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1.  Airway Epithelial Progenitors Are Region Specific and Show Differential Responses to Bleomycin-Induced Lung Injury 
Stem cells (Dayton, Ohio)  2012;30(9):1948-1960.
Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFPhi cells were subdivided by CD24 staining into alveolar (CD24neg) and conducting airway (CD24low) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFPneg and GFPlow progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24lowGFPhi progenitors were present in an overlapping distribution with GFPlow progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24lowGFPhi progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling. Disclosure of potential conflicts of interest is found at the end of this article.
doi:10.1002/stem.1150
PMCID: PMC4083019  PMID: 22696116
Regional; Progenitor; Epithelium; Lung; Bleomycin
2.  Induced pluripotent stem cells with a pathological mitochondrial DNA deletion 
Stem cells (Dayton, Ohio)  2013;31(7):1287-1297.
In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.
doi:10.1002/stem.1354
PMCID: PMC3692613  PMID: 23400930
Induced pluripotent stem cells; Pearson's marrow-pancreas syndrome; Mitochondrial DNA; Heteroplasmy; Human genetics; Hematopoiesis
3.  Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a MELAS patient 
Stem cells (Dayton, Ohio)  2013;31(7):1298-1308.
Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.
doi:10.1002/stem.1389
PMCID: PMC3706526  PMID: 23553816
mitochondria; mitochondrial DNA; induced pluripotent stem cells; MELAS syndrome; regenerative medicine
4.  Pursuing Self-Renewal and Pluripotency with the Stem Cell Factor Nanog 
Stem cells (Dayton, Ohio)  2013;31(7):1227-1236.
Pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for future use in tissue replacement therapies due to their ability to self-renew indefinitely and to differentiate into all adult cell types. Harnessing this therapeutic potential efficiently requires a much deeper understanding of the molecular processes at work within the pluripotency network. The transcription factors Nanog, Oct4, and Sox2 reside at the core of this network, where they interact and regulate their own expression as well as that of numerous other pluripotency factors. Of these core factors, Nanog is critical for blocking the differentiation of pluripotent cells, and more importantly, for establishing the pluripotent ground state during somatic cell reprogramming. Both mouse and human Nanog are able to form dimers in vivo, allowing them to preferentially interact with certain factors and perform unique functions. Recent studies have identified an evolutionary functional conservation among vertebrate Nanog orthologs from chick, zebrafish, and the axolotl salamander, adding an additional layer of complexity to Nanog function. Here we present a detailed overview of published work focusing on Nanog structure, function, dimerization, and regulation at the genetic and post-translational levels with regard to the establishment and maintenance of pluripotency. The full spectrum of Nanog function in pluripotent stem cells and in cancer is only beginning to be revealed. We therefore use this evidence to advocate for more comprehensive analysis of Nanog in the context of disease, development, and regeneration.
doi:10.1002/stem.1384
PMCID: PMC3706551  PMID: 23653415
ESCs; iPSCs; pluripotency; Nanog; self-renewal; reprogramming
5.  IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem Cell Impairment and Tumorigenesis via NFκB Signaling 
Stem cells (Dayton, Ohio)  2013;31(7):1383-1395.
An inflammatory microenvironment may cause organ degenerative diseases and malignant tumors. However, the precise mechanisms of inflammation-induced diseases are not fully understood. Here we show that the proinflammatory cytokines interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) synergistically impair self-renewal and differentiation of mesenchymal stem cells (MSCs) via nuclear factor κB (NFκB)–mediated activation of Mothers against decapentaplegic homolog 7 (SMAD7) in ovariectomized (OVX) mice. More interestingly, a long-term elevated levels of IFN-γ and TNF-α result in significantly increased susceptibility to malignant transformation in MSCs through NFκB–mediated upregulation of the oncogenes c-Fos and c-Myc. Depletion of either IFN-γ or TNF-α in OVX mice abolishes MSC impairment and the tendency toward malignant transformation with no NFκB–mediated oncogene activation. Systemic administration of aspirin, which significantly reduces the levels of IFN-γ and TNF-α, results in blockage of MSC deficiency and tumorigenesis by inhibition of NF-κB/SMAD7 and NFκB/c-FOS and c-MYC pathways in OVX mice. In summary, this study reveals that inflammation factors, such as IFN-γ and TNF-α, synergistically induce MSC deficiency via NFκB/SMAD7 signaling and tumorigenesis via NFκB–mediated oncogene activation.
doi:10.1002/stem.1388
PMCID: PMC3706580  PMID: 23553791
Mesenchymal stem cells; Stem cell-microenvironment interactions; Differentiation; Cancer; Cytokines
6.  NPRA Signaling Regulates Stem Cell Recruitment and Angiogenesis: A Model to Study Linkage Between Inflammation and Tumorigenesis 
Stem cells (Dayton, Ohio)  2013;31(7):1321-1329.
