around 8.5dpc, βH1 was the predominant type of embryonic goblin in CD71high cells. These putative CD71high EryPs were able to go through “maturational globin switching” and exhibited similar expression pattern with YS-derived EryPs. More importantly, we further demonstrated that these CD71high cells matured into Ter119+ enucleated red blood cells when co-culturing with macrophages in vitro. These data thus validate our claim that CD71high population represents an in vitro counterpart of YS-derived EryPs that can mature and enucleate. Notably, enucleation of erythrocytes in vitro has been a challenge in the field of hematopoietic research. It will be of great interest to explore the specific contribution of macrophages may have during this process in the future study.
According to current model, bi-potential megakaryocyte-erythroid precursors (MEPs) exist during primitive hematopoiesis (Klimchenko et al., 2009; Tober et al., 2007). However, it remains unclear how the fate specification of EryPs and megakaryocytes is regulated. We found that Scl promoted the formation of all blood lineages from mESCs as a master regulator of hematopoiesis. In contrast, megakaryopoietic differentiation depends more stringently on Ets family members, such as Erg and Fli1. At the expense of megakaryocytic development, induction of Klf1 favored the commitment of erythrocytes by upregulating CD71high population and transcript levels of βH1 and εy globins. Similarly, Eaf1 depletion blocked the development of erythrocytes as demonstrated by reduced percentage of CD71high cells, thus indicating a critical requirement of Eaf1 in the formation of EryPs. Therefore, our approach to utilize CD71high EryPs from ESCs coupled with genetic modification may represent a valuable model to examine cell-intrinsic regulation of primitive erythropoiesis during early embryonic development.
The following are the supplementary materials related to this article.

Acknowledgment
The Scl−/− ESC line is a kind gift from Dr. Stuart Orkin\’s lab at Harvard Medical School. We would like to thank Dr. Bing Du at East China Normal University for the help in macrophage derivation from BM. This work was supported by grants from the Ministry of Science and Technology of China (2010CB945403 and 2014CB964800), the National Science Foundation of China (31271589 and 30971522), and the Science and Technology Commission of Shanghai Municipality (11DZ2260300 and 13JC1406402).

Introduction
Cell differentiation in adults involves flexible but precise control of the expression of genes specific for each cell fate or pluripotency. Crucial changes in their expression are most likely controlled by specific epigenetic rearrangements (Vincent & Van Seuningen, 2009). Extensive studies conducted to characterise embryonic stem cells (ESC) at the epigenetic level have shown that ESC harbour a permissive chromatin with hypomethylation of their genome compared to differentiated cells (Bibikova et al., 2006) and bivalent histone marks where large regions containing trimethylated K27H3 (gene silencing) colocalise with smaller regions containing methylated K4H3 (gene activation) (Bernstein et al., 2006). This allows rapid transcriptional activation of genes specific for a cell fate. One can hypothesise that the plasticity of adult SC involves similar epigenetic mechanisms. However, only a few studies mostly conducted in vitro have addressed this concept.

The following are the supplementary

The following are the supplementary data related to this article.

Acknowledgments
This work was supported by the Andrew L. Warshaw, MD Institute for Pancreatic Cancer Research, Massachusetts General Hospital, and by NIH-P01CA117969 to SPT. We would like to Thank Dr. Timothy Wang, Dorothy L. and Daniel H. Silberberg Professor of Medicine and Chief, Division of Digestive and Liver Diseases, Columbia University, for kindly providing TKK2 knockout mice.