Natriuretic peptide receptor A (NPRA), the signaling receptor for the cardiac hormone, atrial natriuretic peptide (ANP), is expressed abundantly in inflamed/injured tissues and tumors. NPRA deficiency substantially decreases tissue inflammation and inhibits tumor growth. However, the precise mechanism of NPRA function and whether it links inflammation and tumorigenesis remains unknown. Since both injury repair or tumor growth require stem cell recruitment and angiogenesis, we examined the role of NPRA signaling in tumor angiogenesis as a model of tissue injury repair in this study. In in vitro cultures aortas from NPRA-KO mice show significantly lower angiogenic response compared to wild type counterparts. The NPRA antagonist that decreases NPRA expression, inhibit lipopolysaccharide-induced angiogenesis. The reduction in angiogenesis correlates with decreased expression of vascular endothelial growth factor (VEGF) and chemokine (C-X-C motif) Receptor 4 (CXCR4) implicating a cell recruitment defect. To test whether NPRA regulates migration of cells to tumors, mesenchymal stem cells (MSCs) were administered i.v. and the results showed that MSCs fail to migrate to the tumor microenvironment in NPRA-KO mice. However, co-implanting tumor cells with MSCs, increases angiogenesis and tumorigenesis in NPRA-KO mice, in part by promoting expression of CXCR4 and its ligand, stromal-derived factor 1α (SDF1α). Taken together, these results demonstrate that NPRA signaling regulates stem cell recruitment and angiogenesis leading to tumor growth. Thus, NPRA signaling provides a key linkage between inflammation and tumorigenesis, and NPRA may be a target for drug development against cancers and tissue injury repair.
doi:10.1002/stem.1376
PMCID: PMC3982194  PMID: 23533187
7.  Blockade of peroxynitrite-induced neural stem cell death in the acutely injured spinal cord by drug-releasing polymer 
Stem cells (Dayton, Ohio)  2009;27(5):1212-1222.
Therapeutic impact of neural stem cells (NSCs) for acute spinal cord injury (SCI) has been limited by the rapid loss of donor cells. Neuroinflammation is likely the cause. Since there are close temporal-spatial correlations between the inducible nitric oxide (NO) synthase expression and the donor NSC death after neurotrauma, we reasoned that NO-associated radical species might be the inflammatory effectors which eliminate NSC grafts and kill host neurons. To test this hypothesis, human NSCs (hNSCs: 5×104-2×106/ml) were treated in vitro with “plain” medium, 20 μM glutamate, or donors of NO and peroxynitrite (ONOO-; 100 and 400 μM of spermine or DETA NONOate, and SIN-1, respectively). hNSC apoptosis primarily resulted from SIN-1 treatment, showing ONOO--triggered protein nitration and the activation of p38 MAPK, cytochrome c release, and caspases. Therefore, cell death following post-SCI (p.i.) NO serge may be mediated through conversion of NO into ONOO-. We subsequently examined such causal relationship in a rat model of dual penetrating SCI using a retrievable design of poly-lactic-co-glycolic acid (PLGA) scaffold seeded with hNSCs that was shielded by drug-releasing polymer. Besides confirming the ONOO--induced cell death signaling, we demonstrated that co-transplantation of PLGA film embedded with ONOO- scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) or uric acid (1 μmol/film), markedly protected hNSCs 24 h p.i. (total: n = 10). Our findings may provide a bioengineering approach for investigating mechanisms underlying the host microenvironment and donor NSC interaction and help formulate strategies for enhancing graft and host cell survival after SCI.
doi:10.1002/stem.26
PMCID: PMC4070520  PMID: 19418456
spinal cord injury; neural stem cell; nitric oxide; peroxynitrite; PLGA scaffold; neuroinflammation
8.  IFATS Collection: Combinatorial Peptides Identify α5β1 Integrin as a Receptor for the Matricellular Protein SPARC on Adipose Stromal Cells 
Stem cells (Dayton, Ohio)  2008;26(10):2735-2745.
The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the α5β1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to α5β1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on α5β1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that α5β1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides.
doi:10.1634/stemcells.2008-0212
PMCID: PMC4066418  PMID: 18583538
Adipose stromal cells; Extracellular matrix; Mobilization; Peptide phage display
9.  G9a and Jhdm2a Regulate Embryonic Stem Cell Fusion-Induced Reprogramming of Adult Neural Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(8):2131-2141.
Somatic nuclei can be reprogrammed to pluripotency through fusion with embryonic stem cells (ESCs). The underlying mechanism is largely unknown, primarily because of a lack of effective approaches to monitor and quantitatively analyze transient, early reprogramming events. The transcription factor Oct4 is expressed specifically in pluripotent stem cells, and its reactivation from somatic cell genome constitutes a hallmark for effective reprogramming. Here we developed a double fluorescent reporter system using engineered ESCs and adult neural stem cells/progenitors (NSCs) to simultaneously and independently monitor cell fusion and reprogramming-induced reactivation of transgenic Oct4-enhanced green fluorescent protein (EGFP) expression. We demonstrate that knockdown of a histone methyltransferase, G9a, or overexpression of a histone demethylase, Jhdm2a, promotes ESC fusion-induced Oct4-EGFP reactivation from adult NSCs. In addition, coexpression of Nanog and Jhdm2a further enhances the ESC-induced Oct4-EGFP reactivation. Interestingly, knockdown of G9a alone in adult NSCs leads to demethylation of the Oct4 promoter and partial reactivation of the endogenous Oct4 expression from adult NSCs. Our results suggest that ESC-induced reprogramming of somatic cells occurs with coordinated actions between erasure of somatic epigenome and transcriptional resetting to restore pluripotency. These mechanistic findings may guide more efficient reprogramming for future therapeutic applications of stem cells.
doi:10.1634/stemcells.2008-0388
PMCID: PMC4059405  PMID: 18535151
Neural stem cells; Embryonic stem cells; Reprogramming; Oct4; Histone methyltransferase; Histone demethylase; G9a; Jhdm2a
10.  Injectable Bone Tissue Engineering Using Expanded Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2013;31(3):572-580.
Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, very few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone composed of mesenchymal stem cells (MSCs) and platelet rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out pilot trial cases in patients with long-term follow up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months was significantly higher than the pre-operation baseline (P <0.001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long term follow-up. Injectable tissue-engineered bone restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering.
doi:10.1002/stem.1300
PMCID: PMC4043220  PMID: 23225744
tissue engineering; regenerative medicine; bone; cell transplantation; clinical application
11.  The Sox2-Oct4 Connection: Critical players in a much larger interdependent network integrated at multiple levels 
Stem cells (Dayton, Ohio)  2013;31(6):1033-1039.
The transcription factors Sox2 and Oct4 have been a major focus of stem cell biology since the discovery, more than 10 years ago, that they play critical roles during embryogenesis. Early work established that these two transcription factors work together to regulate genes required for the self-renewal and pluripotency of embryonic stem cells (ESC). Surprisingly, small changes (~2-fold) in the levels of either Oct4 or Sox2 induces the differentiation of ESC. Consequently, ESC must maintain the levels of these two transcription factors within narrow limits. Genome-wide binding studies and unbiased proteomic screens have been conducted to decipher the complex roles played by Oct4 and Sox2 in the transcriptional circuitry of ESC. Together, these and other studies provide a comprehensive understanding of the molecular machinery that sustains the self-renewal of ESC and restrains their differentiation. Importantly, these studies paint a landscape in which Oct4 and Sox2 are part of a much larger interdependent network composed of many transcription factors that are interconnected at multiple levels of function.
doi:10.1002/stem.1352
PMCID: PMC3664101  PMID: 23401375
embryonic stem cells; iPS cells; Sox2; Oct4; transcription factors; proteomics; gene regulatory networks; transcriptional circuitry; DNA repair; reprogramming; self-renewal
12.  Matrix remodeling maintains ESC self-renewal by activating Stat3 
Stem cells (Dayton, Ohio)  2013;31(6):1097-1106.