Introduction
The primary response to inflammation, infection and tissue injury is mediated by the innate immune system via toll-like receptors (TLR1–6). TLRs bind a variety of ligands to transduce signals via receptor-associated kinase systems such as p38 activation, NFκB nuclear translocation and STAT signaling cascades, which trigger the release of pro-inflammatory cytokines like IL1β, IL6 and members of the CCL and CXCL families of cytokines (Kang and Lee, 2011; Brown et al., 2011) that recruit SAR405 of the adaptive immune system if necessary.
The TLR family of receptors is tightly involved in the modulation of functions of mesenchymal and other stem cells (Rolls et al., 2007). Bone marrow-derived mesenchymal stem cells (MSCs) are the principal source of bone regeneration. MSCs have been reported to express TLR1–6 and MSC treatment with a cocktail of pro-inflammatory cytokines especially upregulates TLR2, 3 and 4 expression in mice and humans (Delarosa et al., 2012). The role of TLR in MSC biology has SAR405 not yet been completely unraveled and variably contrasting effects of TLR activation on MSC biology have been reported. TLR2 activation using the ligand Pam3Cys followed by NFκB nuclear translocation completely abolished MSC multipotent differentiation capacity in mice (Pevsner-Fischer et al., 2007), while depending on the time frame and type of activation osteogenic differentiation was either inhibited or enhanced (Huang et al., 2014; Mo et al., 2008; Raicevic et al., 2012; Chang et al., 2013; Zhao et al., 2011). Hence the complete picture of the role of TLR expression in MSC biology is just emerging (Delarosa et al., 2012).
Bone regeneration and bone healing are accompanied by an initial inflammatory reaction following injury and an initial burst of growth factors released by platelets from fracture hematoma (Kolar et al., 2010; Gerstenfeld et al., 2003). Pro-inflammatory stimuli have been reported to enhance osteogenic differentiation and bone healing but may also inhibit bone formation depending on the duration and the specific signaling stimulus (Mo et al., 2008; Raicevic et al., 2012; Chang et al., 2013; Zhao et al., 2011). Moreover, osteogenic differentiation itself stimulates the expression of TLR2 and 4 in hMSC (Kovacevic et al., 2008). In murine and human MSCs TNFα stimulation and NFκB activation have shown very inconsistent results with respect to osteogenic differentiation and bone healing depending e.g. on the origin of MSC (adipose tissue versus bone marrow) and also the recruitment and migration phases compared to later phases of bone healing (Pevsner-Fischer et al., 2007; Huang et al., 2014; Raicevic et al., 2012). Mineralization is an endpoint of osteogenic differentiation and bone formation. However extra-osseous mineralization plays an important role in chronic inflammatory and aging-associated degenerative diseases such as atherosclerosis and sclerosing bone metastases (Hofbauer et al., 2014). We have recently described the expression of WNT5A in TNF and LPS-treated skeletal precursors and a subtle analysis of WNT5A effects on murine MSC described an amplification of pro-inflammatory signals downstream WNT5A (Rauner et al., 2012). Immunohistochemistry analyses of atherosclerotic lesions also revealed marked expression of WNT5A in such areas, consistent with the hypothesis that atherosclerosis is a chronic inflammatory disease (Christman et al., 2008). Given the fact however that treatment with anti-TNF antibodies under certain circumstances could stop arthritis but not the extraosseous bone formation, there is still a different trigger to be found for the inflammatory self-sustaining loop and this pathological form of mineralization.

Our coculture experiments demonstrated that all three

Our coculture experiments demonstrated that all three MSCs showed a similar low in vitro proliferation; for most patients coculture of all three MSCs with primary human AML MAPK Inhibitor Library did not alter MSC proliferation but a minor increase in the proliferation was seen for a minority of patients. No or only minor effects on MSC proliferation were also seen when they were cultured with exogenous cytokines or AML supernatants. These observations suggest that the growth-enhancing effect of primary AML cells on MSCs depends on the overall intercellular crosstalk. It should in addition be emphasized that MAPK Inhibitor Library the MSC growth enhancement was observed for most patients despite the wide variation in cytokine release between patients both in AML cultures and AML–MSC cocultures.
Coculture of MSCs with primary AML cells especially altered the MSC expression of genes involved in TLR-initiated signaling (i.e. genes downstream to the receptors), regulation of NFκB and chemokine/interleukin expression (Fig. 4 and Supplementary Table 3). These three components form an interacting system at different levels of the cells (Bruserud et al., 2007). TLR receptors show transactivation with G-protein coupled receptors (e.g. chemokine receptors) (Abdulkhalek et al., 2012), NFκB is an important downstream target of TLR-initiated signaling and NFκB is in addition an important regulator of chemokine expression/release in various human cells, including primary AML cells. Therapeutic targeting of the NFκB system thus seems to represent an opportunity to modulate the local AML-supporting cytokine network in the bone marrow through inhibition of cytokine release both by the leukemic cells and normal stromal cells.
Ito et al. (2014) demonstrated that MSCs could support the growth of AML cells in cocultures, and our present study shows that there is a bidirectional crosstalk between AML cells and MSCs as the MSC characteristics were altered in our transwell cocultures. However, it is not known whether the cytokine network alone mediates an AML-supporting bidirectional crosstalk between mesenchymal and leukemic cells because the study by Ito et al. questioned the importance of the cytokine network and emphasized the importance of direct cell–cell contact for the MSC-associated growth enhancement of the AML cells (Ito et al., 2014).
The following are the supplementary data related to this article.

Introduction
The induction of cellular plasticity has emerged as a powerful tool for regenerative medicine (Yamanaka and Blau, 2010; Nie et al., 2012; Katsuyama and Paro, 2011). Regeneration seen in a wide range of non-mammalian vertebrates, such as urodele amphibians and teleost fish provides important information about natural mechanisms of using cellular plasticity for renewing large body parts (Brockes and Kumar, 2002; Gemberling et al., 2013). A crucial question for the field of regenerative medicine is whether urodele regeneration can be recapitulated in mammalian cells.
Studies on amphibian or fish regeneration identified reversal and plasticity of the differentiated state as a major mechanism to produce stem cell-like cells during regeneration (Jopling et al., 2010; Echeverri et al., 2001; Kragl et al., 2009). This process, classically called dedifferentiation, reprograms differentiated cells at the wound site back to a progenitor stage, which can then proliferate and replace the missing tissue with appropriate patterning (Stoick-Cooper et al., 2007). Dedifferentiation has been best characterized in salamander muscle cells. It has been shown that upon signals produced in the regenerating tissue, multinucleated postmitotic differentiated myotubes can reverse their differentiated state and give rise to proliferating progenitors (Lo et al., 1993; Kumar et al., 2000; Sandoval-Guzmán et al., 2014). To date, there is not any convincing evidence demonstrating dedifferentiation occurring physiologically in mammalian muscles. Therefore, an intriguing question is whether this process could be engineered into mammalian muscle.

The NAD dependent protein silent information regulator

The NAD-dependent protein silent information regulator 2 (Sir2) is a deacetylase for histones and other proteins and a key regulator of life span in several organisms. Sirtuin (SIRT)1 of the Sirtuin family is the closest homolog of yeast Sir2 in mammals and has critical functions in the regulation of metabolism, genome stability, DNA repair, chromatin remodeling, and stress response (Guarente, 2011; Haigis and Sinclair, 2010). SIRT1 coordinates pluripotency, differentiation, and stress response in mouse embryonic stem Cyanine3.5 alkyne (ESCs) (Han et al., 2008). Whether SIRT1 regulates adult stem cells particularly in the hematopoietic system has been a matter of debate (Leko et al., Cyanine3.5 alkyne 2012; Li et al., 2012; Narala et al., 2008; Singh et al., 2013; Yuan et al., 2012). Despite recent advances in understanding SIRT1 regulation of malignant and stressed hematopoiesis, whether SIRT1 has any function in the control of adult HSC homeostasis or aging remains unknown.
The study of SIRT1 in adult mice and during aging has been hampered by the developmental defects and perinatal death of germline SIRT1 knockout mice (Cheng et al., 2003; McBurney et al., 2003). Using a recently developed adult tamoxifen-inducible SIRT1 knockout mouse model (Price et al., 2012), we show that SIRT1 is essential for the self-renewal and homeostatic maintenance of the HSC pool. Importantly, we show that loss of SIRT1 is associated with anemia and a significant expansion of the myeloid compartment, specifically granulocyte-monocyte progenitors (GMPs), at the expense of the lymphoid compartment. These phenotypic alterations are concomitant with significant modulations of expression of transcription factors implicated in the generation of GMPs and common lymphoid progenitors (CLPs). Notably, we show that the longevity transcription factor FOXO3 mediates SIRT1 homeostatic effects in HSCs. These unexpected results indicate that young SIRT1-deleted HSCs have several overlapping features with normal aged HSCs. Altogether, our studies identify SIRT1 as a key regulator of HSC maintenance under homeostasis. In addition, the evidence supports an essential function for SIRT1 in the regulation of HSC lineage specification. Overall, our findings suggest that SIRT1 might be implicated in delaying HSC aging.