While a variety of natural and synthetic matrices have been used to influence embryonic stem cell (ESC) self-renewal or differentiation, and ESCs also deposit a rich matrix of their own, the mechanisms behind how extracellular matrix affects cell fate are largely unexplored. The ESC matrix is continuously remodeled by matrix metalloproteinases (MMPs), a process that we find is enhanced by the presence of mouse embryonic fibroblast feeders in a paracrine manner. Matrix remodeling by MMPs aids in the self-renewal of ESCs, as inhibition of MMPs inhibits the ability of ESCs to self-renew. We also find that addition of the interstitial collagenase MMP1 is sufficient to maintain long-term LIF-independent mESC self-renewal in a dose-dependent manner. This remarkable ability is due to the presence of endogenously produced self-renewal-inducing signals, including the LIF-family ligand CNTF, that are normally trapped within the ECM and become exposed upon MMP-induced matrix remodeling to signal through JAK and Stat3. These results uncover a new role for feeder cells in maintaining self-renewal and show that mESCs normally produce sufficient levels of autocrine-acting pro-self-renewal ligands.
doi:10.1002/stem.1360
PMCID: PMC3664106  PMID: 23404867
embryonic stem cell; extracellular matrix; matrix metalloproteinase; self-renewal; Stat3
13.  Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells 
Stem cells (Dayton, Ohio)  2013;31(6):1202-1212.
The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In the current study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) over-expressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1−/−, Pim2−/−, Pim3−/− single knockout (KO) mice. HSCs from Pim1−/− KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2−/− or Pim3−/− KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34− HSCs was reduced by ~28-fold in Pim1−/− KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our PCR array and confirmatory RT-PCR studies identified several genes including Lef-1, Pax5 and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.
doi:10.1002/stem.1369
PMCID: PMC3664117  PMID: 23495171
Serine/threonine kinase; Pim kinase; Hematopoietic stem cells; Hematopoietic stem cell transplantation; Apoptosis; Proliferation; Transgenic mouse; Knockout mouse
15.  Mechanically Activated Fyn Utilizes mTORC2 to Regulate RhoA and Adipogenesis in Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2013;31(11):2528-2537.
Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of β-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.
doi:10.1002/stem.1476
PMCID: PMC4040149  PMID: 23836527
Src; Fyn; FAK; RhoA; Mesenchymal stem cells; Adipogenesis
16.  REST regulates oncogenic properties of glioblastoma stem cells 
Stem cells (Dayton, Ohio)  2012;30(3):405-414.
Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self-renewing, glioblastoma-derived stem-like cells (GSCs), the mechanisms that regulate self-renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor REST, suggesting heterogeneity across different GSC lines. Loss- and gain-of-function experiments indicated that REST maintains self-renewal of GSCs. High REST-expressing GSCs (HR-GSCs) produced tumors histopathologically distinct from those generated by low REST-expressing GSCs (LR-GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR-GSCs resulted in increased survival in GSC-transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR-GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR-GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self-renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM.
doi:10.1002/stem.1020
PMCID: PMC4039365  PMID: 22228704
REST; glioblastoma stem cells; neural stem cells; glioblastoma multiforme; invasion; self-renewal
17.  Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium 
Stem cells (Dayton, Ohio)  2008;26(5):1315-1324.
Cumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population. Green fluorescent protein (GFP) driven by the c-kit promoter labels a fraction of the c-kit + cells recognized by antibody labeling for c-kit protein. Expression of GFP by the c-kit promoter and accumulation of GFP-positive cells in the myocardium is relatively high at birth compared with adult and declines between postnatal weeks 1 and 2, which tracks in parallel with expression of c-kit protein and c-kit-positive cells. Acute cardiomyopathic injury by infarction prompts increased expression of both GFP protein and GFP-labeled cells in the region of infarction relative to remote myocardium. Similar increases were observed for c-kit protein and cells with a slightly earlier onset and decline relative to the GFP signal. Cells coexpressing GFP, c-kit, and cardiogenic markers were apparent at 1–2 weeks postinfarction. Cardiac-resident c-kit+ cell cultures derived from the transgenic line express GFP that is diminished in parallel with c-kit by induction of differentiation. The use of genetically engineered mice validates and extends the concept of c-kit+ cells participating in the response to myocardial injury.
doi:10.1634/stemcells.2007-0751
PMCID: PMC4037162  PMID: 18308948
c-kit; Heart; Infarction; Cardiac stem cell
18.  Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells (iPSC) 
Stem cells (Dayton, Ohio)  2012;30(5):997-1007.
Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression which may impact their capacity to differentiate into cell of origin. Here we addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse fetal fibroblasts (MEF-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPS cell lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay and normal diploid karyotype. However, HB-iPSCs were more efficient in directed differentiation towards hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs or mESCs. Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (7 up- and 17 down-regulated genes) typical of the original cells. Continuous passaging of HB-iPSCs erased most of these differences including a superior capacity for hepatic re-differentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development.
doi:10.1002/stem.1074
PMCID: PMC4017241  PMID: 22378611
induced pluripotent stem cells; donor memory; hepatocyte lineage cells; hepatic differentiation
19.  Concise Review: Maturation Phases of Human Pluripotent Stem Cell-Derived Cardiomyocytes 
Stem cells (Dayton, Ohio)  2013;31(5):10.1002/stem.1331.
Human pluripotent stem cell-derived cardiomyocytes (hPS-CM) may offer a number of advantages over previous cardiac models, however, questions of their immaturity complicate their adoption as a new in vitro model. hPS-CM differ from adult cardiomyocytes with respect to structure, proliferation, metabolism and electrophysiology, better approximating fetal cardiomyocytes. Time in culture appears to significantly impact phenotype, leading to what can be referred to as early and late hPS-CM. This work surveys the phenotype of hPS-CM, including structure, bioenergetics, sensitivity to damage, gene expression, and electrophysiology, including action potential, ion channels, and intracellular calcium stores, while contrasting fetal and adult CM with hPS-CM at early and late time points after onset of differentiation.
doi:10.1002/stem.1331
PMCID: PMC3749929  PMID: 23355363
Cardiomyocyte; Pluripotent stem cells; Phenotype; In vitro cell culture; Maturity
20.  Early Embryonic Erythropoiesis … Not so Primitive After All 
Stem cells (Dayton, Ohio)  2013;31(5):849-856.
In the developing embryo, hematopoiesis begins with the formation of primitive erythroid cells (EryP), a distinct and transient red blood cell lineage. EryP play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Progenitors for EryP arise as a cohort within the blood islands of the mammalian yolk sac at the end of gastrulation. As a strong heartbeat is established, nucleated erythroblasts begin to circulate and to mature in a stepwise, nearly synchronous manner. Until relatively recently, these cells were thought to be “primitive” in that they seemed to more closely resemble the nucleated erythroid cells of lower vertebrates than the enucleated erythrocytes of mammals. It is now known that mammalian EryP do enucleate, but not until several days after entering the bloodstream. I will summarize the common and distinguishing characteristics of primitive versus definitive (adult type) erythroid cells, review the development of EryP from the emergence of their progenitors through maturation and enucleation, and discuss pluripotent stem cells as models for erythropoiesis. Erythroid differentiation of both mouse and human pluripotent stem cells in vitro has thus far reproduced early but not late red blood cell ontogeny. Therefore, a deeper understanding of cellular and molecular mechanisms underlying the differences and similarities between the embryonic and adult erythroid lineages will be critical to improving methods for production of red blood cells for use in the clinic.
doi:10.1002/stem.1342
PMCID: PMC3637392  PMID: 23361843
primitive erythropoiesis; transgenic mice; mammalian embryo; erythroid progenitors; hemangioblast; erythroid differentiation; enucleation
21.  Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development 
Stem cells (Dayton, Ohio)  2013;31(5):1010-1021.
Id2 is a helix-loop-helix (HLH) transcription factor essential for normal development and its expression is dysregulated in many human neurological conditions. Although it is speculated that elevated Id2 levels contribute to the pathogenesis of these disorders, it is unknown whether dysregulated Id2 expression is sufficient to perturb normal brain development or function. Here, we show that mice with elevated Id2 expression during embryonic stages develop microcephaly, and that females in particular are prone to generalized tonic-clonic seizures. Analyses of Id2 transgenic brains indicate that Id2 activity is highly cell context specific: elevated Id2 expression in naive NSCs in early neuroepithelium induces apoptosis and loss of NSCs and intermediate progenitors. Activation of Id2 in maturing neuroepithelium results in less severe phenotypes and is accompanied by elevation of G1 Cyclin expression and p53 target gene expression. In contrast, activation of Id2 in committed intermediate progenitors has no significant phenotype. Functional analysis with Id2 over-expressing and Id2-null NSCs shows that Id2 negatively regulates NSC self-renewal in vivo, in contrast to previous cell culture experiments. Deletion of p53 function from Id2-transgenic brains rescues apoptosis and results in increased incidence of brain tumors. Furthermore, Id2 over-expression normalizes the increased self-renewal of p53-null NSCs, suggesting that Id2 activates and modulates the p53 pathway in NSCs. Together, these data suggest that elevated Id2 expression in embryonic brains can cause deregulated NSC self-renewal, differentiation and survival that manifest in multiple neurological outcomes in mature brains, including microcephaly, seizures, and brain tumors.