Results

Discussion

Experimental Procedures

Acknowledgments

Introduction
Mesenchymal stromal cells (MSCs) are a promising cellular therapeutic for numerous disorders because of their anti-inflammatory/regenerative properties and the fact that they are readily expandable ex vivo (Copland and Galipeau, 2011). However, discrepancies in the therapeutic effectiveness of these cells as determined in phase II/III trials have called into question their therapeutic utility (Galipeau, 2013). A common practice in many MSC immunotherapy trials is to expand MSCs ex vivo and then cryogenically bank them until needed. The MSCs are then thawed and administered within a couple of hours to the patient. Until recently, the assumption was that viable post-thaw MSCs have physiological features comparable to those of their noncryopreserved counterparts. We have found that this premise may be flawed. Previously, we demonstrated that cryopreserved MSCs (cryo MSCs) have a blunted indoleamine 2,3-dioxygenase (IDO) response immediately post-thaw, which significantly reduced their immunomodulatory activity (François et al., 2012). In support of this idea are experimental data showing that when renal allograft recipients are treated with the IDO inhibitor 1-methyl-tryptophan or with IDO-deficient MSCs, tolerance is not established (Ge et al., 2010). Thus, MSCs must retain their capacity to make IDO to evoke an immunosuppressive effect.
For MSCs to have an immunosuppressive effect, they must not only actively respond to inflammatory cues but also persist/engraft within the body (Huang et al., 2010; Richardson et al., 2013; Sarkar et al., 2011). Systemic infusion of MSCs in nonhuman primates demonstrated that MSCs can take up residence in many tissues (Devine et al., 2003), but upon sensing an injury signal, MSCs will home to areas of inflammation (Li et al., 2002). Whether the means by which MSCs are prepared for administration impacts their homing/engraftment potential has not been rigorously evaluated. However, Castelo-Branco et al. (2012) demonstrated that transfused cryo MSCs could not migrate to an inflamed colon and had no beneficial effect in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model. This suggests that cryo MSCs may have an engraftment defect. In our efforts to optimize the therapeutic utility of MSCs, we compared the in vitro and in vivo binding/engraftment potential of human MSCs (hMSCs) thawed from cryopreservation with that of MSCs in active culture.

sulfanilamide Intestinal epithelial SCs IESCs are

Intestinal epithelial SCs (IESCs) are roughly categorized as either quiescent or active IESCs based in part on the expression of specific markers, including LGR5, Olfm4, Ascl2, BMI1, MTERT, and LRIG1 (Barker et al., 2007; Montgomery et al., 2011; Powell et al., 2012; Sangiorgi and Capecchi, 2008). They are believed to dynamically switch from one type to the other in response to inhibitory and stimulatory signals caused by cytokines, hormones, or growth factors (Li and Clevers, 2010). Active IESCs, the majority of which are LGR5+ crypt sulfanilamide columnar cells (CBCs), maintain intestinal lineage development and self-renewal with rapid cycling (Barker et al., 2007), and are highly sensitive to intestinal injury (Tian et al., 2011). In contrast, slow-cycling IESCs (label-retaining cells [LRCs]), which are present at the ‘‘+4 crypt position,’’ contribute to homeostatic regenerative capacity, particularly during recovery from injury (Takeda et al., 2011). These LRCs express markers such as BMI1, HOPX, LRIG1, and/or DCLK1, and can convert to rapidly cycling IESCs in response to injury (Yan et al., 2012). Signal transduction pathways, including WNT, NOTCH, TGF-β/BMP, Hedgehog, nuclear hormone receptor, and JAK-STAT, temporally and spatially regulate IESC homeostasis in cell-based tissue self-renewal and regeneration (Crosnier et al., 2006). Recent studies indicated that IESCs can regulate the intestinal homeostatic response to infection and inflammation (Buczacki et al., 2013). However, the mechanisms underlying this cellular regulation remain largely unknown.
JAK-STAT signaling was recently found to mediate IESC self-renewal and differentiation in response to bacterial infection and tissue impairment in Drosophila (Jiang et al., 2009). Compromised JAK-STAT signaling caused loss of IESC quiescence (Buchon et al., 2009), whereas JAK-STAT activation produced extra IESC-like and progenitor cells (Lin et al., 2010). However, the subsequent molecular events by which STAT signaling regulates adult IESCs are poorly defined in mammals. STAT5 activity, as well as its target genes, was predominantly associated with long-term self-renewal and maintenance of hematopoietic (Kato et al., 2005), mammary (Vafaizadeh et al., 2010), and embryonic SC (ESC) phenotypes (Kyba et al., 2003). Temporally controlled STAT5 expression and activation increased mammary SC proliferation, thereby contributing to the functional tissue formation upon chronic inflammatory injury (Vafaizadeh et al., 2010). We previously reported that epithelial STAT5 signaling is required for intestinal epithelial cell (IEC) integrity and homeostatic response to gut injury (Gilbert et al., 2012). Growth hormone (GH) and granulocyte macrophage-colony stimulating factor (GM-CSF) can protect IECs against inflammatory injury through activation of STAT5 (Han et al., 2007, 2010). These findings suggest that STAT5 signaling mediates IEC repopulation through regulation of somatic IESC proliferation or differentiation. Here, utilizing Stat5-modified transgenic mouse models and mouse or human SCs, we characterized the role of STAT5 in IESC homeostasis and response to injury, and deciphered the molecular machineries of STAT5 activation in protecting gut injury. Furthermore, our findings suggest that STAT5 activation could be used as a functional marker for IESC intervention of gut injury.

Results

Discussion
IESCs and intestinal progenitor cells maintain intestinal homeostasis and regeneration in response to gut injury (Zhang et al., 2014). LGR5+ IESCs play a critical role in intestinal homeostasis and regeneration (Metcalfe et al., 2014; Van Landeghem et al., 2012). Interestingly, BMI1+ IESCs are able to replenish LGR5+ IESC upon small-intestinal injury or regeneration (Yan et al., 2012). However, the molecular mechanisms that regulate these two IESC populations remain largely unexplored. Mucosal cytokines regulate IESC responses to inflammation, in part by JAK-STAT signaling (Farin et al., 2014; Jiang et al., 2009). In this study, we investigated whether cytokine-STAT5 signaling plays a role in modulation of these two IESC populations during IEC regeneration. Based on the combined results of our LOF and GOF studies of STAT5 in murine models with cultured mouse or human SCs, we propose a model in which, first, loss of STAT5 impairs rapidly cycling IESCs (Figure 7E-I), and second, genetic activation of Stat5 promotes CBC proliferation and regeneration (Figure 7E-II). However, our current data cannot exclude the potential effects of STAT5 signaling on intestinal progenitors or mucosal cytokine secretion. Interestingly, ChIP analyses identified STAT5 binding to the Bmi1 locus, suggesting that activated STAT5 could directly regulate key genes involved in IESC identity. Collectively, STAT5 controls adult IESC activity upon intestinal injury. PY-STAT5 could be developed as a biomarker for IESC regeneration of inflamed epithelia.