doi:10.1002/stem.1351
PMCID: PMC3637429  PMID: 23390122
neural stem cells; Id2; brain tumor; glioma; medulloblastoma; self-renewal; apoptosis; seizure; CyclinG1; Rett syndrome
22.  Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue 
Stem cells (Dayton, Ohio)  2013;31(5):992-1000.
Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knock-in allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of circumvallate and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue.
doi:10.1002/stem.1338
PMCID: PMC3637415  PMID: 23377989
Lgr5; Adult taste stem cell; progenitor; Taste bud; Lineage tracing
23.  Lmo2 induces hematopoietic stem cell like features in T-cell progenitor cells prior to leukemia 
Stem cells (Dayton, Ohio)  2013;31(5):882-894.
LIM domain Only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells and Early T-cell Precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the hematopoietic stem cell. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.
doi:10.1002/stem.1345
PMCID: PMC3652616  PMID: 23378057
T cells; hematopoiesis; stem cells; leukemia
24.  NOTCH1 is Required for Regeneration of Clara Cells During Repair of Airway Injury 
Stem cells (Dayton, Ohio)  2012;30(5):946-955.
The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the target of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells and repair and reestablishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/Enhancer of Split-5 (Hes5) and a paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.
doi:10.1002/stem.1059
PMCID: PMC4005608  PMID: 22331706
Notch1; PGP9.5; UCHL1; Hes5; Lung morphogenesis; Epithelial repair; Naphthalene; Injury; Clara cells; Pulmonary Neuroendocrine; Stem cells; Progenitor cells; Differentiation; Cell fate
25.  Mesenchymal Stem Cell (MSC) mediated delivery of the Sodium Iodide Symporter (NIS) supports radionuclide imaging and treatment of breast cancer 
Stem cells (Dayton, Ohio)  2011;29(7):1149-1157.
Mesenchymal Stem Cells (MSCs) migrate specifically to tumors in vivo, and coupled with their capacity to bypass immune surveillance, are attractive vehicles for tumor-targeted delivery of therapeutic agents. This study aimed to introduce MSC-mediated expression of the sodium iodide symporter (NIS) for imaging and therapy of breast cancer. Tumor bearing animals received an intravenous or intratumoral injection of NIS expressing MSCs (MSC-NIS), followed by 99mTcO4- imaging 3-14Days (D) later using a BazookaSPECT γ-camera. Tissue was harvested for analysis of hNIS expression by RQPCR. Therapy animals received an intraperitoneal injection of 131I or saline 14D following injection of MSC-NIS, and tumor volume was monitored for 8 weeks. BazookaSPECT imaging following injection of MSC-NIS revealed an image of animal intestines and chest area at D3, with a weak tumor image also visible. By D14, the tumor was visible with a significant reduction in radionuclide accumulation in non-target tissue observed. hNIS gene expression was detected in the intestines, heart, lungs and tumor at early timepoints but later depleted in non-target tissues and persisted at the tumor site. Based on imaging/biodistribution data, animals received a therapeutic dose of 131I 14D following MSC-NIS injection. This resulted in a significant reduction in tumor growth (Mean ± SEM, 236 ± 62mm3 versus 665 ± 204 mm3 in controls). The ability to noninvasively track MSC migration and transgene expression in real time prior to therapy is a major advantage to this strategy. This promising data supports the feasibility of this approach as a novel therapy for breast cancer.
doi:10.1002/stem.665
PMCID: PMC3998644  PMID: 21608083
Sodium Iodide Symporter (NIS); Mesenchymal Stem Cell (MSC); Breast Cancer; Gene therapy; In vivo Imaging; Radiotherapy

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