Whole exome sequencing of tumor DNA

Whole-exome sequencing of tumor DNA from GBM 5 identified a total of 32 SNVs (Table S3). A subset of these SNVs was selected for single-cell analysis based on putative gene function and variant allele fractions encompassing high, low, and intermediate frequencies. The genomic targets selected included SNVs in KCNH5, PLCB2, GDF5, TRMT5, TP53, and PALB2, and CNAs in CDKN2A, TP53, and EGFR.
Single-cell analysis for the simultaneous presence of six SNVs and three CNAs was carried out on flow-sorted neurosphere and xenograft tumor bcr-abl tyrosine kinase inhibitors from GBM 5 (a representative heatmap of the qPCR data from the BioMark HD is given in Figure S3). A comparison of the clonal phylogeny and subclonal architecture of neurosphere and xenograft cells is shown in Figure 4. Homozygous CDKN2A deletion, gain of EGFR (up to four copies), and KCN5, PLCB2, GDF5, and TRMT5 mutations all occurred early and were present in all subclones of the neurospheres (Figure 4A). Loss of one TP53 wild-type allele occurred after EGFR amplification of more than four copies. Heterozygous TP53 and PALB2 mutations occurred after further EGFR amplification. According to the chromosome 7 copy number as assessed by FISH (Figure S4) and single-cell data (not shown), EGFR gain was uncoupled from chromosome copy number at three or four copies of chromosome 7. Subsequently, there was an increasing gain of EGFR, consistent with the formation of extrachromosomal double minutes. All of the cells in the secondary mouse xenograft possessed all of the mutations, including heterozygous TP53 and PALB2 mutations, and most likely derived from two of the most evolved subclones in the neurospheres (being present in only 3.3% and 4%, respectively; Figure 4B). The subclone with one copy of TP53 mutant and more than ten copies of EGFR evolved further in the xenograft cells by acquiring two mutated copies of TP53, and all subclones evolved to show high-level amplification of EGFR (>100 copies) (Figure 4B).
We used secondary transplantation as a more stringent measure of stem cell renewal (Dick et al., 1997). In five cases (GBM 1, GBM 5, GBM 8, GBM 9, and GBM 11), we observed a statistically shorter time to tumor formation in the secondary transplant than in the primary xenograft tumor. This pattern of evolution is consistent with the typical pattern of disease progression seen in patients and would be consistent with the genetically more evolved subclonal structure observed in the neurospheres of GBM 5, GBM 8, and GBM 11, and with the presence of TP53 mutations in GBM 5 and GBM 8.
Clones with EGFR amplification consistently read out after serial transplantation, and usually further evolved with an incremental gain of more copies of EGFR. Other investigators have demonstrated a mosaic pattern of growth factor amplification in GBM tumors, with EGFR, MET, and PDGFRA gain occurring in distinct populations of cells (Snuderl et al., 2011; Szerlip et al., 2012). In the present study, there was one case (GBM 11) with subclones in the neurosphere cells that showed concurrent PDGFRA and EGFR gain in the same cell, as well as subclones with only EGFR gain (Figure 2). However, only the subclones with high-level EGFR amplification repopulated the mouse xenograft; none of the subclones with PDGFRA gain were present. These observations reveal the dynamic complexity of subclonal interactions in GBM and provide deeper insight into the role of PDGFRA. We previously showed that amplification of PDGFRA occurs in the midphase of GBM evolution (Sottoriva et al., 2013) rather than as a primary driver event. Evidence suggests that tumor-propagating clones may arise from a common precursor, with key early events including genetic alterations in EGFR, CDKN2A/B, and TP53 (Goodenberger and Jenkins, 2012; Snuderl et al., 2011; Sottoriva et al., 2013). These observations are supported by data from glioma susceptibility studies that revealed prominent roles for alterations in EGFR, CDKN2A, and TP53 in glioma evolution (Andersson et al., 2010; Egan et al., 2012; Shete et al., 2009; Stacey et al., 2011; Wrensch et al., 2009).

Human liver chimeric mice carrying human

Human liver chimeric mice carrying human pHH can facilitate in vivo study of viral hepatitis infections (Bissig et al., 2010; Carpentier et al., 2014) and liver genetic diseases (Bissig-Choisat et al., 2015; Yusa et al., 2011), and the testing of small molecular drug candidates and biological components including Obeticholic Acid manufacturer (Legrand et al., 2009). Embryonic stem cell (ESC)- or iPSC-derived iHeps have been used to generate chimeric mice by engrafting them into the liver of non-obese diabetic severe combined immunodeficient (NOD-SCID) mice (Chen et al., 2012), NOD/Lt-SCID/IL-2Rγ (NSG) mice (Liu et al., 2011), or uPA transgenic mice (Basma et al., 2009; Carpentier et al., 2014), where iHeps showed further maturation and regeneration potential and displayed key activities of primary hepatocytes. Our study has produced a mouse model that lacks the gene (Ldlr) involved in a human disease (FH) and is engrafted with patient-specific iHeps to mimic the specific human disease condition and perform in vivo drug testing. This approach has significant advantages over studies using Ldlr knockout mice alone (Ishibashi et al., 1993), as endogenous hepatocytes in the latter mouse model are not responsive to drugs modulating the LDLR pathway. A recent study showed engraftment of FH patient-specific iHeps injected subcutaneously rather than into the liver parenchyma of Rag1/Ldlr mice, but the authors did not perform an assay to demonstrate functional recovery (Ramakrishnan et al., 2015). Another report demonstrated the feasibility of directly engrafting FH (due to compound heterozygosity in LDLR gene) pHH into Fah/Rag2/Il2rg mice, and the phenotype of FH was successfully rescued by LDLR gene therapy using adeno-associated virus (Bissig-Choisat et al., 2015). Because of the lack of Fah, these mice had higher repopulation efficiency (>70%) (Bissig-Choisat et al., 2015) than our mice. However, a problem of this approach is that a population of mouse hepatocytes with intact LDLR remains while the engrafted patients\’ pHH did not contain functional LDLR, which can be confounding factors when performing in vivo testing of drugs acting on the LDLR pathway. Moreover, primary FH hepatocytes are difficult to obtain and cannot be expanded in vitro. Although liver repopulation in our chimeric mice is significantly lower than for Fah/Rag2/Il2rg mice engrafted with pHH (Azuma et al., 2007; Bissig-Choisat et al., 2015), it is comparable with results of recent reports describing NSG mice or Gunn rats (a model of Crigler-Najjar syndrome 1) engrafted with iPSC-derived iHeps (2%–17% [Liu et al., 2011] and 5.1% [Chen et al., 2015]). In the future, crossing our LRG mice with Fah knockout mice could help achieve higher liver chimerism, and thus further upgrade in vivo drug testing studies using engrafted FH iHeps. It should also be considered that mice and humans bear differences in lipoprotein metabolism, and therefore it may be desirable to genetically engineer other mammalian species (e.g., rabbits) (Shiomi and Ito, 2009) for more accurate studies using transplanted human FH iHeps. Nevertheless, despite the physiological differences between mice and humans and the moderate chimerism of our own model, the lowering of plasma LDL-C achieved with LDLR-competent iHeps was significant, while FH iHeps were less effective but exhibited good response to statins and particularly to PCSK9 antibodies. Besides plasma LDL-C levels, we measured endothelial function in aortas of LRG mice to assess the efficacy of engrafted iHeps and these two medications in vivo, as this parameter is affected early in patients with FH (Wiegman et al., 2004). Our results showed that cell therapy and the two medications could improve EDV in chimeric mice, indicating that endothelial function can be used as a parameter to evaluate disease progression during preclinical testing in chimeric LRG mice or other similar animal models. Importantly, we also noticed that the engrafted iHeps could be maintained in LRG mouse liver, and were functional, for at least 3 months. Overall, these results highlight the potential relevance of transplanting iPSC-derived iHeps for the treatment of hereditary metabolic liver disorders (Cantz et al., 2015), and the use of chimeric animals humanized with disease-specific iHeps for preclinical evaluation of novel therapies. However, the long-term therapeutic efficacy of iPSC-based approaches remains unclear, and future studies are necessary to address this as well as safety concerns.